Advanced biological active polyfunctional compounds and composites: Health, cultural heritage and environmental protection

ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

ADVANCED BIOLOGICALLY ACTIVE POLYFUNCTIONAL COMPOUNDS AND COMPOSITES: HEALTH, CULTURAL HERITAGE AND ENVIRONMENTAL PROTECTION

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ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

ADVANCED BIOLOGICALLY ACTIVE POLYFUNCTIONAL COMPOUNDS AND COMPOSITES: HEALTH, CULTURAL HERITAGE AND ENVIRONMENTAL PROTECTION

NODAR LEKISHVILI, GENNADY ZAIKOV AND

BOB HOWELL EDITORS

Nova Science Publishers, Inc. New York

Copyright © 2010 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Available upon request ISBN: 978-1-61209-092-4 (eBook)

Published by Nova Science Publishers, Inc.  New York

CONTENTS Preface

ix

Part 1.

Chemistry, Use and Molecular Modelling of Biologically Active Compounds

Chapter 1

Molecular Design and Reactivity of the 1-Hydroxycyclohexyl Hydroperoxide - Alk4nbr Complexes N. A. Turovskij, E. V. Raksha, E. N. Pasternak, I. A. Opeida, and G. E. Zaikov

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Reception of Physiologically Active Substances by Plasmatic Membrane of Vegetable Cell Jaba Oniani, Vladimir Yurin, Ramaz Gakhokidze, Tea Mchedluri, Tamar Oniani and Lela Abadovskaya The Influence of Caffeine Analogues and Antagonists on the Ca2+Accumulation by Sarcoplasmic Reticulum Olga M. Alekseeva, Yuri A. Kim and Vladimir A. Rykov Biologically Active Multifunctional Adamantane-Containing Compounds Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia, Giorgi Lekishvili, Badri Arziani, Iuri Sadaterashvili and Davit Zurabishvili Influence of Some Metal-Cations on the Molecular Organization of DNA N. Vasilieva-Vashakmadze, G. Lekishvili, R. Gakhokidze and P. Toidze State of Lipid Components of Soybean Flour Enzymatic Hydrolyzates during Storage L. N. Shishkina, E. V. Miloradova, E. A. Badichko and S. E. Traubenberg Participation of Aromatic Amines in the Maillard Reaction R. Kublashvili and D. Ugrekhelidze

1

11

25

35

61

69

81

vi Chapter 8

Contents Influence on Oxidation Processes Regulation Is the Reason for Biological Activity of the Ecdysteroid-Containing Compounds L. N. Shishkina, O. G. Shevchenko and N. G. Zagorskaya

Part 2.

Physics, Thermodynamics and Kinetics of Homogeneous and Heterogeneous Nanosystems

Chapter 9

Charge Transfer Mechanisms at Sam-Modified Electrodes Impact of Complex Environments Dimitri E. Khoshtariya, Tina D. Dolidze and Rudi van Eldik

Chapter 10

Chapter 11

Chapter 12

Chapter 13

Chapter 14

Chapter 15

Stability of Drug Delivery PLGA Nanoparticles: Calorimertric Approach Tamaz Mdzinarashvili, Mariam Khvedelidze, Tamar Partskhaladze, Mark Schneider, Ulrich F. Schaefer, Noha Nafee and Claus-Michael Lehr

87

103

129

The Tacticity Governed Stereomicrostructure in Poly(Methyl Methacrylate) (Pmma) as a Way to Explain Its Physical Properties N. Guarrotxena

147

Kinetics of the Fermentative Reaction of H2o2 Decomposition under the Action of Catalase in the Presence of Biosas for the Stationary State A. A. Turovsky, R. O. Khvorostetsky,  L. I. Bazylyak and G. E. Zaikov

155

Thermodynamic and Kinetics of the Metalporphyrin – Base Reactions Tatyana N. lomova

167

Kinetics of the Fermentative Process in Stationary State for Sunflower-Seed Oil Hydrolysis by Lipase in the Presence of Biosas A. A. Turovsky, R. O. Khvorostetsky, L. I. Bazylyak and G. E. Zaikov Dynamic Trend of Energy Exchange Intensity in Brain Under Chronic Stress Nana Koshoridze

Part 3.

New Compounds for Medicine

Chapter 16

Synthesis and Transformation of New Anemia-Opposite Adamantane Derivatives of Ferrocene Oliko Lekashvili, Davit Zurabishvili, Levan Asatiani and Nodar Lekishvili

179

185

195

Contents Chapter 17

Pharmacological Premises of the Creation of New Antitumor Preparations of the Class of Nitrosoalkylurea J. A. Djamanbaev, Ch. Kamchybekova, J. A. Abdurashitova and G. E. Zaikov

Part 4.

Biofibers

Chapter 18

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions C. Vasile, M. Totolin and M. C. Tibirna

Chapter 19

Specific Properties of Some Biological Composite Materials N. Barbakadze, E. Gorb and S. Gorb

Part 5.

Compounds for Antibiocorrosive Covers and Protectors

Chapter 20

Antibiocorrosive Covers and Conservators Based on New Carbofunctional Oligosiloxanes and Biologically Active Compounds Nodar Lekishvili, Khatuna Barbakadze, David Zurabishvili, Tea Lobzhanidze, Shorena Samakashvili, Zurab Pachulia and Zurab Lomtatidze

Chapter 21

Synthesis, Biocide Properties and Structures of Some Arsonium Polyiodides for Antibiocorrosive Covers L. Arabuli, N. Lekishvili and M. Rusia

Part 6.

Environmental Chemistry

Chapter 22

Antimutageni and Anticytotoxic Activity of Bioenergoactivators Ramaz Gakhokidze and Amiran Pirtskhelani

Part 7.

Personally

Chapter 23

The Scientist who Has Outstripped His Time Revaz Skhiladze, Tengiz Tsivtsivadze and Bachana Pichkhaia

Index

vii

201

209 243

275

295

309

319 331

PREFACE During the last decade, researchers working within the field of biologically active compounds were attracted to finding new compounds, materials and methods that could be used toward the protection of human health and environment, along with the preservation of cultural heritage from various microorganisms, viruses and fungi. The actuality of this problem was stressed at the 41st congress of IUPAC devoted to these and related topics. Many actual aspects on creation of new compounds were discussed, materials and methods capable of more effective solving of the aforementioned problems. The topics that are discussed in this book encompass novel bioactive systems for human and environmental protection and preservation of cultural heritage from the microorganisms’ and fungi attacks. Also presented are the results of theoretical and experimental investigations conducted by the experienced scientists from different countries, along with the mechanistic comprehension of their performance under various experimental conditions. Chapter 1 - Chemically activated 1-hydroxycyclohexyl hydroperoxide decomposition in the presence of ammonium salts is proposed to proceed through the complexation stage. Complex structure and reactivity have been investigated by molecular modelling methods. Kinetics of the chemically activated hydroperoxide decomposition in the presence of quaternary ammonium salts (Et4NBr, Pr4NBr, Bu4NBr, and Hex4NBr) has been studied. The correlation between reactivity and structural characteristics of ammonium cations was found. Chapter 2 - Electrical phenomena and cytoplasm movement on an example of seaweed Characea have been briefly described. The results concerning cyclosis nascent mechanism in the cells of seaweed Characea and connection of cytoplasm movement velocity (CMV) with the difference of electrical potentials (DEP) of plasmatic membrane have been adduced. For specification of the appropriateness of interconnection of the cyclosis and DEP, the authors carried out the simultaneous measurement. A suggestion was made for a model in the frame of which it is possible to set up, in every separate case, a way to realize the observed effects by over regulation of the membrane potentials, i.e., finally by changing of ion penetrability of the membrane and in what—due to presence of other influences (for example, by influence on the difference intercellular processes). For ascertainment of the possibility of the presence on the plasmatic membrane of vegetable cell structures, similar to animal receptors and the interaction character of them, effectors, a detailed research had been conducted of acetylcholine acting and some biogenic amines on the velocity of cytoplasm and DEP of cell of seaweed Characea. Nitella flexilis.

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Results show that a reaction of the cell on adrenomimetics, active in respect to α and βreceptors, is discerned by sign. Investigation of combined action of adrenomimetics and αtype blockaders (nor adrenalin-fenitron) and β-type (isadrin – dihydroergotoxin) showed the strongly pronounced antagonistic effect, realizing by concrete mechanism. There were adduced also other proofs allowing for the existence of interacting centers in the cells with molecules of catecholamines, similar to α and β-receptors of animal cells; while this resemblance is spread as on the structure organization, so it is on some functional features. Data about the presence of cAMF, cGMF, enzymes of their synthesis and catabolism (adenilatciclase, gaunilatciclase, phospodietherase) and protein-target – proteincinases, also G-protein, allowed authors of the work to make the conclusion about the specific interaction of acetylcholine and biogenic amines with the specialized structures of plasmatic membrane of vegetable cells and signal transmitting by trigger mechanisms on the G-protein with following participation of secondary intermediary messengers. Following from the above-stated consideration, the authors proposed the presumptive conclusions: There is revealed the phenomenon of receptor regulation of intercellular physiologic processes of vegetable cells. In the experiments with biogenic amines was stated the appropriateness in the direct correlation between alteration of CMV and DEP. The cells of Nitellaceae can be used in the model experiments for testing of biological active substances, inasmuch as there was revealed by them the property to react by constellation receptor system on the exogenous influence of low concentration of testing compounds. All those presuppositions show that in the plant cells is the presence of contractive proteins in view of actinomyozin complexes, otherwise who may answer the question which is open till today: What gives the first push to the cytoplasm particles to move constantly along the perimeter of the vegetable cell? Chapter 3 - This study investigates the actions of caffeine analogues and antagonists to the main Ca2+-depo in the rabbit skeletal muscle - sarcoplasmic reticulum. The efficiency of Ca2+-accumulation by vesicles of heavy fragmented sarcoplasmic reticulum was greatly changed by caffeine analogues and antagonist: cordiamin, camphor, bemegrid, 8methylcaffeine, teophilline, theobromine, midocalm. All tested substances have some similar characteristics at its molecular structure. The carbonyl oxygen and methyl groups are of a great importance for “caffeine effect” of these substances. Chapter 4 - New biologically active adamantane-containing anilides and nitroanilides have been synthesized and studied. By the semiempirical quantum-chemical method, AM1 effective charges on the atoms, bonds lengths and valence angles, enthalpies of formation of initial compounds and probable obtained products of the reaction of nitration of the anilides have been calculated. Based on quantum-chemical calculations and experimental data, the direction of the reaction of nitration has been established. To model physical properties of biologically active 16 anilides and nitroanilides, the authors studied quantitative “structureproperties” relationships (QSPR) on the basis of the experimental data. The authors used several sets of molecular descriptors. The dataset outliers were identified by using Principal Component Analysis (PCA). As a modeling technique, the authors applied Projections to Latent Structures (PLS). To ascertain the quality of models, the authors used cross-validation.

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Based on the author’s research, one concludes that the best models were acquired by the use of GETAWAY. The anthelminthic activity and activity towards various microorganisms of the obtained compounds have been studied. Based on preliminary investigations, it was established that the obtained compounds may be recommended as modifiers of anthelminthic preparations— phenacetyne, trinoine, diamphenetide, raphoxanide—also as a bioactive component for preparation: a) materials with antimycotic properties for prophylaxis and treatment of mycosis and dermatomycosis; b) protective covers stable to biocorrosion from action of some mycopathogenic microorganisms. Chapter 5 - This work deals with the problem of modeling of a possible mechanism of point mutations of DNA under the influence of Ni2+ ions. Two feasible schemes of interaction of Ni2+ with nucleosides are considered. The first scheme presents the formation of the planar complex of Ni2+-G-C, and the second shows the incorporation of Ni2+ between the two neighboring complementing pairs. The authors used the MOPAC package to compare the force constants and energies of intramolecular hydrogen bonds in the complexes and corresponding values in a free G-C pair. The comparison enabled us to make a conclusion implying that the formation of the Ni2+-G-C complex is accompanied by the weakening of a hydrogen bond, nearest the joining point of Ni2+. The incorporation of Ni2+ between the two neighboring complementing pairs of G-C causes the weakening of all the three pairs of hydrogen bonds, but to a lesser extent. It has been demonstrated that in the first case the probability of point mutations, the replacement of G-C by A-T increases, and the probability of divisions, the fallout of triplets of type GGХ or ХGG increases in another case. Chapter 6 - The influence of hydrolysis and centrifugation processes of soybean semifat flour on various indices of the lipid component and dynamics of changes in the composition and characteristics in hydrolyzates within three months of storage were studied. It was shown that processes of hydrolysis and centrifugation, and also storage, cause reliable changes of the physical and chemical characteristics and lipid composition in hydrolyzates. Chapter 7 - Some patterns of the relationship of the interaction between aromatic amines (o-, m- and p toluidin; o-, m- and p-amino phenol; o-, m- and p-amino benzoic acid) and aldoses (D-glucose, D-galactose, D-mannose, L-rhamnose, D-xylose, L-arabinose, Dmaltose, D-lactose) in the Maillard reaction are investigated. In the Maillard reaction, the reactivity of aniline, toluidines and amino phenols increases and the reactivity of amino benzoic acids decreases, with increase of рН of the reaction medium; in comparison with aldohexoses, aldopentoses participate in melanoidin formation more actively. Chapter 8 - The influence of serpisten and inokosterone on the phospholipids composition in liver and blood erythrocytes, intensity of lipid peroxidation in tissues (liver, spleen, blood plasma), catalase activity in liver and general peroxidase activity of white outbreed mice has been studied. A biological activity of ecdysteroid-containing compounds is shown to be associated with an influence on the parameters of the physicochemical regulatory system of lipid peroxidation (LPO). Possessing pronounced membrane-tropic properties due to alterations in the exchange of predominantly choline-containing fractions of phospholipids, ecdysteroid-containing preparations are capable of modifying a cell membrane phase state. A substantial dependence of a biological effect of the compounds on a dose, duration of their

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application as well as on an intensity of the LPO processes in the tissues and an animal’s sex require a more detailed research on the properties of the given ecdysteroids. Chapter 9 - Nanoscopic electrochemical devices composed of SAM-modified electrodes and redox probes (RP) of different kind (complex ions, proteins, organic molecules, etc.), were proven to be suitable systems for studying intrinsic electron transfer (ET) mechanisms and interplay between them. In the present review the authors consider the author’s recent results on the mechanistic studies of Au/SAM/RP nanoscale devices in which the RP were either redox-active protein cytochrome C (CytC) dissolved in aqueous solution, or the complex compound ferrocene (Fe(Cp)2]0/+, Cp=cyclopentadienyl) dissolved in a room temperature ionic liquid (RTIL), [bmim][NTf2]. The SAM composition was either [-S(CH2)n-OH] with n=2,3,4,6 and 11, or [-S-(CH2)n-CH3] with n=1,2,3,5,7,11 and 17, respectively. The modern electrochemical methodology including fast scan cyclic voltammetry and data processing techniques were applied to extract ET rate constants and an impact of the variation of ET distance, viscous additives (or temperature) and high pressure was determined, allowing for a rigorous discrimination of intrinsic ET mechanisms. In particular, at short electrode-reactant separations, n=2-3 for Au/SAM/CytC and n=1-3, for Au/SAM/Fe(Cp)2]0/+, the adiabatic mechanism of ET controlled by the viscosity-related relaxational modes of the RP environment, found to be operating. At larger electrode-reactant separations, n=6-11 for Au/SAM/CytC and n=7-11 for Au/SAM/Fe(Cp)2]0/+, the nonadiabatic ET mechanism can be observed manifested through the exponential decay of rate constant with the increase of n. At n=4 and n=5, the intermediate (mixture) regime of ET can be detected. Furthermore, for the case of n=17 for Au/SAM/Fe(Cp)2]0/+ in [bmim][NTf2], dynamical arrest (broken ergodicity) for ET has been demonstrated. In overall, together with other matching proceedings, the reviewed results, despite of essentially diverse nature of complex environments invoked within two different series of congruent systems (protein/aqueous solution versus RTIL) demonstrated common general patterns of the mechanism changeover, in a nice agreement with the predictions of a generalized theory of ET. Chapter 10 - The spherical PLGA nanoparticles (NP) calorimetric investigation is presented in this paper. Such nanoparticles is used for biological active substances (drugs) encapsulating inside of them with the purpose of medicine transferring into the cell. It is clear that without determination of particle stability it is impossible their practical usage. From calorimetric study of PLGA nanoparticles with PLA/PGA ratio 70:30 it was determined the entirety conditions of such particles and the temperature interval, where the particle destructions take place. It was unambiguously shown that for noncoated PLGA NP and for chitosancoated PLGA NP the stability temperature are equal to 370C and less than physiological temperature, which exclude their practical application. Also it was determined that hermiticity destroy temperature depends on heating rate. At the same time it was established that strongly alkaline and acid area (pH2 – pH9) do not destroy noncoated PLGA NP and chitosancoated PLGA NP what gives possibility for their using orally. Chapter 11 - Three industrial samples of Poly(methyl methacrylate) (PMMA), prepared under different conditions, have been extensively analyzed by means of 1H-NMR spectroscopy. Starting from the mm, rr and mrandrm triad contents, as given by the spectra, the type of tacticity statistics distribution has been deduced. Sample X appears to be completely Bernoullian, while samples Y and Z deviate somewhat from this behaviour

Preface

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exhibiting a tiny trend towards Markovian statistics. The fraction of mmrm and rrrm pentads and that of pure heterotactic and atactic triad moieties has been calculated by assuming either a Markovian statistics for samples Y and Z or a Bernoullian statistics for all the samples. On the other hand, the fraction of the same pentads has been determined by deconvoluting the overall triad signals of the spectra into the corresponding pentad signals. An appreciably good agreement with the values obtained assuming Bernoullian statistics for all the samples appears evident. As a result, the evolution of every pentad content from sample X to Sample Z could be stated. Thus the samples prove to be appropriate models to study the relationship between any physical property and the stereomicrostructure of PMMA as was done previously for Poly(vinyl- chloride) (PVC) and Polypropylene (PP). Chapter 12 - It was investigated the fermentative stationary kinetics of hydrogen peroxide decomposition under the action of catalase in the presence of bioSAS. It were obtained the kinetic parameters of this process. It was shown, that the bioSAS have influence on the fermentative process, which can be explained by the change of the fermentative center activity or by the change of substrate concentration. It was determined that the temperature of a process has an insignificant influence on the value of kinetic parameters. Chapter 13 - The results of studying the axial coordination of tetraphenylporphyrin complexes of high-charged metal cations (AcO)CrTPP, O=Mo(OH)TPP and O=W(OH)TPP with molecular ligands (hydrogen sulfide, imidazole and pyridine) in toluene are discussed. The thermodynamic and the kinetic characteristics of reactions between metalloporphyrin and molecular ligand were obtained by the method of spectrophotometric titration and chemical kinetics. Correlations between the molecular ligand basicity and the molecular complex stability are discussed. Chapter 14 - The catalytic rate constants for the process in the presence of bioSAS by different concentrations have been obtained. It was shown, that the constants some increase at bioSAS concentration increasing up to their micelle−formation beginning. The temperature has a slight influence on the value of catalysis constants, that can be explained by practically zero activation energies and depend on activation entropy. Chapter 15 – The authors studied dynamic trend of changes in the activity of creatinу kinase, aldolase and succinatdehydrogenase in brain cells under 30-day long stress induced by isolation and violated diurnal cycle. It was shown that these enzymes heterogeneously responded to 30-day long stress. Particular sensitivity was recorded by succinatedehydrogenase that showed the decline of activity in various sections of the brain at 60-80% on average. Unlike succinatedehydrogenase, aldolase activity increased on the 10th day of stress and then declined. Similar results were seen in relation to phosphokinase activity. It was observed that the change in the activity of the enzymes in question was accompanied by quantitative changes in nitric oxide-NO. In accordance with experimental data the authors suggested that the main signal molecule causing changes in enzyme activity should be NO. Chapter 16 - Hydrazides are characterized with different pharmacological activity, amongst, compounds with inhibitory action of hydrophobia virus and human immunodeficiency virus. Therefore, the authors considered as perspective synthesis of adamantine containing hydrazides and anaemia opposite ferroocenes admixtures. FerroceneA(ferrocenyl-1-phenyl-dioxy-1,4-butin-2) hes significant antitumour and antibacterial properties. The derivatives of adamantine have broad pharmacological activity, low toxicity,

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high membranotropic and antibacterial properties. The search new biologically active compounds, the authors have synthesized ferrocene- and adamantine-containing derivatives. Chapter 17 - Perspectives in the field of creation of highly effective anticancerogenic preparations have been evaluated. For their creation is offered a new regio-selective method of glycosylation of alkylurea in conditions of nucleophilic catalysis with some following nitrosing of glycosyl carbamides of the D- and L-rows. This method opens principally new possibilities for modification of compounds by means of glycosylamides bond leading to preparations possessing small toxicity and high selectivity. Chapter 18 - Physical and biochemical functionalisation of bast fibres are ways to improve thermo- and moisture regulation, anti-bacterial anti-allergies, hygiene, creating “smart” textile. Enhancing natural properties of vegetable fibres is an intermediary step in the obtaining of new products with special applications. The vegetable fibers are biodegradable, can be recycled, and in natural state are highly polar and hydrophilic. To improve the properties of the cellulosic fibers, the chemical and/or physico – chemical modifications were applied. The surface esterification of the natural polymer with acids can be carried out to obtain biodegradable materials, novel fibres with tailored functionalities for special applications. In this paper, starting from Spanish broom (Spartium junceum, syn. Genista juncea) fibers, under action of cold plasma discharges, and using different kinds of carboxylic acids, cellulose esters with short and long side chains have been synthesized. The new grafted polymers were characterized by FT – IR spectroscopy (ATR), XPS and SEM in order to assess the existence of incorporated functional groups. The thermal characterization of the obtained fibres reveals their particular behaviour. Chapter 19 - Miniaturisation of technical systems creates the need for today’s science and engineering to assess the mechanical properties of small volumes of material. A specific feature of the structure and the combination of the desirable properties across several different length scales are fascinating by the many examples in biology. Determining the extraordinary properties of natural materials at the nanometer scale is regarded as a very attractive target for materials science. Mechanical behavior of various biological materials such as insect and plant cuticles was studied by applying experimental approaches of material science in order to explain their structural design and working principles. Experiments were performed on the head articulation cuticle of the beetle designed for friction minimisation and on the wax covered plant surfaces adapted for attachment prevention. Both insect and plant cuticles are multifunctional composite materials and have a multilayered structure. Gula cuticle of the beetle Pachnoda marginata is a part of the head articulation, which is a micromechanical system similar to a ball bearing. The surfaces in this system operate in contact and must be optimised against wear and friction and provide high mobility within the joint. The measurements on the gula cuticle were performed in order to understand structure and mechanical behavior of the material working for friction minimizing. The wax layer on the plant surfaces is a barrier for the attachment system of insects. Antiattachment function could be caused by contamination of attachment pads of insect with the wax crystalloids. Increase in roughness due to location of the wax crystals on the plant surface causes decrease in the real contact area between the plant surface and attachment pads of the insect. These are two of the hypotheses why insects cannot walk on the plant surfaces structured with wax. Nanoindentations on different plant surfaces were performed in order to understand the deformation behavior of the wax layer. This study is believed to be one of the first for

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mechanically testing insect cuticle and the very first for wax-coated plant surfaces in native condition. Chapter 20 - New carbofunctional oligosiloxanes containing trifluorinepropyl and methacrylic groups at silicon atoms have been synthesized and studied. Biological active nitroanilides with spatial adamantane-containing groups and cadmium complex compounds based on them have been obtained. By using the data of IR and NMR spectral analyses the composition and the structure of synthesized compounds have been established. New composite materials of multifunctional application for individual and environmental protection, based on the obtained silicon-organic carbofunctional oligomers and complex compounds, have been created. It was shown that the created composites could be used as: a) protective covers (film materials and impregnating compositions) stable to biocorrosion; b) materials with antimycotic properties for prophylaxis and treatment of mycosis; c) biologically active polymer materials for protection of archaelogical and museum exhibits; d) for human protection during their contact with microorganisms. Preliminary investigations have shown that the synthesized compounds have also a real perspective to be utilized as accessible antioxidants towards the cancer. Chapter 21 - Quaternary arsonium triiodides [(Ph3AsCH2I]I3 and [(i-Bu)3AsCH2I]I3 have been synthesized and studied. The x-ray structures of [(Ph3AsCH2I]I3 and [(i-Bu)3AsCH2I]I3 have been determined. Crystals belong to the monoclinic (comp.1) system, space group P 21/n (No. 14) with a = 10.97 (1)Å, b=13.152 (1)Å, c=16.882 (1)Å , β=93.01 (1)o and to the triclinic system (comp.2), space group P-1 (No. 2) with a=8.413 (1)Å, b=9.109 (1)Å, c=15.876 (1)Å, α = 76.24 (1)o , β=75.60 (1)o, γ=75.26 (1)o. The structures were refined to an R value of 0.063 from 4082 (comp. I) and 0.091 from 4475 (comp.2) observed reflections. The As atom is coordinated tetrahedraly to the substituents and the anion has a linear structure. The synthesis of [R2(R')AsCH2I]I3 (where R= R' or R≠ R') are described. The possibility of the perspective application of synthesized compounds has been shown. Chapter 22 - Pollution of the environment is caused by human industrial and agricultural activity. It poses a harmful factor to the genetic apparatus of organisms to which are connected hereditary diseases, premature aging, cardiovascular problems, etc. Numerous experimental investigations have shown that many chemical factors are characterized by a mutagenic influence. Following these discoveries, the author’s laboratory elaborated many prophylactic measures, directed at prevention of genetic damage from the influence of harmful mutagenic agents on the organism. In the laboratory, the antimutagenic and anticytotoxic effect of bioenergoactivators (biorag, ragozan, ematon and ragil) on mice have been studied during mutation and cytotoxicity induced with the fertilizer ammonium nitrate and the pesticides (phosphamide, trichlorfon and celtan). The cytologic and genetic methods of investigation have been applied in the study. Experiments showed that tested bioenergoactivators exerted highly effective automutagenic and anticytotoxic action. Environmental pollution, which basically is caused by agricultural and industrial activity of human beings, comes back to them as factors harmful to organisms and their genetic apparatus, resulting in not only hereditary illness, malignant tumors and premature senescence, but also illnesses such as cardiovascular and digestive system disease, neural, allergy, and others.

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In connection with an annual increase of chemical pollutants, science stands in the front of genetic danger. Among those problems whose solutions are first and foremost, the protection of organisms and their progenies from chemical mutagens existeing in the environment is the most actual one. Wide application of chemical preparations in medicine, agriculture, industry and everyday life, as well as the existence of a great amount of chemical pollutants in the soil, water and atmosphere, allow us to talk about a sharp alteration of the ecologic situation [1]. Chapter 23 - This article is dedicated to the 100 years anniversary of birth of Georgian prominent scientist, Professor Akaki Gakhokidze. He is one of outstanding representatives of Georgian chemist’s school. High theoretical preparation, mastery of experiment conduction, unusual scientific flair and intuition allow him to leave the great and light footstep for the posterity on the way of scientific research and pedagogical activity. Fundamental investigation of A.Gakhokidze won the international recognition. His woks was published and broadly considered in the special literature, monographs and manuals of chemistry. There are created the specific paragraphs as “Synthesis of Gakhokidze”, “method of DanilowGakhokidze” etc.

PART 1. CHEMISTRY, USE AND MOLECULAR MODELLING OF BIOLOGICALLY ACTIVE COMPOUNDS

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 1

MOLECULAR DESIGN AND REACTIVITY OF THE 1-HYDROXYCYCLOHEXYL HYDROPEROXIDE ALK4NBR COMPLEXES N.A. Turovskij11, E.V. Raksha1, E.N. Pasternak1, I.A. Opeida2, And G.E. Zaikov23 1 Donetsk National University, Donetsk, Universitetskaya str. 24, 83055, Ukraine; Ukrainian-American Laboratory of Computational Chemistry, Kharkov, Ukraine, Jackson, USA

2 Institute of Physico-Organic and Coal Chemistry of the National Academy of Sciences of Ukraine, R. Luxemburg 70, 83114 Donetsk, Ukraine 3 Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygina, 117334 Moscow, Russian Federation

ABSTRACT Chemically activated 1-hydroxycyclohexyl hydroperoxide decomposition in the presence of ammonium salts is proposed to proceed through the complexation stage. Complex structure and reactivity have been investigated by molecular modelling methods. Kinetics of the chemically activated hydroperoxide decomposition in the presence of quaternary ammonium salts (Et4NBr, Pr4NBr, Bu4NBr, and Hex4NBr) has been studied. The correlation between reactivity and structural characteristics of ammonium cations was found.

1

E-mail: [email protected]. 2 E-mail: [email protected].

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INTRODUCTION Peroxides and hydroperoxides are widely used as initiators as well as components of binary initiating systems in the processes of vinyl monomers polymerization, polymers modification [1-3], oxidation of organic substances by molecular oxygen [4, 5]. Initiating systems on the base of quaternary ammonium bromides were found to be the most effective in the case of the liquid phase oxidation of isopropyl benzene by O2 [5]. 1-hydroxycyclohexyl hydroperoxide - Et4NBr system initiates the reaction of liquid phase isopropyl benzene oxidation at 308-340 K [6] while the hydrocarbon oxidation in the presence of only one component of the binary system was not occurred. The present work presents the study of 1hydroxycyclohexyl hydroperoxide decomposition activated by the tetraalkylammonium bromides to investigate the role of ammonium salt cation in the process of chemical activation of peroxide bond.

EXPERIMENTAL 1-hydroxycyclohexyl hydroperoxide has been prepared from cyclohexanone and H2O2 in anhydrous ether according with [7]. Tetraalkylammonium bromides (Et4NBr, Pr4NBr, and Bu4NBr) were recrystallized from the saturated acetonitrile solution by addition of diethyl ether excess. The solvent (acetonitrile) purity was controlled by electrical conductivity value, which was within (8.5 ± 0.2)·10-6 Оm-1 sm-1 at 303 K. Reactions were carried out in the glasssoldered ampoules in argon atmosphere. Hydroperoxide kinetic concentration was controlled by the iodometric titration with potentiometric fixation of the equivalent point.

CALCULATION METHODS Quantum chemical calculations of hydroperoxides molecules and corresponding radicals were carried out by AM1 semiempirical method implemented in MOPAC package [8]. The RHF method was applied to the calculation of the wave function. Optimization of structure parameters of hydroperoxide and hydroperoxide complexes was carried out by Eigenvector Following procedure. The molecular geometry parameters were calculated with boundary gradient norm 0.01. Solvent effect was considered in COSMO approximation [9].

RESULTS AND DISCUSSION It was shown recently that cyclohexanone peroxides decomposition in the presence of Et4NBr proceeds with lower activation barrier as compared with thermolysis. This fact can be explained by the complexation in the studied system [6]. We assume that the activation extent of the peroxide bond in the complex correlates with hydroperoxide reactivity in the reaction of radical decomposition. Thus we investigated the molecular design and reactivity of the complexes in the system 1-hydroxycyclohexyl hydroperoxide – Alk4NBr.

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MOLECULAR DESIGN OF THE 1-HYDROXYCYCLOHEXYL HYDROPEROXIDE - ALK4NBR COMPLEX To obtain the structure information the molecular modelling of the ROOH-Alk4NBr ionmolecular complexes has been carried out for the case of Et4NBr. In contrast to the aralkyl hydroperoxides (like isopropylbenzene hydroperoxide) the molecule of 1-hydroxycyclohexyl hydroperoxide does not contain the aromatic ring but there is the hydroxide group OH in their structure that also can participate in the intermolecular hydrogen bonds formation. We assume the model of complex formation with combined action of cation and anion (Figure 1) such as previously proposed model of substrate separated ion pair (SubSIP) [10, 11]. Formation of such type associate is accompanied by considerable conformation changes of the hydroperoxide fragment. Association between hydroperoxide molecule and the salt ions occurs by the intermolecular hydrogen bonds formation. It is confirmed by charge increasing on the hydrogen atoms of the corresponding bonds C-H3, O-H1, and O-H2 and elongation of these bonds from 1.11 Å to 1.17 Å and from 0.96 Å to 1.08 Å correspondingly. Calculations in COSMO approximation show that the electron density transfer from bromideanion to the hydroperoxide fragment is lower in the case of solvent effect taken into account (Table 1). Stage determining the rate of the hydroperoxide decomposition is that of O-O bond cleavage. Thus the reaction activation energy will be determined by the energy of peroxide bond homolytic decomposition. Some possible elementary stages of the complex decomposition with the free radical formation are listed in Table 2. It can be seen from the Table 2 that the electrone transfer from the bromide ion to the peroxide bond is improbable in this system because it is needed more energy inputs as compared with homolysis. We assume that SubSIP decomposes with free radicals formation due to homolytical rupture of the peroxide bond. On the Figure 1 the enthalpies of complexation are shown. In the case of SubSIP we assume that the complex is formed according to the exchange mechanism (1) while possibility of the threemolecular reaction (2) is low. Solubility of the Et4NBr in acetonitrile is sufficiently high, and reactivity of the ions and ion pairs in peroxide bond activation is similar. The threemolecular reaction (2) determines the second reaction order on salt while this value changes from 1 (at small salt concentrations) to 0 (at salt abundance) in experimental conditions. Thus the reaction (1) is rather preferable. Table 1. Electron and stereochemical characteristics of COOH-fragment in ROOH – Et4NBr complexes parameters qOα, e qOβ, e qH1, e qH2, e qH3, e ∠COαOβH qBr-

ROOH - 0.18 - 0.20 0.20 0.22 0.10 90.8 -

Et4N+…ROOH…Br- 0.24 - 0.26 0.25 0.27 0.17 33.4 - 0.81

ROOH…Br- 0.18 - 0.29 0.25 0.27 0.11 68.3 - 0.85

ROOH… - 0.20 - 0.23 0.22 0.04 0.10 77.5 -

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N. A. Turovskij, E. V. Raksha, E. N. Pasternak et al. Table 2. Heats of some elementary stages of ROOH – Et4NBr complex decomposition

ROOH → RO· + ·OH Et4N+…ROOH…Br− → RO· + ·OH + Et4N+ + BrEt4N+…ROOH…Br− → RO· + ·OH + Et4NBr Et4N+…ROOH…Br− → RO- + ·OH + Et4N+ + Br· Et4N+…ROOH…Br− → RO· + OH- + Et4N+ + Br· ROOH…Et4N+ → RO· + ·OH + Et4N+

ΔH0, kJ mol-1 gas phase 148 481 65 615 435 192

acetonitrile 151 239 108 354 249 179

ROOH…Br- → RO· + ·OH + BrROOH…Br- → RO- + ·OH + Br· ROOH…Br- → RO· + OH- + Br·

242 188 376

208 219 323

Reaction

Figure 1. Complexes of 1-hydroxycyclohexyl hydroperoxide bromide.

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Et4N+…Solv…Br− + ROOH → Et4N+…ROOH…Br− + Solv (ΔHr(2) = - 53.7 kJ·mol-1)

(1)

Et4N+ + ROOH + Br− → Et4N+…ROOH…Br− (ΔHr(3) = - 88.1 kJ·mol-1)

(2)

Corresponding models like Et4N+…ROOH…Br− were obtained for tetraalkylammonium salts (Pr4NBr, Bu4NBr, и Hex4NBr). Changes in peroxide bond energy (i.e. total of electronic and nuclear energies of two-center term O-O atom pair) can be suggested as extent of the peroxide bond activation. Stable peroxide structure has the lowest O-O bond energy (-12.08 eV for free hydroperoxide) and less stable (or activated) peroxide conformation has the highest one (-11.78 eV for complex Et4N+…ROOH…Br−). Difference between energy of stable and activated peroxide structure increases in the catalyst row: Hex4NBr, Bu4NBr, Pr4NBr, and Et4NBr indicating weakening of the O-O bond in the ROOH-Alk4NBr complex. From this point of view the O-O bond activation in the case of Et4N+…ROOH…Br− is the highest and in complex with Hex4NBr is the lowest. This suggestion is in correspondence with experimental data.

KINETIC FEATURES OF THE ACTIVATED HYDROPEROXIDE DECOMPOSITION Reaction of 1-hydroxycyclohexyl hydroperoxide (ROOH) decomposition activated by the tetraalkylammonium bromides (Alk4NBr) has been investigated at 323 – 353 K under conditions of ammonium salts abundance ([ROOH]0 = 5·10-3 mol·dm-3, [Alk4NBr]0 = 2.5·102 – 1·10-1 mol·dm-3). Kinetics of ROOH activated decomposition on these conditions could be described by the first order reaction proceedings low. The reaction was carried out up to 50 % hydroperoxide conversion and the products did not effect on the reaction proceeding as kinetic curves anamorphouses are linear in the corresponding first order coordinates. The effective rate constant (kef, seс-1) was found to be independent from the hydroperoxide initial concentration within [ROOH]0 = 2.5·10-3 – 1·10-2 mol·dm-3 and constant amount of Alk4NBr (5·10-2 mol·dm-3). Nonlinear character of the dependence of reaction rate effective constants from the ammonium salt initial concentration (Figure 2) in the present conditions points out onto the occurrence of complexation stage between the ROOH and Alk4NBr. These facts are in an agreement with reaction scheme for the isopropyl benzene hydroperoxide activated decomposition recently proposed [12]. Thermolysis contribution into the total reaction rate the ROOH activated decomposition was found to be negligibly small because the thermal decomposition rate constant [6] was two orders lower then corresponding kef values. Some experiments were carried out under conditions of ROOH excess as compared with salts concentration ([Alk4NBr]0 = 5·10-3 mol·dm-3, [ROOH]0 = 5·10-2 mol·dm-3) at 323 K. Even at present concentration ratio of ROOH and Alk4NBr the rate of hydroperoxide activated decomposition is several orders higher then ROOH thermolysis rate. The Brconcentration during reaction proceeding is unaltered. These facts point out onto catalytic character of Alk4NBr action in the process. Catalytic scheme of the hydroperoxide decomposition is supported also in works [13, 14] in which it was noted that O-O bond

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cleavage in the hydroperoxide – catalyst complex proceeds homolytically; catalyst is not consumed and deactivated in the system.

Figure 2. Dependence of kef from the catalyst initial concentration in the direct (a) and inverse (b) coordinates. [ROOH]0 = 5.0·10-3 mol·dm-3, 333 K, 1 – Hex4N+, 2 – Bu4N+, 3 – Pr4N+, 4 – Et4N+.

The kinetic scheme is proposed for the chemically activated ROOH decomposition. It includes the complexation stage between ROOH and salts ions as well as stage of the complex-bonded hydroperoxide decomposition with catalyst regeneration: kef dependence on the salt concentration can be expressed by the relationship (3):

1 1 1 = + k ef k d K C [ Alk 4 NBr ] k d

(3)

The dependence of kef on Alk4NBr concentration is linear in double inverse coordinates (Figure 2). It is in the agreement with the proposed kinetic scheme. The values of rate constants of complex-bonded peroxide decomposition (kd) obtained for the investigated salts decrease in the following order: Et4N+ > Pr4N+ > Bu4N+ > Hex4N+. The values of equilibrium constants of complexation between ROOH and Alk4NBr ions change similarly (Table 3). Isokinetic relationship between complexation parameters in the system leads to the insignificant changes in free energy of complexation for different alkyl substituent in ammonium cation. Considering the intermolecular bonds energy the strongest complex is formed between hydroperoxide and Et4NBr, the weakest - in the case of Hex4NBr (see the corresponding ΔHcom values in Table 3). The reactivity of complex-bonded ROOH also decreases with increasing of alkyl substituent size. Isokinetic relationship for the obtained activation parameters points out upon the unified mechanism of ROOH - Alk4NBr complex decomposition. Changes of the peroxide bond activation in the complex in the case of different ammonium cations point out the role of steric factor at the stage of complex formation as well as at the formation of their decomposition transition state. In the simplest case the own

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volume of the investigated cations could describe the steric effect. A good correlation between activation parameters of the complex decomposition and calculated values of Vander-Waals volumes of cations has been obtained (Figure 3). VVDW values for the tetraalkylammonium cations were calculated in HyperChem package. Calculated values are in agreement with corresponding own volumes of cations listed in [15]. Linear relationship has been observed between the complex heat of formation - ΔH0f and experimental activation parameters - ΔH≠ (Figure 4). Table 3. Kinetic parameters of 1-hydroxycyclohexyl hydroperoxide decomposition in the presence of thetraalkylammonium bromides parameters kd·104, sec-1

KC, dm3mol-1 Ea, kJmol-1 lgA, (A, c-1) ΔHcom, kJmol-1 ΔScom, Jmol-1K-1

T, K 333 343 353 333 343 353 333353

ROOH + Et4NBr 1.14 ± 0.04 2.5 ± 0.1 5.1 ± 0.2 36 ± 2 29 ± 2 23 ± 1 73 ± 1 7.5 ± 0.6 -20 ± 1 -30 ± 4

ROOH + Pr4NBr 0.8 ± 0.1 1.80 ± 0.09 4.20 ± 0.09 28 ± 3 24 ± 1 20 ± 1 81 ± 2 8.6 ± 0.3 -16 ± 2 -21 ± 5

ROOH + Bu4NBr 0.59 ± 0.06 1.51 ± 0.04 3.48 ± 0.08 23 ± 2 20 ± 1 18 ± 2 87 ± 3 9.4 ± 0.2 -12 ± 2 -10 ± 4

ROOH + Hex4NBr 0.34 ± 0.01 0.97 ± 0.02 2.40 ± 0.06 18 ± 3 16 ± 2 15 ± 2 96 ± 2 10.5 ± 0.3 -9 ±1 -3 ± 3

Figure 3. Relationship between activation parameters of the complex decomposition and Van-derWaals volume of the tetraalkylammonium cations.

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Figure 4. Relationship between the complex heat of formation (ΔH0f) and experimental activation parameters (ΔH≠).

Obtained experimental facts have shown that the salt cation participates both in complexation stage and in stage of complex ROOH - Alk4NBr decomposition. Ammonium cation has the regulating action upon the catalytic reactivity of the halide-anion in the reaction of catalytic decomposition of the 1-hydroxycyclohexyl hydroperoxide in the presence of Alk4NBr. Thus the cation structure influences on the reactivity of the hydroperoxide complex and on the extent of peroxide bond activation. The molecular modeling of the hydroperoxidecatalyst reactive complex can be used to preliminarily predict the reactivity of the system.

CONCLUSIONS Summarizing, obtained experimental facts have shown that the chemical activation of the peroxide bond is observed in the presence of Alk4NBr. The kinetic parameters of the hydroperoxide catalytic decomposition have been obtained for the Et4NBr, Pr4NBr, Bu4NBr, and Hex4NBr. Catalysis of the1-hydroxycyclohexyl hydroperoxide decomposition has shown to occur through accompanied action of the ammonium salt cation and anion.

REFERENCES [1] [2] [3]

E.T. Denisov, T.G. Denisova, T.S. Pokidova. Handbook of Free Radical Initiators. John Wiley and Sons Inc.: Hoboken, New Jersey, 2003, 879 p. N.A. Turovskij, I.O. Opeida, O.V. Kush, E.L. Baranovskij. J. Appl. Chem. 2004, 77, 1887. C.J. Perez, E.M. Valles, M.D. Failla. Polymer, 2005, 46, 725,

Molecular Design and Reactivity… [4] [5] [6] [7] [8] [9] [10]

[11] [12] [13] [14] [15]

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I.A. Opeida, N.M. Zalevskaya, E.N. Turovskaya. Kinetika i Kataliz, 2004, 45, 776, I.A. Opeida, N.M. Zalevskaya, E.N. Turovskaya, U.I. Sobka, Petroleum Chemistry, 2002, 42, 6. 460. M.A. Turovskyj, I.A. Opeida, E.N. Turovskaya, O.V. Raksha, N.O. Kuznetsova, G.E. Zaikov. Oxid. Commun., 2006, 29, 249. N.A. Milas, S.A. Harris, P.C. Panagiotacos. J.Am.Chem.Soc., 1939, 61, 9, 2430. J.J.P. Stewart. MOPAC 2000.00 Manual; Fujitsu Limited: Tokyo: Japan, 1999. A. Klamt. J.Chem. Soc. Perkin Trans., 1993, 2, 799. M.A. Тurovskyj, I.O. Оpeida O.M. Turovskaya, O.V. Raksha, N.O. Kuznetsova, and G.E. Zaikov. Order and Disorder in Polymer Reactivity. Howell New York: Nova Scince Publishers, Inc. 2006, pp. 37-51. N.A. Turovskij, S.Yu. Tselinskij, Yu.E. Shapiro, A.R. Kal’uskij. Teor. Eksp. Khim., 1992, 28, 4. 320. M.A. Turovskyj, A.M. Nikolayevskyj, I.A. Opeida, V.N. Shufletuk. Ukrainian Chem. Bull., 2000, 8, 151. L.M. Pisarenko, O.T. Kasaikina. Russ Chem Bull, 2002, 3, 419. L.M. Pisarenko, V.G. Kondratovich, O.T. Kasaikina. Russ Chem Bull, 2004,10, 2110. Y.Marcus. Ion salvation. Chichester etc.: Wiley. 1985, 306 p.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 2

RECEPTION OF PHYSIOLOGICALLY ACTIVE SUBSTANCES BY PLASMATIC MEMBRANE OF VEGETABLE CELL Jaba Oniani1, Vladimir Yurin2, Ramaz Gakhokidze11, Tea Mchedluri3, Tamar Oniani1, and Lela Abadovskaya1 1

Iv. Jvakhishvili Tbilisi State University, Georgia 2 Minsk State University, Belarus 3 Telavi State University, Georgia

ABSTRACT Electrical phenomena and cytoplasm movement on an example of seaweed Characea have been briefly described. The results concerning cyclosis nascent mechanism in the cells of seaweed Characea and connection of cytoplasm movement velocity (CMV) with the difference of electrical potentials (DEP) of plasmatic membrane have been adduced. For specification of the appropriateness of interconnection of the cyclosis and DEP, we carried out the simultaneous measurement. A suggestion was made for a model in the frame of which it is possible to set up, in every separate case, a way to realize the observed effects by over regulation of the membrane potentials, i.e., finally by changing of ion penetrability of the membrane and in what—due to presence of other influences (for example, by influence on the difference intercellular processes). For ascertainment of the possibility of the presence on the plasmatic membrane of vegetable cell structures, similar to animal receptors and the interaction character of them, effectors, a detailed research had been conducted of acetylcholine acting and some biogenic amines on the velocity of cytoplasm and DEP of cell of seaweed Characea. Nitella flexilis. Results show that a reaction of the cell on adrenomimetics, active in respect to α and β-receptors, is discerned by sign. Investigation of combined action of adrenomimetics and α-type blockaders (nor adrenalin-fenitron) and β-type (isadrin – 1 E-mail: [email protected].

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Jaba Oniani, Vladimir Yurin, Ramaz Gakhokidze et al. dihydroergotoxin) showed the strongly pronounced antagonistic effect, realizing by concrete mechanism. There were adduced also other proofs allowing for the existence of interacting centers in the cells with molecules of catecholamines, similar to α and βreceptors of animal cells; while this resemblance is spread as on the structure organization, so it is on some functional features. Data about the presence of cAMF, cGMF, enzymes of their synthesis and catabolism (adenilatciclase, gaunilatciclase, phospodietherase) and protein-target – proteincinases, also G-protein, allowed authors of the work to make the conclusion about the specific interaction of acetylcholine and biogenic amines with the specialized structures of plasmatic membrane of vegetable cells and signal transmitting by trigger mechanisms on the G-protein with following participation of secondary intermediary messengers. Following from the above-stated consideration, we proposed the presumptive conclusions: 1. 2. 3.

There is revealed the phenomenon of receptor regulation of intercellular physiologic processes of vegetable cells. In the experiments with biogenic amines was stated the appropriateness in the direct correlation between alteration of CMV and DEP. The cells of Nitellaceae can be used in the model experiments for testing of biological active substances, inasmuch as there was revealed by them the property to react by constellation receptor system on the exogenous influence of low concentration of testing compounds.

All those presuppositions show that in the plant cells is the presence of contractive proteins in view of actinomyozin complexes, otherwise who may answer the question which is open till today: What gives the first push to the cytoplasm particles to move constantly along the perimeter of the vegetable cell?

As long ago as XIX century by known physiologist of animals Claude Bernard, the idea had been stated by what the irritability is one of the main properties of living organisms and for which the common mechanisms of perception and quick reaction on the outer influence are inherent. But the mechanisms of irritability, including the perception of outer stimulus, information on its transmission and in return reaction had been studied in the XX century [13]. Plasmatic membrane is the primary target of influence of exogenous factors, realizing the perception of their influence and transmission of the signal into the cell. Let us consider the approach developed by us of description of the physiological active substances reception with the structure elements of the plasmatic membrane of the vegetable cell on the base of analysis of regularity of an alteration of electrical characteristics of cyclosis speed. With this purpose, we will characterize phenomena and movement of cytoplasm on the example of seaweed Characea. Ability to generate electrical potential is one of the universal properties of the living system which plays an important role in their viability. It is known that bioelectrical potential today is subdivided on the two basic types: potential of immovability (PI) and potential of excitation (PE). PI is the difference of electrical potential (DEP) in the condition of immovability, PI-alteration of PE at the irritation [4-6]. PI or membrane potential is the main electrical characteristic of the plant cell and corresponding to it is the condition at physiologic

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immovability when the metabolism is in the balance condition. Disturbance of normal physiologic conditions of the cell inevitably leads to the change of PI value. On the membrane of any compartment of a living cell, there exists DEP, the absence of which would be amazing. It would mean the absolute equality of electrolyte concentration in all the cells, organs, outer solutions or complete coincidence membrane penetration value to all the cations and anions. Some examples of membrane potential value of the plant cell is shown in the table. On the base of PI all types of electro physiologic reactions of the cell, in particular excitation potentials, are formed. In the plants, the excitation potentials are represented by two types: potential of action (PA) and potential of variability (PV) [6,7]. PA is the temporary alternation of DEP between the excited section and the section being in the condition of immobility. It appears due to irritation and is spread as excitation waves. PA appearing as a result of the definite irritation force of the plant can be divided on the spreading—i.e. transmitting signal from cell to cell in the limits of the organ or tissue—and the local one, transmitting only in the limits of irritating cells. Table. Membrane potential of the plant cells (Klarkson D. 1978; Lutge Y., Khiginbotam H 1984) Species Tissue Crustof of root Pisum sativum Epicotel Caleoptil Avena sativa Root Zea mays Root Vicia sativa Root Nitella Internodal cell Nitellopsis obtusa Internodal cell Halicystis Cell Valonia Cell

Value of DEP, mV –110 –119 –102 – –109 –71 – –84 –76 – –104 –130 –120 – –150 –200 – –250 –80 +17

Elaborations of corresponding techniques and the interest of investigators in the electrical process in the plants allowed researchers, in the beginning of the past century, to register PA at their irritation. It was that the membrane potential of many vegetable cells, like those of animal origination, at the irritation is displaced to the side of depolarization. At the definite value of membrane potential displacement to the positive side till some critical level there is observed PA. Volume of displacement of membrane potential from initial level to critical one is called the threshold potential. The lower the threshold potential, the higher is the cell irritability. In comparison with animal objects PA, the potential of activity of vegetable cells is developed slowly. Another distinguished feature is that the PA of plant cells is developed on the two membranes—plasmalemma and tonoplast [9]. In its turn PA on the tonoplast is developed more slowly in comparison with plasmalemma. PA on the plasmalemma precedes the PA on the tonoplast. Conductivity of the membrane at the PA generation is increased, reaching its maximum in the peak of PA, then returns to the initial level. As plasmalemma so tonoplast behave, it is

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shown in figure 1. But for first of them it is characterized the considerable alternation of potential and conductivity.

Figure 1. Development of the wave of PA on the chariophytes cell: a) between vacuole and external media, b) on the tonoplast, c) on the plasmolemme.

Independent of outer conditions (environment composition, temperature, light, etc.) and also of physiologic conditions of the cells, the potential of action has a different amplitude and duration. Likely in the case of nerve fiber, PA can exceed potential zero value, i.e., there can be observed overshoot. Cells, independent of medium conditions, physiologic state, etc. can generate PA difference form and value [8,9]. At the end of the XVIII century, it was stated that at a weak electrical current the movement stops in the cytoplasm of seaweed Characea [10]. For the first time, the movement of the cytoplasm of plant cells, which later received the name cyclosis, was observed by Corti in 1774 [11]. At the accumulation of facts they came to the conclusion that cytoplasm movement occurs in the many intact cells, and the character of movement is quite various [12-16]. Reports stated the presence of intercellular movement cytoplasm along plasmodesm, in particular in the cells of parenchyma of different Allium sativum tissues. It is supposed that with the cytoplasm current there is carried away such low molecular substances as sugar amino acids and inorganic salts, etc. [12]. There exists a dependence between growth and movement direction of cytoplasm. Most strong growth of fungi floccus is marked when cytoplasm moves to the top direction. Probably moving cytoplasm carries the substances necessary for floccus growth. If the movement had been stopped, the growth practically was being discontinued [12]. It is supposed that the translocation of generative and vegetative nucleuses at the pollen tube of covered semen plants happens due to movement of cytoplasm. It is clear that in this case the cyclosis has a great importance for impregnation [17]. In the experiments series the movement intensification of cytoplasm serves as an index of particular condition of the cell or

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proceeds of cell transition in this condition. Researchers observed the activation of the cyclosis in the cell of some plants after implantation of parasite fungus [18]. As an example of interconnection of cytoplasm movement intensity and physiologic state in the natural condition can be served the locked cells of stomas. At the closed stoma in the locked cells of the leaf Vicia there had been observed intensive movement, and in the open ones the cytoplasm did not move or there was revealed only oscillatory movement in a separate section ]12]. In accordance with the works of some researchers, the cells and tissues of different plants have different physiological and physical and chemical characteristics depending on the age and closeness to apical and basal ends of the plant. It brings us to the thought about the presence of distinctions in speed of cyclosis of those cells. In reality, it is shown that the most high value of cytoplasm movement speed is characterized for upper growing cells (about 40 mkm/s) of seaweed Characea Nitella, the lowest is for inferior ones. Difference in the values between first and fourth cells was the rate of 10 mkm/s, the tendency of cyclosis speed decrease from upper to down was expressed quite distinctly. These results, apparently, can be interpreted as an effect of metabolism of different level in the cells of different age; the young cells have most high cyclosis velocity —as a reflection of high activity of metabolic processes [19]. Consequently, the velocity of cytoplasm movement is connected with physiologic conditions of the cells and metabolic processes flowing in them. An electrical field influencing itself on the physiologic processes in the cells, affects the different kinds of movement into the plant [20] and as it was mentioned, on the cyclosis velocity also. Current influence on the cytoplasm movement strongly depends on its intensity, durability, etc. At the effect of significant outer electrical current and at the appearing of PA, cyclosis stoppage occurred. At first this phenomena was revealed on the seaweed Characea Nitella cells [21]. Interconnection between PA and cytoplasm movement stoppage were stated for different kinds of seaweed Characea [22-25]. It was revealed that cytoplasm movement stopping takes place after 1–2 sec. of PA beginning or through 1 sec. after PA peak [25-27]. Complete restoration of cyclosis velocity occurs after 5–10 min [26]. This time depends on the kind of object and external conditions [24,25]. For example, in the autumn cells, the movement speed had decreased sharply at the cell excitation, was not stopped completely and then was being turned to the initial level [25]. It must be noted that appearance of PA is always accompanied by stopping or deceleration of the cyclosis but it is not necessary to have a sharp stopping of movement for the appearance of PA. Velocity of cytoplasm movement depends on contents of ATP and is inhibited by intercellular Ca2+ [28, 29]. At the injection of protein of akvorin in the cytoplasm of cells of Chara and Nitella, luminescence intensity of which is proportional to the concentration of free Ca2+ showed that the peak of intensity of luminescence was corresponding to the moment of cyclosis stopping and it had place at the maximum of the sluggish phase of PA. Concentration of free Ca2+ was being changed from 0.1–0.2 mcM to 6.7 mcM, in the peak of PA for the Chara cells, but for Nitella cells—from 0.44–1.1 to 43.0 mcM [28-30]. Increase of inter-cellular concentration of Ca2+ from 10-7 to 10–6 M at the absence of tonoplast notably slowed down the cyclosis in the cells of Nitella; discontinuance of the movement begins in at 10-3 M Ca2+. For the analogous fragments of Chara cells of critical concentration, the inhibited movement was 5·10-4 M. At the calcium removal had been observed the partial restoration of movement velocity [28,31,32].

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Due to the data mentioned above, we can suppose that the factor causing cytoplasm movement stopping at the actuation of membrane is Ca2+. This is confirmed that the fragments of the cells, without tonoplast at the introduction of EDTA complexon, connecting the Ca2+ was not observed at decrease of cytoplasm movement at the excitation of the cell [33]. It has been stated in the early works that in the cells of seaweed Characea the motive force is formed at the interaction between flowing endoplasm and immovable cortical layer and for the motion of cytoplasm it is necessary to have a definite organization of the cortical layer [34-35]. Afterwards were obtained a number of data touching on the presence of subcortical fibrils and endoplasmatic filaments. The last, as it was shown, are the branches of subcortical fibrils [36-39]. By such a method, it is possible to have a double action of calcium ions on the cyclosis. Such action at first is the reversible inhibition Ca2+ of sensitive mechanisms. Secondly is the inhibition through it acting on the filament [32]. Supposition about cytoplasm movement in the vegetable cells connecting with functioning of retractive proteins partially was confirmed after disclosure of actin in the higher plant Ammaryllis [40]. But the earlier works [41] indicated the Myozin similar character of extract obtained by cells of Nitella. More careful identification of protein extract from cells of Nitella has been conducted in the work [42]. At the significant ion force, the activity of identified ATphase protein complex by acting of Ca2+ or EDTA was being increased, but under influence of Mg2+- decreased. Purified preparation of the moizin from the cells of Nitella has had a higher molecular mass as compared to myosin, extracted from skeleton muscles. At last time myosin was identified also to the cells of other high plants, for example, Elodea [43]. Lucopersicon [44]. Extracted from all kind of plants myosin possessed the ability to activate actinstimulated ATphase and formed biopolymer aggregate [45-47]. One of the problem at the research of cytoplasm movement mechanism is an elucidation of intercellular myosin localization and construction its acting model. There was realized the separate processing of endoplasm and cortical layer by specific agents [48,49]. With this goal there were conducted the soft centrifugation intermodal cells of seaweed Characea, at which the cytoplasm was collected to the end of cell and the cortical layer, including the chloroplast and subcortical fibrils remained intact on the opposite (centripetal) end of the cell. Then one end of cell (cortical layer or endoplasm) were processed by corresponding agent and accomplished repetitive centrifugation, at which treated cytoplasm came into contact with non processed cortical layer. Processing of cellular cortical layers with N-ethylmaleinimide (EMI) inhibited with Factin activated myosin of ATphase did not influence the cyclosis. At processing of endoplasm there was not observed the movement. Differentiated holding of separate cell fragments during two minutes at the 47.5°C temperature was being led to acting analogous EMI. This can be explained that myosin is easy denaturizing at the temperature increase, while the actin is more thermostable. At application of cytohalasin B, inhibiting action of which on the cytoplasm movement in the vegetable cells is investigated well [50-53], an opposite effect was being noted as compared to above description. Cytoplasm movement was being stopped only after processing of cortical layer of cell by cytohalasin [54] at the acting of it on endoplasm the cyclosis was being continued. Adducted experimental data allowed authors to consider that cytohalasin, most likely inhibits the microfilaments of subcortical fibrils. Moreover they had

Reception of Physiologically Active Substances by Plasmatic Membrane…

17

stated the supposition that movable filaments of endoplasm do not play any role in the mechanism of protoplasm movement. But this contradicts the stated facts; there is shown by direct observation that the particles move in a quite similar way along endoplasmatic filaments and subcortical fibrils [13]. In connection with the abovementioned, most probable process providing generation of motive forces of cyclosis, is interaction, oriented to the movement direction and polarized actin filaments with citoplamatic oligomers of myosin [55]. Source of movement energy of cytoplasm is the conversion of chemical energy of hydrolyzes ATP by actinomyosin complex in conformation alteration of oligomers of myosin [55-58] which lead to the mechanical directed movement of cytoplasm. By analogy with acsoplasm there are considered that in the cells of seaweed Characea the places of filaments attachment to the excitable membrane are the protein molecules with high affinity to ions Ca2+In result of calcium ion entrance and their tying with active centers, there is weakened the connection between protomers of actin filaments, occur their unfastening from membrane, what leads to the cyclosis stopping. So there was pointed earlier that PA or potassium depolarization in the akson was being caused unfastening membrane connected filaments of cytoskeleton. Following removal of ions Ca 2+leads to fastening of actin filaments [59-60]. Such, there was appeared the circumstantial results, touching to mechanism of cyclosis beginnings in the vegetable cells and with DEP cells. For detailing of regularity of cyclosis interconnection and DEP, it was under-taken by us the attempt of their simultaneous measuring. As it is shown in fig.2, changing of DEP value, entail for them displacement of cytoplasm movement [61].

Figure 2. Kinetics of depolarisation (a) displacement of protoplasm movement (b) under the action of 3.0 mM (1) and 10 mM KCl (2).

For revealing of the qualitative character of this dependence, as an influencing factor on DEP, there was selected K+ ion. It is known that in the physiologic conditions, content this ion in the cytoplasm (10-150mM) of the vegetable cells are 2-3 orders above then in the environment. Therefore the little changes its concentration in the outer solutions (from 0,1 to

18

Jaba Oniani, Vladimir Yurin, Ramaz Gakhokidze et al.

10,0 mM) will not influence essentially on the concentration in the cytoplasm and the same it do not act directly on the structure, responsible for cyclosis process. Considering this displacement appearing in this conditions of DEP (ΔΨ) as the single factor, causing cyclosis speed changing (ΔV), there can be supposed as first approximation, that velocity induced ΔΨ alteration V will be proportional of it. But it must be taken into consideration the presence of opposite, adaptation tendency, proportional to the current displacement ΔV: dV/dt = k1ΔΨ (t) – k2ΔV(t)

(1)

Or after integration t ΔV(t)= k1 / k2 exp( k2 t) ∫ ΔΨ (τ)exp( k2 τ) dτ

(2)

0

Kinetics of Changing DEP and cytoplasm movement speed were approximated by equation of (2). Obtained equation describes well the experimental dependence (see fig. 2), what allows us to use it for estimation of potential dependence of cyclosis speed displacements, coming by acting of different factors [61,62]. In the frame of this model, in case, when experimental points on diagram (ΔV, ΔΨ) is placed on the straight line, passing on the origin of the coordinate system, the reaction is strongly potential dependent (Fig.3).

Fiigure 3. Connection between means of displacement ΔV and corresponding them displacement ΔΨ in case of potential dependence of VPM depending on relation (2).

Reception of Physiologically Active Substances by Plasmatic Membrane…

19

If the condition of analyzing set of points essentially declines from described dependence – this means that in considered case there has place the effects stipulated by interaction of effector with regulating cyclosis velocity by structures directly. Those, there is possible in the every separate case to determine how the observed effects are realized by overregulation of membrane potential, i.e. in sum it is realized by changing of membrane penetration, and in which - due to presence other acting way (for example, through influence on the different intercellular processes). For clarification of possible presence on the plasmatic membrane of vegetable cells structures, similar animal receptors and character of interaction with them effector, by us were conducted fundamental research of acetylcholine acting and number of biogenic amines on the speed of cytoplasm movement and DEP of cells of seaweed Characea [62,63]. Acetylcholine with concentration of 5·10-7 - 5·10-6M caused cyclosis velocity decrease and drop of DEP, which gradually was being turned to the initial level. More expressed action of acetylcholine was being provided at the concentration 5·10-5 M. Reaction of the cell was reversible i.e after removal it from solution was restored the initial movement velocity of cytoplasm. At cumulative acting of increased concentration of acetylcholine on the cell in the interval 5·10-7-5·10-5 M the concentration caused plasmolysis. Serotonin was being provided the opposite action of acetylcholine on the cyclosis speed. Process of cytoplasm movement at the serotonin action bore the oscillatory character. Concentration of serotonin in the medium till 10-5 M caused cyclosis speed raise while at the action of more high one, (10-4 M) the velocity decreased and the DEP meaning was being fallen. Adrenaline and noradrenaline was being begun its influence on the cyclosis and DEP of Nitella cells at the concentration 10-7 M and above it, at that the changes bore oscillatory character. Cell treatment by isodrin, which is stimulator of β adrenoreceptors in the animal organism at the concentration 10-6 M was being led to the increase of cytoplasm movement speed and DEP of the cell. Inderal (blockader of β adrenoreceptors) at concentration 10-6 M caused rapid decrease of cyclosis and DEP velocity. At the increase of direct effect (acting of testing agent on the intercellular system, responsible for keeping of cyclosis) on the movement of acetylcholine cytoplasm and biogenic amines arrange in the row: phenitron
20

Jaba Oniani, Vladimir Yurin, Ramaz Gakhokidze et al.

Figure 4. Threshold concentration (O) and tested ranges of acting concentration (10-8-10-4 g/m) of biogenic amin: S – irritation stimulus; A and B – rapid and slow phases of PA.

Definite confirmation of this hypothesis serves results obtained in the experiments of joint action of activators and blockaders α and β-adrenoreceptors. Preliminary processing of the cells with comparatively low concentration of dihydroergotoxin (5·10-8 M) not influencing on the registering parameters, had prevented the cell reaction on the noradrenaline at concentration 2·10-6 M, causing the authentic answer of intact cells. However the concentration of dihidroenergotoxin did not lead to the complete loss of sensitivity to adrenaline. This is explained by activity of adrenaline regarding as α, so β- receptors. There had been observed the similar picture at the series application of the blockader (Inderal) and activator (isodrin) of β-receptors. In this case the cell threatened by low concentration of (5·10-7 M) Inderal, was losing the sensitivity to isodrin (5·10-7M). The change of order of agent application in this couples: “antagonist –agonist” led to inhibition in both cases of reaction, appearing under agonist influence. The whole adducted data can be explained on the base of hypothesis about existence in the cell the centers of interaction with catecholamines molecules, similar to α and β-receptors of animal cells, at that this similarity spread as on the structure organization, so on some functional features. The basic conclusions in favor of such analogy are the following: -

-

Action of adrenoreceptors blockaders on the cytoplasm movement speed reveals through DEP displacement; in case of adrenoreceptors activators such influence is not expressed. Adrenomimetics, active relating only α or β-receptors, cause opposite, by sign, reaction of the cell.

Reception of Physiologically Active Substances by Plasmatic Membrane… -

-

21

Effect of noradrenaline, activator of α-receptors, is taken away by blockaders of those receptor by dihydrotoxins; Similarly the action of β-receptors activators of isodrin is not revealed in presence of inderal—typical blockaders of β-receptors. For taking away of adrenaline action, active regarding both types of receptors, it is necessary to combine application of dihydroergotoxin and Inderal.

It must be noted that the fact of direct action of a compound on the cyclosis may be explained as a sign of its penetration in cytoplasm. So the assumption that blockaders acting always through DEP, either is not carried by transport system through membrane, or getting into cytoplasm did not influence on the processes stipulating the cyclosis, is quite right. It must be paused on the possible system of signal transmitting into cell, particularly by way of chemical influence. We note that any transmitting system, considering the secondary messengers must be insert the receptor, secondary messengers, enzymes of their synthesis and catabolism. Above adduced results serve as a proof of structure presence of similar acetylcholine receptors and biogenic amines of animal cells. Although it is more typical for the plant to transmit signal with participation calcium ions through complex formation with special, calmodulin proteins (KaM); Ca2+ depended on the proteinkinases and Ca2+/KaM-depending on the proteinkinases [64-67]. But there was stated the presence in the plants also such secondary messengers as c-AMF and cGMF, enzymes of their synthesis and catabolism (adenylaticyclase, guanilatcyclase phosphodiesterase) and protein target – proteinkinases. There is detected also in the plasmatic membrane of vegetable cells the G-protein playing the central role in the mechanism of signal transmission from effectors to secondary messengers [68-73]. Careful analysis of the existing literature and private results in the frame of real physical models with corresponding mathematic apparatus, given in the work [74], allows us at first to conclude that molecular mechanism of cytoplasm movement is stipulated by “ridge” movement of myosin oligomers, fastened to actin filaments. The lasts serve for increase of square and even distribution of motive force at the cytoplasm value. In the second we can speak about specific interaction of acetylcholine and biogenic amines with special structures of plasmatic membranes of the vegetable cells and signal transmission on the G-protein with next participation of secondary messengers. Our conclusion can be confirmed by the work of authors [75] where is shown the participation of a protein in the signal transmitting in the cells of the green seaweeds.

REFERENCES [1] [2] [3] [4] [5]

D.Ch. Boss. Collection of Works at Irritability of Plants. Ed. A.M. Siniukhin., Moscow, 1964, 1, 2. (Russ.). I.I.Gunari, A.M.Siniukhin, I.A. Ozolina, Izvestia. ТСХА. 1965, 2, 68-86. (Russ.). A.M. Siniukhin, E.A. Britikov. Fiziologia Rastrenii, 1967, 14, 463-476. (Russ.). A.B. Hope, N.A. Walker. The Physiology of Giant Algal Cells. New York, 1975. 201p. V.M. Iurin, M.N. Goncharik, S.G. Galaktionov. Transfer of Ions through Membrane of Vegetable Cells. Minsk, 1977, p. 60 (Russ.).

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Jaba Oniani, Vladimir Yurin, Ramaz Gakhokidze et al. V.A. Opritov, S.S. Piatigin, V.G.Retivin. Bioelctrogenesis of Higher Plants. Moscow, 1991, 216 p. (Russ.). S.S. Medvedev. Electrophysiology of the Plants. Moscow, SPb, 1998, 184 p. (Russ.). G.A. Volkov, L.V. Platinova. Different Peaks of Potential Action of Nitella flexlis ́Cells. Seaweeds Characea and their Applications in the Investigation of Biologic Processes in Cell. Vilniius, 1975, p.206-210 (Russ.). V.A. Sakalauskas. Mechanism of Intercellular Transmission of Irritation of Seaweed Characea. Dissertation.Vilniius, 1988, 184 p. (Russ.). W.Velten. Flora, 1876,. 59, 177-182, 193-199, 209-217. B.Corti. Osservazioni Microscopiche sulla Tremella e sulla Circolazione del Fluido in Una Planto Acquaguola. Lucea, Italy. 1774. N.Kamya.Movement of Protoplasm. Moscow, 1962, 306p. (Russ.). N.S. Allen, R.D.Allen. Ann. Rev. Biophys. Bioeng., 1978, 7, 497–526. K. Kuroda. Cytoplasmic Streaming in Plants. Int. Rev. Cytol., 1990, 21, 267-307. T. Shimmen. J. Plant Res., 2007, 120, 1, 31-43. T. Shimmen, E.Yokota. Curr. Op. Cell Biol. 2004, 16, 227-232. A.Lovy–Wheeltr, L.Cardenas, J.G.Kunkel, P.K. Hepler. Cell Mutil. Cytoskeleton. 2007, 64, 3, 217-232. N.Moreno, S.Bougourd, J. Haseloff, J.A. Feijo. Imaging Plant Cells. In.: Handbook of Biological Confacal Microscopy. Ed. J.B. Pawley, New York, 2006, p. 769-787. V.L. Kudasova. Dependence of Biologic Potentials of the Cell of Nitella flexis on their Age. Coll. Characea Seaweeds and their Application in the Investigation of biologic processes of the cell. Vilnius, 1975. p. 201-205. P.J. Simons. The Role of Electricity in Plant Movements. New Phytologist. 1981, 87, 1, 11-37. G. Hormann. Studien uber die Protoplasmstroming bei den Characeaen. Jena, 1896. U. Kishimoto, H.Akabori. J. Gen. Physiol.,1959, 42, 1167-1178. K.Umrath. Elektrophysiologische Phenomene. In: Encyclopedia Plant Physiology. Ed. W. Ruhland, Berlin, 1956, 2, 747-778. F.Auger. Lactivite Protoplasmique des Cellules Vegetales. Actualites Scientifiques et Industrielles, Paris. 1939, 823. T.Sibaoka, K.Oda. Sci. Rep. Tohoku Univ., 4th ser. Biol., 1956, 22, 157-166. Franck F., Auger D. Cr. Fcfd. Sci., 1932, 195, 1321-1322. M.Tazawa, U.Kishimoto. Plant Cell Physiol., 1968, 9, 361–368. R. E. Williamson. B. J. Cell Sci., 1975, 17, 655–668. T. Kohno , T. Shimmen . J. Cell. Biol. 1988, 106, 5, 1539-1543. R.E.Williamson, C.C. Ashley. Chara. Nature, 1982, 296, 647–651. T. Hayama, T. Shimmen, M. Tazawa. Protoplasma, 1979, 99, 305–321. Y. Tominaga, M. Tazawa. Protoplasma, 1981, 109, 103-111. H. Tazawa, M. Kikuyama, T.Shimmen. Electric Characteristics and Cytoplasmic Streaming of Chaesceae Cells Lacking Tonoplast. Cell Struct. Funct., 1976, 1, 165-176. K.Linsbauer. Protoplasma. 1929, 5, 565-621. S.P.Nichols. Bull. Tirrey Bot. Club. 1930, 57, 153-162., 1925 N.S.Allen. J. Cell. Biol. 1974, 63, 270–287. N.S. Allen, R.D. Allen, Biol. Bull. 1976, 151, 398. E. Kamitsubo. Proc. Japn. Acad. 1996, 42, 640–643, 1972.

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P.K. Hepler, B.A. Palevitz. Ann. Rev. Plant Physiol. 1974, 25, 309-362. J.S. Condeelis. Exp. Cell Res., 1974, 88, 435–439. I.A. Vorobev, B.F. Biophys., 1963, 8, 427-429. T. Kato, Y. Tonomura. J. Biochem., 1975, 78, 583–588. K. Ohsuka, A. Inoue. J. Biochem.,1982, 85, 375–378. M.Vaney, S.P. Scordilis. Can. J. Bot., 1980, 58, 797–801. G.Wagner. Actomyosin as a Basic Mechanism of Movement in Animals and Plants. Physiol. Movements, Berlin e.a., 1979, p.114-126. K.Yamamoto, S. Hamada, T. Kashiyama. Cell. Mol. Life Sci. 1999, 56, 227–232. K. Yamamoto , Shimada K., Ito K., Hamada S., Ishijima A., Tsuchiya T., Tazawa M. Chara. Plant Cell Physiol. 2006, 47, 10, 1427-1431. J.C.W. Chen, N. Kamiya. Cell. Struct. Funct., 1975, 1, 1-9. N. Kamiya. Ann. Rev. Plant Physiol., 1960, 11, 323–340. M.O. Bradley. J. Cell. Sci., 1973, 12, 327–343. J.C.W. Chen. Protoplasma, 1973, 77, 427-435. H.-U. Koop, O. Kiermayer. Protoplasma, 1980, 102, 295-306. R.E. Williamson,. Nature, 1974, 248, 801-802. R. Nagai, N.Kamiya. Exp. Cell Res., 1977, 108, 231-237. M.Yano. J. Biochem., 1978, 83, 1203–1204. M. Yano, T. Yamada, H.Shimizu. J. Biochem., 1978, .84, 277–283. M.Yano, T.Yamada, H. Shimizu. J. Biochem., 1978, 84, 1087–1092. M.Yano, T.Yamada, H.Shimizu. J. Biochem., 1978, 84, 1093–1102. M. Denesh. Biophysics, 1986, 31, 634-367. P. Pant, S.Terakwa, J. Baumgold. Bioshim. Biophys. Acta, 1978, 513, 132-140. D.A.Oniani, A.V. Plaks, E.S. Koeva. Biophysics, 1986, 31, 361-362. D.A. Oniani. Physical and Chemical Mechanism of Regulation of Vegetable Cell Cyclosis. Dissertation. Tbilisi.1987, 200 p. D.A. Oniani. Regulation of Cyclosis of Characea Cells. Tbilisi, 1997, 246 p. B.W. Poovaiah. Critic. Rev. Plant Sci. 1993, 12, 185-211. A.J.Trewavas, R. Malho. Cur. Opin. Plant Biol., 1998, 1, 428-433. J. Sheen. Science. 1996, 274, 1900-1902. U.Zentgraf, V.Hemleben. Progress Botany. 1996, 57, 218-234. N.P. Korolev, E.I. Viskrebentskova. Do Function the Cyclical nucleids System in the Higher Plants? In: Growth of Plant. Primary Mechanisms. Мoscow, 1978, p.178-205. V.V.Roshina. Biomediators in Plants. Acetylcholine and Biogenic Amines. Pushchino, 1991, 191 p. R.P. Newton, C.J. Smith. Photochemistry. 2004, 65, 2423-2437. L.V. Dubovskaia, O.V. Molchan, I.D. Volotovski. Fiziologia Rastenii, 2002, 49, 216220 (Russ.). A.Moutinho, P.J.Hussey, A.J.Trewavas, R.Malho. PNAS, 2001, 98, 10481-10486. P. Thuleau, J.I. Schroeder, R. Ranjeva. Cur. Opin. Plant Biol. 1998, 1, 424-427. E.B. Cherniaeva. Dissertation. Moscow, 1984, 205 p. M. Calenberg, U. Brohsonn, M. Zedlacher and G. Kreimer. Plant cell,1998, 10, 91-103.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 3

THE INFLUENCE OF CAFFEINE ANALOGUES AND ANTAGONISTS ON THE CA2+-ACCUMULATION BY SARCOPLASMIC RETICULUM Olga M. Alekseeva1, Yuri A. Kim2, and Vladimir A. Rykov2 1

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences1,. Moscow, 119334, Russia 2 Institute of Biophysics of Cell, Russian Academy of Sciences, Pushchino, Russia

ABSTRACT This study investigates the actions of caffeine analogues and antagonists to the main Ca2+-depo in the rabbit skeletal muscle - sarcoplasmic reticulum. The efficiency of Ca2+accumulation by vesicles of heavy fragmented sarcoplasmic reticulum was greatly changed by caffeine analogues and antagonist: cordiamin, camphor, bemegrid, 8methylcaffeine, teophilline, theobromine, midocalm. All tested substances have some similar characteristics at its molecular structure. The carbonyl oxygen and methyl groups are of a great importance for “caffeine effect” of these substances.

ABBREVIATIONS DTT - dithiothreitol; PMSF - phenylmethylsulfonyl fluoride; EGTA - ethylene glycol-bis [β-aminoethylether]-N,N,N’,N’-tetraacetic acid; SR – sarcoplasmic reticulum; FSR - fragmented SR; RyR - ryanodine receptor.

1 Kosygin str., 4, Moscow, 119334, Russia; Fax: (495) 137-41-01; E-mail: [email protected].

26

Olga M. Alekseeva, Yuri A. Kim and Vladimir A. Rykov

INTRODUCTION Caffeine is one of the most popular food stimulator of a number processes in organism. Administration of caffeine by animals improved task performance through the enhancement of central nervous system activity [1]. In case that caffeine acts in combination with another nutrition, caffeine may have a different pharmacological profile to those containing caffeine alone [2]. It is known that caffeine activates the muscle contraction too. Caffeine acts to the ryanodine receptor (RyR). RyR is the Са2+-regulated channel, which releases the most amount of Са2+, stored at the intracellular Са2+-depo. The Ca2+ release channel of the intracellular Са2+-depo of muscle cells has been purified using ryanodine, a plant alkaloid that binds to the 450-kDa-channel protein with nanomolar affinity. Electron microscopy indicates that the ryanodine receptor is a tetramer and forms the ‘feet’ structures described in muscle triads. This protein complex plays a central role in inter-membrane signal transduction during excitation-contraction coupling [3]. At least 80 mutations have been identified in the gene encoding the skeletal muscle ryanodine receptor and linked to several neuromuscular disorders, whose common feature appears to be a deregulation of calcium homeostasis. These mutations affect the functional properties of the ryanodine receptor that resalt to malignant hyperthermia, central core disease and multiminicore disease [4]. Central core disease is a variable autosomal dominant disorder, which has been linked principally to the gene for the skeletal muscle calcium release channel (RYR1) [5]. There are 3 isoforms of RyR. RyR1 and RyR2 are isoforms that expressed in the skeletal and cardiac muscles, respectively. RyR3 is isoform that expressed at brain and embryo muscle. Their missions mutations, which are clustered in three regions that correspond to each other, cause hereditary disorders such as malignant hyperthermia in skeletal muscle and central core disease, and catecholaminergic polymorphic ventricular tachycardia in cardiac muscle. Phenotypes with RyR1 and RyR2 mutations are mainly caused by misalignment of their functions through the interdomain interaction and luminal Ca2+ [6]. Caffeine hasn’t some influences to the Са2+-АТРаse, which accumulate Са2+ to the lumen of Са2+-depo. The sarcoplasmic and endoplasmic reticulums are the main intracellular Са2+depo at the skeletal muscle and non-muscle cells, respectively. Two preparations of fragmented sarcoplasmic reticulum (FSR): heavy FSR and light FSR are used for Са2+-depo testing. Heavy FSR is prepared from terminal cisterns that release Са2+ by RyR. Light FSR is prepared from longitudinal tubes, which accumulated Са2+ by Са2+-АТРаse [7]. Heavy FSR has RyR and Са2+-АТРаse molecules. Light FSR has negligible quantity of RyR molecules. We investigated the specificity of caffeine analogues and antagonists effects to the membranes by using of the model “heavy-light FSR”. But the heavy fraction is contaminated with adult mitochondria (50%). The light fraction not contains the adult mitochondria; it is contaminated by the mitochondria fragments (5-10 %) only [8]. For that we deal with light fraction only, which separated in 2 sub-fractions: caffeine-sensitive and caffeine-insensitive [9,10]. The RyR and Ca2+-ATPase are present in the membranes of the light caffeinesensitive subfraction. The caffeine-insensitive subfraction not contains RyR. The protein content in this subfraction is presented by the Ca2+-ATPase over 90%, while in the heavy fraction - 50-60% only. The difference of Ca2+-ATPase activity of light and at heavy fractions, was nearly twice as much [11].

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27

The main goal of our study is to find the key groups in caffeine analogues molecules that cause interaction with RyR. We recorded the efficiency of Са2+-accumulation by heavy and light FSR under the tested substances applications. We found that the existence of carbonyl oxygen neighboring with methyl groups is of great importance for interaction with RyR.

MATERIALS AND METHODS The materials: KCl, KH2PO4 (Merck); histidine, imidasol, caffeine (Merck); NaCl, MgCl2 (Merck); DTT (Serva); glycerol (Serva); CaCl2 (Merck); sucrose (Merck); EGTA (Serva); PMSF (Helicon). We used the standard procedures of the isolation and the purification of FSR [9,12,13] with small modifications. The first step was realised in the presence of DTT, PMSF and 10 mM caffeine. The protein concentration was determined by the fast method [14]. Efficiency of Са2+-accumulation by heavy FSR, and light FSR was recorded by pHmetric method [15]. FSR vesicles (3-4 mkg/ml) were incubated in 4 ml medium contains 5mM sodium oxalate, 0,1M NaCl, 4 mM MgCl2, 1,9 mM ATP, 2,5 mM imidasol (pH 6,8) 37o C with intensive mixing. Reaction was stimulated by additions of 80 nmoles CaCl2.

RESULTS AND DISCUSSION We selected caffeine analogues: cordiamin, camphor, bemegrid, 8-methilcaffeine, teophilline, theobromine, and antagonist – midocalm, on the basis of similarity of its structure (figure1). All tested substances have certain pharmacological effects: 1) cordiamin, camphor, bemegrid, and 8-methylcaffeine, teophilline, theobromine – cardiovascular medicines; 2) midocalm – anti-epileptic drug. We used a few criterions for the rating of specificity of caffeine center activation. The first: the substance must decrease the Ca2+-accumulating effectiveness. The rate of Ca2+-ATPase function must be not changed at the beginning of reaction, but must activated at the middle and last phases, because all amount of Ca2+accumulated just was released from FSR through the activated RyR. And Ca2+ cannot inhibit Ca2+-ATPase activity from FSR lumen. Second: Ca2+-accumulating effectiveness must be decreased only for caffeine-sensitive FSR, because caffeine-insensitive FSR haven’t any RyR. Third: procaine or tetracaine and ruthenium red must block the influence of substance to the caffeine-sensitive FSR as for caffeine effects. The standard data of Ca2+-accumulating by FSR are shown at figure 2. The kinetic curves represent the recording of medium pH changes that correlated with Ca2+-accumulating process to the lumen of FSR with the aid of the Ca2+-ATPase Ca2+-pumping activity. There is large Ca2+-ATPase activity at the start of reaction (by Ca2+ addition), because at the first seconds there are large Ca2+ concentrations at recording medium, and there is not the essential quantitative of Ca2+ at FSR lumen. This is the optimal condition for Ca2+-accumulation by Ca2+-ATPase. When Ca2+-ATPase pumped Ca2+ to the lumen, H+ was released to the recording medium and ATP was hydrolyzed. Thus the kinetic curves are rapidly rise upward, and we record the acidification of medium. But, when all excess of Ca2+ was pumped to the FSR lumen, the Ca2+-ATPase activity became small and constant, and the kinetic curves went

28

Olga M. Alekseeva, Yuri A. Kim and Vladimir A. Rykov

out to the plateau. The presence of caffeine (4mM) at the recording medium changes the kinetic curve type for heavy FSR and caffeine-sensitive FSR greatly. The kinetic curves of light FSR and caffeine-insensitive FSR are changed by caffeine slightly. Procaine or tetracaine and ruthenium red block the caffeine effects at the caffeine-sensitive FSR (figure 3). At the present work the efficiency of Са2+-accumulation by heavy FSR, and light FSR was investigated under the tested substances applications. For the calculation of efficiency value of Са2+-accumulation we use the “Ca2+/ATP”, that means the ratio between quantity of pumped Ca2+ ions and quantity of hydrolyzed molecules of ATP. The plateau of kinetic curves means that all Ca2+ ions (certain quantity) are pumped to the FSR lumen. And we calculated from kinetic curves with calibration by addition of certain quantity of KH2PO4 the quantity of hydrolyzed ATP molecules.

Figure 1. The structure formulas of caffeine and analogues: cordiamin, camphor, bemegrid, 8methilcaffeine, teophilline, theobromine, and antagonist – midocalm.

The Influence of Caffeine Analogues and Antagonists…

29

Figure 2. The kinetic curves of Ca2+-accumulation by FSR heavy (A) and light (B). 1 - control; 2 - 5 mM caffeine; 3 - 5 mM caffeine + 10 mM procaine.

Figure 3. The kinetic curves of Ca2+-accumulation by FSR heavy (A) and light (B). 1 - control; 2 - 5 mM caffeine + 0,2 mM tetracaine; 3 - 5 mM caffeine + 3mkM ruthenium red; 4 - 5 mM caffeine; 5 - 10 mM caffeine.

In the figure 4 and table 1 are shown the data of camphor influence to the Ca2+-transport by light and heavy FSR. Camphor 4mM decreases for 3 times the efficiency of Са2+-

30

Olga M. Alekseeva, Yuri A. Kim and Vladimir A. Rykov

accumulation by heavy FSR. Camphor no changes Ca2+/ATP for light FSR, but the velocity of ATP hydrolyzes was not changed for both FSR. All camphor effects are prevented by ruthenium red and procaine additions. Therefore, the effects of camphor and caffeine are similar, according to our 3 criterions.

Figure 4. The kinetic curves of Ca2+-accumulation by FSR. The camphor influence to the transport Ca2+-ions by heavy (A) and light (B) FSR. 1 - 3mkM ruthenium red + 4 mM camphor + 10 mM procaine; 2 - control; 3 – 1,6 mM camphor; 4 - 4 mM camphor; 5 – 5 mM caffeine; 6 – 4 mM camphor + 10 mM procaine.

The mutual element of structural formulas of camphor and caffeine are carbonyl oxygen and alkyl group located nearly. Apparently, the presence of carbonyl oxygen is very important critical point at structure of camphor and caffeine molecules for its specific effects. Because the analog of camphor – borneol, that contained the alcohol group instead of ketogroup, doesn’t exert the uncoupling influence to Ca2+-transport with ATP hydrolysis for heavy FSR. There are 3 methyl groups at structure of camphor and caffeine molecules, as shown at figure1. We tested some cyclic ketones for the determination - what of the methyl groups has a great importance for the caffeine effect. We tested the possibility of caffeine center activation for methylksantins that have certain pharmacological effects. 8methylcaffeine, 3,7-dimethylksantin (theobromine), 1,3-dimethylksantin (teophilline) were tested with aid light and heavy FSR. As shown at the table1, all tested methylksantins have the ability to uncoupling the Ca2+-transport with ATP hydrolysis for heavy FSR. Procaine and ruthenium red eliminate its effects. So, all tested methylksantins exert the caffeine-imitated effect. From the comparison of its structural formulas we concluded that the methyl groups at the 1 and 7 locations are not essential groups for the existence of caffeine-imitated effect.

The Influence of Caffeine Analogues and Antagonists…

31

Probably, the methyl group at the 3 location and the carbonyl oxygen at the 2 location are the active groups for caffeine-imitated effect. Table 1. The influence of caffeine analogues and antagonists to the transport Ca2+-ions by heavy fractions of sarcoplasmic reticulum substance

mM

Ca2+/ATP

Effect (%)

control caffeine 8-methylcaffeine,

4 2,5 5 5 5 4 15 5 10 15

1,1 0,19 0,24 0 0,20 0,20 0,17 0,65 1,1 0,85 0,64

83 87 100 80 80 85 41 0 23 45

theobromine teophilline camphor cordiamin bemegrid

Ca2+/ATP Substance + 3mkM ruthenium red 1,1 1,1 1,1 1,1 1,1 1,1 -

Ca2+/ATP Substance + 10mM procaine 1,1 1,1 1,1 1,1 1,1 1,1 1,1 -

Figure 5. The kinetic curves of Ca2+-accumulation by FSR. The influence of anti-epileptic drug, midocalm (1-piperidino-2-methyl-3paratolyl-propanoil-3-hydrochlorid) to the transport Ca2+-ions by heavy FSR and caffeine effect. 1 - control; 2 - 5 mM caffeine; 3 - 5 mM caffeine + 10 mM midocalm.

32

Olga M. Alekseeva, Yuri A. Kim and Vladimir A. Rykov

Analeptic drugs - cordiamin and bemegrid, exert the caffeine-imitated effect too, as shown at the table 1. The anti-epileptic drug, midocalm (1-piperidino-2-methyl-3paratolylpropanoil-3-hydrochlorid), prevents caffeine effect to the Ca2+-transport by heavy FSR completely (figure 5). The addition of midocalm recovers the Ca2+-transport to the normal value and even increased efficiency of Са2+-accumulation. Our investigations may be interesting for the substance test at the simple FSR- light heavy model.

CONCLUSION The substances, which have the carbonyl oxygen nearly alkyl group in their molecules, activate RyR. It decreases the efficiency of Са2+-accumulation by heavy FSR, and no changes ones by light FSR. The carbonyl group at the 2 location and methyl group at the 3 location are the most important groups at the analogue molecules for the caffeine-imitated effect. This fact illustrates the some similarity of “caffeine center” and esterophilic part of acetylcholine receptor. But the caffeine antagonist – midocalm, blocked the caffeine action. The using of this model allowed us to predict the pharmacological effect of tested food substances.

REFERENCES [1]

[2]

[3] [4]

[5]

[6]

[7]

S. Ataka, M. Tanaka, S. Nozaki, H. Mizuma, K. Mizuno, T. Tahara, T. Sugino, T. Shirai, Y. Kajimoto, H. Kuratsune, O. Kajimoto and Y. Watanabe. “Effects of oral administration of caffeine and D-ribose on mental fatigue”, 2008, 24, 3, 233-238; C.F. Haskell, D.O. Kennedy, A. L. Milne, K. A. Wesnes and A. B. Scholey: Biological Psychology “The effects of l-theanine, caffeine and their combination on cognition and mood” 2008, 77, 2, 113-122. M. Fill and R. Coronado. Ryanodine receptor channel of sarcoplasmic reticulum. 1988, Trends in Neurosciences , 453-457; S. Treves, A.A. Anderson, S. Ducreux, A. Divet, C. Bleunven, C. Grasso, S. Paesante and F. Zorzato “Ryanodine receptor 1 mutations, dysregulation of calcium homeostasis and neuromuscular disorders”, 2005, Neuromuscular Disorders, 15, 9-10, 577-587; M.R. Davis, E. Haan, H. Jungbluth, C. Sewry, K. North, F. Muntoni, T. Kuntzer, P. Lamont, A. Bankier, P. Tomlinson, A. Sánchez, P. Walsh, L. Nagarajan, C. Oley, A. Colley, A. Gedeon, R. Quinlivan, J. Dixon, D. James, C.R. Müller, et al. “Principal mutation hotspot for central core disease and related myopathies in the C-terminal transmembrane region of the RYR1 gene”, 2003, Neuromuscular Disorders, 13, 2, 151157; Y. Ogawa, “Distinct mechanisms for dysfunctions of mutated ryanodine receptor isoforms“, 2008, Biochemical and Biophysical Research Communications Article in Press; V.B.Ritov, O.M.Vekshina and N.B. Budina. Bulletin experimentalnoy biologii i medizini. 1984, 9, 317-320.

The Influence of Caffeine Analogues and Antagonists…

33

[8]

O.M Vekshina and N.L.Vekshin. International symposium. «Biological motility: new trends in research». Pushchino. August 20-26. 2001, p.166. [9] V.B. Ritov, N.B. Budina and O.M.Vekshina. Bulletin experimentalnoy biologii i medizini. 1985, 1, 53-54. [10] V.B. Ritov. Bulletin experimentalnoy biologii i medizini. 1985, 4, 445-447.

[11] O.M.Vekshina and N.L. Vekshin. Mol. Biol. (Moscow). 1989, 23, 4, 10411050. [12] O.M Alekseeva., and V.B. Ritov. Biochimia. 1979, 44, 1582-1593. [13] N.Ikemoto, D.H. Kim, and B. Antoniu. Methods Enzymol. 1988, 157, 469-480. [14] N.L. Vekshin. Photonics of biopolimers. Springer “Biological and Medical Physics Series”. 2002. [15] V.B. Ritov. Biochimia. 1971, 36, 393-399.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 4

BIOLOGICALLY ACTIVE MULTIFUNCTIONAL ADAMANTANE-CONTAINING COMPOUNDS Khatuna Barbakadze1, Nodar Lekishvili1, Zurab Pachulia1, Giorgi Lekishvili2, Badri Arziani2, Iuri Sadaterashvili1, and Davit Zurabishvili1 1

Ivane Javakhishvili Tbilisi State University1 Tbilisi, Georgia 2 Tbilisi State Medicinal University2, Tbilisi, Georgia

ABSTRACT New biologically active adamantane-containing anilides and nitroanilides have been synthesized and studied. By the semiempirical quantum-chemical method, AM1 effective charges on the atoms, bonds lengths and valence angles, enthalpies of formation of initial compounds and probable obtained products of the reaction of nitration of the anilides have been calculated. Based on quantum-chemical calculations and experimental data, the direction of the reaction of nitration has been established. To model physical properties of biologically active 16 anilides and nitroanilides, we studied quantitative “structure-properties” relationships (QSPR) on the basis of the experimental data. We used several sets of molecular descriptors. The dataset outliers were identified by using Principal Component Analysis (PCA). As a modeling technique, we applied Projections to Latent Structures (PLS). To ascertain the quality of models, we used cross-validation. Based on our research, one concludes that the best models were acquired by the use of GETAWAY. The anthelminthic activity and activity towards various microorganisms of the obtained compounds have been studied. Based on preliminary investigations, it was established that the obtained compounds may be recommended as modifiers of anthelminthic preparations— phenacetyne, trinoine, diamphenetide, raphoxanide—also as a bioactive component for preparation: a) materials with antimycotic properties for 1 2

1, Ilia Chavchavadze Ave., 0179 Tbilisi, Georgia; *e-mail: [email protected]. Vazha Pshavela Ave., 333, 0177 Tbilisi, Georgia E-mail: [email protected].

36

Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al. prophylaxis and treatment of mycosis and dermatomycosis; b) protective covers stable to biocorrosion from action of some mycopathogenic microorganisms.

1. INTRODUCTION Treatment of a population fallen ill from especially dangerous helminthes (echinococcosis, trichinellosis, fascioliasis, etc.) is becoming an important issue of the day [13]. Anthelminthic preparations of a wide spectrum of action are used in veterinary and medical practices for dehelminthization of animals and people. Research of derivatives of aminophenoles and salicylic anilides has led to the creation of the great group of highly effective anthelminthics (hexiqole, acemidophene, etc.) [4,5]. However, during the protracted course of therapy by the abovementioned preparations, they gradually lose efficiency owing to the helminths becoming accustomed to them. Some of preparations have shown embryo toxicity, teratogenicity and mutagen properties. Therefore, researching preparations that are effective and safe for the person and the environment but without the collateral properties of anthelminthic preparations is an actual problem for modern pharmacology [5]. Modification of various biologically active compounds containing HO- and NH2-groups by O,N-adamantylation and O,N-adamantoylation leads to improvement of their hydrolytical stability, membranotropic and immunotropic properties [6,7]. Compounds of this type are characterized with minimal therapeutic dose, a high therapeutic index and a wide spectrum of action. According to the aforementioned, the modification of existent preparations by lipophilic and membranotropic adamantane radical have a great potential [8]. Besides, in many regions of the world some diseases of agricultural plants are extensive. They are caused by various phytopathogenic microorganisms [9,10]. For example, roots cancer is caused by A. tunefacicus. Tumors, halles and nodes are formed as a result of intensive division of affected cells of meristem plant tissues. Roots and fruit-trees cancers are provoked by—А. tunefacicus; a cancer of root crops, beets is provoked by X. campestris pv. beticols, etc. These diseases distractively damage plants and significantly decrease harvesting efficiency. They also deteriorate quality of grape, water-melons, melons and gourds and other agricultural plants [10]. Adamantane-containing compounds are interesting in this area, too [11, 12]. Therefore synthesis of new compounds with high biological activities as plant protectors and effective means against phytopathogenic microorganisms, as well as conservators and compounds for antibiocorrosive covers of various natural, synthetic and artificial materials and cultural heritage is also extremely significant and requires further development [13].

2. EXPERIMENTAL Synthesis of adamantane-containing anilides: to the benzene solutions of hydrochlorides of initial amines [7] and basic agents (triethylamine, NaHCO3 or NaOH) was added a dropwise benzene solution of the chloroanhydride of various carbonic acids (adamantane-1carboxylic acid, acetic acid, benzoic acid or phenylacetic acid, correspondingly). The mixture

Biologically Active Multifunctional Adamantane-Containing Compounds

37

was heated and stirred for 1.5–3 hrs. The precipitated crystals were separated by filtration and washed with H2O; they were dried and physical constants of obtained compounds were determined. Synthesis of adamantane-containing nitroanilides: to the solution of adamantanecontaining anilides in acetic anhydride and acetic acid was added drop-wise 56–58% HNO3. The mixture was cooled to 5–10ºC and stirred for 0.5–2 hrs. The reaction mixture was dispersed (by being poured out on ice water). The precipitated crystals were separated by filtration and washed with H2O; they were dried and physical constants of the obtained compounds were determined.

METHOD OF ANALYSIS Spectral analysis: IR spectra were obtained with a spectrophotometer Nicollet Nexus 470 machine with MCTB detector [14]. NMR spectra were obtained with an AM-400 (Brucker®) and Talsa BS-467 instrument at an operating frequency of 400 MHz using CDCl3 as a solvent and tetramethylsilane as an internal standard [15]. Mass-spectrograms were obtained with a mass-spectrometer MX-1321A, energy of ionized electrons’, 70 eV [16]. Thin-layer chromatography: the reaction course and the purity of the obtained compounds were monitored by thin-layer chromatography on silufol UV-254 and alufol plates. Quantum-chemical calculations were performed on PC with AMD processor with the built-in coprocessor by using Mopac 2000 and CS Chem3D Ultra, v8. We gave the following key-words to guide each computation: EF GNORM = 0.100 MMOK GEO-OK AM1 MULLIK LET DDMIN=0.0 GNORM=0.1 GEO-OK. QSPR calculations: We used MDL Isis Draw 2.5.SP4 to build molecular models. Afterwards, we concatenated the models into the dataset by use of EdiSDF 5.02. The textual format of the dataset was SDF. We used VCC-Lab e-Dragon web application for calculation of molecular descriptors. Statistica 6.0 was a tool of our choice for building PCA and PLS models.

3. DISCUSSION OF RESULTS 3.1. Synthesis and Quantum-Chemical Investigation of AdamantaneContaining Anilides and Nitroanilides We synthesized and studied adamantane-containing anilides and nitroanilides with various organic radicals in benzene ring (Scheme 1) [17,18]. In order to select these compounds, we considered the availability of their synthesis and possibility of their perspective wide commercialization. Adamantane-containing (Ad) anilides we synthesized in two stages according to the Scheme 1. We carried out the adamantylation of phenols with 1-adamantanole and 1-bromadamantane and the nitration of the obtained intermediate products. The reduction of the 4-(1adamantoxy)nitrobenzene with various systems we carried out by molecular hydrogen in

38

Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al.

presence of Rene’s nickel in dry ethanol and ethylacetate in two-phase system [Fe:NH4Cl:H2O:aromatic solvent (benzene, toluene, xylene); Fe:CH3COOH:CH3OH], by acylation of obtained alkyl(aryl)oxyanilines with carboxylic acid anhydrides and chloroanhydrides in the presence of base agents (triethylamine, NaOH, Na2CO3 or CH3COONa): O2N

OH

OH

X

+

X = Br, OH.

Fe / NH4Cl, H2O

NO2

O

arom. solv.

O3 HN

O (a)

O

/

R COCl

NH2

O

NH

O

C

R/

R/ = CH3 (III); R/ = 1-Ad (IV); R/ = Ph (V); R/ = CH2Ph (VI).

R/ O

NO2

R

Fe / NH4Cl, H2O arom. solv.

R

/

O

NH2

AdCOCl

R/ O R

R

(b)

NHCO

R = H: R/ = CH3 (I); R/ = C2H5 (II); R = Cl, R/ = y-ClC6H4 (VII).

Scheme 1. Synthesis of some adamatane-containing anilides.

Adamantane-containing nitroanilides we synthesized by nitration of obtained anilids with 56–58% HNO3 in the media of CH3COOH, at the 5–10oC, according to the following general reaction scheme: R/

NHCOR//

O

HNO3 CH3COOH

R/

NHCOR//

O

R

R I-VII /

NO2 VIII-XIV

//

/

//

R=H, R =CH3, R =Ad (VIII); R=H, R =C2H5, R =Ad (IX); R=H, R/=Ad,

R//=CH3 (X); R=H, R/=Ad, R//=Ad (XI); R=H, R/=Ad, R//=Ph (XII); R=H, R/=Ad, R//=CH2Ph (XIII); R=Cl, R/=y-ClC6H4, R//=Ad (XIV).

Scheme 2. Synthesis of some adamantane-containing nitroanilides.

The composition and structure of the synthesized compounds (Scheme 1 and Scheme 2) we established by IR, NMR and mass spectral data (Figure 1a and Figure 1b, Figure 2a and Figure 2b).

Table 1. Some physical constants of synthesized correspond-containing anilides and nitroanilides, IR and NMR spectral data Rf Hexane : diethyl ether, 1:1 0.60

IR spectra, ν, cm-1

NMR 1H (DMSO-D6)

NMR 13C (DMSO-D6)

3290 (NH), 3050 (C-H, ar.), 1640 (C=O), 1230, 1035 (C-OC) 3290 (NH), 3050 (C-H, ar.), 2930, 2908, 2834 (C2H5, Ad), 1640 (C=O), 1230, 1035 (C-OC) 3330(N-H), 3100, 3030 (C-H, ar.), 2908, 2845 (CH, Ad),1650 (C=O), 1210(C-O-C) 3420 (NH), 2950, 2930, 2850(Ad), 1670 (C=O), 1210, 1050 (C-O-C)

δ=8.95 (s, 1H), 7.52 (d, J=9.1 Hz, 2H), 6.84 (d, J=9.1 Hz, 2H), 3.71 (s, 3H), 2.01 (s, 3H), 1.89 (m, 6H), 1.70 (s, 6H)

δ=175.4,155.0,132.3, 121.8, 113.4, 55.0, 40.6, 38.3, 36.0, 27.6

δ=9.01 (s, 1H), 7.52 (d, J=8.9 Hz, 2H), 6.85 (d, J=8.8 Hz, 2H), 2.12 (s, 3H), 2.01 (s, 3H), 1.89 (m, 6H),1.76(m, 6H), 1.70(m, 6H), 1.56(m, 6H) δ=10.18 (s, 1H), 7.94 (d, J=7.0Hz, 2H), 7.67 (d, J=8.8Hz, 2H), 7.58 (t, J =7.1Hz, 1H), 7.52 (dd, J=7.0Hz, 2H), 6.94 (d, J=8.8Hz, 2H), 2.13 (s, 3H),1.79 (m, 6H),1.57 (m, 6H) δ=10.07 (s, 1H), 7.48 (d, J=8.8Hz, 2H), 7.33 (m, 4H), 7.24 (m, 1H), .87 (d, J=8.8Hz, 2H), 3.60 (s, 2H), 2.10 (s, 3H), 1.75 (m, 6H), 1.55 (m, 6H)

δ=175.5, 148.8, 135.0, 124.3, 120.7, 76.5, 42.2, 40.7, 38.3, 35.9, 35.5, 30.1, 27.6

#

m.p., 0 C

I

178179

II

206-207

0.40

III

165-168

0.50

IV

240-241

0.77

V

160

0.41

3390(N-H),3050(C-H, ar.), 2908, 2845 (C-H, Ad),1650 (C=O)

VI

181-182

0.53

3290 (N-H), 3030 (C-H ar.), 2908, 2845 (C-H, Ad),1650 (C=O)

VII

176-177

0.45

3290 (N-H), 3030 (C-H, ar.), 2908, 2845 (C-H, Ad), 1650 (C=O), 1530 (Cl), 1250 (COC)

δ=165.2, 149.3, 135.0, 134.8, 131.3, 128.2, 127.5, 124.5, 120.9, 76.7, 42.2, 35.5, 30.1

δ=168.6, 148.9, 136.0, 134.9, 128.9, 128.2, 126.4, 124.6, 119.5, 76.6, 43.2, 42.2, 35.5, 30.1

Table 1. (Continued) VIII

134135

0.65

IX

133-135

0.73

X

119-122

0.42

XI

172-174

0.86

XII

151-153

0.80

XIII

127-130

0.65

XIV

168-170

0.91

3278 (N-H), 3090 (C-H, ar.), 2908, 2846 (CH, Ad),1650 (C=O), 1581, 1350 (N-O), 1288, 1249 (C-O-C) 3379 (N-H); 3090 (C-H, ar.), 2930, 2908, 2854 (C2H5, Ad); 1650 (C=O); 1581, 1350 (N-O); 1280, 1056 (C-O-C) 3090 (C-H, ar.), 2916, 2854 (CH, Ad), 1650 (C=O),1350 (NO), 1250 (C-O-C) 3448, 3371 (N-H), 3090 (C-H ar.), 2928, 2850 (CH, Ad), 1689(C=O), 1342 (N-O), 1265, 1242 (C-O-C) 3447 (N-H); 3090 (C-H, ar.), 2915, 2854 (CH, Ad), 1681(C=O), 1342 (N-O), 1265, 1234 (C-O-C) 3317 (N-H); 3108, 3070 (C-H ar.), 2908, 2854 (CH, Ad), 1697(C=O), 1334 (N-O), 1272, 1234 (C-O-C) 3290 (N-H), 3030 (C-H, ar.), 2908, 2845 (CH, Ad),1650 (C=O), 1530 (Cl), 1342 (N-O), 1250(C-O-C)

δ=9.62 (s, 1H), 7.65 (d, J=9.0 Hz, 1H), 7.49 (d, J=3.0Hz, 1H), 7.30 (dd, J=9.0, 3.0Hz, 1H), 3.83 (s, 3H), 2.02 (s, 3H), 1.88 (m, 6H), 1.70(m, 6H) δ=9.61 (s, 1H), 7.65 (d, J=9.0Hz, 1H), 7.46 (d, J=2.9 Hz, 1H), 7.29 (dd, J=9.0, 2.9Hz, 1H), 4.10 (d, J=6.9Hz, 2H), 2.02 (s, 3H), 1.88 (m, 6H), 1.70 (m, 6H), 1.34 (t, J=6.9Hz, 3H)

δ=175.6, 155.7, 142.8, 127.3, 125.0, 120.3, 108.8, 55.9, 40.5, 38.2, 35.9, 27.5

δ=9.75 (s, 1H), 7.75 (d, J=8.8Hz, 1H), 7.51 (d, J=2.6Hz, 1H), 7.34 (dd, J=8.8, 2.6Hz, 1H), 2.15 (s, 3H), 2.03 (s, 3H), 1.76 (m, 24H)

δ=211.1, 175.8, 130.4, 128.0, 125.8, 120.0, 78.6, 41.9, 40.6, 38.4, 38.1, 35.9, 35.9, 35.3, 30.1, 27.4, 27.3

δ=175.6, 155.0, 142.7, 127.2, 124.9, 120.7, 109.3, 64.0, 40.5, 38.2, 35.9, 27.5, 14.3

Biologically Active Multifunctional Adamantane-Containing Compounds

41

In the IR spectra of the obtained compounds we observed the characteristic absorption bands for the following groups: νas NH (3430-3130cm-1), νas C–H of aromatic ring (31203030 cm-1), νas C–H of adamantyl groups (2910–2830 cm-1), νas >C=O carbonyl group (1670– 1640 cm-1), νas NH, C–N (1540–1500 and 1360–1330 cm-1), NO2 (1330–1350 cm-1) and C– O–C (1270–1230 cm-1) groups [14]. In the 1H NMR spectra of the synthesized anilides (experimental; Figure 2a) one can observe singlet signal with chemical shifts within the range 9.01–10.07 ppm for the protons in the NH groups. In the spectra we could observe also quartet signals with chemical shifts at 7.30–7.94 ppm for protons in phenyl groups. In the spectra we found also multiple signals with chemical shifts at 1.57–2.01 ppm related to the 15 protons in adamantyl groups and the singlet signal with chemical shifts within the range 3.71–3.83 ppm corresponded to three protons in methyl groups (I, VIII). The singlet signal with chemical shifts 3.60 ppm is related to protons of the methylene group (VI). In the 1H NMR spectra of the synthesized nitroanilides (Experimental; Figure 2b) one can also observe doublet signal with chemical shifts within the range 7.46–7.51 ppm for the protons C(3)H; two doublet signal with chemical shifts within the range 7.29–7.65 ppm is related to protons C(2)H and C(5)H. The value of the constant of spin-spin interaction J=2.8 confirms the substitution of the nitro-group in the position 2 (Scheme 2) [15]. In the 13C NMR spectra one can observe the signal with four chemical shifts within the range 27.6–40.7 ppm typical for adamantyl groups and the chemical shift 55.0 ppm related to the carbon atom of CH3 group (I). In the 13C NMR spectra we also observed chemical shifts within the range 165.17–175.57 ppm and the chemical shifts within the range 113.37–155.73 ppm related to carbon atom of C=O groups and carbon atom of phenyl group, correspondingly.

Figure 1a. IR spectrum of 4-Methoxy-N-(1-adamantoyl)anilide.

42

Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al.

Figure 1b. IR spectrum of 4-Methoxy-N-(1-adamantoyl)-2-nitroanilide.

Figure 2a. 1H NMR spectrum of 4-Methoxy-N-(1-adamantoyl)anilide.

Biologically Active Multifunctional Adamantane-Containing Compounds

43

Figure 2b. 1H NMR spectrum of 4-Methoxy-N-(1-adamantoyl)-2-nitroanilide.

The mass-spectrogram data for the synthesized anilides and nitroanilides (Figure 3a and Figure 3b, Table 2) show that the masses of molecular (M+) and fragmental ions correspond with obtained structures of aforementioned compounds (Scheme 1 and Scheme 2). Table 2. Mass-spectral data for some synthesized anilides and nitroanilides Compounds* I IV VIII XI

Data of mass-spectra, m/z (Irel., %) 285.2 [M+] (75), 257.2 (6), 214.1 (2.4), 200.1 (13.7), 149.0 (5.4), 135.1 (100) 330.2 [M+] (18.7), 284.2 (3.6), 210.1 (1.8), 135.1 (100) 405.2 [M+] (10.1), 255.2 (13.7), 212.1 (1.8), 135.1 (100) 450.3 [M+] (8.1), 316.1 (1.8), 270.2 (1.8), 227.2 (1.7), 152.1 (1.9), 135.1 (100)

*) Scheme 1, 2.

By the semiempirical quantum-chemical method of AM1 we calculated geometric parameters (valency angles and bond lengths) and effective charges on the atoms of initial anilides (Figure 4, Tables 3-5). Quantum-chemical calculations were performed by using CS MOPAC (Chem3D Ultra-version 8.03, method AM1 - Austin Model 1) [19, 20]. Such calculations are useful as the basic reference data of the aforementioned parameters for analogical structures.

44

Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al.

Figure 3a. Mass-spectrum of 4-Methoxy-N-(1-adamantoyl)anilide.

Figure 3b. Mass-spectrum of 4-Methoxy-N-(1-adamantoyl)-2-nitroanilide.

Biologically Active Multifunctional Adamantane-Containing Compounds

45

As the initial anilides contain several feasible reaction centers we performed series of quantum-chemical calculations to determine the direction of the reaction of nitration (Scheme 1). We have also calculated the heat of formations (enthalpy, ΔHf) and reaction heat effects (ΔHreac.) for probable reaction products. As a model reaction we selected the nitration of 4-(p-chlorophenoxy)-3-chloro-N-(1-adamantoyl)anilide (AH): O Cl O O

Cl

NH

C

O

NH Cl

HNO3

I

NO2 NO2

(b)

Cl

(a)

Cl

O

C

NH

O C

Cl II

Scheme 3. The model system of nitration of 4-(p-chlorophenoxy)-3-chloro- N-(1-adamantoyl)anilide.

The 3D model of the optimized structure of 4-(p-chlorophenoxy)-3-chloro-N-(1adamantoyl)anilide give in the Figure 4.

Figure 4. 3D model of 4-(p-chlorophenoxy)-3-chloro-N-(1-adamantoyl)anilide.

We have calculated valence angles between carbon atoms in benzene ring of the 4-(pchlorophenoxy)-2-nitro-3-chloro-N-(1-adamantoyl)anilide (118-120o) what correspond to the sp2-hybridization state of carbon atoms in same arylen systems [18]. The quantum-chemical calculations show that in benzene ring (a) the value of effective charges (Table 4) at C(2) and C(6) carbon atom are -0.23315 and -0.20288, correspondingly. During the reaction of nitration the NO2+ group (Figure 3) attacks C(2) carbon atom. The results of the calculation show that formation of the product I (ΔHf = – 175.60 kJ/mole) is

46

Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al.

slightly more probable than the product II (Sceme 3) (ΔHf = –166.90 kJ/mole) (for the initial anilide [4-(p-chlorophenoxy)-3-chloro-N-(1-adamantoyl)anilide] ΔHf = – 220.16 kJ/mole). Table 3. Bond distances in the molecule of 4-(p-chlorophenoxy)3-chloro-N-(1-adamantoyl) anilide Atoms C(1)-N(8) C(2)-C(3) C(2)-H(35) C(3)-C(4) C(3)-Cl(21) C(4)-C(5) C(4)-O(7) C(5)-C(6) C(5)-H(33) C(6)-H(34) O(7)-C(22) N(8)-C(9) N(8)-H(36) C(9)-O(10) C(9)-C(11) C(11)-C(12) C(11)-C(16) C(11)-C(17)

Bond length, Å 1.401 1.388 1.103 1.404 1.713 1.406 1.392 1.371 1.103 1.102 1.394 1.390 1.004 1.246 1.537 1.537 1.533 1.536

Atoms C(12)-C(13) C(12)-H(37) C(12)-H(38) C(13)-C(14) C(13)-C(20) C(13)-H(39) C(14)-C(15) C(14)-H(40) C(14)-H(41) C(15)-C(16) C(15)-C(19) C(15)-H(42) C(16)-H(43) C(16)-H(44) C(17)-C(18) C(17)-H(45) C(17)-H(46) C(18)-C(19)

Bond length, Å 1.525 1.118 1.118 1.525 1.527 1.120 1.526 1.118 1.118 1.517 1.527 1.121 1.119 1.119 1.528 1.118 1.118 1.505

Atoms C(18)-C(20) C(18)-H(47) C(19)-H(48) C(19)-H(49) C(20)-H(50) C(20)-H(51) C(22)-C(23) C(22)-C(27) C(23)-C(24) C(23)-H(29) C(24)-C(25) C(24)-H(30) C(25)-C(26) C(25)-Cl(28) C(26)-C(27) C(26)-H(31) C(27)-H(32)

Bond length, Å 1.548 1.121 1.118 1.118 1.118 1.119 1.400 1.405 1.389 1.102 1.397 1.101 1.399 1.708 1.390 1.102 1.102

The calculated activation energy of the obtained nitroanilides shows that the minimal activation energy (71.2 kJ/mole) corresponds to nitroanilide obtained by nitration in 2position (ring a). For position 6 activation energy is 100.8 kJ/mole (Scheme 3). We have established that the reaction of nitration of benzene ring (b) has lower probability than the reaction of nitration of benzene ring (a). Activation energies for identical positions in benzene ring (b) are 98.4 kJ/mole and 104.7 kJ/mole, correspondingly. Reaction of nitration in this position takes place only in rigid condition (in the media of concentrated H2SO4, at the18-20oC) in comparison with reaction of nitration of benzene ring (a). The obtained results are in accordance with the NMR spectral data of the corresponding product [7, 12]. By the AM1 method we also calculated the heat of formation of AH, its nitro-compounds and intermediate products (complex of AH with nitrating mixture). The reaction of nitration of AH we described by following scheme: AH + NO2+ + HSO4- → A–NO2 + H2SO4

Biologically Active Multifunctional Adamantane-Containing Compounds

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Table 4. Effective charges on the carbon atoms in the molecule of 4-(p-chlorophenoxy)-3-chloro-N-(1-adamantoyl)anilide Atoms C(1) C(2) C(3) C(4) C(5) C(6)

Charge 0.10404 -0.23315 -0.04126 0.03545 -0.16977 -0.20288

Atoms C(9) C(11) C(12) C(13) C(14) C(15)

Charge 0.38497 -0.08632 -0.23775 -0.16102 -0.24443 -0.16405

Atoms C(16) C(17) C(18) C(19) C(20) C(22)

Charge -0.22828 -0.23910 -0.16030 -0.24526 -0.24527 0.05010

Atoms C(23) C(24) C(25) C(26) C(27)

Charge -0.18751 -0.16605 -0.08462 -0.16718 -0.19257

Table 5. Valence angles in the molecule of 4-(p-chlorophenoxy)3-chloro-N-(1-adamantoyl ) anilide Atoms C(2)-C(1)-C(6) C(2)-C(1)-N(8) C(6)-C(1)-N(8) C(1)-C(2)-C(3) C(1)-C(2)-H(3) C(3)-C(2)-H(3) C(2)-C(3)-C(4) C(2)-C(3)-Cl(21) C(4)-C(3)-Cl(21) C(3)-C(4)-C(5) C(3)-C(4)-O(7) C(5)-C(4)-O(7) C(4)-C(5)-C(6) C(4)-C(5)-H(33) C(6)-C(5)-H(33) C(1)-C(6)-C(5) C(1)-C(6)-H(34) C(5)-C(6)-H(34) C(5)-C(6)-H(34) C(4)-O(7)-C(22) C(4)-O(7)-C(22) C(1)-N(8)-C(9) C(1)-N(8)-H(36) C(9)-N(8)-H(36) N(8)-C(9)-O(10) N(8)-C(9)-C(11) O(10)-C(9)-C(11) C(9)-C(11)-C(12) C(9)-C(11)-C(16) C(9)-C(11)-C(17) C(12)-C(11)-C(16) C(12)-C(11)-C(17) C(16)-C(11)-C(17) C(11)-C(12)-C(13) C(11)-C(12)-H(37)

Valence angle,o 117.845 118.065 124.090 120.857 119.335 119.808 121.085 118.015 120.898 117.316 122.348 120.321 122.541 118.887 118.571 120.347 121.458 118.192 118.192 113.937 113.937 130.901 111.117 117.982 123.046 115.177 121.777 109.913 110.202 109.677 109.477 108.980 108.648 109.886 109.950

Atoms C(11)-C(12)-H(38) C(13)-C(12)-H(37) C(13)-C(12)-H(38) H(37)-C(12)-H(38) C(12)-C(13)-C(14) C(12)-C(13)-C(20) C(14)-C(13)-H(39) C(20)-C(13)-H(39) C(13)-C(14)-C(15) C(13)-C(14)-H(40) C(13)-C(14)-H(41) C(15)-C(14)-H(40) C(15)-C(14)-H(41) H(40)-C(14)-H(41) C(14)-C(15)-C(16) C(14)-C(15)-C(19) C(14)-C(15)-H(42) C(14)-C(15)-C(16) C(14)-C(15)-C(19) C(14)-C(15)-H(42) C(16)-C(15)-C(19) C(16)-C(15)-H(42) C(19)-C(15)-H(42) C(11)-C(16)-C(15) C(11)-C(16)-H(43) C(11)-C(16)-H(44) C(15)-C(16)-H(43) C(15)-C(16)-H(44) H(43)-C(16)-H(44) C(11)-C(17)-C(18) C(11)-C(17)-H(45) C(11)-C(17)-H(46) C(18)-C(17)-H(45) C(18)-C(17)-H(46) H(45)-C(17)-H(46)

Valence angle,o 111.048 109.805 109.129 106.973 109.473 109.534 109.479 109.364 109.018 110.163 110.159 110.213 110.284 109.997 106.990 109.439 109.355 109.997 109.439 109.355 109.812 108.758 109.462 109.571 110.187 110.225 109.761 109.973 107.092 109.932 110.965 109.955 109.193 109.765 106.980

Atoms C(17)-C(18)-C(19) C(17)-C(18)-C(20) C(17)-C(18)-H(47) C(19)-C(18)-C(20) C(20)-C(18)-H(47) C(15)-C(19)-C(18) C(15)-C(19)-H(48) C(15)-C(19)-H(49) C(18)-C(19)-H(48) C(18)-C(19)-H(49) H(48)-C(19)-H(49) C(13)-C(20)-C(18) C(13)-C(20)-H(50) C(13)-C(20)-H(51) C(18)-C(20)-H(50) H(50)-C(20)-H(51) O(7)-C(22)-C(23) O(7)-C(22)-C(27) C(23)-C(22)-C(27) C(22)-C(23)-C(24) C(22)-C(23)-H(29) C(24)-C(23)-H(29) C(23)-C(24)-C(25) C(23)-C(24)-H(30) C(25)-C(24)-H(30) C(24)-C(25)-C(26) C(24)-C(25)-Cl(28) C(26)-C(25)-Cl(28) C(25)-C(26)-C(27) C(25)-C(26)-H(31) C(27)-C(26)-H(31) C(22)-C(27)-C(26) C(22)-C(27)-H(32) C(26)-C(27)-H(32)

Valence angle,o 109.526 109.167 109.294 108.909 110.080 110.076 110.186 110.095 109.109 110.385 106.935 109.483 110.156 110.054 110.319 106.912 120.202 120.814 118.981 121.179 119.111 119.710 119.524 119.518 120.958 119.813 120.269 119.918 120.515 120.592 118.888 119.979 119.274 120.741

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Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al.

The initial distance (RCN) between carbon atom and nitrogen atom of nitronium ion in AH was 2.50 Ǻ. The approach of NO2+ to molecule of AH was realized by step equal to 0.05 Ǻ. In the hydrosulphat-anion the distance between the oxygen atom and substituted hydrogen atoms of AH molecule decreased synchronously with the changing of RCN. The dependence of the changes of enthalpy (ΔHf) on RCN showed that in both cases the character of the changing of ΔHf is the same (Figure 5 and Figure 6).

Figure 5. The dependence of enthalpy (ΔΗf) on the reaction coordinate (RCN) for 6-nitroanilide.

Figure 6. The dependence of enthalpy (ΔΗf) on the reaction coordinate (RCN) for 2-nitroanilide.

Biologically Active Multifunctional Adamantane-Containing Compounds

49

3.2. Models of Physical Properties of Adamantine-Containing Anylides and Nitroanylides The technique of quantitative structure-activity relationship (QSAR) has been in use for establishing reliable models of biological activities and physical-chemical properties of organic and element-organic molecules. The aim is to assist organic chemists to plan and to carry out purposeful synthesis of compounds with the given spectra of desirable characteristics. The aforementioned approach is based on representation of molecular structures with numeric quantities. They are calculated via straightforward algorithms and are known as molecular descriptors. Among the latter, considerable attention is granted to the autocorrelation based descriptors [21], GATEWAY [22], WHIM WHIM [23], Burden Eigenvalues, and traditional graph-theoretical invariants [24]; they were selected for our research. Our data set [25] contained 16 compounds. In order to detect outliers, i.e., the compounds, which did not belong to the modeling population, we performed PCA [26]. However unlike to our previous contribution [Geprg. Chem. J], we did not identify any of the investigating compounds as outliers. O R

NH

O

C

R/

R// where R=Ad, R/= CH3,R//=H (1); R=Ad, R/=C6H5,R//=H (2); R=Ad, R/=CH2C6H5,R//=H (3); R=Ad, R/=Ad, R//=H (4); R= CH3,R/=Ad, R//=H (5); R= C2H5,R/=Ad, R//=H (6); R=ClC6H4, R/= CH3,R//=H (7);R=Ad, R/= CH3,R//=NO2 (10);R=Ad, R/=C6H5,R/=NO2 (11);R=Ad, R/=CH2C6H5,R//=NO2 (12);R=Ad, R/=Ad, R//=NO2(13); R= CH3,R/=Ad, R//=NO2 (14); R= C2H5,R/=Ad, R//=NO2 (15);R=ClC6H4,R/= CH3,R//=NO2 (16).

Br

OH O C

NH

X

Br where X = - (8); X = C6H4 (9). Scheme 4.

As one can see (Fig 7), compounds 7, 8, 13-16 are situated somehow farther than the main group. This alone does not allow for their removal. For example, the Burden eigenvalues reveal that only compound 8 is an outlier, see Fig 8. When we used the Randic type invariants, none of the compounds left the main group (Fig 9).

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Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al.

Figure 7. The outlier detection by means of PCA. The descriptor used is GATEWAY.

Figure 8. The outlier detection by means of PCA.

Therefore, we decided to keep all of the compounds in the training dataset.

Biologically Active Multifunctional Adamantane-Containing Compounds

51

It is noteworthy that the Randic type invariants clearly output several clusters of compounds. Our final step was establishment of relationships between these descriptors and the retentions factors measured experimentally. Our studies show that best model was achieved by employing, again, the GATAWAY descriptors. We used PLS [27] as the number of predictors was much higher than that of cases. We used cross-validation to define the optimal number of latent variables. In our study, we used 13 compounds in training set and 3 for the cross-validation tests. Of course, the prediction power was lower in case of cross-validation. We modeled both melting points (mp) and retention factors (Rf) within the same model, which, therefore, had 2 responses. The descriptors used are the Burden eigenvalues.

Figure 9. The outlier detection by means of PCA. The descriptors used are the Randic type invariants.

The results of modeling look impressive as the square of the average correlation coefficient was a high as 0.92. The PRESS was also good enough (Table 6). One can examine the experimental and calculated values (Table 7). A reader should take into account that compounds 4, 13, 16 produced the test (validation) set.

Table 6. The Statistical Parameters of the Model Latent Var. № 1 2 3 4 5 6 7 8

Increase R2 on Y 0.391833 0.286881 0.033169 0.041860 0.121957 0.028227 0.025273 0.023788

Average R2 on Y 0.391833 0.678714 0.711883 0.753743 0.875700 0.903927 0.929201 0.952989

Increase R2 on X 0.286528 0.275862 0.206076 0.109336 0.023599 0.048229 0.030173 0.004540

Average R2 on X 0.286528 0.562390 0.768466 0.877802 0.901401 0.949629 0.979802 0.984342

R2 for mp 0.577891 0.611797 0.666069 0.670454 0.886302 0.910597 0.912324 0.939416

R2 for Rf 0.205776 0.745632 0.757697 0.837033 0.865099 0.897258 0.946077 0.966562

Sc. PRESS, mp 2.254355 2.094057 1.780881 1.763578 1.268438 1.211559 1.198989 1.389769

Sc. PRESS, Rf 1.727976 1.922039 1.833227 1.837925 1.445234 1.240578 1.180627 1.137673

Average Sc. PRESS 1.991166 2.008048 1.807054 1.800751 1.356836 1.226068 1.189808 1.263721

Biologically Active Multifunctional Adamantane-Containing Compounds

53

Table 7. Experimental vs. calculated R/s

№ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

mp, calc

Rf, calc

mp, exp

Rf, exp

184.9834 126.5095 142.8738 102.3756 154.5519 128.6926 171.7885 167.9010 193.2231 166.1550 178.3934 170.2936 198.3374 175.8185 226.3155 183.8285

0.518455 0.704182 0.705937 0.591046 0.776873 0.633454 0.867685 0.917788 0.447994 0.486902 0.473237 0.525245 0.549713 0.426382 0.555867 0.146642

178.5 134.5 134.5 121.5 152 128.5 173 169 206.5 166.5 160 181.5 240.5 176.5 226.5 194.5

0.60 0.65 0.73 0.42 0.80 0.65 0.86 0.91 0.40 0.50 0.41 0.53 0.77 0.45 0.55 0.17

3.3. Preliminary evaluation of the bioactivity of the synthesized anilides In order to study of biological activity of adamantine-containing anilides we carried out virtual screening by two programs of internet-systems – Molinspiration Chemoinformatics (Calculation of Molecular Physicochemical Properties) and PASS CandT [28-30]. The program, wich are prepared by Molinspiration Cheminformatics (www.molinspirationcheminformatics), were studied compounds in following direction: I)

Calculation of Molecular Properties – characterized compounds in following properties: 1. Octanol-water partition coefficient logP; 2) Molecular polar surface area (PSA) (Molecular Polar Surface Area MPSA); 3) Molecular volume; 4) Rule of 5" Properties; 5) nrotb. II) 1. 2. 3. 4.

Calculation of Drug-likeness – characterized compounds in following properties: GPCR ligand; Ion channel modulator; Kinase inhibitor; Nuclear receptor ligand.

In this direction the activities of some compounds are obtained to the following values: GPCR ligand – maximum sense – 0.50: 0.43 IV(1), 0.30 IV(2), 0.29 II(1).

54

Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al. Kinase inhibitor – maximum sense – 0.75 (0.50-1.00). Ion channel modulator – maximum sense – 0.25 (0.00-0.50). Nuclear receptor ligand – maximum sense – 0.75 (0.50-1.00).

On the based of studied of compounds (1-7, scheme 4) by method of Molinspiration Cheminformatics and calculation of its physical-chemical properties showed, that they may be alike of known preparations. By means of prognosis PASS CandT are possible to study of feasible pharmaceutical effects, mechanism of action, mutagenesis, cancer genesis, teratogenesis and embryotoxic properties based on structural formulas of compounds (http://www.ibmc.msk.ru/pass). Estimation of probability activity of compounds carried out by parameters Pa (active) da Pi (inactive). If Pa>0.7, compound experimental shows this activity too and possible may be alike of known preparations. Compounds, which were studied by program PASS CandT:

R

O NH

R2 O

R1

where R= CH3, R1=Ad, R2=H (1); R= C2H5, R1=Ad, R2=H (2); R=Ad, R1=CH3, R2=H (3); R=Ad, R1=C6H5, R2=H (4); R=Ad, R1=CH2C6H5, R2=H (5); R=Ad, R1=Ad, R2=H (6); R=ClC6H4, R1=Ad, R2=Cl (7). Scheme 5.

In case of the Pa>0.7 adamantane-containing amides may be detection this activity (Table 8): -

Lipid metabolism regulator Pa = 0.973 (3), 0.971(6), 0.970 (4), 0.966(5); 5 Hydroxytryptamine release inhibitor Pa = 0.900(1), 0.891(2), 0.876(6), 0.843(7) Urologic disorders treatment Pa = 0.872(1), 0.855(2), 0.818(6), 0.788(7). Antiviral (Influenza) Pa = 0.791(1), 0.781(2), 0.775(6), 0.752(7); Antiviral (Picornavirus) Pa = 0.705(1, 6); Dependence treatment Pa = 0.741(1), 0.720(7); Membrane integrity agonist Pa = 0.838(4), 0.792 (7), 0.770(3), 0.752(2), 0.703(1); Prolyl aminopeptidase inhibitor Pa = 0.894(2); Cardiovascular analeptic Pa = 0.730 (2); Membrane integrity agonist Pa = 0.752(2), 0.703 (1). CYP2B5 substrate 0,745(2); CC chemokine 2 receptor antagonist Pa = 0.789(4). Muramoyltetrapeptide carboxypeptidase inhibitor Pa = 0.795(5).

Biologically Active Multifunctional Adamantane-Containing Compounds

55

As it is showed from the aforemantioned data, compounds of this group may be characterized Pa>0.7 for the most part the following activities: Lipid metabolism regulator, 5 Hydroxytryptamine release inhibitor, Urologic disorders treatment, Prolyl aminopeptidase inhibitor, Antiviral (Influenza, Antiviral (Picornavirus). Table 8. Relative activity of some adamantane-containing amides (Scheme 4)

2

-–

3

0,973 0,003 0,970 0,003 0,966 0,004 0,971 0,003 -–

4 5 6 7

Dependence treatment

Antiviral (Picornavirus)

0,900 0,004 0,891 0,004

0,872 0,010 0,855 0,013

0,791 0,004 0,781 0,004

0,705 0,004 -–

0,703 0,101 0,752 0,083

0,741 0,014 -–

-–

-–

-–

-–

-–

-–

-–

-–

-–

-–

-–

-–

-–

0,770 0,075 0,838 0,043 -–

0,876 0,005 0,843 0,005

0,818 0,021 0,788 0,030

0,775 0,005 0,752 0,005

0,705 0,004 -–

-–

-–

0,792 0,065

0,720 0,017

Membrane integrity agonist

Antiviral (Influenza)

-–

Urologic disorders treatment

1

5 Hydroxytryptamine release inhibitor

Compounds

Lipid metabolism regulator

Pa / Pi *

-– -–

*) Pa – green color, Pi – red color.

3.4. Study of Bioactive Properties of Adamantane-Containing Anilides and Nitroanilides We carried out the preliminary calculations of bioactivity of synthesized anilides. We have tested the anthelminthic properties and biological activity of adamantanecontaining anilides and nitroanilides towards different microorganisms. We have studied fasciolocide activity of obtained compounds from anilide series I-VII on artificially infected (Fasciola hepatica) experimental animals (white rats) (Table 9 and Table 10) by the method described in ref. 18. We have established that synthesized compounds in dose 65, 150 and 200 mg/kg in the form of suspension have sufficiently high activity – intensive efficiency (IE) 60 and 80%, extensive efficiency (EE) – 80 and 90%, correspondingly (Table 6 and Table 7) [18]. We have tested the anthelminthic activity (fascioliasis, strongyliatosis and triqocefalosis) some of alkoxyanilides (Scheme 1, I-VII) on sheeps (Table 11). As the table 8 shows 4methoxy-N-(1-adamantoyl)anilide has the best polyhelmithic activity (80-100%). At the same time, the used compounds did not have the poisonous action on the experimental animals

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Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al.

224 312 265 266 105 92 92 95 95 96 260 290 256 260

Standard Experimental __ Standard

3 3 3 3

270 303 313 305

Number of days after ill

5 4 5 5 5 5 5 5 5 5 3 3 3 3

200, bolus 65, bolus 200, bolus __ 200,suspension __ __ 200, Curd’s paste __ 65,suspension 65, bolus __ __ 200, bolus

60 60 60 60 54 54 54 54 54 54 57 57 57 57

2 3 2 0 2 2 2 2 5 0 0 2 2 3

40 75 40 0 40 40 40 40 100 0 0 66 66 100

14 0.25 1.6 2.6 0.6 1.0 1.0 1.0 0 2.6 3.3 2.3 2.3 0

46.15 90.4 38.4 0 76.9 61.5 61.5 61.5 100 0 37.7 56.6 75.4 100

__ 200, bolus ___ ___

57 35 35 35

0 0 0 0

0 0 0 0

5.3 4.0 2.0 5.0

0 20 60 0

Dose, mg/kg and form

Animals middle mass

Experimental __ __ Standard Experimental __ __ __ __ Standard Experimental __ __ __

Determination of effectivity by method of dissection Healing EE,% Discovery IE, % animals fasciole number middling in heart

Preparation

Testing Groups

Animals number in group

Table 9. Relation efficiency of compounds I, II, III, IV, V and hexiqole by rats’ fascioliasis

I II II __ II I II I II __ III IV V hexiqole __ II V __

70 70 70 66 86 68 70

5 3 3 3

68 283 305 305

Standard Experimental _ _ Standard

II III III __ V VII phenacetyne __ IV V __

Number of days after ill

5 5 5 5 5 5 5

Dose, mg/kg and form

Animals middle mass

Experimental __ __ Standard Experimental _ _ __

Preparation

Testing Groups

Animals number in group

Table 10. Relation efficiency of compounds II, III, IV, V, VII and phenacetyne by rats’ fascioliasis Determination of effectivity by method of dissection Healing EE, Discover IE, animals % y % number fasciole middling in heart

150,suspension __ __ __ 150, suspension __ __

13 13 13 13 20 20 20

3 3 0 0 1 4 4

60 20 0 0 20 80 80

0.4 2.4 0.18 2 1.6 0.2 0.8

80 30 91 _ 20 90 90

__ 200, bolus __ __

20 25 25 25

0 0 0 0

0 0 0 0

2.0 3.0 4.0 5.1

0 34 20 0

Biologically Active Multifunctional Adamantane-Containing Compounds

57

Table 11. Anthelminthic activity of 4-methoxy-N-(1-adamantoyl)anilide (sheep)

Compound

Dose, mg/kg

I 100 Fascioliasis Strongyliatosis Triqocefalosis

Helminth-scopy, %

Dissection, %

EE

IE

EE

IE

80.0 80.0 100.0

98.5 99.7 100.0

66.5 66.6 100.0

70.5 99.7 100.0

Biocide properties of synthesized compounds were performed by using procedures described in ref. 18 and 21. We have tested the bactericidal and fungicidal activity of adamantane-containing compounds I, VIII and IX (Schemes 1 and 2). As test cultures we have selected the fungi Fusarium proliferatum and Agrobacterium tumefaciens (on the basis of buter-penton agar) damage some important agricultural plants, and Actinomyces violaceus, destructed some water-soluble polymers. As the testing results have shown, the compound IX reveals relative high activity in comparison with compound VIII and neutralizes Fusarium proliferatum at the concentration 0.01 g/l and 0.1 g/l (The zones of neutralization 1.5±0.064 mm and 1±0.04 mm, correspondingly). Compound I reveals relative high activity towards Agrobacterium tumefaciens (zone of neutralization 2±0.04 mm at the concentration 0.1 g/l). Based on preliminary investigations, we also established that the obtained compounds may be recommended as a modified anthelminthic preparation—phenacetyne, trinoine, diamphenetide, raphoxanide—also as a bioactive component for preparation: a) materials with antimycotic properties for prophylaxis and treatment of mycosis and dermatomycosis; b) protective covers (film materials and impregnating compositions) stable to biocorrosion from action of some micopathogenic microorganisms [31,32].

REFERENCES [1]

[2] [3] [4] [5] [6] [7]

D. Wakelin. “Helminths – Review Article”, Current Opinion in Infectious Diseases, 2000,13, 5, 465-469. I. Feirweather. “Triclabendazole: new skills to unravel an old(ish) enigma”, J. Helminthol, 2005, 79. 3, 227-234. Q.A. McKellar, F. Jackson. “Veterinary anthelmintics: old and new”, Trends Parasitol., 2004, 20,10, 456-461. M.L. Mottier, L.I. Alvarez, M.A. Pis, and C.E. Lanusse. Experimental Parasitology, 2003, 103, 1-7. I.S. Tsizin, A.M. Bronshtein. “Successes in the field of creation new anthelmintics”, Chem.-Pharm. Journal, 1986,10, 1171-1190. I.S. Morozov, V.I. Petrov, S.A. Sergeeva. Pharmacology of Adamantanes, Volgograd medical academy, Volgograd 2001. V.I. Kovtun, V.M. Plakhotnic. “Use of adamantane carbonic acids for modification of pharmaceuticals and biological active compounds”, Chem.-Pharm. J. 1987, 21, 8, 931940.

58 [8] [9]

[10] [11] [12]

[13] [14] [15] [16]

[17] [18]

[19] [20] [21] [22] [23] [24] [25] [26] [27] [28]

[29]

Khatuna Barbakadze, Nodar Lekishvili, Zurab Pachulia et al. D. Lednicer, W. Heyd, E. Emmert. “Hypobetalipoproteinemic Agents. 2. Compounds Related to 4-(1-Adamantyloxy)aniline”, J. Med. Chem., 1979, 22, 1, 69-77. Kh. Barbakadze, Sh. Chipashvili, L. Nanuashvili, K. Revia, D. Zurabishvili. “Synthesis of O-adamantyl and N-adamantoylanilides and biological activity”, Proceedings of Ivane Javakhishvili Tbilisi State University, Chemistry (Editor N. Lekishvili), 2006, 361, 35-39. E.Z. Koval and L.P. Sidorenko. “Mycodestructores for the industrial articles”. Kiev: Naukova Dumka, 1989, 192p. N.G. Artsimovich, T.S. Galushina, T.A. Fadeeva. “Adamantanes – medicinals if XXI sentury”, International Journal on Immunorehabilitation, 2000, 2, 1, 54-60. Kh. Barbakadze, N. Lekishvili, T. Lobzhanidze, R. Vardiashvili, D. Zurabishvili, Z. Pachulia. Proceedings of Ivane Javakhishvili Tbilisi State University, Chemistry (Editor N. Lekishvili), 2008, 362, 19-28. 41st IUPAC World Chemistry Congress. Program and abstracts. Turin (Italy), August 511, 2007. K. Nakamoto. IR and combination scattering spectra of inorganic and coordinating compounds. MIR, 1991. H. Friebulin. “Bask one- and two-dimensional NMR spectroscopy”, VCH, Germany 1991. K. Amirakhanashvili, N. Bogveradze, R. Gakhokidze, M. Labartkava, R. Macharadze, L. Svanidze. Use of the Mass-spectrometry in Bioorganic Chemistry. Ivane Javakhishvili Tbilisi State University Press (Georgia), 2006. D. Lednicer, Portage, Mich. US Patent, #4 200 588, Int CI. CO7 C 93/14, 1980. Kh. Barbakadze, D. Zurabishvili, M. Lomidze, I. Sadaterashvili, T. Lobzhanidze, N. Lekishvili. Proceedings of the Georgian National Academy of Sciences, Chemistry, 2008, 34 1, 45-52. J.S. Michael Dewar, Eve G. Zoebisch, Eamonn F. Healy and James J.P. Stewart. J. Am. Chem. Soc., 1985,107, 3902-3909. N.L. Allinger. J. Am. Chem. Soc., 1977, 99, 8127-8134. M. C. Hemmer, V. Steinhauer, J. Gasteiger. vibrat. Spectr., 1999,19, 151-164. V. Consonni, R. Todeschini, L. Pavan. J. Chem. Inf. Comput. Sci., 2002, 42, 682-692. R. Todeschini, M. Lesagni, E. Marengo. J. Chemom., 8, 263-273 (1994). R. Todeschini, V. Consonni. Handbook of Molecular descriptors, Wiley-VCH, Weinheim, 2001. L. I. Denisova, V. M. kosareva, K. E. Lopukhova, I. G. solonenko. Kh. F. Zh., 1975, 9, 18-21. M. Otto. Chemometrics, Wiley-VCH, Weinheim, 1999. P. geladi, B. R. Kowalski. Anal. Chim. Acta, 1986,185, 1-17. P. Ertl, B. Rohde, P. Selzer, Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties. J. Med. Chem. 2000, 43, 3714-3717. C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug. Delivery Rev. 1997, 23, 4-25.

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[30] D.F. Veber, S.R. Johnson, H.-Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple, Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615-2623. [31] E. Markarashvili, N. Lekishvili, Z. Lomtatidze, Sh. Samakashvili, Izvestia VUZOV. Khimia i Technologia, 2006, 48, 5, 117 -119. [32] N. Lekishvili, Kh. Barbakadze, D. Zurabishvili, T. Lobzhanidze, Sh. Samakashvili, Z. Pachulia , Z. Lomtatidze. Oxid. Commun. (submitted).

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 5

INFLUENCE OF SOME METAL-CATIONS ON THE MOLECULAR ORGANIZATION OF DNA N. Vasilieva-Vashakmadze, G. Lekishvili, R. Gakhokidze and P. Toidze Iv. Javakhishvili Tbilisi State Universityg ,Tbilisi, Georgia

ABSTRACT This work deals with the problem of modeling of a possible mechanism of point mutations of DNA under the influence of Ni2+ ions. Two feasible schemes of interaction of Ni2+ with nucleosides are considered. The first scheme presents the formation of the planar complex of Ni2+-G-C, and the second shows the incorporation of Ni2+ between the two neighboring complementing pairs. We used the MOPAC package to compare the force constants and energies of intramolecular hydrogen bonds in the complexes and corresponding values in a free G-C pair. The comparison enabled us to make a conclusion implying that the formation of the Ni2+-G-C complex is accompanied by the weakening of a hydrogen bond, nearest the joining point of Ni2+. The incorporation of Ni2+ between the two neighboring complementing pairs of G-C causes the weakening of all the three pairs of hydrogen bonds, but to a lesser extent. It has been demonstrated that in the first case the probability of point mutations, the replacement of G-C by A-T increases, and the probability of divisions, the fallout of triplets of type GGХ or ХGG increases in another case.

INTRODUCTION Metals commonly get into a human or animal body as soluble compounds – salts, which, when dissociated, yield cations, the metal ions in the body. The results of numerous experiments have enabled us to fix a regular dependence between the high concentration of g I, Ilia Chavchavadze Ave., 0128 Tbilisi, Georgia, [email protected].

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some metals (Zn, Ni, Co…) in the animal body and the tendency of cells for invasive growth – malignant transformation [1, 2]. A number of experiments have demonstrated that the aforementioned metals may result in carcinogenic activity [3, 4]. A notorious example of the metal influence on the DNA structure is the transformation of guanine G→G, resulting in a possible replacement of G-C pair by A-T pair. As it is known, there are data evidencing the special role Ni2+ plays in the process of malignation of cells. The transformation of nucleosides under the influence of nickel is a process that takes place at an electronic level. Therefore, in order to study the mechanism of this process, the peculiarities of the electronic structure of nickel and its complexes are to be taken into account. The total content of nickel in the earth’s crust is ~0,003%. It is a permanent constituent element of an animal body (~10-2%). However, biochemical nickel regions of a plant contain much more nickel. Besides, they often have a deformed shape evidencing the disturbance of their genetic apparatus. Ni in microscopic concentrations, as a microelement is included in liver, skin and endocrine glands; it is the activator of arginase in the body; it is a constituent element of insulin and it has an effect on the oxidation processes [5,6]. In nature, nickel is spread as minerals - pentlandin (FeNi)9S8, nickeline (NiAs), garnierite (NiMg)3Si4O10(OH)10·4H2O and others. Many of the nickel compounds are complex ones, e.g. hexamine [Ni(NH3)4·(H2O)2]·X2 among them, where X is a monobasic anion. There are nickel compounds known as dimmers, having a square-planar structure, e.g. [Ni(CN)3]2-. Nickel is characterized by a square-planar form of coordination (with the coordination number of 4), as well as octahedral form (with the coordination number of 6). A relatively better-studied specimen of square complexes of nickel is the salicyl-al’diminat complex.. Nickel is attributed to the group of transition metals (Z=28) and has an open 3d shell. Its fundamental state is …3d94S1. Bivalent ion (sulphion) Ni2+ has an electronic configuration of … 3d8, which depending on the symmetry of the ligand field may result in different configurations of nickel complexes – octahedral spatial structures or almost planar squares [7]. Many metallic complexes of purine nucleotides were studied by the method of X-ray structure analysis. For instance, a compound of a hydrated nickel with nucleotides is known, with five water molecules and purine nucleotide, bonded in the state N(7), forming an octahedral coordination sphere of the metal [1]. However, the formation of nickel complexes with a square-planar coordination is also possible, like natural compounds of a tetracoordinated nickel. It is known that the formation of complexes has an effect on the configuration of systems of the constituent components and their chemical properties - when the power levels of the higher occupied orbits of the metal are cut in between the levels of valence states of ligands, they may participate in the rearrangement of these states, which in its turn may change the properties of ligands.

Influence of Some Metal-Cations on the Molecular Organization of DNA

63

RESULTS AND DISCUSSION When studying the metallocomplexes in water solutions, it is reasonable to apply the theory of ligand substitution [8, 10]; it considers the electronic structure of all atoms included in the complex, viewed as a unitary quantum-mechanical system. The transient period metals, with Ni among them, being hydratable in water solutions, capture the polarized water molecules. Besides, as it is known, the radius of a hydration shell and the coordination number characterizing the local electrostatic field, eventually are the result of the properties of a central cation, the complex-former. As the coordination number of such compounds exceeds the valency (the number of coordination bonds is more than the number of the valence electrons), the electron density at the bonds is less than one. This results in the lability of bonds which becomes apparent in that the coordination bonds spontaneously break up and are re-formed, creating favourable conditions for the process of the ligand replacement. Besides, a significant role is attributed to stereo-specific interactions when the radius of a hydration shell and the coordination number of a cation ensure the steric conformity between the active center of the molecule and the coordination sphere of a cation. It is known that the energy of the higher occupied orbital defines the electron-donating properties of the molecules. Guanine has the highest heterocyclic base filled with the highest energetic level: E= - 0,307β [8], and relatively more electronegative atoms of guanine are N(7) [qπ = -0,571e] and O(10) [qπ = - 0,46e]. Atoms of guanine nitrogen may be arranged in the following order according to the value of the electronic charge π [8,9]: N(7)>N>(3)>N(1)=N(2)>N (9) (Figure 1) As it is known, the reaction of interaction of the ligand with the electron-seeking reagent is possible, if the energy of the highest occupied level of ligand is higher than or equal to the energy of the lowest free level of the reagent. Therefore, it is the guanine of purines and the cytosine of pyrimidines that are considered most inclined to the electrophilic reactions. The ability of the metals to interact with guanine is reduced in the range of Cu>Ni>Zn>>Mn>>Mg. It has been proved experimentally that bonding of metals often takes place along the imidazole ring of histidine N(7) and N(9) or pyrimidine ring in nucleotides N(3) and N(1). Bonding in nucleosides takes place via N(7) and O(10) of guanine and N(1), N(7) of adenine (Figure 1). The works indicate [2, 3] that it is possibile the incorporating of the metal between two neighbouring guanines in one chain or joining the metal between N(1) purine and N(3) pyrimidine. Analyzing different schemes of interaction of nickel with nucleosides, we dwelt on the two most typical cases. These are the interaction of nickel with a complementing pair G-C and a respective formation of 4- and 6-coordinated complex with Ni2+. We consider that a 4-coordinated hydrocomplex of nickel, which is in fact a planar system, when interacting with purine, which also has a planar structure, must stabilize as a planar system Incorporation of nickel between two neighboring purines, located along a polynucleotide chain, must result in the formation of an octahedral complex with a spatial configuration. as

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presented in Figure 2. When designing the schemes of a complex-formation, mean and interatomic distances and angles were taken of the data gained through the results of the Xray analysis of similar complexes, as well as of the relevant tables. The scheme of incorporation of Ni2+ between two neighboring pairs of G-C was designed by considering the data on the configuration of 6-coordinated complexes of Ni2+ and based on the data on the most electronegative ligands O(10), N(7), N(3) and N(17), O(20), N(13) (Fig 1, Figure 2)

Figure 1.

Figure 2.

Influence of Some Metal-Cations on the Molecular Organization of DNA

65

The present work deals with the influence of the formation of complex Ni-G-C on the stability of the hydrogen bonds in complementing pair G-C. As the calculation has shown, in the process of complex formation according to scheme 1 (Figure 1), the binding energy E[O(10)-H] ≈0,06 ev, i.e. ≈1,38 kcal/mole corresponds to van der Waals interaction which is less than the energy of the hydrogen bonds, whose value ≈3÷8 kcal/mole. This evidences the weakening of the hydrogen bonds allocated near the jointing point with metal. Besides, the values of force constants for three hydrogen bonds Pkp, π-electronic charges for Qπ atoms and binding energy Ekn were found (numbering is referred to in Figure 1). For the sake of convenience, we introduced the numbering of hydrogen bonds in Figure 1 with Roman numerals (I, II, III). PI=0.024

Qπ[O(10)]= - 0.310

E1=0,060 ev

P1=0.057

Qπ[O(1)]= - 0.200

E1=0,087 ev

PIII=0.068

Qπ[O(2)]= - 0.200

E1=0,090 ev,

where: Pi is the force constants and Ei is the binding energy. Thus, at forming a planar 4-coordinated nickel complex with G-C pair, the hydrogen bond O(10)…H-N is destabilized (Figure 1) while other two H-bonds are quite strong. The destabilization of H-bonds O(10)…H-N in the formed complex Ni…G-C increases the probability of the cytosine easily splitting off guanine at the subsequent replication. Thus, it may be supposed that such kind of complex-formation at the subsequent replication reduces the ability of normal formation of a complementing G-C pair, whose stabilization needs the formation of three hydrogen bonds. The through this process, the ability of forming two hydrogen bonds is preserved, the transformed guanine G* may join thymine at replication. Thus, G* acquires the similarity to adenine, capable of forming two hydrogen bonds with thymine. At the subsequent replication, adenine must join thymine, i.e. a point mutation takes place which is the replacement of a pair G-C by A-T is secured during the subsequent replications. Thus, as the analysis shows, joining of a hydrated 4-coordinated Ni2+ to guanine in the complementing pair G-C causes the transformation G→G*, whose result is the possibility of replacing the pair G-C by A-T. Unlike a 4-coordinated Ni2+, the 6-coordinated Ni2+, when incorporated between two neighboring complementing pairs (G-C …Ni…G-C) causes changes of a somewhat different nature. Similarly to the method used by us to carry out the preliminary analysis to design a planar tetra-coordinated complex Ni2+ with a complementing G-C pair, a corresponding scheme was drawn up for the complex formed by 6-coordinated Ni2+ incorporated between two neighboring pairs of G-C, and the calculus by using the program MOPAK was used.

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For the sake of convenience, instead of numbering the atoms as in Figure 1, for hydrogen bonds we used numbering by Roman numerals. The values of force constants Pkn and binding energies between guanine and cytosine in both complementing pairs of complex G…Ni2+…G (Figure 2) calculated by us are referred to below: P(I)=P(IV)=0.039

E(I)= - 0.12 ev = E(IV)

P(II)=P(VI)=0.042

E(II)= - 0.12 ev = E(V)

P(III)=P(VI)=0.042

E(III)= - 0.13 ev = E(VI)

The comparison of the force constants of the hydrogen bonds evidences a simultaneous destabilization of all the three pairs of H-bonds between guanine and cytosine in both complementing pairs of the bases. However, such destabilization is observed to a lesser extent than in case of formation of a planar complex. This evidences the reduction of the ability of forming complementing pairs during the replication at the points, where triplets of type G-G-H or H-G-G were located. We may conclude that the incorporation of Ni2+ between the neighboring complementing pairs (G…Ni2+…G-C) may cause deletions, the fallout of triplets of type G-G-H or H-G-G in DNA. On the basis of the accomplished analysis, the following conclusions may be made: 1) The formation of complex Ni-G-C causes the transformation of guanine G→G*, during which the destabilization of the hydrogen bond in the complementing pair G*C, being the nearest to the jointing point of Ni takes place. As a result, in the phase of replication, guanine loses the ability to form three complete hydrogen bonds to form a normal complementing pair with cytosine. At the same time, the formation of the G-T pair is also possible. At the subsequent replication, thymine joins adenine, i.e. in place of G-C pair, there may appear A-T pair. This means point mutation. 2) In another case, the incorporation of Ni2+ between the complementing pairs causes the destabilization of all the three hydrogen bonds in both neighboring pairs. At a subsequent replication this may cause divisions (fallout) in triplets of a type G-G-H or H-G-G.

REFERENCES [1] [2]

[3]

Metal Ions in Biological Systems. Collection edited by Zigel H. Moscow, MYR, 1982 E Andronikashvili, Esipova I. The Role of Metal Ions in the Initiation and Development of Malignant Transformations, Tbilisi, the Academy of Sciences of Georgian SSR, 1982 F. Schmidt, Coordination-chemical Bases of Metal-organic Catalysis. Irkutsk, Irkutsk State University, 1981

Influence of Some Metal-Cations on the Molecular Organization of DNA [4]

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I. Berasper. The Electronic Structure and Properties of Coordination Compounds. Leningrad, Khimia, 1976 [5] K. Balhaussen, Introduction to the Ligand Field Theory, Moscow, Myr, 1989 [6] K. Lengford, G. Grey, Ligand Replacement Processes. Moscow, Myr, 1969 [7] O. Neyland, Organic Chemistry. Moscow, Visshaya Shkola, 1990 [8] Metal Ions in Biological Systems. Collection edited by Zigel H. Moscow, MYR, 1982 [9] N. Kochetkov The Organic Chemistry of Nucleic Acids, Moscow, Khimia, 1970 [10] N. Vasilyeva-Vashakmadze Destabilization of Intermolecular Hydrogen Bonds in the Metal-nucleotide Complex. Reports of the Academy of Sciences of Georgian SSR. 1986, 122, 629.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 6

STATE OF LIPID COMPONENTS OF SOYBEAN FLOUR ENZYMATIC HYDROLYZATES DURING STORAGE L.N. Shishkina1, E.V. Miloradova2, E.A. Badichko2, and S.E. Traubenberg2 1

N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciencesh, Moscow 119334, Russia 2 Moscow State University of Food Production, Volokolamskoe shosse, 125080 Moscow, Russia

ABSTRACT The influence of hydrolysis and centrifugation processes of soybean semifat flour on various indices of the lipid component and dynamics of changes in the composition and characteristics in hydrolyzates within three months of storage were studied. It was shown that processes of hydrolysis and centrifugation, and also storage, cause reliable changes of the physical and chemical characteristics and lipid composition in hydrolyzates.

AIMS AND BACKGROUND Quality of the lipid content products is defined by safety of their lipid component [1,2]. Many factors promote oxidation of lipids, involving change of the consistency, colour and taste of a product, loss of vitamins and essential fatty acids. Thus, oxidation of lipids is accompanied by loss not only of tasting quality, aroma and nutritional value of a product, but also leads to formation of unhealthy compounds [1-3]. Because the accumulation of primary and secondary products of oxidation is due to the interaction of lipids with oxygen in the air, there is a need to monitor the lipid component in the process of storage.

h

4 Kosygin str., Moscow 119334, Russia.

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It is necessary to be aware that the type and kind of packing material, and also storage period, is of great importance in safeguarding the integrity of the products’ quality. The polyethylene film is one of the most used packing materials as it helps maintain the moisture and appearance of products, interferes with and prevents oxidation [4]. Data about significant changes of the lipid component of wheat flour [5], dry yeast envelopes [6], dried doughnut mixes [7], lecithin enriched soybean fat-free flour in various types of packing [8] during storage, which indicate complicated hydrolytic and oxidative processes in their lipid component during storage of dry food objects, are known. However, data about the stability of the lipid component of dried up soybean enzymatic hydrolyzates are practically absent. The enzymes are used more often to receive hydrolyzates, whose application allows creating and improving existing foodstuff [9], and also when the process of centrifugation and drying are used, which also make a significant impact on a state of lipid component of the initial sample [10-12]. The aim of the given work is the study of the influence of hydrolysis and centrifugation of semi-fat soybean flour on various indices of lipid components and the dynamics of change in the composition and characteristics of its lipid hydrolyzates during storage.

MATERIALS AND METHODS Objects of research were lipids isolated from soybean enzymatic hydrolyzates, received from soybean deodorized semi-fat flour (GOST 3898-56). The proteolytic enzyme Beerzym Chill is used for hydrolysis of soybean flour for 8 hours at its optimal conditions which have been chosen before (temperature - 50° С, рН = 8) [13]. The received liquid hydrolyzate is divided into two parts, one of which is clarified by centrifugation. The clarified part (supernatant) and non clarified liquid hydrolyzate (partly hydrolyzed soybean flour) is dried at spray drying “Mobile minor”. After that, samples are tightly simultaneously packed into single-layered transparent packing and kept in a dry place at room temperature. The analysis of samples is carried out before packing and later at 1; 1.5 and 3 months of storage. The content of products interacted with 2 - thiobarbituric acid (TBA-reactive substances, TBA-RS), in suspension of hydrolyzates was determined by method [14]. The amount of protein in soybean hydrolyzates was analyzed by using modified microbiuretic method [15]. Lipids were isolated from the samples using the Blay and Dyer methods in the Kates modification [16]. The separated lipids were used for studying the content of peroxides and composition of lipids. The amount of peroxides in lipids was determined using the method of iodometric titration according to the procedure specified in GOST 26583-85. The ability of lipids to decompose peroxides (their antiperoxide activity, APA) was evaluated by method [17]. The qualitative and quantitative composition of phospholipids (PL) were determined using the thin-layer chromatography method with the use of silica gel, type G (Sigma, USA) and glass plates measuring 9×12 cm [18]. The chloroform—methanol—glacial acetic acid - water mixture in the ratio of 50:30:8:4 was used as the solvent system. The development of

State of Lipid Components of Soybean Flour Enzymatic Hydrolyzates during Storage 71 chromatograms was performed by iodine vapour. The quantitative analysis of the PL composition was determined after the removal of fractions from the plate and after PL perchloric acid digestion to inorganic phosphate. For the colour reaction to phosphorus we used ammonium molybdate and ascorbic acid, produced by Serva (Germany) and also perchloric acid of chemically pure grade. The amount of inorganic phosphorus was judged according to the optical density of the solutions at the λ=800 nm wavelength as measured with spectrophotometer KFK-3 (Russia). Plotting of the calibration curve was carried out on the monosubstituted potassium phosphate of especially pure grade. Besides the quantitative analysis of different fraction of PL, the generalized parameters of the lipid composition were evaluated: amount of PL of total lipid composition (%PL), the ratio between the sums of the more easily oxidizable to the more poorly oxidizable PL fractions (ΣEOPL/ΣPOPL) and the phosphatidyl choline/phosphatidyl ethanolamine (PC/PE) ratio. The value between ∑EOPL /∑POPL was calculated by the formula [19]: ∑EOPL /∑POPL = (PI+PS+PE+PG+CL+PA)/(LPC+SM+PC), where PI is phosphatidyl inositol, PS is phosphatidyl serine, PG is phosphatidylglycerol, CL is cardiolipin, PA is phosphatidic acid, LPC are lysoforms of PL, SM is sphingomyelin. The more easily oxidizable phospholipids are the fractions in the composition of which there are predominantly unsaturated fatty acids and the more poorly oxidizable phospholipids are the fractions in the composition of which there are predominantly saturated fatty acids in the hydrocarbon part molecules. That ratio makes it possible to judge about the oxidizability of lipids [19]. The content of sterols was determined spectrophotometrically at 625 nm according the methods, described in [20]. The measurements in each from independent samples were made three - six times. The experiment data were processed with a commonly used variational statistic method [21]. The experimental data are presented in the tables and figures in form of arithmetic means with the indication of the mean square errors of the arithmetic mean (M ± m).

RESULTS AND DISCUSSION During the first stage of work, the influence of technological process to the content of total lipids (TL), TBA-reactive substances, amount of PL and sterols in the composition of total lipids in investigated samples were studied. The results are shown in Table 1. Their analysis testifies that the reliable decrease of TL in 1 g of absolutely dry matter (ADM) is observed during the hydrolysis of flour and the following process of centrifugation and drying. Besides, the quantity of TBA-reactive substances in samples increases in inverse consequence by 1.6 times in not clarified hydrolyzate and by 3.4 times in supernatant, correspondingly. It allows to assume that process of hydrolysis and centrifugation intensify lipid peroxidation (LPO) because it is well known that LPO intensity is evaluated by the TBA-reactive substances content in a complex biological system [22].

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Table 1 also is possible to conclude that the process of centrifugation is reduced the amount of PL in the total lipid composition by 2 times. However, the content of sterols after centrifugation, on the contrary, increased by 3 times. Table 1. The influence of enzymatic hydrolysis and centrifugation processes on biochemical characteristics of soybean hydrolyzates Parameters

Soybean semi fat flour (n*=3)

Clarified soybean hydrolyzate (n=6)

86.2 ±11.6

Not clarified hydrolyzate (partly hydrolyzed soybean flour) (n=6) 27.8±2.8

[TL]/ADM, mg/g [TBA-RS], nmole/mg of protein

0.109±0.023

0.179±0.029

0.370±0.071

[TL]/[protein]

0.257±0.034

0.049±0.007

0.0371±0.0024

[sterols]/[PL]

0.544±0.027

0.70±0.13

3.08±0.79

% PL

24.1±1.6

24.8±1.3

12.4±1.4

% sterols

7.3±1.2

9.1±1.25

26.4±1,6

14.8± 0.7

*n – number of independent measurements

It is known that peroxide compounds are primary products of the lipid oxidation [1]. It is necessary to note heterogeneity of the investigated samples on the given parameter. So, lipids in soybean semi fat flour mainly had the ability to compose peroxides, and only have been found peroxides in the amount of 1.8 μmol/g of lipids in one variant. The amount of peroxides is 4.9±0.8 μmol/g of lipids in clarified soybean hydrolyzate, and 6.8±2.8 μmol/g of lipids in not clarified soybean hydrolyzate. However, lipids of hydrolyzates have APA in solitary cases. The composition of PL of soybean flour and its hydrolyzates are shown in Table 2. It’s clear, that the main fractions of PL are PC and PE in all samples. However, the processes of hydrolysis and centrifugation produce a significant change in quantitative ratio of Pl fractions. Thus, the reliable growth in the share of PC by 15.7% and the decrease of relative amount of CL+PA by 46.9% in PL of not clarified hydrolyzate is observed compare to composition of PL in soybean flour. Besides, the appearance of the additional minor fraction is observed, which can be identify as oxidized PE (PE’). The portions of SM and CL+PA are reliable increase by 3,1 and 2,1 times correspondingly, at reduction of the relative content of PC by 30.9% and PS by 1.9 times in PL composition of the clarified hydrolyzate compared with their amount in PL of not clarified hydrolyzate. The amount of the oxidized PE’ is showed the tendency to further growth. The next step of this work was to study the lipid component stability during 3 months of storage. The dynamic of TBA-RS active substances in hydrolyzates is shown in Figure1. The data of Figure1 shows that this parameters in not clarified hydrolyzates is quite stable, while the clarified hydrolyzates is revealed only a trend of decline, due to the high variability of the

State of Lipid Components of Soybean Flour Enzymatic Hydrolyzates during Storage 73 intensity of LPO in the initial sample. In addition, the content of oxidation products in the not clarified hydrolyzates significantly lower than in clarified during the whole period of storage. Perhaps, this is due to the presence of SH-containing amino acids which can inhibit the oxidation processes in not clarified hydrolyzates [23]. Table 2. Composition of phospholipids of soybean flour and its hydrolyzed products Fraction, %Р

Soybean flour (n*=15)

LPC

semifatty

Not clarified hydrolyzate (partly hydrolyzated flour) (n*=29)

Clarified soybean hydrolyzate (n*=21)

3.9±0.6

3.55±0.50

6.60±1.85

SM

4.25±0.65

3.8±0.45

11.85±0.9

PC

31.4±1.2

36.3±0.9

25.1±1.45

PS

15.3±0.95

14.1±0.6

7.40±0.65

PI

5.2±0.85

5.65±0.45

6.3±0.70

PE

24.45±1.45

23.6±0.85

19.15±1.65

PE′

-----

3.2±0.5

5.25 ±1.0

PG

3.6±0.4

3.5±0.3

4.8±0.8

CL+PA

12.0±1.8

6.35±0.8

13.55±1.8

n – number of measurements.

Figure1. Changes in the intensity of LPO in hydrolyzates of soybean flour during storage

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L. N. Shishkina, E. V. Miloradova, E. A. Badichko et al.

Peroxides only were found in lipids of both hydrolyzates during storage. However, in not clarified hydrolyzates the peroxide content is roughly decreased after 1 month from the beginning of the experiment, but is increased during subsequent periods of storage. In the centrifuged hydrolyzates amount of peroxides in lipids chances at phages: a minimal degree of lipid oxidation is detected after 1 month and a maximal – after 1.5 months of storage. The dynamics of the ratio [TL] [protein] in both hydrolyzates during storage is similar: the maximal value of this parameter was found after one month of storage (Figure 2).

Figure 2. The ratio of the [total lipid]/protein in hydrolyzates during storage.

Table 3. The phospholipid composition of clarified soybean hydrolyzate during storage Fraction, % Р

Duration of storage, month 0

1

1.5

3

LPC

6,60±1,85

6,77±0,40

9,55±1,00

6,75±1,05

SM

11,85±0,9

6,7±0,75

5,01±0,9

11,85±2,30

PC

25,1±1,45

24,85±1,7

21,35±1,45

25,3±3,6

PS

7,41±0,65

19,65±2,3

7,2±1,0

20,4±2,6

PI

6,3±0,70

6,6±0,6

4,13±0,47

2,6±0,8

PE

19,15±1,65

17,2±2,1

18,45±1,7

14,15±2,2

PE′

5,25 ±1,0

------

0,56±0,30

-------

PG

4,8±0,8

2,35±1,15

12,4±0,8

5,55±1,2

CL+PA

13,55±1,8

15,9±2,5

20,45±2,9

13,4±3,3

State of Lipid Components of Soybean Flour Enzymatic Hydrolyzates during Storage 75 Changes in the intensity of oxidative processes and the ratio [TL] / [protein] in dried hydrolyzates are due to quantitative change in the ratio of the various kinds of lipids. The analyses of the PL composition in hydrolyzates during storage are presented in Tables 3 and 4. The main fractions are still a fraction of PC and PE in both hydrolyzates during the whole experiment. The proportion of PC in the clarified hydrolyzates did not change significantly, and on the contrary this parameter increases in not clarified hydrolyzate during 3 month of storage. Table 3 shows that the most significant changes in the ratio of PL fractions in clarified hydrolyzates detected after 1.5 months of storage. Thus, a reliable drop in the relative content is found for the SM and PI, with a significant increase of PG and the sum of CL+ PA. Phase changes in the ratio of PS in PL of clarified soybean hydrolyzates, is revealed their maximal value is detected after 1 and 3 months of storage. Changes in the PL composition of not clarified hydrolyzate (Table 4) are different during storage. Authentic fall of the share of SM and PI is detected in a 1.5 and 3 months storage, but the most significant increase is found the relative amount of the CL+ PA during 1 month after the beginning of the experiment (Table 4). Besides, low amounts of oxidized PE’ in PL of both hydrolyzates are found only at the beginning and after 1.5 months. Table 4. The phospholipid composition of not clarified hydrolyzate during storage Fraction, % Р LPC

Duration of storage, month 0 1 3.55±0.50 2.75±0.55

1.5 3.9±0.60

3 5.85±0.85

SM

3.8±0.45

4.6±0.95

2.3±0.25

2.25±0.3

PC

36.3±0.9

36.7±1.3

40.25±1.1

41.35±0.95

PS

14.1±0.6

16.45±1.25

17.15±0.8

16.4±1.0

PI

5.65±0.45

3.2±0.45

1.57±0.30

2.3±0.35

PE

23.6±0.85

20.95±1.0

22.6±0.75

22.35±1.05

PE′

3.2±0.5

----------

1.35±0.25

--------

PG

3.5±0.3

4.5±0.9

2.85±0.35

2.25±0.65

CL+PA

6.35±0.8

10.9±1.35

8.0±1.1

7.3±0.90

Such significant changes of quantitative ratio of different fractions within the PL composition cause the changes of relationship of generalized parameters of PL composition in hydrolyzates during storage. Interestingly, as in the original sample, so within the first 1.5 months value PC/PE (Figure 3) in PL of clarified hydrolyzates significantly lower than in not clarified hydrolyzate. But at the end of the experiment, this ratio is almost identical in PL of both hydrolyzates and significantly higher than in the initial samples.

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Figure 3. The dynamic of changes of the PC/PE ratio in PL of soybean hydrolyzates during storage.

Figure 4. Changes in the ratio of sums of the more easily oxidizable to more poorly oxidizable phospholipid fractions of hydrolyzates during storage.

The ΣEOPL/ΣPOPL ratio characterizing the ability of lipids to oxidation is increased by 1.5 times in the clarified hydrolyzates during 1.5 months (Figure 4). This is due to the increased share of the more easily oxidizable fractions in PL composition. However, when stored for 3 months, the decrease of this ratio in the clarified hydrolyzates, to the initial value, but it significantly lower than the control in not clarified hydrolyzate. Figure 5 presents the change of PL shares in the TL composition of hydrolyzates during storage. It’s seen that the share of PL in not clarified hydrolyzates during 1.5 months of storage is lower than in the initial samples. However, after 3.0 months, the share of PL is 1.4 times greater than in control. PL amount in the TL composition of clarified hydrolyzates

State of Lipid Components of Soybean Flour Enzymatic Hydrolyzates during Storage 77 varies within the limits of variability for the original sample. It may be noted only significant difference value between this parameter after 1 and 1.5 months. The ratio [sterols]/[PL], in contrast, nearly 4.4 times higher after centrifugation, this provide some evidence for the advantages of Pl absorption, compared with sterol, during centrifugation process of hydrolyzed soybean flour (Figure 6). A higher ratio of [sterols]/[PL] in clarified hydrolyzates retained during the whole period of storage. Besides, this parameter in the total lipids of not clarified hydrolyzate remained unchanged during storage. Molar ratio [sterols]/[PL] in lipid of clarified hydrolyzates changes at stages reaching a maximum after 1 and 3 months and decreased 2,3-times after 1.5 months of storage (Figure 6).

Figure 5. Changes of phospholipid content in the total lipid composition in hydrolyzates during storage.

Figure 6. The Molar ratio of [sterols]/[PL] in the lipid hydrolyzates during storage .

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L. N. Shishkina, E. V. Miloradova, E. A. Badichko et al.

The data obtained suggests that the high labiality of lipid component favours the proceeding of the hydrolytic processes. The results of the study allow us also to conclude that not only technological processes but storage is due to a reliable change in lipid composition in both types of hydrolyzates. It sets up a need for finding a means for stabilizing and protecting the hydrolyzate lipids during storage in the case of their further application.

REFERENCES [1] [2] [3] [4]

[5] [6] [7] [8]

[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]

I.M. Emanuel, J.N. Lyaskovskaya. Inhibition of Fat Oxidation, Moscow: Pishepromizdat, 1961, 355 pp. (Russ.). F.M. Rzhavskaya. Lipids of fishes and marine mammals, Moscow: Pishepromizdat, 1976, 470 pp. (Rus.). Min-Hsing Pan, Chi-Tang Ho. Chem. Soc. Rev., 2008, 37, 2558-2574. V.B. Spirichev, L.N. Shatnyuk, V. M. Poznyakovskii. Enrichment of foodstuff by vitamins and mineral substances. Novosibirsk Publishers Sib. Universiti, 2004, 548 pp. (Russ.). V.L. Kretovitch. Biochemistry of grain and bread, Moscow: Nauka, 1991, 136 pp. (Russ.). V.A. Menshov, L.N. Shishkina, Z.N. Kishkovskii. Applied biochemistry andMicrobiology, 1993, 29, 6, 675 - 683. L.N. Shishkina, M.A. Klimova, G.F. Dremutcheva, S.E. Traubenberg. Applied biochemistry and microbiology, 2000, 36, 4, 503-508. L.N. Shishkina, S.E Traubenberg, E.V. Miloradova, I.V Vialtseva, A.A. Kozlova. New Trends in Biochemical of Physics Research. Eds S.D. Varfolomeev at al. Nova Science Publishers: New York, 2007, рр. 101-109. M.L. Domoroshenkova. Food industry, 2001, 4, 5-11. (Russ.). V.A. Menshov, L.N. Shishkina, E.B. Burlakova, Z.N. Kishkovskii, I.I. Samoiilenko, E.V. Idrisova // Applied biochemistry and microbiology, 1993, 29, 3, 334-339. L.N. Shishkina, A.A. Kozlova, E.V. Miloradova. Storage and Processing of Farm Products, 2006, 1, 25-27. (Rus.). L. N. Shishkina, S. E. Traubenberg, E. V. Miloradova, I. V. Vialtseva, A. A. Kozlova. Storage and Processing of Farm Products, 2006, 3, 13-17 (Russ.). S.E Traubenberg, E.V. Miloradova, E.V. Alekseenko, E.A. Badichko. Storage and Processing of Farm Products, 2007, 5, 62-65 (Russ.). T. Asakawa, S. Matsushita. Lipids, -1980, 15, 3, 137-140. R. Itzhaki, D.M. Gill. Anal.Biochem, - 1964, 9, 401-409. M. Kates. The technique of lipidology, Moscow: Mir, 1975, 322. pp. (Russ.). L.N. Shishkina, N.V. Khrustova. Biophisics, 2006, 51, 2, 340-346. Biological membranes. A practice approach. Eds J. B.C. Findlay, W.H. Evans – Moscow: Mir, 1990, 424 pp. (Russ.). L.N. Shishkina, E.V. Kushnireva, M.A. Smotrjaeva. Radiat. biology. Radioecology, 2001, 41, 3, 301-306 (Russ.). W.M. Sperry, M. Weeb. Biol. Chem., 1950, 187, 1, 97-106.

State of Lipid Components of Soybean Flour Enzymatic Hydrolyzates during Storage 79 [21] G.F. Lakin. Biometry. 3rd publication, Moscow: High school, 1990, 293 pp. (Russ.). [22] [22] Endogenous Produkcts, Itogi nauki I tekhniki VINITI Akad. Nauk SSSR. Ser. Biofizika, 1986, 18, 136 (Rus.). [23] E.T. Denisov, T.J. Denisova Handbook of Antioxidants. Bond Dissociation Energy, Rate Constants, Activation Energy and Enthalpies, of Reactions (2uaed). Boca Raton, New York, Washington: CRC Press, 2000, 290 pр.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 7

PARTICIPATION OF AROMATIC AMINES IN THE MAILLARD REACTION R. Kublashvili and D. Ugrekhelidzei Iv. Javakhishvili Tbilisi State University, 0179 Tbilisi, Georgia

ABSTRACT Some patterns of the relationship of the interaction between aromatic amines (o-, mand p toluidin; o-, m- and p-amino phenol; o-, m- and p-amino benzoic acid) and aldoses (D-glucose, D-galactose, D-mannose, L-rhamnose, D-xylose, L-arabinose, Dmaltose, D-lactose) in the Maillard reaction are investigated. In the Maillard reaction, the reactivity of aniline, toluidines and amino phenols increases and the reactivity of amino benzoic acids decreases, with increase of рН of the reaction medium; in comparison with aldohexoses, aldopentoses participate in melanoidin formation more actively.

INTRODUCTION Non-enzymatic reaction between reducing sugars and amines, for the first time described by L.C. Maillard [1], includes interaction of reducing sugar with any of compound having a free amino group, as a result of which the colored high-molecular weight melanoidines are formed. Compounds formed as a result of this reaction actually determine aroma and taste of thermally processed products [2]. It was shown that Maillard reaction proceeds as well in living organisms. It is supposed that in an organism excess glucose reacts with proteins according to Maillard reaction, that causes changes in structure and function of proteins, and this is the reason of such diseases as premature aging, atherosclerosis, diabetes and others [3, 4]. Participation of amino acids and proteins in Maillard reaction is investigated thoroughly; however aromatic amines in this respect are investigated insufficiently.

i E-mail: [email protected].

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EXPERIMENTAL The reaction mixtures for melanoidin formation were prepared in the 1/15M phosphate buffer; in 20 ml of phosphate buffer 1mM of an aldose and 1mM of aromatic amine were dissolved. These reaction systems were studied at pH 4.9, 7.0 and 9.2, at 100oC and reaction time 4 hr. After exposure, the reaction mixtures were cooled with ice water up to 20oC, and their optical density was measured. Distribution of a melanoidin pigment (λ = 470 nm) between low-molecular weight (< 3500 dalton) and high-molecular weight (> 3500 dalton) fractions was studied in reaction mixture formed by interaction of m-amino benzoic acid and D-glucose (conditions of reaction: the phosphate buffer, рН 7.0; 0.1 M solutions, a molar ratio - m-amino benzoic acid/D-glucose = 1:1, 100ОC, duration 20, 40, 60, 80, 100 and 120 minutes. A dialysis against distilled water within 72h, at 15-18 °C, in dialysis sacks SERVAPOR® (retains proteins with M.W. > 3500). Spectrophotometric measurements were carried out on the device Specord UV Vis. Hydroxymethyl furfural was determined by square-wave polarography on dropping mercury electrode on the background of 0.1 M solution of lithium hydroxide, on the device OH-104 (Radelkis) [11].

DISCUSSION OF RESULTS It is shown, that аromatic amines actively participate in the Maillard reaction with formation of appropriate melanoidins, and an initial stage of this process, as well as in a case of aliphatic amines, is formation of N-glycosides [5, 6]. From the viewpoint of dependence from pH of the reaction medium, aromatic amines (aniline, toluidines and amino phenols) are similar to aliphatic amino acids – their reactivity is increased with increase of рН. In these conditions, the isomeric aromatic amines are subject to the certain regularity. For example, by interaction of D-glucose with o-, m- and p-amino phenols in acid, neutral and alkaline medium, the amount of formed melanoidin is increased with increase of the pH of the reaction medium, and thus the meta-isomer is always least active. In this respect, in a case of amino benzoic acids opposite regularity is observed - their activity in Maillard reaction decreases with increase of рН, and reactivity of p-isomer is rather low (Figure 1). The analogous regularity is observed at the monitoring of reaction by quantity of the free amino acid and by amount of formed hydroxymethyl furfural (Table 1). According to the obtained data, interacting of amino benzoic acids with D-glucose is subject to the certain pattern – by an increase of temperature the quantity of the reacted amino benzoic acid increases. Thus, in acid, neutral and alkaline mediums, the m-amino benzoic acid reacts much more actively, than the p-isomer, and in this respect o-isomer occupies an intermediate position. Presumptively, such regularity is conditioned by various values of pKa of isomeric amino benzoic acids (Table 2). The fact that a reactivity of amino benzoic acids in Mailladr reaction is reduced by decrease of рН of reaction medium is common regularity and is confirmed in case of all aldoses investigated by us. In comparison with aldohexoses, aldopentoses participate in process of formation of melanoidins more actively. For example, aryl amines including

Participation of Aromatic Amines in the Maillard Reaction

83

amino phenols, so actively react with aldopentoses (a xylose, an arabinose) that in the reaction conditions indicated above the great bulk of an end product - a melanoidin transfers in an insoluble condition. A number of researches specifies that the melanoidin complex formed as a result of interacting of sugars and amino acids, consists not only of high-molecular weight compounds, but the significant part of its colored (λ = 470 nm) compounds is rather lowmolecular. Study of the products formed by thermal treatment of a reaction mixtures of glucose/glycine (pH 5,5; 55ОC) has shown that the high-molecular weight part of formed products makes only about 10 % of total yield of the melanoidin complex, and the remaining part is a share of low-molecular weight fraction (<3500 dalton) [Leong, Wedzicha, 2000]. Similar research was carried out on reaction systems glucose/glycine and glucose/αalanine in a phosphate buffer at pH 7 and temperature 95ОC within 4 hours. It was found out that in these conditions the share of the colored high-molecular fraction (> 3000 dalton) is only insignificant part of a yield, and melanoidin coloring basically is determined by a lowmolecular weight fraction [Hofmann, 1998].

Figure 1. Formation of melanoidin at interacting of o-, m-, p-amino phenols and o-, m-, p-amino benzoic acids with D-glucose in acid, neutral and an alkaline medium (reaction conditions: a phosphate buffer, 0.1М solutes, a molar ratio – aromatic amine/glucose = 1:1, temperature 100oC, reaction time 2 h. λ = 470 nm).

Martins [2003] studied a high-molecular weight fraction (>3500 dalton) of the melanoidin complexes formed by interacting of glucose and glycine at various temperatures and рН. It was found out that from compounds stipulating absorbance at 470 nm, the share of the high-molecular fraction is no more than 20 %. Using a similar technique, we investigated distribution of a melanoidin pigment (λ = 470 nm) between high-molecular weight (> 3500 dalton) and low-molecular weight (< 3500 dalton) fractions of the reaction mixture received as a result of interaction of m-amino benzoic acid and D-glucose (Figure 2).

R. Kublashvili and D. Ugrekhelidze

84

Table 1. Melanoidin reaction between amino benzoic acids and D-glucose (concentration 10-3М, molar ratio 1:1; 0.1 M phosphate buffer, reaction time 4 hours)

Reacting components

o-Amino benzoic acid + D-glucose

m-Amino benzoic acid + D-glucose

p-Amino benzoic acid + D-glucose

Reaction conditions pH t °C

Unreacted amino benzoic acid (10-3 M)

Hydroxymethylfurfural

4.9

25

0.43

-

4.9 4.9 7.0 7.0 7.0 9.2 9.2 9.2 4.9 4.9 4.9 7.0 7.0 7.0 9.2 9.2 9.2 4.9 4.9 4.9 7.0 7.0 7.0 9.2 9.2 9.2

50 80 25 50 80 25 50 80 25 50 80 25 50 80 25 50 80 25 50 80 25 50 80 25 50 80

0.36 0.21 0.47 0.38 0.23 0.55 0.42 0.33 0.36 0.24 0.11 0.38 0.30 0.17 0.49 0.33 0.25 0.57 0.43 0.40 0.51 0.44 0.40 0.56 0.55 0.48

+ + + + + + + + + + + + -

The note: - it is not detected; + it is detected and identified.

Тable 2. pKa Values of substituted phenyl amines [7] Compound o-Toluidin m-Toluidin p-Toluidin o-Amino phenol m-Amino phenol p-Amino phenol o-Amino benzoic acid m-Amino benzoic acid p-Amino benzoic acid

pKa 4.39 4.60 5.12 4.72 4.17 5.17 2.11 3.12 2.41

Participation of Aromatic Amines in the Maillard Reaction

85

Figure 2. Distribution of a melanoidin pigment (λ = 470 nm) between low-molecular weight (< 3500 dalton) and high-molecular weight (> 3500 dalton) fractions as a result of a dialysis of the reaction mixture formed by interaction of m-amino benzoic acid and D-glucose (conditions of reaction: the phosphate buffer, рН 7.0; 0.1 M solutions, a molar ratio - m-amino benzoic acid/D-glucose = 1:1, 100ОC, duration 20, 40, 60, 80, 100 and 120 minutes. A dialysis against distilled water within 72h, at 15-18 °C, in dialysis sacks SERVAPOR® (retains proteins with M.W. > 3500). 1 – Low-molecular weight (< 3500 dalton) fraction 2 - High-molecular weight (> 3500 dalton) fraction

In result it was shown that in a melanoidin, formed from m-amino benzoic acid and Dglucose, the contents of high-molecular weight fraction (> 3500 dalton) was higher (22-30 %), than in melanoidines from the above-stated systems (glucose/glycine and glucose/αalanine). It is necessary to note that in contrast to glycine and α-alanine, which react with glucose with low intensity, amino benzoic acids react with glucose more actively; especially active is m-amino benzoic acid, which reacts with aldoses with high intensity, forming in most cases melanoidines, insoluble in water and buffer systems. Its reactivity is especially high in acid medium.

REFERENCES [1] [2] [3] [4] [5]

L-C Maillard. Action des acides aminés sur les sucres. Formation des Mélanoidins par voie méthodique. Compt. Rend., 1912, 154, 66-68. H.D. Belitz, W. Grosch Food chemistry. Berlin, Heidelberg, New York. SpringerVerlag, 1999, 263-318. V.M. Monnier. Nonenzymatic glycosylation, the Maillard reaction and the aging process. J. Gerontol., 1990, 45, 105-111. M.A. Van Boekel The role of glycation in aging and diabetes mellitus. Mol. Biol. Rep., 1991,15, 57-64. R. Kublashvili, N-Glucosides of Aminobenzoic Acids and Aminophenols. Chemistry of R. Kublashvili, M. Labartkava, K. Giorgadze, D. Ugrekhelidze Synthesis and

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characterization of N –tolyglycosylamines. Chemistry of Natural Compounds, 2008, 44, 413-415. [6] A. Albert, E.P. Serjeant, 1984. The Determination of Ionization Constants: A Laboratory Manual. Chapmand and Hall: London, NY [7] L.P. Leong, B.L Wedzicha. A critical appraisal of the kinetic model for the Maillard browning of glucose and glycine. Food Chemistry, 2000, 68, 21-28. [8] T. Hofmann. Studies on the relationship between molecular weight and the color potency of fractions obtained by thermal treatment of glucose/amino acid and glucose/protein solutions by using ultracentifugation and color dilution techniques. J. Agric. Food Chem., 1998, 46, 3891-3895. [9] I. F. S Martins. Unravelling the Maillard reaction network by multiresponse kinetic modelling. Ph.D. Thesis, Wageningen University. The Netherlands. 2003. [10] E. Koen. A polarographic method of determining the microlevels of furfural in the air of the work area. Probl Khig. 1989, 14, 127-35.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 8

INFLUENCE ON OXIDATION PROCESSES REGULATION IS THE REASON FOR BIOLOGICAL ACTIVITY OF THE ECDYSTEROID-CONTAINING COMPOUNDS L.N. Shishkina, O.G. Shevchenko*and N.G. Zagorskaya* N.M. Emanuel Institute of Biochemical Physics Russian Academy of Sciences,j Moscow, Russia * Institute of Biology, Komi Scientific Center, Ural Division of Russian Academy of Sciences,k Syktyvkar, Russia

ABSTRACT The influence of serpisten and inokosterone on the phospholipids composition in liver and blood erythrocytes, intensity of lipid peroxidation in tissues (liver, spleen, blood plasma), catalase activity in liver and general peroxidase activity of white outbreed mice has been studied. A biological activity of ecdysteroid-containing compounds is shown to be associated with an influence on the parameters of the physicochemical regulatory system of lipid peroxidation (LPO). Possessing pronounced membrane-tropic properties due to alterations in the exchange of predominantly choline-containing fractions of phospholipids, ecdysteroid-containing preparations are capable of modifying a cell membrane phase state. A substantial dependence of a biological effect of the compounds on a dose, duration of their application as well as on an intensity of the LPO processes in the tissues and an animal’s sex require a more detailed research on the properties of the given ecdysteroids.

j k

4 Kosygin st., Moscow 119334, Russia. 28 Kommunisticheskaya st., 167610 Syktyvkar, Russia.

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AIMS AND BACKGROUND Well-known biological properties, in particular, tonic and adaptogenic qualities of either ecdysteroids or ecdysteroid-containing plant preparations [1-5] are similar to those of a number of natural and synthetic antioxidants (AO) [6-8]. This allows us to assume that a biological activity of ecdysteroids should be derived from their influence on the parameters of the lipid peroxidation (LPO) system in tissues of hematothermal animals. It is known, that regardless of a reason for LPO intensification, changes in the oxidation rate are associated both with a reduction in the amount of bioantioxidants and changes in the membrane phospholipid (PL) composition [9] due to either a more rapid degradation of oxidized lipids or the acceleration of lipid transport by transferring proteins. Therein, the role of PL in the oxidative processes is multifunctional, since on the one hand they are substrates for oxidation, on the other hand PL are capable of modifying oxidative processes acting as antioxidants, their synergists or antagonists [9, 10]. LPO activation is associated with the vital physical and chemical membrane properties—penetrability, viscosity and phase state [9-12]. A state of the membrane lipid phase has an effect on the activity of membrane-associated enzymes and the cell sensitivity to hormonal and nervous regulation [10, 13, 14]. Phospholipids either maintain the work of the vital cell mechanisms such as ion exchange, biological oxidation or influence both an activity of the mitochondria enzymes and the oxidative phosphorylation [15[. In biomembranes, a lipid component that is formed as a functionally active template integrates an external influence and participates in triggering the cell control programs [16–18]. A plasma membrane possesses the unique receptive and signaling functions of the vital cell processes regulation whose lesions could lead to a cell death. A mechanism of the origin and development of the majority of pathological states are due to the disturbances in a structure and properties of membranes [9, 10, 19]. The data on membrane-stabilizing effect of ecdysteroids are available in the current literature [5, 20, 21]. Noteworthy, that phytoecdysteroids were revealed to influence a lipid exchange, in particular, cholesterol synthesis and catabolism [22-24]. Moreover, hematoreologic activity of ecdysteroid-containing plants and ecdysterone which can be ascribed to a likely modification of an erythrocyte membrane lipid phase was shown previously [25]. Rising interest in a research study on erythrocyte membranes has been caused by participation of these cells in the processes associated with maintaining homeostasis at the whole organism level. Regularities of changes in the erythrocyte membrane structure and functions can be extrapolated to other membrane systems with a definite correction due to the species specificity of cells [19, 26]. A visible simplicity of the erythrocyte structure enables one to investigate into functional properties of a plasma membrane without any hindrances exerted by intracellular membrane formations or organelles [27[. Structural and functional peculiarities of the erythrocyte as well as its availability for investigations enable one to consider it a universal model for a research study on the changes in cytoplasm membranes and cell metabolism of an organism The aim of the present research is an experimental verification of a hypothesis on a relation between a biological activity of ecdysteroid-containing preparations and their action on the parameters of the LPO physicochemical regulatory system in animal tissues.

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EXPERIMENTAL The experiments were carried out on white outbreed mice—males and females (2-3 months age)— which lived at standard vivarium conditions. Since an antioxidant status of animal tissues substantially depends on a season [28], all the experiments were carried out from March to May. Serpisten and inokosterone were isolated by V.V. Volodin and a coworker in a laboratory from Serratula coronata [29] and politely given to us for research. A range of the tested serpisten doses under a single per os preparation administration was from 10 to 3000 mg/kg of body weight. A dose was calculated individually for each animal with regard to body weight. The animals were decapitated 10 days after the preparation administration. To study a chronic serpisten action, a serpisten solution in distilled water was given to the animals instead of drinking water for 10 or 30 days. The concentrations were selected so that the total doses of serpisten were equal to 5; 50 and 500 mg/kg. For inokosterone, the total doses were 5 mg/kg of the body weight for 10 days or 5 and 15 mg/kg for 30 days. The calculation was performed taking into account animals’ weights and a volume of the consumed liquid. The control animals were given distilled water. Following decapitation of the mice, their organs were placed on ice. The blood was collected in test tubes treated by 5% solution of sodium citrate. The blood plasma was separated from the blood corpuscles by centrifugation. The content of the LPO secondary products reacting with 2-thiobarbituric acid (TBA-reactive substances, TBA-RS) was determined using the method described in [30]. The catalase activity in liver was measured spectrophotometrically at the wavelength of 410 nm according to formation of a colored complex of ammonium molybdate in the presence of hydrogen peroxide [31]. The protein content was evaluated by the modified micro-biuretic method described in [32]. The total peroxidase activity (TPA) of blood which is characteristic of the intensity of LPO processes and reflects the ratio between pro-oxidative and antioxidative (AO) blood systems [33, 34] was determined by a photometrical registration of a drop in indigocarmine concentration that is oxidized by hydrogen peroxide in the presence of peroxidases [35]. Lipids from liver and erythrocytes were isolated by the method of Blay and Dyer in the Kates modification [36]. The qualitative and quantitative composition of phospholipids (PL) was analyzed by the thin-layer chromatography [37] with the use of silica gel of type G (Sigma, USA) and glass plates measuring 9 × 12 cm. We used a mixture of chloroform – methanol - glacial acetic acid - water in the ratio of 50:30:8:4 as the mobile phase. A quantitative analysis of the PL composition yielded after removal of pellets from a plate and burning them to an inorganic phosphate with perchloric acid was performed spectrophotometrically at the wavelength of 800 nm and according to formation of complex in the presence of ascorbic acid. A more detailed description of the PL composition determination is presented elsewhere [38]. In addition to a quantitative analysis of a ratio between the different PL fractions, generalized parameters of the lipid composition were evaluated: the PL amount in the total lipid composition (% PL); the phosphatidyl choline/phosphatidyl ethanolamine (PC/PE) ratio; the ratio of the sums of the more easily to the more poorly oxidizable PL fractions (ΣEOPL/POPL). The latter were calculated according to the formula [37]: ΣEOPL/POPL = (PI + PS + PE + CL + PA) (LPC + PC + SM), where PI is phosphatidyl inositol, PS is phosphatidyl serine, CL is cardiolipin, PA is

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phosphatidic acid, LPC are lysoforms of PL (mainly PC), and SM is sphingomyelin. The LPC/PC ratio which is characteristic of a PC degradation rate and its lysoform utilization was also assessed [39, 40]. The obtained experimental data were processed with the commonly used methods of the variational statistics [41]. The experimental data are presented in tables and figures in the form of arithmetic means with the indication of the mean square errors of the arithmetic means (M ± m).

RESULTS AND DISCUSSIONS First of all, it is necessary to note that within the observed range of doses, serpisten possesses neither acute (10–3000 mg/kg) nor chronic (5–500 mg/kg for 30 days) toxicity [42]. An extreme low toxicity of other phytoecdysteroids even at very high doses was shown in the experiments on hematothermal animals by a number of authors [3, 42]. . LPO process regulation in cells and tissues is known to be realized either with participation of low molecular antioxidants and metabolites or due to functioning of enzymes of the antioxidant defence [9, 10, 17, 38, 43, 44]. Parameters for the physicochemical system of LPO regulation, that include the phospholipid composition directly associated with LPO intensity, as well as an activity of the antioxidant defence enzymes in murine liver and blood have been studied in the present work. According to our data (Table 1), a single administration of serpisten at average and high doses (100–3000 mg/kg) led to no reliable changes in the quantitative composition of the male liver PL regarding most of the already investigated indicators 10 days after the preparation administration. Nevertheless, serpisten administration to an animal organism at a dose of 2000 mg/kg resulted in a reliable reduction of the PC share in liver PL. Table 1. The phospholipid composition (%P) and the generalized parameters of the lipid composition (relative units) in liver of mice (males) after 10 days under a single administration of serpisten Phospholipid fraction, parameter

LPC SM PC PC + PI PE CL+PA % PL PC/PE ΣEOPL/POPL LPC/PC

Dose, mg/kg Control

100

2000

3000

5.05±0.11 2.09±0.96 53.84±0.51 3.68±1.53 30.56±3.29 4.79±0.40 22.91±1.20 1.86±0.22 0.65±0.04 0.094±0.003

5.40±0.23 4.40±0.36 52.08±2.38 7.54±2.62 26.04±0.50 4.56±0.39 26.28±0.13 2.00±0.05 0.63±0.07 0.105±0.009

4.85±0.11 4.82±0.10 46.47±0.15*** 4.39±1.09 34.11±1.00 5.37±0.15 21.58±4.86 1.37±0.04 0.79±0.00 0.104±0.003

5.07±0.24 3.97±0.10 49.84±2.03 5.00±0.81 27.52±0.04 8.61±1.60* 27.94±2.36 1.81±0.07 0.71±0.07 0.102±0.001*

Note: here and thereafter the significant differences from the control are * P < 0.05, ** P < 0.01, *** P < 0.001.

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Moreover, a trend to an increase in the ratio LPC/PC was observed for phospholipids of all the groups of mice, the sum portion of CL and PA in the PL of animal liver rising upon introduction of the preparation at a dose of 3000 mg/kg (Table 1). Acid phospholipids (PS, PI, CL and PA) are known to regulate the activity of Ca+2- and Na+,K+- ATPases and required for maintaining the cell ion balance [14, 45]. Modulation of the activity of these enzymes is a response of a cell to the factors disturbing its homeostasis. In addition, an increase in the CL share in liver PL is likely to be associated with the energy exchange activation, since the main portion of this PL synthesizes and localizes at mitochondria. A long-term (30 days) administration of low and average doses of serpisten (5-500 mg/kg) to an animal organism caused sufficient changes both in the quantitative ratio of different PL fractions and generalized indices for the liver PL composition (Table 2). The analysis of the obtained data testifies to the fact that a long-term administration of high doses of serpisten causes a trend to a reduction of the PC proportion in liver PL in parallel to a growth of a relative LPC content both in males and females. Application of serpisten at a dose of 50 mg/kg evoked a decrease of the ratio PC/PE in the PL composition of liver both in males and females. That indicates a diminution in rigidity of the liver membrane structures. However, it must be noted, that murine females have a higher sensitivity to serpisten, which might be attributed to the existing in norm distinctions in the quantitative composition of the tissue membrane lipids with regard to a sex. In females, a long-term application of serpisten at a dose of 500 mg/kg led to a reliable growth of the ratio LPC/PC, the sum PC content and its lysoforms being unchanged. In addition to an increase of a lysoform share in liver PL of the females that received serpisten we observed a reliable decrease in the relative content of SM that resulted in the total reduction of a share of choline-containing fractions, thus leading to a rise in the content of easily oxidizable PL fractions. A long-term administration to a female organism of serpisten at any dose also resulted doth in a reliable enhancement of a relative content PI+PS and decrease of the PL proportion in the total lipid composition of liver. Table 2. The phospholipid composition (%P) and the generalized parameters of the liver lipid composition (relative units) of the mice females under a chronic application of the serpisten solution for 30 days (decapitation 15 days after the preparation administration) Phospholipid fraction, parameter

Dose, mg/kg Control

5

50

500

LPC SM PC PI + PS PE CL + PA % PL PC/PE ΣEOPL/POPL LPC?PC

5.65±1.60 10.63±0.37 37.27±2.31 9.96±0.03 25.35±0.76 11.14±1.12 46.61±0.37 1.47±0.05 0.86±0.02 0.175±0.050

8.70±0.24 2.47±0.19*** 33.21±3.47 17.74±2.68* 22.90±3.16 14.98±4.27 10.68±0.53*** 1.56±0.34 1.25±0.12* 0.271±0.030

6.40±3.17 1.07±0.54* 38.16±0.15 14.20±1.00** 29.67±0.42*** 10.51±2.21 27.18±2.26*** 1.29±0.01* 1.23±0.14 0.167±0.08

15.65±1.72*** 5.38±1.72** 30.07±2.95 14.30±1.24** 23.35±0.68 11.25±0.29 33.79±3.95** 1.28±0.09 0.96±0.03* 0.564±0.110**

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The next step of our work was to evaluate how lower serpisten doses affect LPO parameters in murine tissues. Figures 1 and 2 present the data on certain parameters of the lipid composition in liver and blood erythrocytes of the mice (males) which received serpisten at the doses of 5 and 50 mg/kg for 10 days. The animals were decapitated immediately after the preparation application had been stopped. Application of serpisten at either dose (especially at 5 mg/kg) caused a rise in the total PL content in lipids of blood erythrocytes (Figure 1). In liver lipids, an increase in the PL proportion was observed only in case of exposure to the preparation at a dose of 50 mg/kg. Noteworthy, that along with an increase of the total PL content in blood erythrocyte lipids, serpisten at a dose of 50 mg/kg causes a reliable reduction of lipid oxidizability that can be assessed with regard to the ratio of more easily to more poorly oxidizable PL fractions: ΣEOPL/ΣPOPL = 0.265 ± 0.020 in the experimental group and 0.372 ± 0.030 in the control. Also observed is a simultaneous enhancement of a rigidity of erythrocyte membranes that can be derived from a growth of the ratio PC/PE: 5.78 ± 1.00 in the experimental group and 3.22 ± 0.14 in the control. Application of serpisten at a dose of 50 mg/kg also resulted in a reliable diminution of the SM share in blood erythrocyte PL. Serpisten administration at a dose of 5 mg/kg led to a drastic rise in a share of lysoforms in PL of both blood erythrocytes and liver (Figure 2). However, a 10-fold increase in the preparation dose caused a reverse effect, mainly in erythrocytes (Figure 2). It is known LPC is required for the normal cell functioning, participates in the activity regulation of the majority of membrane-associate enzymes, and is a secondary mediator of trans-membrane transmission of a signal within a cell [46-49]. At the same time, the LPC amphiphylicity and “wrong” structure underlie either its detergent, fluidizing action or ability to disturb the membrane topography, thus making it possible to employ a level of PL lysoforms for estimating an extent of pathological processes [46, 48]. The growth of the lysoform share in the phospholipids of the blood erythrocytes and liver of the animals which received serpisten represents a powerful factor of modifying the properties of either a lipid bilayer or integral membrane proteins and can result from both an rise of the phospholipase A2 activity and lysophospholipase and acyltransferase inhibition [46, 48]. Meanwhile, phospholipase A2 activation is accompanied by not only an increase in the amount of LPC, but also a growth of an intracellular pool of arachidonic acid [50]. Activation of an arachidonic cascade is considered by some authors [51] an integrated negative unit for autoregulating the activity of the phospholipid-depending signaling cell system, whereas ecdysteroids are believed to be possible effective modulators of intracellular eicosanoid pools in tissues of hematothermal animals. Modification of a phase state of the cell membrane system under administration of ecdysteroids can be conditioned not only by the PL lysoform action but also by a reduction in PL relative content of SM that along with cholesterol favors an enhancement of microviscosity and rigidity of the membrane lipid phase due to a predominant presence in its molecule of saturated fatty acid residues [52]. Metabolism of SM and cholesterol in cells is known to have an integrated character [53]. Cholesterol building in among PL molecules limits their mobility and significantly predetermines fluidity and viscosity of a red blood cell membrane, thus influencing on a lateral diffusion of receptors, ion transport, and penetrability for dissolved substances [52]. A phase state of the cell membrane exerts a remarkable influence on the processes of membrane transport, the systems of a trans-membrane

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information transmission, and the activity of membrane-bound enzymes. Changes of the viscous properties of membranes emerging as a result of alterations in the quantitative ratio of different fractions affect not only a form of the erythrocytes, but also their ability to be deformed. The changes in a relative content of PL lysoforms and SM are most likely to cause those changes of a shape and osmotic resistance of erythrocyte membranes that were detected after administration of serpisten and other ecdysteroid-containing compounds in the experiments on the laboratory animals [54, 55]; the observed by the authors effect depending to a considerable extent on an animal’s sex, compound dose, or a period of its application. It was shown [25] that ecdysteroid-containing extracts exhibited hematoreologic activity in model experiments at the concentrations under which ecdysterone constituting them displayed neither antiradical nor antioxidative activity. These authors later reported [56] that the extracts from Lychnis chalcedonica and Raponticum sarthamoides containing ecdysteroids actually prevented a pathological modification of erythrocytes shape under administration to animals through their influence on the content and ratio of different fractions of lipids and phospholipids in erythrocyte membranes.

Figure 1. The content of phospholipids in the total lipid composition of the blood erythrocytes and liver of mice which received serpisten at the doses of 5 and 50 mg/kg for 10 days.

L. N. Shishkina, O. G. Shevchenko and N. G. Zagorskaya

LPC, % P

94

6

control 5 mg/kg

5

50 mg/kg

4 3 2 1 0 erythrocytes

liver

Figure 2. The share of lysoforms in phospholipids of the blood erythrocytes and liver of mice which received serpisten at the different doses for 10 days.

It is unanimously accepted that sphingolipids influence not only a structural state of biological membranes but also play a part of secondary messengers in the array of the most important cell processes and are the mediators of some extracellular stimuli [57-59]. SM exchange modulation is considered one of the effective mechanisms of an early (pregenomic) action of ecdysteroids [60]. At the same time, there is evidence [61, 62] that ecdysteroids and ecdysteroid-containing plant extracts are able to influence a functional activity of the endocrinal system organs (pancreas and, most likely, adrenal gland and testicles). Obviously, the revealed alterations in a relative SM content in the liver and blood erythrocyte PL are attributed to the changes in the thyroid gland functional activity, since the SM metabolism in different tissues is controlled by thyroid hormones [63] Indeed, that histomorphological study of thyroid gland of the mice which received serpisten for 10 days points to a diminution of the colloid volume density, that can testify to a diminution in the thyroid gland functional activity [64]. The fact that biological activity of serpisten depends in many respects on the presence in its content of inokosterone [42] caused a necessity of a more detailed research on the properties of the latter. In this connection, we studied the influence of inokosterone on the parameters reflecting an intensity of the LPO processes in murine tissues. Analysis of the LPO secondary product content showed a dependence of the obtained effects both on the preparation dose and the tested tissue (Table 3). Application of low doses of inokosterone (5 mg/kg) at both a short-termed (10 days, variant 1) and long-termed (30 days, variant 2) administration to the organism led to a reliable 2-4-fold diminution of the LPO intensity in the blood plasma. To the contrary, unidirectional changes in the TBAreactive substances content were detected in liver and spleen: a growth of the LPO intensity at 5 mg/kg and the absence of reliable differences from the control at a dose of 15 mg/kg. It is significant that an increase of the inokosterone dose up to 15 mg/kg also resulted in normalization of this parameter in the murine blood plasma. In all variants of the experiments, no reliable differences in the activity of catalase in the liver of intact mice and animals which

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had received both serpisten and inokosterone were revealed due to a high individual variability. The total peroxidase activity (TPA) of blood of the animals that had received inokosterone was associated both with a scheme of the application of the preparation (for 10 or 30 days) and an initial level of activity in the control groups of animal (Figure 3). Interestingly, that under inokosterone application at a dose of 5 mg/kg a growth of the blood TPA was observed only in the group of mice with a low level of the enzymatic activity in the control. An analogous dependence of the influence on the antioxidative system is described for other biologically active substances in articles [65, 66]. Investigations of the blood erythrocyte lipids of the mice which received inokosterone according to different schemes have showed a sufficient influence of inokosterone on a exchange of choline-containing PL fractions. The preparation was shown to change the LPC/PC ratio towards the intensification of the acylation reaction of LPC to PC (Figure 4, A), thus inducing a reduction in the lysoform proportion in PL of the blood erythrocyte. Some increase of a relative SM content has also been noted. Analysis of generalized parameters for the PL composition (PC/PE and ΣEOPL/ΣPOPL) shows that application of inokosterone at the investigated doses can evoke either some augmentation of the erythrocyte membrane rigidity or reduction of its lipid oxidizability (Figures 4.A and 4.B) thus interfering with a further LPO intensification. Sufficient differences in an individual reaction of animals to inokosterone administration causing a significant variability of parameters in the experimental group are in accord with the data of other researchers who tested this compound [42]. A high individual and tissue variability of the effects detected under inokosterone application to animals is likely to result from the fact that development of a cell response under administration of the biological active substances depends on the state of its antioxidant and pro-oxidant systems [66, 67]. Table 3.The content of TBA- reactive substances (nmol/mg of protein) in the murine tissues after application of the inokosterone solution at different doses (decapitation after 5 days under finishing of the preparation administration) Variant of experiment Control Inokosterone 5 mg/kg, 10 days

Blood plasma 0.140±0.017 0.035±0.008***

Liver 0.152±0.017 0.175±0.031

Spleen 0.366±0.038 0.751±0.094***

Control Inokosterone 5 mg/kg, 30 days Inokosterone 15 mg/kg, 30 days

0.152±0.031 0.078±0.012* 0.200±0.010

0.101±0.031 0.171±0.017 0.122±0.041

0.472±0.061 0.541±0.035 0.435±0.035

L. N. Shishkina, O. G. Shevchenko and N. G. Zagorskaya

μmole × min/ ml of blod

96

250

control

240

inokosterone 5 mg/kg

230

inokosterone 15 mg/kg

220 210 200 190 180 170 160 150

The first variant

The second variant

Figure 3. The total peroxidase activity of blood of mice which received inokosterone at thedoses of 5 and 15 mg/kg for 10 (the first variant) and 30 (the second variant) days

Thus, a biological activity of the preparation significantly depended on the tested dose in the experiments with both inokosterone and serpisten. In a number of cases a 3-10-fold increase of a dose either led to an enhancement of the effect or induced a normalization of parameters and even changed the sign of the effect. The absence of the augmentation of the action with an increased dose of ecdysterone was also observed when studying haemoreological properties of ecdysteroid-containing extracts [25]. Evidence on a possible antiradical and antioxidative activity of ecdysteroids is rather contradictory [51, 68, 69]. Model experiments [70, 71] and tests on animals [51] established that ecdysteroids possess both pro-oxidant and antioxidant properties depending on their concentration and intensity of oxidative processes. Such dualism of a response of the system of the organism antioxidant defense was recorded under administration of numerous biologically active compounds, including oxysterols, ά-tocopherol, carotinoids, ascorbic acid, and other natural and synthetic antioxidants [8, 10, 43, 67, 72, 73]. The total results of the study allow us to conclude that a biological activity of both serpisten and inokosterone is associated with the action on the parameters of the physicochemical system of LPO regulation. The most remarkable effect is observed under a long-term application into the organism of compounds at low doses. Possessing pronounced membrane-tropic properties, due to alterations in the exchange of, predominantly, cholinecontaining fractions of phospholipids, ecdysteroid-containing preparations are capable of modifying a phase state of the membrane system of tissues. A remarkable dependence of a biological effect of these preparations on a dose, duration of administration to the organism, as well as on the intensity of LPO processes in tissues and a sex of an animal require a more detailed research on the properties of the given ecdysteroid-containing compounds. The work was supported by the Program of Fundamental Research of Presidium of Ural Division of Russian Academy of Sciences “Fundamental Sciences for Medicine”.

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97

control inokosterone 5 mg/kg inokosterone 15 mg/kg

0,4 0,3 0,2 0,1 0

4 3,5 3

LPC/PC

a

ΣEOPL/ΣPOPL

control inokosterone 5 mg/kg inokosterone 15 mg/kg

2,5 2 1,5 1 0,5 0

PC/PE b

Figure 4. The generalized parameters of the phospholipid composition of the blood erythrocytes of mice which received inokosterone at the different doses for 30 days: a - phospholipid lysoforms/phosphatidyl choline ((LPC/PC) ratio and the ratio between the sums of the more easily to the more poorly oxidizable phospholipid fractions (∑EOPL∑POPL); b – phosphanidyl choline/phosphatidyl ethanolamine (PC/PE) ratio.

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[26] V.V. Novitsky, N.V. Ryazantseva, E.A. Stwepovaya et al. Bull. Sib. Med., 2006 2, 6269 (Russ.). [27] Yu.V. Postnov, S.N. Orlov “Primary hypertension as the pathology of cell membranes” Moscow, Meditsina, 1987, 192 pp. (Russ.). [28] L.N. Shishkina, E.B. Burlakova “The Value of Antioxidant Properties of Lipids in Radiation Damage and Membrane Repair”, pp. 334 – 364, in the book: “Chemical and Biological Kinetics. New Horizons” Vol. 2: Biological Kinetics. Eds. E.B. Burlakova, S.D.. Varfolomeev. Leden. Boston, VSP, 2005. [29] V.V. Volodin, S.O. Volodina Patent 2153346, Russia, MKIS A 61 K 35/78 “Method of the ecdysteroids production”, Institute of Biology Komi SC, Ural Division of Russian Academy of Sciences, N 99106351/14, Publ. 27.07.2000, BI N 21. [30] Asakawa, S. Matsushita. Lipids, 15, 1137-1140, 1980. [31] M.A. Korolyk, L.I. Ivanova, I.G. Maiorova. Labor. Practice, 1, 16-19, 1988 (Russ.). [32] R. Itzhaki, D.M. Gill. Anal. Biochem. 1964, 9, 401–410. [33] I.A. Goroshinskaya, L.V. Mogil’nitskaya, L.A. Nemashkalova, A.A. Khodakova. Radiobiology, 1972, 12, 232–235(Rus.). [34] L.A. Tiunov, E.A. Zherbin, B.N. Zherbin “Radiation and poisons”. Moscow, Atomizdat, 1977, 144 pp. (Russ.). [35] T. Popov, L. Neikovskaya. Hygiiene and Sanitation, 1971, 10, 89-91 (Russ.). [36] M. Kates “The Technology of Lipidology”. Moscow, Mir, 1975, 322 p.(Russ.). [37] Biological membranes: A practice approach. Eds. J.B.R. Findlay, W.H. Evans. Moscow, Mir, 1990, 424 pp. (Russ.). [38] L.N. Shishkina, Ye.V. Kushnireva, M.A. Smotryaeva. Oxid. Commun., 2001, 24, 276286. [39] T.P. Kulagina, I.K. Kolomiitseva, V.I. Arkhipov. Bull. Exper. Biol. Med. 2000, 130, 292-294 (Russ.). [40] A.E. Lychkova, V.M. Smirnov. Bull. Exper. Biol. Med., 2005, 133, 364-366 (Russ.). [41] G.F. Lakin. Biometry. Moscow, Vysshaya shkola, 1990, 352 p. (Russ.). [42] K. Slama, R. Lafon // Eur. J. Entomology, 1995, 92, 355-377. [43] N.K. Zenkov, V.Z. Lankin, E.B. Men’shchikova “Oxidative Stress. Biochemical and Pathophysiological Aspects”. MAIK-Interperiodika, 2001, 343p. (Russ.). [44] L.N. Shishkina, E.V. Kushnireva, M.A. Smotryaeva. Radiat. Biology. Radioecology, 1995, 44, 289-295 (Russ.). [45] A.A. Boldyrev. Ukr. Biochem. J., 1992, 64, 5-10 (Russ.). [46] G.A. Gribanow. Voprosy Med. Khimii, 1991, 37, 2-10 (Russ.). [47] N.V. Prokazova, N.D. Zvezdina, A.A. Korotaeva. Biochemistry, 1998, 63, 38- 46 (Russ.). [48] T.I. Torkhovskaya, O.M. Ipatova, T.S. Zakharova et al. Biochemistry, 2007, 72, 149157 (Russ.). [49] A.A. Kunshin, V.I. Tsyrkin, N.V. Prokazova. Bull. Exper. Biol. Med., 2007, 143, 604607 (Russ.). [50] M.G..Sergeeva, A.T. Varfolomeeva. Cascade of Arachidonic Acid. Moscow, Narodnoye obrazovaniye, 2006, 256 p. (Russ.). [51] A.V. Kotsyryba, O.N. Bukhnevich, S.S. Tarakanov. Ukr. Biochem. J., 1995, 67, 45-52 (Russ.). [52] E.V. Dyatlovitskaya. Biochemistry, 1995, 60, 843-850 (Russ.).

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[53] H. Ohvo-Rekila, B. Ramstedt, P. Leppimaki, J.P. Slotte. Prog. Lipid Res., 2002, 41, 6697. [54] N.A. Moiseenko, Zh.E. Ivankova, A.S. Tsvetkova. Influence of 20-hydroxiecdydone on properties of the red blood components of rats during 24 hours after injection”, pp. 242256, in the book Radioecological and biological consequences of low intensity actions. Ed. A.I. Taskaev. Syktyvkar, 2003 (Russ.). [55] Zh.E. Ivankova, N.A. Moiseenko, S.A. Moiseenko “Role of 20-hydroxiecdydone in the development of post-hemorrhagic anemia at rabbits”, pp. 257–273, in the book: Radioecological and biological consequences of low intensity actions” . Ed. A.I. Taskaev. Syktyvkar, 2003 (Russ.). [56] M.Ya. Plotnikov, O.I. Aliev, A.S. Vasil’eva et al. Bull. Exper. Biol. Med., 2006, 146, 50-53 (Rus.). [57] AV. Alesenko. Biochemistry, 1998, 63, 75-82 (Russ.). [58] E.I. Ostashkin, Yu.B. Bespalova, I.M. Molotkovskaya et al. Dokl. Acad. Nauk Russia, 2000, 371, 406-409 (Russ.). [59] O.M. Ipatova, T.I. Torkhovskaya, T.S. Zakharova, E.M. Khalilova. Biochemistry, 2006, 71, 882-893 (Russ.). [60] A.V. Kotsyryba, A.V. Tuganova, O.N. Bukhnevich, S.S. Tarakanov. Ukr. Bioch. J., 1995, 67, 53-58 (Russ.). [61] I.N. Todorov, Yu.I. Mitrokhin, O.I. Efremova, L.I. Sidorenko. Chem.-Pharm. J., 2000, 34, 3-9 (Russ.). [62] I.N. Todorov, G.I. Todorov. Stress, aging and its biochemical correction. Ed. S.M. Aldoshin. Moscow, Nauka, 2004, 479 p. (Russ.). [63] N.A. Babenko, Yu.A. Natarova. Biochemistry, 64, 1085-1089, 1999 (Russ.). [64] O.V. Raskosha, O.V. Ermakova, A.V. Selezneva, O.V. Strekalovskaya. Morphological Newspaper, 1-2, Suppl. 1, pp. 243-245, 2006 (Russ.). [65] E.V. Ryabikina, Z.I. Mikashinovich, V.N. Zhenilo, Yu.A. Kalmykova. BLOOD.RU, 2007 [66] L.N. Shishkina, Yu.P. Taran, S.V. Eliseeva, V.G. Bulgakov. Izv. Acad. Nauk SSSR, Ser. Biol., 1992, 3, 350-357 (Russ.). [67] E.M. Treshchalina “Antitumor activity of substances of the natural origin”, Moscow, Practicheskaya Meditsina, 2005, 270 pp. (Rus.). [68] L.F. Osinskaya, L.M. Saad, Yu.D. Kholodova. Ukr. Biochem. J., 114-117 1992 (Russ.). [69] A.I. Kuz’menko,R.N. Morozova, I.N. Nikolaenko et al. Biochemistry, 1997, 62, 712715 (Rus.). [70] L.N. Shishkina, Ye.V. Kushnireva, V.V. Volodin “The study of the 20hydroxiecdyzone antioxidant properties in the model system”, p.125, in Abstracts of Workshop on Phytoecdysteroids, Syktyvkar, 1996. [71] L.N. Shishkina,, A.G. Kudyasheva, N.G. Zagorskaya et al. “Antioxidative Properties of ecdysteroids in systems in vitro and in vivo”, pp. 632-633 in Abstracts of papers of IV Inter. Conf. Bioantioxidants, Moscow, 2002 (Russ.). [72] L.N. Shishkina, E.B. Burlakova. Chem. Phys. Report., 1996, 15, 43-53. [73] G.P. Zhizhina, T.M. Zavorykina, E.M. Mill’, E.B. Burlakova. Radiat. Biology. Radioecology, 2007, 47, 414-422 (Russ.).

PART 2. PHYSICS, THERMODYNAMICS AND KINETICS OF HOMOGENEOUS AND HETEROGENEOUS NANOSYSTEMS

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 9

CHARGE TRANSFER MECHANISMS AT SAM-MODIFIED ELECTRODES IMPACT OF COMPLEX ENVIRONMENTS Dimitri E. Khoshtariya***, Tina D. Dolidzel** and Rudi van Eldik *** * Ivane Javakhishvili Tbilisi State University, Faculty of Exact and Natural Sciences, Department of Physics,m Tbilisi, Georgia ** Institute of Molocular Biology and Biophysics, *** Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg

ABSTRACT Nanoscopic electrochemical devices composed of SAM-modified electrodes and redox probes (RP) of different kind (complex ions, proteins, organic molecules, etc.), were proven to be suitable systems for studying intrinsic electron transfer (ET) mechanisms and interplay between them. In the present review we consider our recent results on the mechanistic studies of Au/SAM/RP nanoscale devices in which the RP were either redox-active protein cytochrome C (CytC) dissolved in aqueous solution, or the complex compound ferrocene (Fe(Cp)2]0/+, Cp=cyclopentadienyl) dissolved in a room temperature ionic liquid (RTIL), [bmim][NTf2]. The SAM composition was either [-S(CH2)n-OH] with n=2,3,4,6 and 11, or [-S-(CH2)n-CH3] with n=1,2,3,5,7,11 and 17, respectively. The modern electrochemical methodology including fast scan cyclic voltammetry and data processing techniques were applied to extract ET rate constants and an impact of the variation of ET distance, viscous additives (or temperature) and high pressure was determined, allowing for a rigorous discrimination of intrinsic ET mechanisms. In particular, at short electrode-reactant separations, n=2-3 for Au/SAM/CytC and n=1-3, for Au/SAM/Fe(Cp)2]0/+, the adiabatic mechanism of ET controlled by the viscosity-related relaxational modes of the RP environment, found to be operating. At larger electrode-reactant separations, n=6-11 for Au/SAM/CytC and n=7-11 for Au/SAM/Fe(Cp)2]0/+, the non-adiabatic ET mechanism can be observed manifested l

[email protected] 1, Ilia Chavchavadze ave., Tbilisi, Georgia.

m

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through the exponential decay of rate constant with the increase of n. At n=4 and n=5, the intermediate (mixture) regime of ET can be detected. Furthermore, for the case of n=17 for Au/SAM/Fe(Cp)2]0/+ in [bmim][NTf2], dynamical arrest (broken ergodicity) for ET has been demonstrated. In overall, together with other matching proceedings, the reviewed results, despite of essentially diverse nature of complex environments invoked within two different series of congruent systems (protein/aqueous solution versus RTIL) demonstrated common general patterns of the mechanism changeover, in a nice agreement with the predictions of a generalized theory of ET.

INTRODUCTION Intrinsic charge-transfer (CT) mechanisms of even simple redox-active species such as ferrocene (Fe(Cp)2]0/+), or small and well-characterized proteins such as cytochrome c (CytC), despite of continuous efforts, is difficult to recognize conclusively because of diverse and extremely inhomogeneous structural and dynamic properties of the reaction environment [1,2]. Among other assemblages, the artificial nanoscopic electrochemical devices made of electrode-deposited self-assembled monolayer (SAM) films of variable composition and thickness, and redox probes (RP) attached or freely diffusing to the SAM terminal groups (also subject of broad variations), were proven to be systems with most well-controlled variable parameters, and, hence, suitable for fundamental studies [3-12], in addition to the promises for versatile nano-technological applications [13,14]. The most systematic previous investigations that exploited the Au/SAM/RP composite systems, with freely diffusing or irreversibly (covalently or electrostatically) immobilized RPs, resolved two kinds of phenomenological behavior. For the case of thicker SAMs (longrange ET) these studies indicated an exponential decay of the ET rate constant, ket, with the ET distance, Re, in accordance with the non-adiabatic ET (long-range tunneling) theory [312,15-17]:

k et ∝ exp [− β (Re − Ro )]

(1)

where β is the decay parameter normally of the order of ca. 1 Å-1 (within the series of congruent systems). This behavior is similar to one found for a few of other systems involving bound or free non-biological redox species (i.e., complex ions), for which the longrange ET was allowed through the variation of Re. In few studied cases where nano-devices with thin SAMs and irreversibly bound CytC were invoked, the ET rate constant was shown to exhibit a power-law dependence on the solution viscosity, η, normally varied by change of solvent or addition of viscous compounds such as sugars or glycerol [3-12,18-21]:

k et ∝ η −δ

(2)

where δ is an "empirical" solvent-protein coupling parameter with values between 0 and 1. Such a manifestation, according to the general theory is indicative of the frictional control for a barrier-crossing process in general, and for the particular case of elementary ET processes (including biological ET) can be a signature of the adiabatic (viscosity-controlled)

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mechanism. An alternative pattern implies so-called "conformational gating", also discussed for the particular heterogeneous systems involving CytC [8]. Generally speaking, for biological ET (suppose the intrinsic stage is unequivocally identified), both non-adiabatic and adiabatic mechanisms are controlled by proteins' intrinsic dynamics, but in different manner. In all cases the role of hierarchically organized multiple conformational sub-states, outlined first by Frauenfelder et al. should be taken into the account. Specific manifestations may take place through Eqs. 1, 2, or other dependencies with temperature or pressure as variables (vide infra). In another breakthrough development the new type of reaction media, along with biomolecules, the room temperature ionic liquids (RTIL) have been suggested as an advantageous alternative to aqueous/organic solvents, due to a number of their favorable physico-chemical properties (negligible vapor pressure, low toxicity, high chemical and electrochemical stability, high conductivity) and the ability to dissolve wide range of organic and inorganic compounds [22]. In the present review we consider our recent results on the mechanistic studies of Au/SAM/RP nanoscale devices in which the RP were either redoxactive protein cytochrome C (CytC) dissolved in aqueous solution, or the complex compound ferrocene (Fe(Cp)2]0/+, Cp=cyclopentadienyl) dissolved in a room temperature ionic liquid (RTIL), [bmim][NTf2]. The SAM composition was either [-S-(CH2)n-OH] with n=2,3,4,6 and 11, or [-S-(CH2)n-CH3] with n=1,2,3,5,7,11 and 17, respectively. The modern electrochemical methodology including fast scan cyclic voltammetry and data processing techniques were applied to extract ET rate constants and an impact of the variation of ET distance, viscous additives (or temperature) and high pressure was determined, allowing for a rigorous discrimination of intrinsic ET mechanisms. We have taken the advantage of independent variation of the ET distance (affecting electronic coupling), solution viscosity (externally affecting protein's intrinsic friction, and hydrostatic pressure (directly affecting the RTIL’s bulk or protein's intrinsic friction).

METHODOLOGY Materials and Preparation of SAM-modified Electrodes. Horse heart CytC was purchased from Sigma Chemical Co (96 % with a water content of 4 %) and used either as received, by directly dissolving it in 0.5 M Tris-HCl (Sigma, pH 7.4), or 0.002 M phosphate buffer (Sigma, pH 7.4) containing 0.5 M KCl (Fluka), or after exhaustive ultra-filtration against the same buffer solutions. All other chemicals were of highest available purity and used as received. The working concentration of CytC was 5 -10 mg ml-1, that is (4 - 8) x 10-4 M, throughout all the experiments. ω-hydroxy alkanethiols [HO(CH2)nSH], n = 2,3,4,6,11, were purchased from: 2-mercapto-ethanol, 99 % (Across), 3-mercapto-1-propanol, 95 %, 4mercapto-1-butanol, 95 %, 6-mercapto-1-hexanol, 97 %, 11-mercapto-1-undecanol, 97 % (Aldrich). Other chemicals were: decyl-trimethylammonium bromide (Across), glucose (Aldrich). Millipore MilliQ water was used throughout de-aerated with argon after the solution preparations. Gold disk electrodes of different diameter, 1.6 mm (BAS), 2 mm, and 3 mm (Metrohm) were used as SAM-deposited substrates in bio-electrochemical kinetic experiments. The Au surface was cleaned with successive exposures to 60 oC sulfochromic acid and 5 % HF just

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before the immersion into 30 mM ω-hydroxy alkanethiol solutions as previously described. In the case of 11-mercapto-1-undecanol SAMs, the latter solution contained in addition 50 mM decyl-trimethylammonium bromide. The electrodes were kept in these solutions overnight to allow complete formation of SAM films.

Figure 1. Schematic representation of the arrangement of cytochrome c at electrode surfaces modified by the self-assembled monolayer films with terminal -OH groups.

INSTRUMENTATION, HIGH-PRESSURE UNIT AND ELECTROCHEMICAL MEASUREMENTS Electrochemical measurements (CV and steady-state) were carried out with an Autolab Electrochemical Analyzer PGSTAT30 (Eco-Chemie, The Netherlands) equipped with the General Purpose Electrochemical System (GPES) software for Windows (version 4.9). The pressure vessel and electrochemical cell were similar to those described earlier with the difference that the working electrode was a 1.6 mm Ø gold disc electrode sealed in a Teflon cylinder (BAS). The working electrode, together with the auxiliary electrode (platinum wire) and the reference electrode (Ag/AgCl/4M KCl) were sealed into the cell cap by two O-rings. The working volume of the high pressure electrochemical cell was 5 ml [see Ref. (18) for more details]. The assembled pressure vessel containing the cell was placed in a thermostated water jacket equilibrated at 25.0 ± 0.1 oC. Heterogeneous standard rate constants and diffusion coefficients for electrodes modified by the ω-hydroxy alkanethiols [HO(CH2)nSH], n = 2,3,4,6, were calculated from the peak-topeak separation and peak current values, respectively, according to the method of Nicholson and subsequent procedures [20,21,23-25]. In the case of a non peak-shaped CV (n = 11), the rate constant was calculated using data from the low overpotential region (initial portions of CV curves) where the mass transport effect and other effects resulting in nonlinearity of the dependence of log(khet) on ΔE=E-Eo (Tafel plot) are negligible. All the HP experiments were performed using the Tris-HCl buffer, adjusted at pH 7.4 (pH-meter reading for working solutions containing CytC). Tris buffer is known to withstand pressure induced pH changes. A high ionic strength was necessary to exclude artifacts due to the uncompensated cell resistance. The latter was minimized (contribution to peak-to-peak separation less than 1 %) as could be seen from the fact that the obtained kinetic constants in each case were essentially

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107

independent of the CV scan rate, electrode surface and CytC concentration in the solution. A phosphate buffer of the same pH was also used in some cases for comparison purposes.

Figure 2. Cyclic voltammograms for the CytC electron exchange at Au electrodes modified by OHterminated n-alkanethiol SAMs of variable thickness, (1): n=3, (2): n=6, (3): n=11. Ambient pressure, scan rate: 0.05 V s-1.

THEORETICAL BACKGROUND General. For freely diffusing reactant species such as CytC, the experimentally determined standard heterogeneous rate constant within the framework of the conventional encounter pre-equilibrium model, can be written as [20,21]: o k het = K A k eto

(3)

where KA is a statistically averaged equilibrium constant, and koet is the intrinsic unimolecular ET rate constant representative of either non-adiabatic or adiabatic process. Theoretical work that accounts for both mechanisms and the smooth turnover between them is available. The recently updated expression reads [8-12]:

k eto

=

( H if ) 2 h

⎛ π 3 RT ⎞ ⎜ ⎟ 1 + g ⎜⎝ λ ⎟⎠

ρm

1/ 2

⎛ ΔG a exp ⎜ − ⎜ RT ⎝

⎞ ⎟ ⎟ ⎠

(4)

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where Hif is the electronic coupling matrix element, λ is the reorganization free energy and ρm is the density of electronic states in the metal (electrode). The activation free energy is defined as [15-17]:

ΔG a =

(λ − ΔGo ) 2 − H if 4 λ

(5)

where ΔGo is the free energy gap (throughout the present work ΔGo = 0 by definition, vide infra). The adiabaticity criterion, g, that acts as a control parameter, is given by the following expression [8,18-20]:

g=

π 3 RT (H if

)2 ρ m

hν eff λ

,

(6)

where the effective frequency νeff is related to a single or several relaxation process(es) in the vicinity of the reaction zone, that is (are) intrinsically coupled to electron transfer.

Figure 3. Schematic illustration of role for the key physical parameters that determine the probability and mechanistic shape of thermally activated electron transfer under the condition of zero driving force ( Go = 0). The status of effective frequency of nuclear motion within the parabolic wells, eff, is not indicated here.

When g << 1, one arrives at the following equation (non-adiabatic case):

Charge Transfer Mechanisms at SAM-Modified Electrodes…

k eto ( NA)

⎛ ΔG a = k O ( NA) exp ⎜ − ⎜ RT ⎝

⎞ ⎟ ⎟ ⎠

109

(7)

where kO(NA) = A (Hif)2 (in which A is the constant), and hence

⎡ β ⎤ H if = H ifo exp ⎢− (Re − Ro )⎥ ⎣ 2 ⎦

(8)

leading to Eq. 1. When g >> 1, one arrives at the equation:

k eto ( AD )

⎛ ΔG a = k O ( AD ) exp ⎜ − ⎜ RT ⎝ ⎛ε ⎞

⎞ ⎟ ⎟ ⎠

(9)

RT

in which kO(AD) = B νeff =B ⎜⎜ s ⎟⎟ [18]. Here, B is the constant, η is the solvent ⎝ ε∞ ⎠ 3ηVm viscosity; εs and ε∞ are static and high-frequency dielectric constants, respectively, and Vm is the molar volume. From this formalism Eq. 2 can be deduced with δ =1. Actually, the frictional mechanism is unavoidably manifested through the viscosity control, irrespectively whether one or several relaxations are involved (that is true for non-Debye solvents, mixed solutions, and actual proteins). In addition to conventional approaches, high-pressure (HP) kinetic studies provide unique information about the activation volumes for various processes. Together with the equilibrium (reaction) volumes, they enable the construction of reaction volume profiles, adding a new dimension to the development of fundamental mechanistic understanding. It was suggested in earlier work (18), and will be demonstrated below again that HP kinetic studies of heterogeneous bio-electrochemical processes seem to be advantageous compared to the "homogeneous" ET analogs (with the participation of the same redox protein) with respect to the identification of the intrinsic reaction mechanism. Let us consider pressure effects on the CT rate constant for both the non-adiabatic and adiabatic regimes. A general expression for the activation volume of any kind of microscopic barrier-crossing process, including ET can be defined as [23]:

⎡ ∂ (ln k ) ⎤ ΔVa = − RT ⎢ ⎥ ⎣ ∂P ⎦ T

(10)

After the substitution of Eq. 4, skipping minor terms, and taking into account Eqs. 5 and 8 applicable in the non-adiabatic case, one obtains [9]:

Dimitri E. Khoshtariya, Tina D. Dolidze and Rudi van Eldik

110

1 ⎛ ∂λ ⎞ ⎛ ∂R ⎞ ΔVa ( NA) = βRT ⎜ e ⎟ + ⎜ ⎟ ⎝ ∂P ⎠ T 4 ⎝ ∂P ⎠ T

(11)

Eq. 11 indicates that when the pre-equilibrium constant (Eq. 3) is not affected by pressure (it contains some terms that weakly depend on pressure and which contributions tend to cancel each other) and the nonadiabatic (long-range tunneling) mechanism, Eqs. 1,7,8, is operative, the experimentally measurable volume of activation, Eq. 11, may originate from the effects of pressure on the ET distance due to shrinking of the reactive (here SAM/protein) system and/or change in the medium (SAM/protein/solvent) reorganization energy (Franck-Condon factor). Now if we consider the effect of pressure on the adiabatic (viscosity-dependent) ET process, Eqs. 1 and 9, and skipping again the minor terms, it may be written [9]:

1 ⎛ ∂λ ⎞ ⎛ ∂ ln η ⎞ ΔVa ( AD ) = RT ⎜ ⎟ + ⎜ ⎟ ⎝ ∂P ⎠ T 4 ⎝ ∂P ⎠ T

(12)

from which it follows that in the case of full dynamic (viscosity) control, Eq. 9, viscosity changes due to increasing pressure may result in a large positive contribution, provided that viscosity is affected by pressure. This is the case for all known liquids except water [see (18,54), and references cited therein]. Actually, for most solvents η increases exponentially with pressure, and yields the maximum net viscosity-related contribution as high as +20 cm3 mol-1 [as upper limit, see also Discussion]. In addition, on the grounds of general expression [26], we shall assume that in the case of composite multi-component systems like in the present case, the overall medium reorganization energy can roughly be reproduced by the summation over individual components:

⎛ 1 1 − ⎜ ε o(k ) ε s (k ) ⎝

λ = ∑ Ck ⎜ k

⎞ ⎟ ⎟ ⎠

(13)

where k indicates different contributing space domains (here solvent, protein and SAM interiors) each described by the characteristic geometrical constants, Ck, and bulk dielectric constants, εo(k) and εs(k). Hence, the overall value can be given by λ = λsolv + λprot + λSAM, where the three contributions represent the solvent, protein and SAM interior, respectively.

EXPERIMENTAL RESULTS (CYTC) Dependence of the Rate Constant on the CT Distance for CytC. Changes in the CV response for bio-electrochemical systems composed of Au modified with the ω-hydroxy alkanethiols [HO(CH2)nSH] (n = 2,3,4,6,11) with respect to the SAM thickness, are presented in Figure 3 (for clarity, only CVs for n = 3, 6 and 11 are presented). Peak separation of CVs

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111

increases on variation of the potential scan rate (for n = 3 and 6, respectively), under the highest pressure applied (up to 150 MPa). For SAMs with n = 2, 3, 4 and 6, for which the peaked curves were observed, an increase in the peak-to-peak separation indicated the onset of a kinetic factor, the values of which where used for the accurate determination of heterogeneous standard rate constants, according to the method of Nicholson. The dependence of the logarithm of the experimental standard heterogeneous rate constant, kohet, on the number of SAM methylene units is presented in Figure 4. For clarity, Figure 4 depicts only the data obtained with solutions in Tris-HCl buffer (pH 7.4). The data set obtained with solutions in phospate buffer (plus 0.5 M KCl, pH 7.4), did not differ notably from the former one. The upper curve, clearly distinguishable for SAMs with n = 2, 3 and 4, represents kinetic data obtained in the absence of a viscose additive, and the two other curves that merge with the former one for SAMs with n = 6 and 11, represent kinetic data obtained at a higher solution viscosity, in the presence of 200 and 400 g l-1 glucose, respectively. Figure 3 for larger electrode-reactant separations for all three cases depicted here, at least for SAMs with n = 6 and 11, demonstrates an exponential decay of the rate constant with increasing distance (increase of the methylene unit number), Eq. 1, with a slope of ca. 1 per CH2. This is in excellent agreement with the previous results obtained for various model systems (involving bound or freely diffusing redox species), and for CytC attached to the SAM terminal groups in different manner. Meanwhile, at least SAMs with n = 2 and 3 obviously demonstrate a plateau region. The data for SAM with n = 4, at least in the high viscosity domain, fall in the intermediate region. This becomes clearer when considering the series with the maximum solution viscosity applied Figure 4. Dependence of the Rate Constant on the Solution Viscosity for CytC. Figure 5 represents kinetic data for the logarithm of the standard heterogeneous rate constant, kohet, vs. the logarithm of the relative solution viscosity for the case of SAMs with n = 2, 3, 4 and 6. Phenomenologically these dependencies can be approximated by eq. 2 with power indexes (actual slopes in Figure 5) of δ = 1 (n = 2, 3), 0.7 (n = 4), and 0 (n = 6, 11) (±0.05 in each case; the latter case is not shown in Figure 5). The observation of a slope of unity, formally corresponding to "full" dynamic control, was to some extends a surprise. Normally, biochemical processes display less sensitivity to the external viscosity, which results in slopes less than unity. On considering CytC in different environments, the "plateau" value of ca. 0.6 was detected in the case of specific adsorption on the pyridinal-terminated thin SAMs (plateau region). Some greater slopes were found in the case of an ET within the "homogeneous" system involving zinc-substituted CytC, and wild-type and mutant cupriplastocyanin, ranging from 0.7 to 0.9 (±0.1 for each case). Somewhat unexpectedly high values of δ for the present case will be discussed below (see Discussion). In conclusion, we note that both the above-considered dependencies of Ln (kohet) on n and Lnη, along with the HP kinetic results, point to the existence of two different mechanisms for electrode CT and a rather smooth transition between them (vide infra). Dependence of the Rate Constant on the Hydrostatic Pressure for CytC. The dependence of Ln(kohet) on the hydrostatic pressure up to 150 MPa for SAMs with n = 3, 4 and 6, are depicted in Figure 6, respectively. It can be seen that for the case of SAM with n = 3, the value of Ln(kohet) decreases linearly with pressure, yielding a positive volume of activation of +6.7 ± 0.5 cm3 mol-1, Figure 6. This value is very close to +6.1 ± 0.5 cm3 mol-1 found in the case of 4,4'-bipyridyl- and 4,4'-bipirydyl-disulfide modified Au electrodes (also to be

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considered as thin SAMs). On the contrary, for a much thicker SAM with n = 6, we found for the first time that the value of Ln(kohet) increases linearly with increasing pressure to yield a negative volume of activation of −5.5 ± 0.5 cm3 mol-1, Figure 5 In the case of a SAM with n = 4, an intermediate behavior is displayed with no effect of pressure on kohet, yielding ΔVa ≈ 0, Figure 6.

Figure 4. Logarithm of the heterogeneous standard rate constant for the CytC electron exchange at Au electrodes modified by hydroxy-terminated n-alkanethiol SAMs (n = 2,3,4,6,11) vs. methylene unit number under variable solution viscosity. Upper curve (filled circles): no viscous additive, η =1; middle curve (open circles), η =1.80; lower curve (asterisks) η =3.96 (note all three curves merge at n = 6 and 11, and form a single sloped line).

MECHANISTIC PATTERNS FOR CYTC Frictional Control vs. Conformational Gating for CytC. The distance dependence of the kinetic data for ET in Figure 4 a clearly indicates the mechanism turnover, but there is definitely no replacement of the rate-determining CT step by any other kinetic event that may precede or succeed CT. This is also obvious solely from the nature of the voltammetric response, viz. the change in the classical shape of the CVs recorded for both thinner and thicker SAMs, is caused by the increase/decrease of the peak-to-peak separation due to the variation of the CV scan rate (for

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a given n and kohet), or n and kohet (for a given scan rate). In addition, with this and other arguments discussed elsewhere, it should be noted that the hypothetical "conformational gating" mechanism (that can be opposed to a viscosity-sensitive adiabatic ET mechanism advocated in this work), as suggested for electrostatically bound (SAM-deposited) CytC, implies rate-determining large-scale (rotation, sliding, etc.) SAM/protein rearrangements. This kind of rearrangements, most probably, can be excluded for the case of freely diffusing CytC. All in all, the general similarity of kinetic patterns regarding the SAM thickness (exhibition of plateau and sloped segments and gradual turnover between them) for the bound and unbound (Figure 5) cases, strongly suggests that the conformational dynamics behind the friction control, although is dependent on the external viscosity ("slaved") [27-30], however can be viewed as "intrinsic" (effective) dielectric relaxation operating through Eq. 2 rather than large-scale rearrangements like docking, surface diffusion, or similar.

Figure 5. Logarithm of the heterogeneous standard rate constant for the CytC electron exchange at Au electrodes modified by hydroxy-terminated n-alkanethiol SAMs vs. logarithm of solution viscosity, (1): n = 2 (crosses) and n = 3 (open circles; (2): n = 4 (closed circles); (3): n = 6 (crosses).

Pressure Affects Internal Friction of CytC. It has been found before that due to the unique property of water not to change its viscosity with pressure, HP kinetic studies of biochemical processes at room temperature provide a unique possibility to vary the proteins' intrinsic viscosity without altering the viscosity of external non-solvating (bulk) water (although the solvating or "bound" water should be considered as part of the protein molecule, vide infra) [31].

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Figure 6. Logarithm of the heterogeneous standard rate constant for the CytC electron exchange at Au electrodes modified by hydroxy-terminated n-alkanethiol SAMs (n = 3, 4, 6)) vs. hydrostatic pressure, (1) n = 3; (2) n = 4; (3) n = 6.

Indeed, according to numerous experimental and theoretical studies, the proteins' interior, especially peripheral regions forming the unified fluctuating dynamic system together with, and slaved by (vide infra) the bound water, can be viewed as a highly heterogeneous liquid rather than a dissolved, solid-like macromolecular substance [27-35]. The effective viscosity of such a complex "liquid", like any other liquid mixtures, should increase with pressure due to a universal mechanism of increasing intrinsic friction. Bulk water around room temperature has a unique mechanism to avoid an increase in viscosity. This is probably due to the unique multi-component nature of neat water. Moderately high pressure affects those structural components that are not responsible for transport properties including fluidity and diffusion [36,37]. On the basis of the arguments presented above, we are confident that our HP kinetic experimental data nicely agree with the theoretical predictions for adiabatic and non-adiabatic bio-electrochemical CT made above. Indeed, Eq. 12 predicts a substantially positive activation volume due to the increase in viscosity (friction) in the vicinity of the redox center. In the case of a protein globule as reaction medium, that mainly has properties of a viscous liquid or a mixture of such liquids [31, 34], a large positive contribution due to the first term in Eq. 12 can be expected. Pressure Effect on the Franck-Condon Factor for CytC. The second term due to variation of the outer-sphere reorganization energy (Franck-Condon) factor, when considered as originating from the effect of pressure on the bulk dielectric properties of external water, can be predicted to amount to ca. −(4 to 6) cm3 mol-1, assuming that λ amounts to ca. 1 eV (∼20 to 25 kcal mol-1), and totally originates from the bulk properties of water (68). In the case of

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CytC reactions, the overall value of λ = 0.6 to 0.8 eV is roughly composed of ca. 50 % "outersphere" (solution) component that can contribute about half of the activation volume, and another ca. 50 % of the "inner-spere" protein reorganization component (Franck-Condon term) found to be negligibly altered by pressure, and therefore does not contribute to the activation volume (69). Note that the same can be concluded for the contribution from the SAM interior (λSAM ∼ 0), since it can be assumed that εs = ε∞ [26]. Thus, the most probable overall contribution of the second term of Eq. 12 would be −(2 to 3) cm3 mol-1. This value, together with the overall experimental value of +6.7 cm3 mol-1 (Table 2) yields the value of the frictional term, viz. +(8 to 10) cm3 mol-1. We stress again that this contribution originates from the direct influence of hydrostatic pressure on the intrinsic protein friction (viscosity). On considering the non-adiabatic mechanism, Eq. 11, it is seen that the second term due to the Franck-Condon factor may again contribute about −(2 to 3) cm3 mol-1. This value together with the overall experimental value of −5.5 cm3 mol-1, yields for the first term (related to the effect of squeezing the system) the value of ca. −(2 to 3) cm3 mol-1. This kind of contribution to the overall volume of activation has actually been predicted for protein systems (70). On going to the pressure effect for a system with n = 4, tentatively denied to fall into the intermediate regime, it is found that the experimental activation volume is virtually zero (ΔVa ∼ 0). This value is almost halfway between the values found for n = 3 and n = 6, viz. 0.6 ± 1 cm3 mol-1. Table 1 summarizes the ranges of estimated values for the individual contributing factors to experimental ΔVa, for the cases of both observed mechanisms and in the intermediate regime, which are in a good mutual agreement. Consequently, we conclude that our activation volumes obtained from HP kinetic experiments are in good agreement with the results obtained through other approaches, and therefore have a predictive power of high confidence. Table 1. Experimental volumes of activation (± 0.5, in cm3 mol-1) for the CytC electron exchange at Au electrodes modified by hydroxy-terminated n-alkanethiol SAMs (n = 3,4,6) under variable hydrostatic pressure, and estimated values of different contributing terms (the same units, see text for details) SAM n=3 n=6 n=4

ΔVa(exp) + 6.7 − 5.5 ∼0

1/4 (∂λ/∂P)T − (2 to 3) − (2 to 3) − (2 to 3)

RT (∂lnη/∂P)T + (8 to 10) (0) + (4 to 5)

β RT (∂Re/∂P)T (0) − (2 to 3) − (1 to 2)

External Friction and Dynamic Slaving for CytC. Turning now to the effect of external viscosity (Figure 5), one can mention that for SAMs with n = 2 and 3, formally "full" frictional control is realized (δ = 1, Eqs. 2,9), normally characteristic for redox species in which the redox-active (typically metal) center is directly solvated by the aqueous medium. In the case of biochemical processes occurring inside the protein environment, the viscosity control takes place through Eq. 2, in which normally 0 < δ < 1. As mentioned above, in the present case there is no doubt about the uncomplicated frictional origin of the observed dependence, Figure 4. To explain the observed deviation of δ from unity (provided the intermediate regime is excluded), earlier workers suggested that the overall "effective"

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friction that controls the process, additively originates from both solvent and protein components, and operates through the equation [35]:

k AD =

C ⎛ ΔGa ⎞ exp⎜ − ⎟, σ +η ⎝ RT ⎠

(14)

in which C is constant, and σ represents the proteins' intrinsic viscosity. However, from this approach it follows that for cases where δ ≈ 0.5 - 0.8, e.g., the external (solution) and protein interior contribute comparably (the ratio σ /η amounts only to ca. 2 to 4), which is highly questionable because the protein interior is known to be very viscous compared to solvating water, or in most cases, even compared to aqueous solutions containing conventional viscous additives. At the same time, for the cases where δ ≈ 1 (as in the present case), this model suggests that σ ≈ 0, which implies that there is no frictional effect from the protein interior. Obviously, at least in this particular case, such a conclusion does not agree with our HP kinetic results that convincingly revealed an essential role of the protein's intrinsic. Also, earlier work allowed the reliable estimation of an intrinsic effective relaxation time of CytC that controls adiabatic ET, viz. ca. 200 ps. This rather slow relaxation time roughly corresponds to a ca. 1000-fold enhanced intrinsic viscosity compared to the bulk aqueous environment. A question emerges on how the moderate variation of external viscosity can alter the highly exceeding internal viscosity so effectively? The answer can be found in a recently introduced concept of dynamic "slaving" that implies that "solvent fluctuations dominate protein dynamics and function". Indeed, for the most well-studied "model" protein, myoglobin, it has been established that several of its dynamic features (including the rate constants of functional elementary processes, kF (T)) change in parallel with the rate coefficient of the solution's dielectric fluctuations, kD (T), such that [29-30]:

k D (T ) k F (T ) ≈ m = Const

(15)

where, for different events, m varies within the range of 3 × 102 to 3 × 104 (in average ca. 103). The origin of such a slaving of intrinsic conformational dynamics to external viscosity, should lie in a cooperative effect of simultaneous multi-site interaction of the solvent components (water, sugar, glycerol, etc.) with charged and polar groups on the protein surface. Indeed, numerous studies indicated the essential role of protein solvation in triggering and further tuning of their dynamic characteristics and proper functioning. Our finding that CytC under the present conditions (weak interaction with SAM terminal groups) exhibits the totally slaved behavior (δ = 1), compared to the case where it is (specifically) tightly adsorbed at the surface (δ ≈ 0.6), confirms such a conclusion. Furthermore, it suggests that when the external solution is screened due to the tight protein/SAM contact (that mimics the protein-membrane or protein-protein multi-site contact), the slaving effect is not complete due to the lack of the full protein-solution interaction.

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BROKEN ERGODICITY FOR ET General Assay. Recent advances in the perception of liquid-phase charge-transfer processes focused on the phenomenon of broken ergodicity that leads to the kinetic regime of dynamically arrested electron transfer (ET) [38-41]. Such an odd pattern seems to be typical for cases of slowly relaxing media that are of current bio-medical and/or nano-technological interest (biomolecules and their arrays, liquid crystals, ionic liquids etc.), and can be disclosed when the variation of the ET rate within the series of reactions of a given category is possible, such that the ET characteristic time (reciprocal of intrinsic rate constant), upon decreasing, approaches and eventually traverses the characteristic time scale(s) of the relaxation of the medium. Within the conventional charge transfer theory, dynamic solvation is characterized by the key parameter defined as “outer-sphere reorganization energy” (vide infra). In the situation mentioned above, the reactive system does not completely explore its phase space, and “equilibrated” dynamic solvation (full reorganization) along some degrees of freedom is not established. However, rather scarce experimental data is available that demonstrates this pattern [42-45]. In the ergodically “normal” ET regime, typical for sufficiently fluid liquid-phase media, for particular heterogeneous (electrode-implicated) processes proper unimolecular rate constants can be presented by the unified expression in Eq. 4. When the ergodicity is broken, the statistically equilibrated reorganization along some slow degrees of freedom of the solvent can not be achieved and the corresponding part of the reorganization free energy is turned off. The effective (“reduced” with respect its “full” value) reorganization free energy in the simplest case can be expressed by Eq. 16:

λ(effI ) (ΔGo ) = f ( I ) λo

(16)

where, in general, the value of f (I) may vary between 0 and 1. Eq. 16 and similar expressions predict anomalous kinetic manifestations, viz., significant deviation from the normal pattern; however, Eqs. 4-9, and similar should preserve their physical meaning under “abnormally” variable λ, vide infra. At the same time, one may note that several mechanistic aspects of even “traditional” ergodic patterns, especially connected with the solvent control (frictional) limit, vide infra, are still under thorough corroboration. Specifically, the theoretical model of Zusman predicts freezing out of a part of the full reorganization energy due to the solvent controlled (yet ergodic) mechanism, Eq. 17:

λ(effII ) (ΔGo ) = f ( II ) λo

(17)

However, in contrast to the non-ergodic regime, in this particular case, lowering of λο should be expected at the cost of freezing out of the medium’s fastest degrees of freedom (vide infra). Again, only a few experimental reports are available that confirms this prediction [7,12,18]. RTIL as the ET Environment. Since ET events are at the core of many biological and nano-technological processes, exhaustive experimental verification of existing theoretical

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models, along with a search for novel mechanistic properties beyond the conventional models, are of exceptional importance for further developments within these fields. Accordingly, the design and testing of a series of congruent systems, allowing the coverage of broad ranges of ET rate constants and traverse all the known mechanistic regimes, seemed to be an especially challenging experimental task. In order to reach this goal, we selected among other variants electrochemical devices that consist of gold electrodes coated by selfassembled alkanethiol SAMs of variable thickness, a ferrocene/ferrocenium redox probe, and a typical room-temperature ionic liquid (RTIL), [bmim][NTf2] as a unique reaction medium. These components were favored because of the following advantageous prerequisites: (a) Alkanethiol self-assemblies have been established as electronically well-behaved, almost defect-free insulating films allowing for a gradual and controllable variation of the electrode-to-redox probe (i.e., charge-transfer) distance [3-12]. In this work, alkanethiol SAMs of different chain length, HS−(CH2)n−CH3 with n = 1, 2, 3, 5, 7 and 11, i.e., with a total number of carbon atoms (hereafter defined as: c(n+1)) of 2, 3, 4, 6, 8 and 12, were applied that allow inputs for the ET distance from 4.14 to 22.06 Å, respectively (Table 2). (b) The ferrocene/ferrocenium couple, [Fe(Cp)2]0/+ (Cp = cyclopentadienyl) is widely used as redox probe, especially sufficient for outer-sphere (electrode) ET studies due to its potentially low impact on interfacial motifs (because of the low charge type) and negligible inner-sphere reorganization (validating the predominant role of solvent reorganization) within the redox process [3,6,20,21]. (c) RTILs, [bmim][NTf2] in particular, are known as liquids of exceptional physical properties [46-56]. First of all, a broad distribution of relaxational characteristics, involving both the solvent’s fast and slow degrees of freedom, should be mentioned. The results of recent experimental (dielectric and solvation spectroscopy, NMR, Kerr relaxations, etc.) and MD simulations indicated essentially heterogeneous nanostructural organization of RTILs in which relaxation dynamics occur over a broad time range of 100 fs to 10 ns, and slower. The response functions were found to be biphasic, consisting of a sub-picoseconds component (100 fs to 200 ps) of modest amplitude (10 to 20 %), and a dominant slower component stretched down to tens and, presumably, hundreds of nanoseconds. In addition, the broadband dielectric spectroscopic experiments, probed ultraslow, extremely large-scale (highly cooperative) structural fluctuations in RTILs that presumably occur in the electrode-adjusting interfacial layer (diffusive Stern zone) [53,54]. Herein, we review ET studies with the implication of interfacial Au/SAM/RTIL junctions (Figure 7) that allowed a variation of the intrinsic unimolecular rate constant within the range of 0.1 to 2 × 107 s-1, and disclosure of multiple patterns and mechanisms of ET. Among a series of unique details (vide infra), we have disclosed an amazing tenfold drop in the full (“equilibrated”) value of the medium reorganization free energy of ca. 1 eV to the actual (effective) value of 0.1 eV upon traversing the ET regimes. Variation of SAM thickness resulted in a spectacular variation of the parameter Hif within the range of 10-3 to 10-10 eV (0.1 to 10-8 kJ mol-1). At the same time, the value of ΔGo varied from 0 down to −0.6 eV, and the relaxational properties of the reaction medium, [bmim][NTf2], reflected by the matching macroscopic parameter viscosity, were varied over a 20-fold range through the variation of temperature (278 – 308 K) and pressure (0.1 – 150 MPa). Some earlier work reported on ET kinetics at bare Au electrodes in RTILs and a brief preliminary report on the extension of that work at SAM modified electrodes, are available. Our previous analysis based on kinetic data

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involving bare Au and glassy carbon electrodes indicated an adiabatic (solvent friction) mechanism controlled by the most probable value of νeff for [bmim][NTf2] as solvent, as low as ca. 100 ns with the reactive site near the Au electrode, ascribed mostly to the slow diffusive motion of the RTIL constituting ions (vide infra).

Figure 7. A sketch for the arrangement of the Au/SAM/RTIL junction (interfacial region) for electrochemical systems under present study (see text for the details).

RESULTS AND DISCUSSION FOR ET IN RTIL Methodological Aspects. The advantage of modern, well-established electrochemical methodology (two complementary approaches, vide infra) and related analysis was taken to extract intrinsic ET rate constants under standard conditions (i.e., at zero overpotential, ΔGo = 0), and at electrode potentials different from the standard value of our redox probe, [Fe(Cp)2]0/+, at ΔGo ≤ 0. Fast-scan cyclic and steady-state voltammetry, global computeraided and Nicholson’s basic two-point fitting, Savean’s deconvolution procedure and Gaussian curve analysis, were applied to determine heterogeneous rate constants and/or reorganization energies, respectively. Unimolecular ET rate constants were calculated by scaling with the encounter (heterogeneity) factor of 10−9 cm. Voltammetric response for Au/SAM/RTIL/Fc devices comprising alkanethiol SAMs with c(n+1) = 3, 8 and 18 as representative systems, under standard experimental conditions (v = 0.1 V s-1, T = 298 K, P = 0.1 MPa), is depicted in Figure 8. Figure 9 presents the dependence of the pre-equilibrium-corrected ET rate constant on the carbon number of the SAM constituent alkanethiol chains (c(n+1) = 2, 3, 4, 6, 8, 12 and 18). The cyclic voltammograms including their fast scan extension (FSCVs) for c(n+1) = 2, 3 and 4 were essentially identical at all scan rates yielding virtually the same standard rate constants within the experimental error limits (Table 2). Some minor divergence was detected for the corresponding activation parameters under variable temperature conditions (vide infra). With a further increase in carbon number, the

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Faraday current and corresponding ET rate constants started to decrease gradually, indicating the onset of the non-adiabatic regime, Eqs. 1.7 (that is the outcome of Eq. 4 under the condition of g << 1).

Figure 8. Cyclic voltammetric response of the [Fe(Cp)2]+/0 couple dissolved in [bmim][NTf2] at Au electrodes coated with alkanethiol [CH3–(CH2)n–SH] films. Selected cases with n = 2 (1), 7 (2) and 17 (3), matching total carbon number of 3, 8 and 18, respectively (of a total of 8 systems studied).

Disclosure of Dynamically Arrested ET: RTILs are almost unexplored substances as ET environments, in terms of the validity of traditional charge-transfer models. Especially, the issue how respective fast and slow modes contribute to the parameter λo, seems most challenging. In order to gain more decisive information about this issue in the context of the ET mechanisms, we applied two complementary approaches, both allowing for the deduction of values for λo or λeff at different c(n+1) (that is under variable ET donor/acceptor separation). These included: (a) the Savéant analysis implying elimination of the mass transport contribution from the experimental voltammograms and subsequent Gaussian fitting and (b) a simple determination of standard rate constant (extracted through the Nicholson or similar analysis) under variable temperature conditions. Figure 10 presents the dependencies of pre-equilibrium- and mass-tranport-corrected rate constants on the overvoltage (ΔGo) and their Gausian fits for two representative systems containing SAMs with c(n+1) = 12 and 18. Despite of the limited span of experimental points (in the case of sluggish liquids, i.e. low diffusion coefficients for the redox probe, the method is restricted to maximal overvoltages less than ca. 50−60 % of λο), the Gaussian fits yielded decisive results regarding both the band maximum and bandwidth samplings, allowing for the reliable extraction of λο (or λeff). By plotting the data on semi-logarithmic coordinates, Figure 10, indicates that the experimental kinetic data within the whole range of methodologically available overpotentials are very well represented by Gaussian curves.

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Figure 9. Unimolecular rate constants determined from the cyclic and steady-state voltammetric data (see text, closed blue circles) vs. the SAM carbon number. Open dark red circles represent values that are independent of the ergodicity regime (kinetic frequency factors corrected for the corresponding experimental reorganization energies deduced from the Savéant analysis). The broken line represents extrapolation of the non-adiabatic frequency factor to the value of the “maximal approach” (see text for further details).

Figure 10. Representative semi-logarithmic curves for uni-molecular ET rate constants (symbols) determined through the Savéant analysis and their Gaussian fits (broken lines) vs. the electrode overvoltage (reaction driving force).

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Figure 11. Experimental reorganization free energies (open red squares) determined through the Savéant analysis vs. the SAM carbon number. Broken green line – prediction of the Marcus and related theoretical “continuum” models. The brown and dark blue arrows indicate the onset of dynamically arrested and friction control regimes, respectively (see text for details).

In the case of c(n+1) = 18, the fitting procedure yielded λο = 1.0 ± 0.1 eV, a value that is close to that deduced theoretically and found experimentally for the ferrocene/ferrocenium couple in aqueous and other molecular (polar) solvents. We note that neither the classical Marcus equation (see Supporting Information),[18,20] nor its extensions to the three-phase (metal/SAM/solution) configuration, can be a priori applied in the case of RTILs as reaction medium. At least one reason is the important contribution of slow translational degrees of freedom in actual dielectric polarization properties. As a result, “classical” models underestimate the values of full (“equilibrated”) λο in RTILs. On the other hand, the Marcus equation and related expressions always predict a moderate and very smooth decrease of λο with decreasing ET distance (Supporting Information) that is solely due to geometric properties of the model rather than intrinsic dynamic characteristics of the solvent (see bold dashed curve in the top of Figure 3). In contrast to this prediction for the case of c(n+1) = 12, Gaussian analysis yielded the value λ = 0.50 ± 0.05 eV (Figures 10, 11). This value is about 50 % that could be expected from the “classical” Marcus behaviour shown in Figure 5. Furthermore, the effective value of λ drops successively with decreasing ET distance (Table 2) and, finally, reaches the value of 0.10 ± 0.02 eV for c(n+1) ≤ 4 (including the case of a bare Au electrode)! Figure 6 presents the values of λ/4 (in kJ mol-1; λ determined from the Savéant analysis, Figures 10, 11) to match the values of ΔGa, implying that the intrinsic activation entropy (ΔSa) of ET is negligible, and the values of Hif at sufficiently short ET distances are still rather small (Table 1), therefore, ΔGa = ΔHa = λ/4 for c(n+1) ≥ 4.

Table 2. Experimental and calculated kinetic parameters for ET within the Au/SAM/RTIL/[Fe(Cp)2]0/+ systems (see text for details). SAM Thickness (Input to the ET Distance), Å 22.06

k0ET, s-1 (at ΔGo = 0)

ΔHa(COR), kJ mol-1

ΔVa(EXP), cm3 mol-1

Hif, eV (kJ mol-1)

eV

ΔHa(EXP), kJ mol-1

0.1

1.0

-

24.0

-

5 × 10−6 (0.0005)

c = 12 Au-S(CH2)11CH3

15.34

1.10 × 104

0.50

-

11.8

−21.7

1 × 10−4 (0.01)

c=8 Au-S(CH2)7CH3

10.86

6.67 × 105

0.40

11.9

10.0

−4.2

5 × 10−4 (0.1)

c=6 Au-S(CH2)5CH3

8.62

6.70 × 106

0.25

16.3

6.0

+2.4

1 × 10−3 (0.3)

c=4 Au-S(CH2)3CH3

6.38

2.46 × 107

0.12

31.4

2.9

+22.6

1 × 10−2 (1.0)

c=3 Au-S(CH2)2CH3

5.26

2.45 × 107

0.12

30

-

+25.3

3 × 10−2 (2.9)

c=2 Au-S(CH2)CH3 c=0 Bare Au

4.14

2.58 × 107

0.12

29.6

-

-

5 × 10−2 (5.2)

∼3

5.77 × 106

0.10

22.5

2.4

-

9 × 10−2 (8.7)

SAM Assembly

c = 18 Au-S(CH2)17CH3

λo or λeff,

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The values of ΔHa(EXP) which, seemingly, in the case of c(n+1) = 8 and 12 almost coincide with those of λ/4, are also presented. In contrast, for cases of c(n+1) ≤ 4, the values of ΔHa(EXP) are very close to the corresponding values for the viscosity of the RTIL, ΔHa(η), vide infra, whereas for c(n+1) = 6, the intermediate pattern is noticeable (see Table 2 and Figures 10, 12).

Figure 12. Arrhenius-like plots for the reduced ET standard rate constants for cases of Au/SAM/[bmim][NTf2]/Fe(Cp)2]0/+ composite systems involving alkanethiol SAMs with carbon number of c = 2, 4, 6 and 8. “Reducing” within this series implies dividing by the standard rate constant determined at 25 oC; “standard” implies condition of ΔGo = 0.

Taking into account the broad distribution of slow diffusion/conduction-related relaxational degrees of freedom in [bmim][NTf2], spanning the range of 1 to tens of ns or slower, and the recent disclosure of ultra-slow collective relaxational motion in electrode adjusting layers occurring within the time domain from 0.1 ms to a few hundreds of μs (even slower motion has been detected very recently by the method of dynamic light scattering) we conclude that the observed effect of at least the first drop in λο is due to the onset of an ergodically broken regime. In the meantime, the value of λ = 0.45 ± 0.10 eV holds for the cases of c(n+1) = 8 and 12, for which the dependence of koET on c(n+1) seems to obey Eq. 1, with the decay parameter (β) of ca. 1 per methylene unit. This is indicative of a non-adiabatic ET mechanism. Importantly, for systems with c(n+1) = 8 and 12, the complementary studies through the Savéant analysis and the Arrhenius treatment, Figure 12 (latter yielding values of ΔHa(EXP)) provided completely matching results (Table 2), in essential agreement with Eq. 5. Hence, we conclude that at least within the range of a SAM thickness of 10 to 20 Å (Table 2), the non-adiabatic ET mechanism (Eq. 7) operates, however, under the dynamically arrested condition, Eq. 16. As can be deduced from the data analysis shown in Figure 9, the characteristic time of ultraslow “locking” (large-scale) relaxation should be of the order of ca.

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50 μs. This is what can be expected for the moderately viscous RTIL, [bmim][NTf2]. Indeed, for the RTILs [bmim][BF4] and [mmim][Me2PO4], with viscosities of 104.2 and 363 cP at 300 K, respectively (vs. 44 cP for [bmim][NTf2]), the ultraslow relaxation times of ca. 100 μs (280 K) and 0.3 to 1 ms (270 K; the band maximum position depends somewhat on the electrode material), respectively, can be deduced [53,54]. The estimated value of 50 μs (300 K) [12] nicely fits this sequence. These relaxation times can be ascribed to the large-scale density fluctuations of RTILs occurring in the electrode-adjusting interfacial layer (diffusive Stern zone) in broadband dielectric relaxation experiments, or in the analogous zone of the Au/RTIL or Au/SAM/RTIL junctions, in our current experiments. To our knowledge, this is the first documentation of dynamical arrest in the case of interfacial ET.

Figure 13. Pressure dependencies for the reduced ET standard rate constants for cases of Au/SAM/[bmim][NTf2]/Fe(Cp)2]0/+ composite systems involving alkanethiol SAMs with carbon number) of c = 3, 4, 6, 8 and 12. “Reducing” within this series implies dividing by the standard rate constant determined at 5 MPa; “standard” implies condition of Go = 0.

Further disclosure of the solvent control pattern for ET within the regime of broken ergodicity: As mentioned above, for c(n+1) ≤ 4, the values of ΔHa(EXP) are very close, with minor variation, to the corresponding values for the viscosity of the RTIL, ΔHa(η). A similar result was found in earlier work for the case of a bare Au electrode. Furthermore, for c(n+1) = 6 (and to some extent for c(n+1) = 8), the intermediate pattern is noticeable (Table 2 and Figure 11). Figure 6 also shows the values of contributions from the hypothetical enthalpy term of the type δΔHa(η) (depicted in green), implying that the adiabatic mechanism, Eqs. 2 ,9, or the intermediate (with respect of a non-adiabatic one) may operate at shorter distances. (Note, from Eq. 2 it follows that, in general, koET ∝ η−δ, where 0 ≤ δ ≤ 1). The values of such an enthalpy term, along with the actual values of Hif were estimated through Eq. 5 and by

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resembling the experimental values of λ and ΔHa(EXP) (Table 2). We note again, in a fully adiabatic regime, where g >> 1, Eqs. 4, 9 is operative whereas in the non-adiabatic regime (g << 1), formally, koET(NA) ∝ η−0, i.e., there is no contribution due to direct solvent (viscosity) control. The secondary drop of the effective value of λ from the value of 0.50 ± 0.05 eV to 0.10 ± 0.02 eV for c(n+1) ≤ 4 (including the case of a bare Au electrode) obviously can be ascribed to the elimination of another part of the full reorganization energy due to the onset of the adiabatic mechanism, however, at the cost of freezing out of the medium’s fastest degrees of freedom, Eq. 17. It follows that fast relaxational degrees contribute about 40 % to the total reorganization energy of 1.0 ± 0.1 eV detected for the present system (vide supra). Seemingly, the latter finding is the most convincing demonstration of a very specific and less recognized signature of the adiabatic mechanism predicted by Zusman [18].

CONCLUSIONS In summary, we reviewed the published results for two types of nanoscopic multicomponent electrochemical systems with a general shape: Au/SAM/RP, with essentially different redox probes and their immediate environments involved. In one case the redoxactive Fe-heme containing protein, CytC, was dissolved in aqueous electrolyte solution and freely diffusing to/from the SAM interface comprising terminal –OH groups. In another case the redox-active metallocomplex, Fe(Cp)2]0/+, was dissolved in a room temperature ionic liquid, [bmim][NTf2], and freely diffusing to/from the SAM interface comprising terminal – CH3 groups. By further tuning the overvoltage, the intrinsic ET rate constant can be varied over eight orders of magnitude and more! These kinds of systems allow for a very accurate gradual variation of the metal-reactant ET distance, hence the strenghth of the electronic coupling, as well as the relaxational properties of the reactants’ immediate environment (that might be itself rather complex) through the application of variable temperature, pressure, or viscous additives. The modern electrochemical methodology including fast scan cyclic voltammetry and data processing techniques allows for a very accurate determination of intrinsic ET rate constants for the electron exchange that occurs between the electrode and redox probe with the latter situated in diffusive Stern zone, near the SAM “surface”. As a result, highly remarkable multiple interplay of inherent ET mechanisms can be observed and analyzed on the grounds of contemporary charge-transfer theory and its recent modifications. In-depth comprehension of a variety of microscopic ET mechanisms and conditions for their interplay may play decisive role in the further progress towards the nanotechnological applications of composite nanoscopic electrochemical assemblies, encompassing molecular electronics, sensor/biomedical array design, solar energy conversion, etc.

ACKNOWLEDGMENTS Financial support from the Alexander von Humboldt Foundation (DEK), DAAD (TDD), Volkswagen Foundation (RvE and DEK), and the Deutsche Forschungsgemeinschaft as part of SFB 583 on “Redox-active Metal Complexes” is kindly acknowledged.

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In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 10

STABILITY OF DRUG DELIVERY PLGA NANOPARTICLES: CALORIMERTRIC APPROACH Tamaz Mdzinarashvili1a, Mariam Khvedelidzea, Tamar Partskhaladzea, Mark Schneider2 b, Ulrich F. Schaefer3 c, Noha Nafeec, and Claus-Michael Lehrc a

Ivane Javakhishvili Tbilisi State University, Faculty of Exact and Natural Sciences, Department of Physics, Tbilisi, Georgia b Saarland University, Pharmaceutical Nanotechnology, Germany c Saarland University, Biopharmaceutics and Pharmaceutics Technology, Saarbrücken, Germany

ABSTRACT The spherical PLGA nanoparticles (NP) calorimetric investigation is presented in this paper. Such nanoparticles is used for biological active substances (drugs) encapsulating inside of them with the purpose of medicine transferring into the cell. It is clear that without determination of particle stability it is impossible their practical usage. From calorimetric study of PLGA nanoparticles with PLA/PGA ratio 70:30 it was determined the entirety conditions of such particles and the temperature interval, where the particle destructions take place. It was unambiguously shown that for noncoated PLGA NP and for chitosancoated PLGA NP the stability temperature are equal to 370C and less than physiological temperature, which exclude their practical application. Also it was determined that hermiticity destroy temperature depends on heating rate. At the same time it was established that strongly alkaline and acid area (pH2 – pH9) do not destroy noncoated PLGA NP and chitosancoated PLGA NP what gives possibility for their using orally.

1 [email protected]. 2 [email protected]. 3 [email protected].

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INTRODUCTION Modern pharmaceutical approaches have allowed identifying many potent drugs. Unfortunately, many of those show a small bioavailability because of their limited aqueous solubility. Hence, nanoparticles (NP) offer the potential to overcome not only this obstacle but also promise smaller amounts of drugs delivered to specific cells and tissues [1]. Furthermore, the perspective of using nanoparticles is very interesting for delivering the therapeutic agents unaltered to damaged organs or tissues,. Hereby, crossing natural barriers is the first obstacle for drug delivery. For a broad applicability the consideration of diverse environmental conditions such as high acidic pH, unfavorable metal ions, active enzymes etc. are essential. In general, nanotechnology is considered to have a huge potential for drug delivery, since it offers a suitable transportation of anticancer agents, antihypertensive agents, immunomodulators, hormones, and macromolecules such as proteins, peptides, antibodies or genes [1-9]. Recently, it was shown that nanoparticles with a therapeutic agent of interest encapsulated in their polymeric matrix or adsorbed or conjugated onto the surface can be administered orally [1, 10-23] or injected locally [18, 20, 24]. Among the most common nanoparticles are those made of poly (lactic acid) (PLA), poly (glycolic acid) (PGA), and their copolymers poly (lactic-co-glycolic acid) (PLGA) [20, 2531]. Changing PLGA nanoparticles PLA/PGA ratio (Figure1) gives possibility to obtain the nanoparticle with different physico-chemical properties.

Figure 1.

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Such polymers have controllable biodegradability, excellent biocompatibility, high safety, they are non-toxic, and they are resorbable through natural pathways [27, 32-37]. There are literary data indicating that the drug entrapped in PLGA matrix is released at a sustained rate through diffusion of the drug in the polymer matrix by swelling and degradation of the polymer matrix [25, 38, 39], though there has not been studied completely the mechanism by which the drug entrapped in such nanoparticles releases in time. Hedley et al. have demonstrated protection of DNA from nucleases when encapsulated into PLGA microspheres [40]. By changing the copolymer composition and molecular weight it is possible to vary the release of encapsulated drugs from PLGA nanoparticles from days to months [1, 26, 28, 41-44]. These properties are the reason for PLGA being approved by the FDA [26]. In general, intriguing candidates to be delivered by these nanoparticulate matrials are genetically active residues. These materials are degraded very rapidly and can not penetrate cells very well. The complexation of DNA, oligonucleotides and siRNA onto the surface of NP can protect them from degradation and improve transfection [45,46]. There are literary data confirming that DNA can be condensed on dendrimers [47,48], cationic peptides [49,50], cationic polymers [51-57], cationic lipids [58,59,60,61], as well as on liposomes [39,62-68]. DNA could be transferred to the cells encapsulating in or by adsorption onto the particles surface [45, 46, 59, 69-71]. Chitosan is a promising and often used candidate for surface modification due to its biocompatibility and positive charge. Chitosan-modified PLGA should adsorb better than other lipophilic polymer derivatives, because of its hydrophilicity, and improve transfection rate of the particle-DNA-complexes in vitro and also in vivo experiments [72]. For successful application of nanoparticles as gene delivery systems their interaction with the payload is essential. Therefore a fundamental study was performed regarding nanoparticles and their possible biological active complexes. From this point of view it is important to investigate the influence of each possible environmental parameter on nanoparticle-DNA complexes. One disadvantage of existing delivery systems is the limitation by using organic solvents and relatively harsh formulation conditions. PLGA nanoparticles are generally formulated using emulsion solvent evaporation or by solvent displacement techniques [26], which induce some problems with limited core loading, i.e., <15%, the variable burst release of entrapped drug and organic solvent residues [39]. One of the main drawbacks of colloidal carriers is their thermodynamic instability: they tend to agglomerate during storage. The goal of our study was the biophysical investigation of the thermodynamical properties of PLGA nanoparticles, in particular, how stable are particle and how environment conditions influence on nanoparticle stability. Especially, the examination of the influence on nanoparticles such ambient conditions as pH, temperature, polarity of the solvent. For the purpose of determine how the particle covering with chitosan will influence on nanoparticles stability in experiments two types of nanoparticles have been chosen to study: non-coated and chitosan-coated nanoparticles with main size 148.2 nm and 146.8 nm respectively.

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METHODS Preparation of the Nanoparticles Non-coated and chitosan-coated PLGA nanoparticles were made using solvent evaporation method [71,73,74]. In brief, PLGA 70:30 was dissolved in 5 ml ethyl acetate at room temperature. The organic phase was added dropwise to an equal volume of the aqueous phasecontaining the stabilizer PVA (2.5% w/v) for PLGA particles and PVA and chitosan for chitosan coated particles, under stirring using a magnetic stirrer, at 1000 rpm, for 1 h, at room temperature. The emulsion was then homogenized (Ultra-Turrax T25, Janke and Kunkel GmbH and Co-KG, Germany) at 13,500 rpm for 10 min. Particle formation was obtained by adding MilliQ water dropwise under gentle stirring to obtain a final volume of 50 ml. Stirring is continued overnight at room temperature to get rid of the organic solvent.

Colloidal Characteristics of Particles The size distribution of the particles was determined using photon correlation spectroscopy (ZetaSizer Nano ZS, Malvern Instruments Ltd., Worcestershire, UK). The potential of particles was obtained using the same apparatus by measuring the electrophoretic mobility of the particles. The non-coated PLGA nanoparticle size was found to be d = 148.2 nm (PI=0.03, charge -8.6mV). In the case of chitosan-coated PLGA nanoparticles the size was 146.8 nm (PI=0.12, charge +39.98 mV). There was no detectable amount of chitosan left in solution as is described elsewhere. The study of the thermodynamic features of PLGA nanoparticles and the interaction between DNA and the NP were carried out using a precise DASM-4A microcalorimeter (Russian Academy of Science, Pushchino, Russia), which belongs to high sensitivity type heat flow calorimeters and the resolution is less than 0.5 µW [75]. In addition, this device allows to carry out experiments with low rate of temperature scanning and therefore to perform quasi equilibrium measurements. The spectrophotometric investigation was done using the spectrophotometer HEλIOS β (Thermospectronic, Thermo Fisher Scientific, USA).

RESULTS For nanoparticle application in biological systems it is important to investigate their stability under different environmental conditions such as temperature, pH, various salts, etc. We have studied the thermodynamical properties of nanoparticles and have gained some insights about nanoparticles’ properties using supersensitive differential microcalorimetry. To calculate nanoparticle specific heat capacity it is necessary to know nanoparticle partial volume. According to nanoparticle weight, diameter and its shape, we calculated the partial volume. Our nanoparticles in water were situated in suspended condition, had a spherical shape with approximately 147 nm diameter [74]. This allows to calculate the nanoparticle volume which turned out to be on average 1.7 • 10-15 ml. Based on nanoparticle mean weight

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which was obtained by freeze drying (2.88•10-15g), finally, it is possible to calculate the nanoparticle partial volume which turned out to be ν=0.59ml/g (or density ρ=1.695g/ml). In Figure 2 are shown the chitosan-coated PLGA nanoparticle (a) and non-coated nanoparticle (b) suspension microcalorimetric curves. The heating rate was 2°/min. The enthalpy of this effect is 1.98±0.02 J/g. For both particles the curves have the same appearance. Therefore hereinafter we give only the results for the thermodynamic parameter investigation for one of the nanoparticles, as the results were basically identical for equal conditions. From the microcalorimetric curve four temperature intervals can be concluded: a) 10-23°C; b) 23-35°C; c) 35-130°C; d) 130-140°C. As it can be seen the subsequent hanging of temperature (10-23°C) leads to increase of heat capacity. The change of heat capacity of solution induced by the particles partial volume increase. This is a result of particle swelling, when the particle inside solvent thermal expansion occurs which leads to particles increase in volume.

a)

b) Figure 2. Dependence of the specific heat capacity on temperature: (a) in the case of chitosan-coated PLGA nanoparticles; (b) in the case of non-coated PLGA nano- particles. The heating rate was 2°/min. Solvent was bidistilled water, pH 5.0.

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In 23°C–35°C temperature interval the particles’ structure change occurs and its core becomes reachable for the solvent. At this intertval the particles cracking take place. The phase transition temperature Tm for the chitosan-coated NP is 30°C and in the case of noncoated NP is 28°C. Furthermore, at elevated temperatures (Tm > 50°C) the monotonously decreasing curves indicate that the particles start to aggregate (Fig 2). Further increase of the temperature leads to particles’ aggregate destruction (above 120°C) which appears in sharp increase of particles’ heat capacity. This is the result of separations of the molecules forming nanoparticles having high heat capacity (contact between hydrophobic groups and water molecules causes an increasing specific heat). It was carried out turbidity measurements of nanoparticles with temperature (noncoated; PLA/PLG 70:30). The device of turbidity was constructed in Tbilisi State University by us, where as a source of light it is used blue- light-emitting diode (with wavelength λ=480 nm) and the detector of light is photomultiplier. The suspension of nanoparticles was placed in the glass tube with length 10 centimeter, which was heated by heater which was surrounded around tube. The measurement of temperature was carried out by mercury thermometer, which with good thermal contact was fixed on the glass tube. On the Figure 3 it is given dependence of suspension transparency changes on temperature during scanning the temperature, from which it is obvious that while increasing the temperature of nanoparticles in 15°C -35°C temperature interval the intensive suspension transparency increase takes place (heating of suspension up to higher temperatures (>50°C) was not achieved for this time due to construction of device).

Figure 3. Dependence of non coated nanoparticles’ suspension transparency on heating temperature; PLA/PLG 70:30.

To our opinion obtained curve should be related with volume increase of spherical nanoparticles (in mentioned temperature interval) until it will be destroyed, as a result of this (>30 °C) the transparency of suspension is changed not considerably. We would like to underline the circumstance, that while increasing the temperature of suspension the small (not large) increase of nanoparticles’ volume takes place, which results the decrease of concentration of nanoparticles in suspension (the number of particles in the unit of volume will be less) and consequently the increase of turbidity occurs. This experimental datum is

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explaining the increase of specific heat capacity of nanoparticles in the above mentioned temperature interval which takes place during calorimetric experiments. Spectrophotometric data showing that the so-called Rayleigh scattering (light intensity is proportional to λ-4) is the same for both, “native” and particles after agglomerate desintegration. In other words, the absorption spectrum of the destroyed chitosan-coated nanoparticles, obtained by heating up to 150°C, did not differ from the spectrum of the “native” ones (Figure 4).

Figure 4. The light absorption spectra of chitosan-coated PLGA nanoparticles (the solid line) with 1.238 mg/ml concentration and for denatured particles with the same concentration (the dash line). Solvent was bidistilled water, pH 5.0 and its temperature was 250C.

Figure 5. Dependence of the specific heat capacity of non-coated PLGA nanoparticles on temperature at different heating rates: 1 – 0.5 K/min; 2 – 1 K/min; 3 – 2 K/min; 4 – 4 K/min.

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The same results were obtained for non-coated nanoparticles. To determine whether the nanoparticles melting is a kinetic process or not, the calorimetric experiments with NP suspensions at different heating speeds (0.5°/min, 1°/min, 2°/min, 4°/min) have been carried out (Figure 5). These experiments show that thermodynamic parameters such as Tm and the particle enthalpy (the area of the peak in 23°C–35°C temperature interval) depends on particle heating rate. In particular, the higher the speed of heating, the higher is the particle transition temperature Tm and their melting enthalpy (Table 1). It must be noted that the observation accuracy was increased by multiple determinations carried out. Table 1. Dependence of transition temperature (Tm) and melting enthalpy (ΔH) of non-coated PLGA nanoparticles on different heating rates Heating rate [K/min] 0.5 1 2 4

Transition temperature Tm [0C] 26.5 27.9 29.5 31.7

Melting enthalpy ΔH [J/g] 1.87 1.92 1.98 2.25

For practical application it is important to determine the storage stability of the NP. Especially, the storage in suspension for these biodegradable samples is of high interest. For this reason we carried out calorimetric experiments with nanoparticle prepared three months before and stored at 4°C in refrigerator. The result was compared with the same experiment performed with freshly prepared particles. Figure6 shows calorimetric curves for freshly prepared non-coated nanoparticles (line 1) and the same particles after 3 months (line 2).

Figure 6. Dependence of the specific heat capacity of non-coated PLGA nanoparticles on temperature: 1 - fresh prepared nanoparticles; 2 - the nanoparticles after 3 months.

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As it can be seen from this figure storage time influences only particle aggregation process, in other words, the influence of time on nanoparticles becomes apparent in an attempt to increase particle aggregation (they aggregate at lower temperature: 40-110°C range). In addition, the experiments have been carried out with the so-called nanoparticle annealing by temperature, where the nanoparticles were heated only till 23°C – the temperature where the NP internal architecture is starting to modify (Figure 7). The peak is restored at lower temperature (Tm = 25°C) after particle pre-heating. The result of Figure 7 points out that the particles already change at lower temperature.

Figure 7. Dependence of the specific heat capacity of non-coated PLGA nanoparticles on temperature: first heating till 23°C; second is the totally heating up to 1500C.

It is known that in creation of nanoparticles the existence of hydrophobic forces play an important role. Therefore, we changed the solvent’s polarity to investigate the solvent-particle interaction. The calorimetric experiments in 10% ethanol solution lead to a decreased transition temperature Tm = 24°C (Figure8). In contrast to water, in ethanol no aggregation was observed. Moreover, because of the reduced solvent hydrophobicity no particle aggregation process was observed.

Figure 8. Dependence of the specific heat capacity of chitosan-coated PLGA nanoparticles on temperature: 1 – nanoparticles in bidistilled water; 2 – nanoparticles in 10% methanol solution.

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As it was mentioned above, under the influence of temperature, at first nanoparticles modification (10-25°C) takes place, which is followed by the particle cracking process at Tm=30°C. Hence the particle transition temperature has to be depended on the environmental pressure in all our calorimetric experiments the samples were situated under 6-7 atmosphere pressure to avoid the solution boiling process during heating. To find out if the pressure influences on widening process, the experiments have been performed at 1.5 atmosphere pressure and without excess pressure have been carried out. No differences were obtained. Also experiments were done to determine the stability of nanoparticles in deionized water. Our results emphasize that using deionized water is not so necessary for stability of nanoparticle. For this reason other parameters such as the solvent’s pH were investigated. Figure 9 shows microcalorimetric study of nanoparticles in buffers of different acidity ranging from pH 2 to pH 8.2. The buffer molarity was chosen not too low (0.02M Na2HPO4 and 0.01M citric acid) to obtain a sufficient buffer capacity for maintaining the solution’s pH during the heating process in the wide temperature interval (10-150oC). These data are compared to the calorimetric data of nanoparticles suspension in pure bidistilled water.

Figure 9. The microcalorimetric study of chitosan-coated PLGA nanoparticles, immersed in buffers with different acidity, ranging from pH 2 to pH 8.2. The buffer molarity was 0.02M Na2HPO4 and 0.01M citric acid. The solid line – pH 2; the short dot line – pH 3.8; short dash dot line – pH 5; dash line – pH 8.2.

DISCUSSION From the calorimetric results in Figure2 we can conclude that the coating of nanoparticles with chitosan did not exert an influence on the behavior of the particles. This means that thermodynamic parameters, in particular the profile of the specific heat absorption curves in dependence on temperature for non-coated nanoparticles and chitosan-coated nanoparticles were the same. Also it is clear that the calorimetric curves themselves have a complex shape

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(Figure 2) which is composed out of the heat absorption peak area (23-35°C), the sharp change at T ~ 130°C, and the monotonic sections from 10 – 23°C and 35 – 130°C. Such complicated nature of the calorimetric curves indicates that the temperature has multifarious influences on the nanoparticles and causes significant modification of their structure. It should be mentioned that the influence of temperature on nanoparticles starts right at the beginning of the experiment, namely at 10°C, where an increasing heat absorption is observed which turns into a peak at 23-35°C temperature interval. At the beginning of the experiments respectively at the start of temperature scanning the increasе of particle specific heat absorption might be induced by increasing the particle volume, as it happens typically for solid bodies. We received such conclusion, owing to the calorimetry construction, because the DASM-4A calorimeter (DSC) is a device whose ampule is represented by platinum thin capillary which when it is entirely infilled, measures the sample heat effect for only the half of the filled volume. Other construction calorimeters (calorimeters, whose measuring ampules are hermetically closed) measure the whole investigated sample heat effect and are less sensitive to particle widening effect [75]. In other words, as we have mentioned above, in our case the DASM-4A calorimeter measures only part (half) of the suspension filled up in the capillary and if the volume of the particle changes in this part of the capillary, this change instantly affects the heat capacity of this volume phase. Moreover, based on aforesaid it is clear that in such calorimeters it becomes necessary to know the partial volume for measuring the sample’s heat capacity. The partial volume was obtained from nanoparticles’ weight, diameter and its shape and was found to be 0.59 ml/g (assuming that the nanoparticles suspended in water had spherical shape and a diameter of approximately d = 147 nm resulting in a mean weight of 2.88•10-15g). Finally it can be concluded, that at initial temperatures the growing of particle volume takes place, the particle small swelling, which at 23-350C temperature interval finishes with particle cracking. Because in this case the break in particle existing bonds takes place, the heat absorption peak is springing up. On the other hand it should be mentioned that the shape of the nanoparticles did not change even for such high temperature as 150°C. Hence, cooling them back to room temperature resulted again in the homogeneous suspension which absorption spectrum is analogous to the spectrum of non-heated nanoparticle suspensio. This is supported by spectrophotometric data showing the so-called Rayleigh scattering (the scattering intensity I ~ -4) which is the same for “native” and destroyed particles (Figure 4). Moreover, the spectrum of the particles, obtained by heating up to 150°C, did not differ from the spectrum of the “native” particles. This indicates that the particles (polymer composition) and they are coctostabile; otherwise they would be destroyed entirely at high temperatures. Therefore the optic spectroscopy, in particular turbodimetric method, is not able to distinguish the initial and temperature-induced changed nanoparticles from each other because both spectrums were carried out at room temperature. For increasing temperature, on the surface of the particles additional hydrophobic chemical groups appear and try to connect with the neighbouring particle surface hydrophobic groups. It betokens that particles will aggregate. From calorimetric curve we can see that after the heat adsorption peak, at higher temperature the specific heat capacity curve is decreasing approximately from 50°C to 120°C area (Figure 2), which is the typical case of aggregates appearing. A further increasing of temperature (above 120°C) leads to a sharp increase of the heat capacity curve, which in our point of view, is caused by aggregate/conglomerate dissociation perhaps to suspension of

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nanoparticles. The hydrophobic part of torn nanoparticles falls into the contact with water and with further temperature increase the amount of aggregates raises. The enthalpy and temperature dependence on heating rate (Figure 5) may be explained by nonequilibrium of process when the interior temperature is less than its outer once and this distinction is as more as the heating rate increase. Earlier it has been shown that PLGA (50:50) nanoparticles, with inherent viscosity of 0.69 dL/g coated with PVA, have glass transition temperature (Tg) onset 38°C and endset 45°C [76]. The heating speed in those calorimetric experiments was 10°/min. Because of thermal gradients, the high heating rate caused increasing the transition temperature value. In other words, the closer we can come to equilibrium the exacter the thermal parameters can be measured. This is also supported by the so-called annealing experiments where the nanoparticles first were heated only until 23°C – the starting temperature of the particles’ melting process (Figure 7). The significant changes in the enthalpy and transition temperature (Tm) from 30°C to 25°C in the reheating curve show that if the particles are heated till the temperature, which is required to start their internal transition, the process proceeds spontaneously - no more energy is needed to destroy them up to the end. It is clear that the existence of hydrophobic forces in creation and stability of nanoparticle emphasizes the high profile. The nanoparticles expected destruction temperature depends on the contact forces which originate during production. The main part belongs to hydrophobic forces. However, their strength depends on the solvent properties around the particles. Changing (decrease) the polarity of the solvent would have significant influence (diminution) on particle transition temperature. In other words the stability of the particles must depend on the extent of solvent’s polarity. In Figure 8 we can see Tm of the particles’ diminished right away (from 30°C up to 24°C) when a 10% ethanol solution is used. Moreover because of the solvent’s hydrophobicity decrease there was no particles’ aggregation process observed. An important parameter for future applicability of NP-based delivery systems in pharmaceutics is their stability under relevant environmental conditions to avoid their damage and prematurely drug release. Therefore, the influence of temperature, pH and various salts on nanoparticles needs to be investigated. At first, an unchanged Tm (Figure 9) indicates the stability of the NP in all investigated pH-values .The nanoparticles maintain their structure in deep acid (pH 2) as well as in alkaline (pH 8.2) conditions (heat absorption peak at 30oC, Figure 9), i.e. the particles are not destructed under these conditions. Moreover, the constant heat capacity in the acid range for T > Tm reflects the augmented stability due to electrostatic repulsion, in contrast to water and alkaline ranges. This is valid and expected for cNP. For non-coated NP this would be surprising because of the absence of any pH influence[74]. Therefore besides the particles are stable in buffer, in pH 2 (stomach pH) there is no aggregation process observed what makes them suited for oral delivery as already shown in the literature. The experiments unambiguously show that in a wide pH interval (2-8) the changes in transition temperature did not take place. These results are important for two reasons: such nanoparticles (PLA/PGA ratio in these PLGA nanoparticles is 70:30) could be used in acidic surrounding (for instance, in stomach) for drug transfer and the particles structure, stability and their other properties are less depended on either the particles were in water (bidistilled or deionized) or the suspension of particles were located in buffer (at least in buffer with low molarity).

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ACKNOWLEDGMENTS Authors are thankful of the collaborative program between Tbilisi State University (Georgia) and Saarland University (Germany), which allow carrying out the collaborative research.

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[68] G. Mrevlishvili, B. Kankia, T. Mdzinarashvili, T. Brelidze, M. Khvedelidze, N. Metreveli, G. Razmadze, Liposome-DNA interaction: microcalorimetric study Chemistry and Physics of Lipids, 1998, 94, 139-143. [69] Z. Cui, R. Mumper. Plasmid DNA-entrapped nanoparticles engineered from microemulsion precursors: in vitro and in vivo evaluation. Bioconjug Chem. 2002, 13, 13191327. [70] R. Kumar, U. Bakowsky, C-M. Lehr. Preparation and characterization of cationic PLGA nanospheres as DNA carriers. Biomaterials. 2004, 25, 1771-1777. [71] R. Kumar, S. Mohapatra, X. Kong, P. Jena, U. Bakowsky, C-M. Lehr. Cationic poly(lactide-co-glycolide) nanoparticles as efficient in vivo gene transfection agents. J. Nanosci Nanotechnol. 2004b, 4, 990-994. [72] J. Haas, K. Ravi, G. Borchard, U. Bakowsky, C-M. Lehr. Preparation and characterization of chitosan and trimethyl-chitosan-modified Poly-(ε-caprolactone) nanoparticles as DNA carriers. AAPS Pharm. Sci. Tech. 2005, 6, 22-30. [73] J. Luengo, B. Weiss, M. Schneider, A. Ehlers, F. Stracke, K. König, K-H. Kostka, Lehr C.-M, Schaefer U. Influence of nanoencapsulation on human skin transport of flufenamic acid, Skin Pharmacology and Physiology. Skin Pharmacology and Physiology. 2006,19,190-197. [74] N. Nafee, S. Taetz, M. Schneider, U. Schaefer, C-M. Lehr. Chitosan-coated PLGA nanoparticles for DNA/RNA delivery: effect of the formulation parameters on complexation and transfection of antisense oligonucleotides. Nanomedicine: Nanotechnology, Biology and Medicine 2007, 3, 173-183. [75] P. Privalov, S. Potekchin. Scanning microcalorimetry in studing temperature – induсed changes in proteins. Methods in Enzimology. 1986, 131, 4-51. [76] S. De, D. Robinson. Particle size and temperature effect on the physical stability of PLGA nanospheres and microspheres containing Bodipy. AAPS Pharm. Sci. Tech, 2004, 5, 1-7.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 11

THE TACTICITY GOVERNED STEREOMICROSTRUCTURE IN POLY(METHYL METHACRYLATE) (PMMA) AS A WAY TO EXPLAIN ITS PHYSICAL PROPERTIES N. Guarrotxena1 Instituto de Ciencia y Tecnología de Polímeros (ICTP), Consejo Superior de Investigaciones Científicas (CSIC). C/ Juan de la Cierva 3. 28006 Madrid, Spain

ABSTRACT Three industrial samples of Poly(methyl methacrylate) (PMMA), prepared under different conditions, have been extensively analyzed by means of 1H-NMR spectroscopy. Starting from the mm, rr and mrandrm triad contents, as given by the spectra, the type of tacticity statistics distribution has been deduced. Sample X appears to be completely Bernoullian, while samples Y and Z deviate somewhat from this behaviour exhibiting a tiny trend towards Markovian statistics. The fraction of mmrm and rrrm pentads and that of pure heterotactic and atactic triad moieties has been calculated by assuming either a Markovian statistics for samples Y and Z or a Bernoullian statistics for all the samples. On the other hand, the fraction of the same pentads has been determined by deconvoluting the overall triad signals of the spectra into the corresponding pentad signals. An appreciably good agreement with the values obtained assuming Bernoullian statistics for all the samples appears evident. As a result, the evolution of every pentad content from sample X to Sample Z could be stated. Thus the samples prove to be appropriate models to study the relationship between any physical property and the stereomicrostructure of PMMA as was done previously for Poly(vinyl- chloride) (PVC) and Polypropylene (PP).

1 E-mail: [email protected].

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N. Guarrotxena

INTRODUCTION A considerable amount of earlier work in our laboratory has dealt with the relationships between the physical properties determining processes at molecular level and some tacticity governed stereomicrostructures of poly(vinyl chloride) and polypropylene polymers (1-10). These structures are some repeating stereosequences located at the end of isotactic or syndiotactic sequences, such as mmrm and rrrm tetrads occurring necessarily whenever those sequences break off respectively. The length of the isotactic or syndiotactic sequence associated to mmrm or rrrm is very important too. To these may be added moieties of pure heterotactic and atactic short sequences which both also influence on physical properties. The frequency of those microstructures along the chain obviously depends on the type of tacticity statistics in the polymer and then on the polymerization conditions. It is worth noting that an equal number of r and m placements may give different numbers of each stereomicrostructure depending on the tacticity distribution statistics of the polymer. This principle led us to explore the extent to which that statistics and the resulting microstructures are or not a prominent feature in polymer materials science. The microstructures so defined are important disruptions of the chain regularity, each involving changes in either local free volume or rotation mobility facilities or both (2,7-10). As a consequence, the inter- and intramolecular interactions should be expected to change as the content of the distinct microstructures changes, and hence .the physical properties of the polymer should change too without taking place any change in the chemical composition of the polymer. By producing polymer samples of different overall microstructure, as accurately stated especially through NMR spectroscopy, a straight relation between it and most of the physical properties of PVC and PP materials could be demonstrated in the above quoted work (1-10). In order to extend this knowledge to other polymers of industrial interest, we endeavoured to study the Poly(methyl methacrylate) (PMMA) in a similar way to the used before. The major requirement for such study is to ensure a detailed description of the stereomicrostructure of some polymer samples prepared under different conditions so that some changes in the overall tacticity are produced. This is the objective of the present paper where that stereomicrostructure is assessed for three PMMA industrial samples.

EXPERIMENTAL Materials Commercial PMMA samples, obtained from Atochem were used in this work. PMMA samples were purified using tetrahydrofuran (THF, Scharlau) as solvent and water as precipitating agent, and then washed in methanol and dried under vacuum at 40 ºC for 48 h. THF was distilled under nitrogen with aluminium lithium hydride (Aldrich) to remove peroxides immediately before use.

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149

Characterization of Samples The tacticity of the three distinct PMMA samples was measured by 1H-NMR spectroscopy on a Varian UNITY-500 spectrometer operating at 500 MHz with CDCl3 as solvent at 50ºC. Parameters of 8000 Hz spectral width and 1.9 s pulse repetition rate were used. The delay time was set at 1.9s. The spectra were obtaining after accumulating 64 scans with a sample concentration of 10 wt% solutions. The relative peak intensities were measured from the integrated peak areas, which were calculated with an electronic integrator. The molecular weight distributions were measured by SEC using a chromatographic system (515 Waters Division Millipore) equipped with a Waters Model 410 refractive index detector. THF (Scharlau) was used as the eluent at a flow rate of 1 mL min-1 operated at 25ºC. Styragel packed columns, HR1, HR3, HR4E and HR5E, were used. PMMA standars (Waters Associates) in the range between 1.4 x 106 and 3 x 103 g mol-1 were used to calibrate the columns.

RESULTS AND DISCUSSION Microstructure of the Samples The 1H-NMR spectra of samples X, Y and Z are compared in Figure 1. The indicated resonance assignments for the mm, mr and rr centered pentads are those taken from literature (11). The spectra are typical of predominantly syndiotactic PMMA but showing different isotactic contents.

Figure 1. 500 MHz 1H NMR spectra of a X, b Y and c Z PMMA samples measured in CDCl3 at 50ºC.

N. Guarrotxena

150

Table 1. 1H NMR data for the PMMA samples Sample Mn

mma

mra

rra

Pmb

Prb

Pm/rb

Pr/mb

Pm/mb

Pr/rb

X

45500 8.39

43.16

48.45

29.97

70.03

0.72

0.308

0.28

0.69

Y

43100 15.16

40.86

43.98

35.59

64.41

0.57

0.31

0.43

0.69

Z

44900 20.93

37.92

41.15

39.89

60.11

0.47

0.31

0.53

0.69

mra

rra

Pmb

Prb

Pm/rb

Pr/mb

Pm/mb

Pr/rb

Sample Mn

mma

X

45500 8.39

43.16

48.45

29.97

70.03

0.72

0.308

0.28

0.69

Y

43100 15.16

40.86

43.98

35.59

64.41

0.57

0.31

0.43

0.69

Z

44900 20.93

37.92

41.15

39.89

60.11

0.47

0.31

0.53

0.69

b

Pr/mb

Pm/mb

Pr/rb

Sample Mn

a

mm

mr

a

rr

a

Pmb

b

Pr

Pm/r

X

45500 8.39

43.16

48.45

29.97

70.03

0.72

0.308

0.28

0.69

Y

43100 15.16

40.86

43.98

35.59

64.41

0.57

0.31

0.43

0.69

Z

44900 20.93

37.92

41.15

39.89

60.11

0.47

0.31

0.53

0.69

The quantitative amount of mm, mr and rr triads, as measured on the spectra, are given in Table 1. Since the physical properties of poly(vinylchloride), PVC and polypropylene, PP were demonstrated to relate to the tacticity arrangement along the chain, it appeared of prime importance to examine this point for samples X, Y and Z. To do that we first determined the type of repeating sequence statistics, whether Bernoullian or Markovian, as calculated from the experimental values of mm, mr and rr triads (11). Secondly the likely fraction of each individual sequence was calculated according to the respective type of tacticity distribution. And finally the values so obtained were compared, in a semi-quantitative way to those stemming from direct measurement on the spectra, of some of the pentads. Actually the latter are only approximate because the proximity of signals makes their deconvolution rather complicate. The extent to which each sample fits into, or departs from, Bernoullian statistics can be determined from the mm, rr and mr and rm triad content as measured on the spectra. That quantity is called the persistant ratio and is defined by ρ=P(s)P(i)/P(is), where P(s)=rr+1/2mr; P(i)=mm+1/2mr and P(is)=1/2mr. The results obtained are 0.9725, 1.1220 and 1.2709. These values would seem to indicate that sample X is Bernoullian, while samples Y and Z are apparently non-Bernoullian isotactic. Another criterion for Bernoullian statistics is based on the conditional probabilities of first order Markov statistics, indicating the probability of occurrence of one m or one r diad preceded by one r or one m diad respectively. They are denoted by P(r/m) and P(m/r) (11). If Bernoullian statistics apply, P(m/r) + P(r/m)=1. This sum is 1.02, 0.88 and 0.78 for samples X, Y and Z respectively, so confirming that sample X is Bernooullian and samples Y and Z would tend to be somewhat Markovian. However the values for Y and Z samples depart no sufficiently from unity for them to be considered completely Markovian.

The Tacticity Governed Stereomicrostructure…

151

As a result the probability of forming pentads in sample X may be easily determined (11), e.g. [rrrr]=P(s)4; [rmmr]=P(s)2 P(i)2; [rrrm]=P(s)3 P(i)2, etc. In the case of non-symmetric sequences, like the latter, factor 2 is necessary because both directions should be counted. The probability of forming pentads in samples Y and Z may be also calculated through the conditional probabilities (11). For example an mmrm pentad fraction is given as [mmrm]=[mm] P(m/r) P(r/m)+[mr] P(r/m) P(m/m). The values obtained for the most useful pentads for the purpose of this paper, are given in Table 2. On the other hand, these values have been also determined from the spectra of Figure 1 by deconvoluting the overall triad signals into the individual pentad signals indicated in Figure 1. The Origin Program was used. It allows both that deconvolution and the distribution of every experimental triad percentage into the corresponding pentad percentages, through an internal mathematical treatment. The data so obtained are shown in Table 3. When comparing them to the calculated assuming Markovian statistical tacticity distribution for samples Y and Z, (Table 2), a strong divergence appears evident, especially in the order of changing from sample X to sample Z. This proves the calculation way utilised to be wrong. Table 2.The iso, hetero and syndiotactic pentads values calculated through the conditional of first order Markov statistics probabilities1 for PMMA samples

Triads rr mr

Pentads rrrr mrrr+mrrm rmrr+mmrr mrmr+mmrm

mm rmmr mmmr+mmmm

X 0.24 0.294 0.294 0.125 0.088 0.0457

Sample Y 0.21 0.4545 0.3562 0.2043 0.100 0.1407

Z 0.1959 0.3495 0.3184 0.202 0.094 0.2166

Table 3. The iso, hetero and syndiotactic pentads values obtained by ined from 1H NMR spectra (Fig. 1) of PMMA samples. Sample

rr mr mm

Pentads rrrr

X 0.395

Triads 0.39

Z 0.39

mrrr+mrrm

0.089

0.049

0.0245

rmrr+mmrr mrmr+mmrm rmmr mmmr+mmmm

0.4197 0.0118 0.019 0.064

0.397 0.018 0.0406 0.111

0.3070 0.072 0.0226 0.1866

Since the departure of samples Y and Z from Bernoullian statistics is not so much great, an attempt was made to compare the experimental values, and those obtained assuming Bernoullian statistics for all the samples (Table 4). As can be seen the evolution order of each pentad is satisfactorily coincident so indicating that Bernoullian statistics apply for all the

N. Guarrotxena

152

samples. The small deviations observed only for rrrr and rmmr pentads obey the contribution of some little signals around 0.90 ppm, increasing from X to Z sample and being counted as rrrr pentads, and the experimental uncertainties when deconvoluting the signals at mm region, respectively. It is worth noting that the difference between absolute calculated and experimental values lies within the experimental uncertainties when deconvoluting the signals on the spectra. The fact that the tacticity statistics of samples Y and Z are closer to, but not exactly the Bernoullian statistics might also influence the calculated values. What is of major importance is that there is no change in the evolution order in both sets of values. Table 4. The iso, hetero and syndiotactic pentads values calculated by . assuming Bernoullian statistics for PMMA samples Sample Triads

Pentads rrrr

X 0.24

Y 0.17

Z 0.13

rr

mrrr+mrrm

0.4984

0.3474

0.230

mr

rmrr+mmrr mrmr+mmrm

0.295 0.163 0.0525

0.2882 0.191 0.057

mm

rmmr mmmr+mmmm

0.2939 0.125 0.044 0.0457

0.058

0.0763

Consequently the above results are quite valuable to settle the evolution of any repeating stereosequence from one sample to the other. The sequences that were proved to be the major driving force for the physical properties of PVC and PP according to earlier work, are: i) the average isotactic and syndiotactic sequences length; ii) the mmr-based and the rrm-based local structures which occur necessarily whenever an isotactic or syndiotactic sequences breaks off respectively. Nevertheless, these structures are not active by themselves because it is the occurrence of either one m placement following mmr or one r placement preceding rrm and the length of the -mm…- and –rrr….- sequences connected with them, that were identified as a property determining factor; thence the really important factors are: a) the fraction of mmr followed by one m placement (-mmrm-structures) and the length of the isotactic sequence preceding mmrm. In fact the ratio of –mmrm- repeating stereosequences of at least one heptad in length to the same shorter ones, was proved to be of major importance; and b) the fraction of rrm preceded by one or more r placement, i.e. the –rrrm- structures at the end of syndiotactic sequences; iii) the pure heterotactic –mrmr- sequences and iv) the short atactic moieties like rmrr, mmrr, mrrm and rmmr. The changes of these repeating stereosequences in X, Y and Z samples can be specified in the light of the above quoted both calculated and experimental results (Tables 3 and 4). It may be thus stated that: 1. The average length of isotactic –mmmm…- sequences increases from sample X to sample Z and so does the content of mmrm sequence and the length of the isotactic sequence preceding it. As a result the ratio of mmrm stereosequences longer than one heptad to the shorter ones will increase in the order X
The Tacticity Governed Stereomicrostructure…

153

2. The fraction of –rrrm- structures and the length of the syndiotactic sequence preceding it, will decrease in the order X>Y>Z. 3. As indicated by the individual calculated values, the fraction of pure heterotactic stereosequences, -mrmr.-, hardly changes from one sample to the other. A tiny tendency towards decreasing from X to Z is however observed. 4. The short atactic moieties, mrrm, rmrr and mmrr decrease in the order X>Y>Z, this tendency being significant for mrrm only. The rmmr changes in the reverse order. On the other hand, it has been extensively conveyed (12,13) that mmrm can adopt GTTGTT and GTGTTT conformation, the equilibrium between them being strongly displaced towards the latter conformation. It is worthy to note that in PMMA such a displacement should be much enhanced relative to PVC, because of the more hindered rotation facilities. Nevertheless, the occurrence of GTTG-TT conformation will decrease in a similar way to mmrm, i.e. Z>Y>X. By correlating the changes in all the above repeating stereosequences with those in any physical property of the samples, the understanding of the processes at molecular level, involved in that property should take a step further. A considerable amount of work on PVC and PP makes this prospect quite reliable.

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

N. Vella, A.Toureille, N. Guarrotxena, JL. Millán, J. Macromol. Chem. Phys. 1996, 197, 1301-1309. N. Guarrotxena, N. Vella, A.Toureille, JL. Millán, Macromol. Chem. Phys. 1997, 198, 457-469. N. Guarrotxena, JL. Millán, N. Vella, A. Toureille, Polymer 1997, 38, 4253-4259. N.Guarrotxena, N. Vella, A. Toureille, JL. Millán, Polymer 1998, 39, 3273-3277. N. Guarrotxena, A. Toureille, J. Millán, Macromol. Chem. Phys. 1998, 199, 81-86. N. Guarrotxena, J. Millán, G. Sessler, G. Hess, Macromol. Chem. Phys. 2000, 21, 691696. N. Guarrotxena, G. Martínez, J. Millán, Polymer. 1997, 38, 1857-1864. N. Guarrotxena, G., Martínez, J. Millán, Polymer. 2000, 41, 3331-3336. N. Guarrotxena, J.J. del Val, J. Millán, Polymer Bulletin. 2001, 47, 105-111. N. Guarrotxena, J.J. del Val, A. Elicegui, J. Millán, J. Polym. Sci. Polym. Phys. 2004, 42, 2337-2347. K. Hatada, T. Kitayama. NMR Spectroscopy of Polymers, Chap 3, Springer 2004, ISBN: 3-450-40220-9 N. Guarrotxena, G. Martínez, J. Millán, Eur Polym J. 1996, 33, 1473. and referentes cited therein N. Guarrotxena, F. Schue, A. Collet, J. Millán, Polym. Int. 2003, 52, 420.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 12

KINETICS OF THE FERMENTATIVE REACTION OF H2O2 DECOMPOSITION UNDER THE ACTION OF CATALASE IN THE PRESENCE OF BIOSAS FOR THE STATIONARY STATE *

*

A.A.Turovsky, * R.O.Khvorostetsky1, ** L.I. Bazylyak, *** and G.E. Zaikov

Chemistry and Biotechnology of Combustible Minerals Division; Physical Chemistry of Combustible Minerals Department; Institute of Physical–Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko; National Academy of Science of Ukraine2, Lviv, Ukraine ** Chemistry of Oxidizing Processes Division; Physical Chemistry of Combustible Minerals Department; Institute of Physical–Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko; National Academy of Science of Ukraine, Lviv, Ukraine *** Kinetics of Chemical and Biological Processes Division; Institute of Biochemical Physics named after N. N. Emanuel; Russian Academy of Sciences,3 Moscow, RUSSIA

ABSTRACT It was investigated the fermentative stationary kinetics of hydrogen peroxide decomposition under the action of catalase in the presence of bioSAS. It were obtained the kinetic parameters of this process. It was shown, that the bioSAS have influence on the fermentative process, which can be explained by the change of the fermentative center activity or by the change of substrate concentration. It was determined that the temperature of a process has an insignificant influence on the value of kinetic parameters.

1 E-mail: [email protected]. 2 3а Naukova Str., Lviv–60, 79060, UKRAINE; E-mail: [email protected]. 3 4 Kosygin Str., Moscow, 119991, RUSSIA; E-mail: [email protected].

156

A. A.Turovsky, R. O.Khvorostetsky, L. I. Bazylyak et al.

I. INTRODUCTION Regulation of the ferments activity is the subject of a great interest among scientists from the point of view both of fundamental and applied microbiology and biochemistry. Development of high–effective complex enzymatic preparations is the actual task of the modern biotechnology. Among potential regulators of enzymatic activity the surface–active substances (SAS) call a special attention, since they are characterized with unique physical– chemical properties. Thus, in references, for example, there are data as to stimulative action of the separate synthetic SAS on the activity of the horse–radish peroxidase [1−3]. At the same time, biogenic SAS (or biosurfactants or bioSAS) of the high activity are characterized by a series of advantages in comparison with the synthetic SAS, in particular: they are non–toxic, biodegradable, they are characterized by high efficiency in a wide range of the temperatures and рН [4−8]. Since in references practically absent any data as to studies of bioSAS action on the activity of the ferments, we have studied an influence of the biosurfactants on fermentative activity with following interpretation of obtained results. That is why, the aim of the presented work was to study the influence of bioSAS on kinetics of the fermentative catalysis of the hydrogen peroxide decomposition reaction under the action of catalase.

2. EXPERIMENTAL Synthesized in our lab rhamnolipids and trihalosolipid [9] have been used in presented work as bioSAS. The value of catalase activity was expressed in mCat/l [10], and the activity itself was determined by spectrophotometrically with the use of spectrophotometer of UV–visible diapason Uvmini–1240 (P/N 206–89175–92; P/N 206–89175–38; Shimadzu Corp., Kyoto, Japan) at the wave length λ = 410 nm. Catalase has been purified via the following stages: і) centrifugation of culture broth of bacteria by Bacillus Sp. ShR−05 strain; іі) precipitation of protein from supernathant with acetone and ііі) ion exchange chromatography. In order to carry out the ion exchange chromatography the 50 mg of ferment preparation were re−dissolved in 5 ml of distilled water and after that were drifted on column (1,5 × 25 sm) with DEAE−Toyopearl 650 M (Toyo Soda MFG, Co. Japan). Washing out of proteins was carried out using the following buffers: (і) 20 mM tris−HCl, pH 7,0; (іі) 10 mM Na−acetate buffer, pH 5,5; (ііі) 1 M NaCl in Na− acetate buffer, рН 5,5. With the aim of carrying out more precise investigations, it has been done the eliminating and purification of catalase from the supernathant of culture broth of the Bacillus Sp. ShR−05 strain. Characteristics of separate stages of the ferment eliminating and also the efficiency of catalase purification are represented in Table 1 and on Figure 1. Obtained in mentioned above purification way catalase was used for carrying out the following experimental investigations with the use of bioSAS.

Kinetics of the Fermentative Reaction of H2o2 Decomposition under the Action… 157 Table 1.Characteristic of separate stages of the ferment eliminating and also the efficiency of catalase purification

Stage of purification

Volume, ml

General protein, mg

General activity, mCat/l

Supernathant Acetone DEAE−Toyopearl 650 M

250 80

400 48

79 47

Specific activity, mCat /l·mg 0,16 0,84

60

1,62

5,5

6

Multiplicity purification

Yield upon activity, %

(1) 5,3

(100) 60

36

6

Figure 1. Ion exchange chromatography of catalase on DEAE−Toyopearl 650 M.

3. RESULTS AND DISCUSSIONS In a case when the concentration of substrate exceeds the concentration of the ferment [S0] >> E0, that is usual condition for the study of fermentative reactions kinetics, the rate equation for these reactions can be written as

υ=

k 2 [ E0 ][ S ] , K S + [S ]

(1)

A. A.Turovsky, R. O.Khvorostetsky, L. I. Bazylyak et al.

158

where υ is the reaction rate; k2 is the rate constant of the ferment−substrate complex decomposition; [E0] is the concentration of ferment; [S] is the concentration of substrate; KS is the constant of ferment−substrate complex dissociation. For initial rates of the reaction, when the discharge of substrate can be neglected, that is, when [S] = [S0], we will obtain

υ=

k2 [ E0 ][ S0 ] K S + [ S0 ]

(2)

Equation (2) describes the dependence of rate for the fermentative reaction, proceeding of which is ordered to the scheme (3): k1

kat E + S ⇔ ES ⎯k⎯→ E+P

(3)

k−1

on the initial concentration of substrate and permits practically to determine the constants k2 KS, which are very important characteristics of the enzymatic reactions. In a case, when k2 and KS are effective, in other words, they are depend on рН of medium, presented in a system of inhibitor or activators, k2 is called by «catalytic constant» (kkat), and KS is the Michael’s constant (or КМ(im.)). In this case the equation (2) takes a form

υ=

k kat [ E0 ][ S0 ] K М ( уявн.) + [ S0 ]

(4)

The equation (4) is called by Michael’s−Menten equation. Composition kkat[E0] is maximal rate (Vmax). At the great concentrations of the substrate S, (S0 >> KM), the ferment «saturated» by the substrate and, the rate of the catalytic process kinetically is controlled by chemical transformation of ferment−substrate complex

υ = k[ES ]

(5)

Or

kT − υ= e h

ΔG ≠ внутр . RT

[ ES ]

(6)

Thermodynamic efficiency of the fermentative catalysis is determined by a difference of free energies of external molecular (at the formation of Michael’s complex) and ≠

intermolecular (in the transitional state ( ΔG int . )) of bond formation between the groups of ferment and the substrate. In this case the motive force of the catalysis is a free energy of

Kinetics of the Fermentative Reaction of H2o2 Decomposition under the Action… 159 interaction between the groups of a ferment and the substrate into transition state of the reaction (but not into an intermediate complex). It is necessary to notify, that very often in biology references under the «catalytic activity» it is understand the change of general reaction rate per some period of time. However, it should be remembered that the catalytic process foresees a decrease of the potential reaction barrier that is increasing of their rate constant (k). The reaction rate depends both on the value k, and on the concentrations of the reagents. The value k is accepted as a criterion of the catalytic reaction. In the presented case kkat places this role in biocatalysis. Typical figure in coordinates 1/V on 1/S is represented on Figure 2.

Figure 2. Dependence of the reaction rate on concentration of the substrate in coordinates of the Lainuiver−Berck’s equation.

Kinetic characteristics of the biocatalysis reaction of peroxide hydrogen by catalase (Vmax and kkat) under different conditions are represented in Tables 2.1 − 2.4. Table 2.1. Dependence of the biocatalysis reaction kinetics for peroxide hydrogen by catalase on concentration RL at t = 20 0 Concentration RL, ml/mg control 0,010 0,025 0,075 0,100 0,125

Vmax, mlg/ml·min. 2,35 3,07 4,00 5,00 6,66 5,71

k2, min.−1 0,94 1,22 1,60 2,00 2,66 2,28

160

A. A.Turovsky, R. O.Khvorostetsky, L. I. Bazylyak et al.

Table 2.2. Dependence of the biocatalysis reaction kinetics for peroxide hydrogen by catalase on concentration RL at t = 40 0C Concentration RL, ml/mg control 0,010 0,025 0,075 0,100 0,125

Vmax, mlg/ml·min. 2,55 2,95 4,12 5,40 6,52 5,90

k2, min.−1 1,02 1,18 1,64 2,16 2,60 2,36

Table 2.3. Dependence of the biocatalysis reaction kinetics for peroxide hydrogen by catalase on concentration RL at t = 60 0C Concentration RL, ml/mg control 0,010 0,025 0,075 0,100 0,125

Vmax, mlg/ml·min. 3,00 3,60 4,90 6,10 7,30 5,90

k2, min.−1 1,20 1,44 1,96 2,44 2,92 2,36

Table 2.4. Dependence of the biocatalysis reaction kinetics for peroxide hydrogen by catalase on concentration RL at t = 80 0C Concentration RL, ml/mg control 0,010 0,025 0,075 0,100 0,125

Vmax, mlg/ml·min. 3,10 3,40 3,95 6,00 6,85 5,80

k2, min.−1 1,24 1,36 1,58 2,40 2,74 2,30

We can see from the presented Tables 2.1−2.4, that at the bioSAS (ramnolipid (RL)) concentration increasing, Vmax is increased up to some concentration RL. However, at the bioSAS concentration in a field of the micelle−formation (0,2 мг/мл) the reaction rate is decreased. The values kkat during the reactions proceeding under different concentrations of RL are also increase; this fact proved the some decrease of the process energy activation value depending on the concentration of RL. Tenuous decrease of Vmax at the change of RL concentration can be explained by micelles aggregation that is by decreasing the active surface on which the catalytic reaction takes place. Properly, as to mechanism of the catalysis it is enough in a complicated manner somewhat to affirm − this fact needs a special studies of the role of a series of bioSAS by different nature in catalysis.

Kinetics of the Fermentative Reaction of H2o2 Decomposition under the Action… 161 It is necessary to notify, that the Michael’s−Menten constant is insignificantly decreased depending on the concentration of RL and temperature. Since

K Мен. =

k −1 k kat + k1 k1

(7)

(see. eq. (3)), this means that at least the ratio of the constants

k −1 k1

and

k kat k1

without RL and

in the presence of RL is changed insignificantly, in other words, it can be assumed, that in reactions k1

kat E + S ⇔ ( ES ) ⎯k⎯→ E+P

(8)

k−1

k1

kat E sol + S sol ⇔ ( ES ) sol ⎯k⎯→ E sol + P

k −1

is the equality

k −1 k −' 1 ≈ ' k1 k1

і

with taken into account that

' k kat k kat ≈ ' k1 k1

(9)

.

k kat k = K Міх . That is, k1 = kat . k1 K Міх

If the reaction practically displaced in a side of the Michael’s complex, then there is possibility enough simply to determine the constant rate k1. Interesting phenomena can be observed in the reactions of the peroxide H2O2 decomposition at the temperature variation. We can see from the Tables 2.1−2.4, that Vmax. is somewhat increased at the temperature increasing in the presence of high concentration of bioSAS. At low concentrations of bioSAS, the Vmax. is changed insignificantly. It can be concluded from the data of Tables 2.1−2.4, that the constants kkat. at the temperature variation is changed insignificantly. This means that the activation energy of a process is low and is neared to zero. We can see from Figure 3, that the Arrhenius’s law for above described systems is not realized. Among reasons of its omission is an increasing the ferment denaturations like to protein under higher temperatures. If we take into account the fact, that the denaturation under low temperatures is insignificant and to calculate the activation energy of peroxide decomposition in absence of bioSAS in a range of temperatures 20-40 0С, then it can be obtained the value Е

A. A.Turovsky, R. O.Khvorostetsky, L. I. Bazylyak et al.

162

neared to 1 ccal/mole. If to take into account RL, then the value Е consists of not more than 0.1 ccal/mole, that is, practically, zero.

1,2

lg kkat

1,0 0,8 0,6 0,4 0,2 0,0

0,0029

0,0030

0,0031

0,0032

1/T, K

-1

Figure 3. Dependence of kkat on temperature in Arrhenius’s coordinates.

With taken into account the described above circumstances, the reaction rate constant practically is equal to preexponent, which, in a practical, does not depend on temperature, that is:

KT k =χ e h where

χ

ΔS ≠ R

ΔH

e − RT

(10)

is the transmission coefficient; k is the rate constant; К is Boltzman’s constant; h is

Plank’s constant;

KT k =χ e h

ΔS

≠

is an activation entropy. Since the term e

ΔS ≠ R

−

ΔH ≠ RT

≈ 0 , then (11)

≠

Value ΔS for reactions at lower and higher temperatures is changed slightly. Rate constants at the lowest and at the highest temperatures are differed in 1,2 times: ΔS ≠

KT − χ 1e R k1 h = ≈ 1,2 ΔS ≠ k2 KT2 − R χ e h

(12)

Kinetics of the Fermentative Reaction of H2o2 Decomposition under the Action… 163 It is necessary to notify, that the rate of biocatalytic reaction of hydrogen peroxide decomposition depends on temperature, since the catalytic constant depends on temperature, however its role in presented process is not dominating and increasing the rate with temperature of process increase can be conditioned by conformational transitions of ferment, complex−formation at the expense of hydrogen bonds «ferment−substrate−RL», and also by a role hydrophobic interactions «ferment−H2O2−RL». As to ferment denaturation at different temperatures it can be assumed that: 1. At low concentrations of SAS and low values of temperatures it will be dominated the process of hydrogen bonds formation H2O2−RL−E and denaturation of the ferment will be low; 2. At low concentrations of RL and high temperatures the ferment denaturation will be higher, the stabilization of the substrate will be less, and, respectively, the rate will be less; 3. At high concentrations of RL and low temperatures, the concentration of the ferment and the substrate centers is more; respectively, the denaturation is less and the rate is high; 4. At high concentrations of SAS and high temperatures the denaturation of the ferment is strong, the stabilization is less at the expense of hydrogen bonds, however, the high concentration of SAS is prevail. The rate can be changed slightly or enough greatly. Kinetics of biocatalytic decomposition of H2O2 in the presence of trigalosolipide (TGL) practically is slightly differed from the kinetics of biocatalytic decomposition of H2O2 in the presence of rhamnolipide (see Tables 3.1−3.4). Table 3.1.Dependence of biocatalysis reaction kinetics of hydrogen peroxide by catalase on concentration of Th at t = 20 0C Concentration Th, ml/mg control 0,010 0,025 0,075 0,100 0,125

Vmax, mlg/ml·min. 2,35 3,07 4,00 5,00 6,66 5,71

k2, min.−1 0,94 1,22 1,60 2,00 2,66 2,28

Table 3.2. Dependence of biocatalysis reaction kinetics of hydrogen peroxide by catalase on concentration of Th at t = 40 0C Concentration Th, ml/mg control 0,010 0,025 0,075 0,100 0,125

Vmax, mlg/ml·min. 2,55 2,95 4,12 5,40 6,52 5,90

k2, min.−1 1,02 1,18 1,64 2,16 2,60 2,36

A. A.Turovsky, R. O.Khvorostetsky, L. I. Bazylyak et al.

164

Таble 3.3.Dependence of biocatalysis reaction kinetics of hydrogen peroxide by catalase on concentration of Th at t = 60 0C Concentration Th, ml/mg control 0,010 0,025 0,075 0,100 0,125

Vmax, mlg/ml·min. 3,00 3,60 4,90 6,10 7,30 5,90

k2, min.−1 1,20 1,44 1,96 2,44 2,92 2,36

Table 3.4. Dependence Of Biocatalysis Reaction Kinetics Of Hydrogen Peroxide By Catalase On Concentration Of Th At T = 80 0C Concentration Th, ml/mg control 0,010 0,025 0,075 0,100 0,125

Vmax, mlg/ml·min. 3,10 3,40 3,95 6,00 6,85 5,80

k2, min.−1 1,24 1,36 1,58 2,40 2,74 2,30

It can be seen from the presented Tables, that on all given interval of the bioSAS concentrations the value Vmax is increased not more than in 3 times. Decreasing the values Vmax and kkat at higher concentrations of bioSAS can be explained by aggregation of SAS micelles, and at decreasing the data of values at higher temperatures, possible, it is necessary to take into account the denaturation of biocatalyst (catalase). Probably, that such assumptions demand the carrying out of the additional investigations. Under the temperature reaction proceeding in the presence of TGL the values Vmax and kkat are changed insignificantly. Estimated value of the activation energy of catalytic reaction in absence of bioSAS consists of ~ 1 ccal/mole; in the presence of TGL this value consists of not more 0,1 ccal/mole. It can be considered, that Еact. ~ 0, that is the change of catalytic constants at different temperatures is conditioned by ratio:

KT2 h = T2 , KT χ 1 T1 h

χ

where Т2 is the highest temperature of the experiments carrying out; Т1 is the lowest temperature of the experiments carrying out. Constants rate at the highest and the lowest temperatures are differed in 1,2 times. Generally, it is necessary to notify, that the nature of bioSAS (RL and TGL) in some manner has an influence on catalytic constant of the process, and its insignificant increase is

Kinetics of the Fermentative Reaction of H2o2 Decomposition under the Action… 165 explained by activity of the catalase centers. However, this assumption demands the future experiments carrying out.

REFERENCES [1] [2] [3] [4] [5] [6]

A.N. Eriomin, D.I.Mietielitsa, G. Smietan. Biokhimiya, 1984, 49, 6, 976-984. S. Helenius, K Simons. Biochem. Biophys. Acta, 1975, 415, 1, 29-79. C.A. Nelson J. Biol. Chem., 1971, 246, 2, 3895p3901. G. Bognolo Physical Chemistry and Engineering, 1999, 152, 41-52. Z.Eliora, R.and E. Rosenberg. Env. Microbiology, 2001, 3, 4, 229-236. A.M Bezborodov. Biotechnologiya produktov mikrobnogo sinteza. М.: «Agropromizdat», 1991, 235 p. [7] E.V. Karpenko, N.B. Martynyuk, Shulga A.N. Patent of Ukraine № 71222, bull. № 12, 2004. [8] EV. Karpenko, R.I. Vildanova. Shcheglova. Appl. Biochem. and Microbiology, 2006, 42, 156-159. [9] A.N. Shulga, E.V. Karpenko, S.A. Elysseev, R.I. Vildanova-Martsishyn, Patent of Ukraine № 10467 A, 1996. [10] Korolyuk M.A., Ivanova L.I., Mayorova I.G., Tokaryev V.E. «Laboratornoye dielo», 1988, 1, 16-19.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 13

THERMODYNAMIC AND KINETICS OF THE METALPORPHYRIN – BASE REACTIONS Tatyana N. lomova Institute of the Solution Chemistry of Russian Academy of Sciences,1 Ivanovo, Russia,

ABSTRACT The results of studying the axial coordination of tetraphenylporphyrin complexes of high-charged metal cations (AcO)CrTPP, O=Mo(OH)TPP and O=W(OH)TPP with molecular ligands (hydrogen sulfide, imidazole and pyridine) in toluene are discussed. The thermodynamic and the kinetic characteristics of reactions between metalloporphyrin and molecular ligand were obtained by the method of spectrophotometric titration and chemical kinetics. Correlations between the molecular ligand basicity and the molecular complex stability are discussed.

INTRODUCTION The coordinative capacity of metal cations in metalloporphyrins is not exhausted by the formation of simple metal - porphyrin complexes. Most of the porphyrins are capable of coordinating more molecular or anionic ligands to yield extra complexes, which is one of the most important properties of this class of compounds [1, 2]. The thermodynamics of extra coordination of oxygen or nitrogen ligands has been studied mostly for M2+ porphyrin complexes [3-6]. Preliminary studies [7-11] showed that, among porphyrin complexes of high-charged (z > 2) Group IV-VI d-metal cations, chromium(III), molybdenum(V) and tungsten(V) porphyrins are most sensitive to the action of hydrogen sulfide, imidazole and pyridine in an inert solvent (toluene). This type of reactions has hitherto been poorly understood for metalloporphyrins containing acido ligands, i.e., for complexes of highcharged cations. In this study, we report on the results of studying the rates and equilibria of 1 153045 Akademicheskaya, 1, Ivanovo, Russia, Fax +7 0932 33 62 37, e-mail: [email protected].

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the reactions of (AcO)CrTPP, O=Mo(OH)TPP and O=W(OH)TPP metalloporphyrins and molecular ligands (hydrogen sulfide, imidazole and pyridine) in toluene.

EXPERIMENTAL Materials The complex (AcO)CrTPP was synthesized by the procedure in [12]. (Acetato)chromium (III)tetraphenylporphine. H2TPP and Cr(AcO)3 (taken in a 1 : 5 molar ratio) and benzonitrile were placed in a flask and refluxed at 468 K for 12 h. Then, the mixture was cooled, and the solvent was removed in vacuo. The dry residue was dissolved in a minimal possible amount of CHCl3. This solution was applied to a column packed with activated Al2O3, Brockmann II, and chromatographed with the use of CHCl3. The yield of the complex was 40 %. The electronic absorption spectra of (AcO)CrTPP (λmax, nm (log ε)) were 604 (3.90), 565 (3.99), 525 (3.65), 451 (5.20), 394 (4.34), and 358 (4.18) in CHCl3 and 602 (3.99), 564 (4.05), 525 (3.70), 449 (5.33), 398 (4.63), and 364 (4.45) in acetic acid (the spectrum corresponds to that described in [13]). The IR spectrum in KBr (ν, cm-1) was 798 (γC-H), 1009 (νC-C, νC-N) and 1342 (γC=N) (vibrations of pyrrole ring bonds); 1540 (skeletal vibrations); 702, 755 (γC-H), 1067, 1173 (δC-H), 1483, 1575 (νC=C), and 3050, 3085 (νC-H) (vibrations of benzene ring bonds); 1617 (O-C-O); and 445 (νCr-N). The O=Mo(OH)TPP and O=W(OH)TPP were prepared, as octaethylporphine (H2OEP) complexes [12], by complexing H2TPP with MoO3 and WCl6, respectively. (Oxo)(hydroxo)molybdenum(V)tetraphenylporphine. H2TPP (0.1 g, 0.16 mmol) was refluxed with MoO3 (0.076 g, 0.53 mmol) in 0.8 g phenol at 454 K for 4 h. Dry air was continuously fed to the reactor. To isolate the solid complex, phenol was distilled off in vacuum. Then, a saturated chloroform solution of the complex was prepared and twice chromatographed on activity grade II alumina. The yield of the complex was 60 %. The electronic absorption spectrum in CHCl3 (λmax, nm (log ε)) was 620.0 (2.94), 584.0 (2.92), 456.0 (3.78). (Oxo)(hydroxo)tungsten(V)tetraphenylporphine. H2TPP (0.1 g, 0.16 mmol) was refluxed with WCl6 (0.2 g, 0.53 mmol) in 0.8 g phenol at 454 K for 4 h. The complex was isolated and purified in the same way as O=Mo(OH)TPP. The yield of the product was 60 %. The electronic absorption spectrum in CHCl3 (λmax, nm (log ε)) was 621.0 (2.85), 585.0 (3.11), 449.0 (4.25).

Spectroscopic Measurements UV-Vis spectra were recorded on SF-26 and Specord M-400, and the IR spectrum was recorded on a Specord 75-IRr. The MP reactions with molecular ligand (hydrogen sulfide, imidazole and pyridine) in toluene were studied by spectrophotometric titration at 298 K on a Specord M400.

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Spectrophotometric Studying of Thermodynamics and Kinetics The thermodynamic characteristics of the reactions were determined by the method spectrophotometric titration (Figure 1, 2). Temperature was maintained constant accurate to ± 0.1 K. The reactions of MP with molecular ligand in toluene were studied in a wide range of the extra ligand concentrations (10-6 ÷ 101 M). The stability constants of the (L)MP extra complex were determined by the equation valid for three-component equilibrium systems,

A e -A o A∞ - A o K = A -A 1 - e o A∞ -Ao

.

1 Ae -Ao CL - C oMP A∞ - A o

(1),

where С0МP and СL are the initial concentrations of the metalloporphyrin and extra ligand, respectively, in toluene and Ao, Ae, and A∞ are the optical densities at the wavelengths of measurements for metalloporphyrin and an equilibrium mixture at certain ligand and extra complex concentrations. The K constants were calculated by the method of least squares using the Microsoft Excel program. The relative error of determining K did not exceed 30%.

Figure 1. (a) Electronic absorption spectra of (AcO)CrTPP in toluene (1.70 × 10-5 M) at various hydrogen sulfide concentrations of (1) 0 and (2) – (8) 6.50 × 10-3 - 7.15 × 10-2 M and (b) the corresponding titration curve.

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The kinetics of the reaction was studied spectrophotometrically at 298 K. The rate constants were calculated by a formally first-order kinetic equation taking into account that the base was present in excess with respect to the metalloporphyrin,

A -A∞ 1 k eff = τ ln o Aτ - A ∞

(2)

Here, Ao, Aτ, and A∞ are the optical densities of the metalloporphyrin solution at the wavelength of measurements at time moments 0,τ, and after reaction completion. The relative error in determination of keff was 2 - 10%.

Figure 2. Curves of spectrophotometric titration of the reaction of O=W(OH)TPP with Py in toluene. CPy, M: 4.13 × 10-6 - 1.65 × 10-4 (a), 1.65 × 10-4 - 2.48 × 10-3 (b), 2.48×10-3 - 1.49 × 10-2 (c).

DISCUSSION OF RESULTS Reactions of (AcO)CrTPP with H2S, Im and Py Chromium(III) is known [14] to be a typical complex-forming metal characterized by the formation of complexes with oxygen- and nitrogen-containing ligands. When bound to a

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complex with porphyrin, which occupies four coordination sites in the coordination sphere of Cr(III), chromium(III) is capable of coordinating molecular and anionic ligands [7]. The (AcO)CrTPP reaction with H2S in toluene can be treated as the one-stage replacement of the single-charge anionic ligand AcO- in the coordination sphere of the complex by SH-: (AcO)CrTPP + H2S ↔ Cr(SH)TPP + AcOH

(3)

The equilibrium constant of the reaction at 298 K was 41 M-1. The experimental data were treated in the log ((Ae – Ao)/(A∞ - Ae)) – log CL coordinates by the method of least squares (tan n ≈ 1, ρ = 0.995) to determine the stoichiometry of the reaction (3). The (AcO)CrTPP reactions with Im and Py [8, 9] are more complicated than the preceding reaction with H2S. The reaction was found to be a complex process including reversible and irreversible elementary events (Tables 1 and 2), namely, two-step equilibrium coordination of the Im molecule at the sixth coordination site and the replacement of AcO- by Im in (AcO)(Im)CrTPP with the formation of the [(Im)2CrTPP]+(AcO)- ion pair (K1 = 2300 M-1, rate constant k1 = 2.18 s-1×mol-1×l and K2 = 820 M-1) and irreversible displacement of AcO- from the first coordination sphere of (AcO)(Im)CrTPP (k = 2500 s-1×mol-2×l2) after establishing equilibrium at the first stage (K1). The irreversible step determined the rate of the overall reaction between (AcO)CrTPP and Im(Scheme 1).

Scheme 1.

Processing the log ((Ae - Ao)/(A∞ - Ae)) – log CL relationship supports the stoichiometry of reactions (n1, 2 ≈ 1). The temperature dependences of the equilibrium constants (Figure 3) were used to determined the thermodynamic parameters of both stages of the reaction between (AcO)CrTPP and Im in toluene. At the first stage, the reaction is an exothermic process (∆Ho1 = -10 ± 1 kJ/mol) with a small positive ∆So value [∆So1 = 30 ± 3 J/(mol K)]. The second stage is an endothermic reaction (∆Ho2 = 21 ± 3 kJ/mol) with a large positive ∆So value [∆So2 = 126 ± 10 J/(mol K)].

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Table 1. The equilibrium constants (K1, K2, K3, M-1) for different axial coordination reactions at 298 K Molecular ligand H2S

(AcO)CrTPP K1 = 41

O=Mo(OH)TPP K1 = 83

Py

K1 = 400 K2 = 2.8

Im

K1 = 2300 K2 = 820

K1 = 9140 K2 = 39.3 K3 = 1.0 K1 = 1850 K2 = 480 -

O=W(OH)TPP K1 = 176 K2 = 100 K1 = 13300 K2 = 8400 K3 = 89 K1 = 205500 K2 = 747 -

Table 2. Reaction orders in СL and true rate constants of direct and reverse coordination reactions of extra ligand with metallotetraphenylporphyrin Molecular ligand

Py

Im

(AcO)CrTPP

O=Mo(OH)TPP

O=W(OH)TPP

k1 = 0.197 s-1×mol1/2 1/2 ×l k-1 = 4.92×10-4 s1 ×mol1/2×l-1/2 m1 = 0.5

k1 = 5.25 s-1×mol-1×l

k1 = 1.81 s-1×mol-1×l

k2 = 1.83×10-2 s-1×mol-1×l

k2 = 1.32 s-1×mol-1×l

k3 = 1.19×10-3 s-1×mol-1×l k-1 = 5.75×10-4 s-1 k-2 = 4.65×10-4 s-1 k-3 = 1.20×10-3 s-1 m1, 2, 3 = 1

k3 = 4.44×10-2 s-1×mol-1×l k-1 = 1.36×10-4 s-1 k-2 = 1.57×10-4 s-1 k-3 = 5.01×10-3 s-1 m1, 2, 3 = 1

k1 = 2.18 s-1×mol-1×l k-1 = 9.47×10-4 s-1 m1 = 1 k(1-2) = 2500 s-1×mol2 2 ×l m(1-2) = 2

Because the second reaction stage (Scheme 1) developed in time and equilibrium with constant K1 was established not instantaneously, we were able to spectrophotometrically measure the rates of the direct equilibrium reaction (k1) and of the transformation of the (AcO)(Im)CrTPP product into the [(Im)CrTPP]+(AcO)- ion pair (k(1-2)). The linear dependence log keff = log k + m log CL

(4)

was treated by the method of least squares to determine the true rate constant values and reaction orders in Im concentration (Table 2).

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Figure 3. Dependences of -ln K on 1/T for the reaction between (AcO)CrTPP and Im in toluene; (1) at the first and (2) at the second stage; correlation coefficients 0.990 and 0.981, respectively.

The equilibrium process of pyridine coordination by (AcO)CrTPP is characterized by the equilibrium constants K1 = 400 M-1 and K2 = 2.8 M-1 (Table 1) and the rate constant k1 = 0.197 s-1×mol-1/2×l1/2 (Table 2). At the first stage of the reaction with K1 (Eq. (5)), one pyridine molecule interacts with two (AcO)CrTPP molecules (reaction order in СPy is 0.5) (Figure4). In the second equilibrium (K2) (Eq. (6)), the 1:1 extra complex is formed. 2(AcO)CrTPP + Py ↔ [(AcO)CrTPP]2(Py)

(5)

(AcO)CrTPP + Py ↔ (AcO)(Py)CrTPP

(6)

Figure 4. log keff – log CL for the reactions of (AcO)CrTPP with Py (1) (stage 1, m = 0,5, ρ = 0,996), of O=Mo(OH)TPP with Py (2) (stage 3, m = 1, ρ = 0,995), and of O=W(OH)TPP with Py (3) (stage 3, m = 1, ρ = 0,996).

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The stability of extra complexes, characterized by the equilibrium constants of their formation, changes along the series Im (2300) > Py (400) > H2S (41).

(7)

This sequence coincides with the sequence of the growth in the protonation energy of the molecular ligand (E, kJ/mol) (398, 438, and 520 for Im, Py, and H2S, respectively; are calculated by Zajceva and Zdanovich). K1 changes roughly in proportion to E. In all cases, the coordination of L results in the bathochromic shift of the B(0,0) band of the π → π* transition. This allows us to assume that the stability of the (AcO)(L)CrTPP complexes in series (7) changes in line with an increase in the metalloporphyrin – extra ligand σ-bond strength. The π-acceptor action of Im and Py, as well as the interaction of dπ orbitals of Cr(III) with vacant d orbitals of sulfur in H2S, is immaterial for the coordination of molecular ligands by metalloporphyrins, presumably, due to the high stability of the CrIII t32g configuration in complexes. The reaction of metalloporphyrin with Im [8] is 50 times faster than the reaction with Py [9]. The decrease in rate constants, as well as K1 values, corresponds to an increase in the protonation energy.

Reactions of O=Mo(OH)TPP with H2S, Im and Py The complexes of molybdenum(V) contain anionic ligands, in addition to the porphyrin macrocycle, in their first coordination sphere [15]. In O=Mo(OH)TPP the O2- and OHanionic ligands are trans to the N4 plane. The O=Mo(OH)TPP reaction with H2S leads to the bathochromic shift of the band with λmax = 472 nm [10]. Processing of the log ((Ae – Ao)/(A∞ - Ae)) versus log CL data gives a nearunity value for the H2S stoichiometric index in the reaction in question (n = 1.02) (Figure 5). From these data, the O=Mo(OH)TPP reaction with H2S in toluene can be treated as the replacement of the single-charge anionic ligand OH- in the coordination sphere of the complex by SH- (Table 1): O=Mo(OH)TPP + H2S ↔ O=Mo(SH)TPP + H2O.

(8)

The reaction between Im and O=Мо(OH)TPP was found to be a complex multistage process (K1 =1850, K2 = 480 M-1, n1, 2 ≈ 1): O=Mo(OH)TPP + Im ↔ O=Mo(OH)(Im)TPP

(9)

O=Mo(OH)(Im)TPP + Im ↔ [O=Mo(Im)2TPP]+OH-

(10)

At the first stage, the reaction is an exothermic process (∆Ho1 = -28 ± 2 kJ/mol) with a small negative ∆So value [∆So1 = -30 ± 5 J/(mol K)]. The second stage is an endothermic reaction (∆Ho2 = 9 ± 1 kJ/mol) with a large positive ∆So value [∆So2 = 81 ± 1 J/(mol K)]. The thermodynamic characteristics of reaction (10) agree well with the suggested scheme of chemical transformations in the Mo complex – Im system.

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Figure 5. log ((Ae - Ao)/(A∞ - Ae)) – log CL for the reactions of O=Mo(OH)TPP with H2S (1) (ρ is 0.995), of O=Mo(OH)TPP with Py at the first (2), the second (3), and the third (4) stages (ρ is 0.996, 0.995, and 0.977, respectively).

The exothermic character of the process and the large positive ∆So value at the second step (Eq. (10)) show that the reaction occurs with the formation of less solvated species compared with the reagents because of the desolvation of Im and the initial coordination unsaturated O=Мо(OH)TPP complex. The reaction between Py and O=Мо(OH)TPP was found to be a complex process including three equilibrium elementary events (11) – (13) (Tables 1 and 2). O=Mo(OH)TPP + Py ↔ [O=Mo(Py)TPP]+OH-

(11)

[O=Mo(Py)TPP]+OH- + Py + H2O ↔ [Mo(OH)(Py)2TPP]2+ . 2OH-

(12)

[Mo(OH)(Py)2TPP]2+ . 2OH- + Py ↔ [Mo(Py)3TPP]3+ . 3OH-

(13)

n1, 2, 3 ≈ 1, (Figure 5, ρ1 = 0.996, ρ2 = 0.995, ρ3 = 0,977). The data of the kinetic investigation of the O=Mo(OH)TPP reaction with Py are resulted in Table 2 and at Figure 4. The results of studying equilibria of the reactions (oxo)(hydroxo)molybdenum(V)tetraphenylporphine with hydrogen sulfide, imidazole and pyridine allowed us to reveal some specific features of coordination of bases by the molybdenum(V) porphyrin complex. The stability of extra complexes of O=Mo(OH)TPP does not change along the series (7). The first-step reactions of O=Mo(OH)TPP with H2S and Py in toluene can be treated as the replacements of the single-charge anionic ligand OH- in the coordination sphere of the complex by SH- and Py. The first-step reaction of O=Mo(OH)TPP with Im is coordination of the Im molecule at the eighth coordination site.

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Reactions of O=W(Oh)TPP with H2s, Im and Py In O=W(OH)TPP, like in O=Mo(OH)TPP complex, the O2- and OH- anionic ligands are trans to the N4 plane. The O=W bonds O=W and W-N in O=W(OH)TPP is more stable than in O=Mo(OH)TPP [7, 13]. From the evolution of the O=W(OH)TPP electronic spectra in toluene as a function of H2S concentration at 298 K, the two-stage character of this reaction was inferred. The first stage can be treated as the replacement of the single-charge anionic ligand OH- in the coordination sphere of the complex by SH-: O=W(OH)TPP + H2S ↔ O=W(SH)TPP + H2O

(14)

The equilibrium constant of reaction (14) is 176 M-1 (Table 1). The IR spectra of O=W(OH)TPP complex before and after of its treatment by H2S [7] confirm the nature of the equilibrium reaction (14). The equilibrium with the K2 (15) corresponds probably to the replacement of the two-charge ligand O2- by two anionic SH- ligands. O=W(SH)TPP + H2S ↔ W(OH)(SH)2TPP

(15)

The equilibrium constant of the reaction (15) is 100 M-1 (Table 1). n1, 2, ≈ 1. The reactionы between Im and O=W(OH)TPP are similar to reactions (9) and (10).At the first stage, the reaction is an exothermic process (∆Ho1 = -47 ± 2 kJ/mol) with a small negative ∆So value [∆So1 = -57 ± 5 J/(mol K)]. The second stage is an endothermic reaction (∆Ho2 = 24 ± 1 kJ/mol) with a large positive ∆So value [∆So2 = 134 ± 4 J/(mol K)]. The reaction between Py and O=W(OH)TPP was found to be a complex process including three equilibrium elementary events, namely, the coordination of the Py molecule to the W atom with increasing its coordination number to 8 (K1 = 13300 M-1, k1 = 1.81 s-1×mol1 ×l), the coordination of the second Py molecule with the displacement of the OH- group into the second coordination sphere (K2 = 8400 M-1, k2 = 1.32 s-1×mol-1×l), and the coordination of the third Py molecule due to breaking of the M=O double bond and protonation at the oxygen atom (K3 = 89 M-1, k3 = 4.44 × 10-2 s-1×mol-1×l) (Tables 1 and 2). n1, 2, 3 ≈ 1. The nature and the sequence of the elementary reactions have been explained by the high bond strength of the O2- ligand with the W atom. O=W(OH)TPP + Py ↔ O=W(OH)(Py)TPP

(16)

O=W(OH)(Py)TPP + Py ↔ [O=W(Py)2TPP]+OH-

(17)

[O=W(Py)2TPP]+OH- + Py + H2O ↔ [(OH)W(Py)3TPP]2+ . 2OH-

(18)

The linear dependence (4) was treated by the method of least squares to determine the true rate constant values and reaction orders in Py concentration (Table 2). The rate constants of the reverse reactions were 1.36×10-4, 1.57×10-4, and 5.01×10-4 s-1×mol×l-1, respectively. The stability of extra complexes of O=W(OH)TPP changes along the series

Thermodynamic and Kinetics of the Metalporphyrin – Base Reactions Im (205500) > Py (13300) > H2S (176)

177 (19)

CONCLUSION The results of studying equilibria of the reactions of the metalloporphyrins with extra ligands allowed us to reveal some specific features of coordination of bases by the metalloporphyrin complexes. The stabilities of WV, MoV and CrIII extra complexes with H2S and Py, characterized by the equilibrium constants of their formation (K, M-1), change along the series (20) for H2S, and along the series (21) for Py. The series (20) and (21) coincide with the kinetic stability series [7, 13]. O=W(OH)TPP (K = 176) > O=Mo(OH)TPP (83) > (AcO)CrTPP (41)

(20)

O=Mo(OH)TPP (K = 9100) ≈ O=W(OH)TPP (8400) > (AcO)CrTPP (2.8)

(21)

ACKNOWLEDGMENTS This work was partially supported by Russian Foundation for Fundamental Research (grants no. 06-03-96343 and 07-03-00639). The author thanks Tipugina M.Yu. for cooperation.

REFERENCES [1] [2]

[3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

B.D. Berezin, O.I. Koifman Usp. Khim. 1980, 49, 2389. J.K.M Sanders., N. Kampos, Z. Clyde-Watson, et. al. In The Porphyrin Handbook, vol. 3. K.M Kadish., K.M Smith., Guieard R. (ed.). Academic Press: San Diego, San Francisco, N.-Y., Boston, London, Sydney, Toronto, 1999, 1. O.I Koifman., T.A Koroleva., B.D. Berezin Koord. Khim. 1977, 3, 1811. T.Takayanagi, H.Yamamoto, T. Kwan. Bull. Chem. Soc. Jpn. 1975, 48, 2618. Izatt RM, Bradshaw JS, Pawlak K, et al. Chem. Rev. 1992, 92, 1261. T.V.Karmanova, O.I.Koifman, B.D. Berezin. Koord. Khim. 1983, 9, 919. M.Yu.Tipugina, T.N. Lomova. Zh. Fizich. Khim. 2002, 76, 653. M.Yu. Tipugina, T.N.Lomova. Zh. Inorg. Khim. 2002, 47, 1085. M.Yu. Tipugina, T.N. Lomova. Zh. Inorg. Khim. 2004, 49, 1051. M.Yu. Tipugina, T.N.Lomova. Zh. Inorg. Khim. 2004, 49, 1. J.W. Buchler, K.Rohbock. Inorg. Nucl. Chem. Lett. 1972, 8,1073. T.N. Lomova, N.I. Volkova, B.D.Berezin. Zh. Inorg. Khim. 1985, 30, 626. A.A.Grinberg Vvedenie v khimiyu kompleksnykh soedinenii (Introduction to the Chemistry of Complex Compounds), Leningrad: Khimiya, 1971. W.R. Scheidt in The Porphyrins. D. Dolphin (ed.), Academic Press: New York, 1978.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 14

KINETICS OF THE FERMENTATIVE PROCESS IN STATIONARY STATE FOR SUNFLOWER-SEED OIL HYDROLYSIS BY LIPASE IN THE PRESENCE OF BIOSAS *

A.A. Turovsky, * R.O. Khvorostetsky, ** L.I. Bazylyak, *** and G.E. Zaikov

*Chemistry and Biotechnology of Combustible Minerals Division; Physical Chemistry of Combustible Minerals Department; Institute of Physical–Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko; National Academy of Science of Ukraine1 Lviv, Ukraine **Chemistry of Oxidizing Processes Division; Physical Chemistry of Combustible Minerals Department; Institute of Physical–Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko; National Academy of Science of Ukraine2 Lviv, Ukraine ***Kinetics of Chemical and Biological Processes Division; Institute of Biochemical Physics named after N. N. Emanuel; Russian Academy of Sciences3 Moscow, Russia

ABSTRACT The catalytic rate constants for the process in the presence of bioSAS by different concentrations have been obtained. It was shown, that the constants some increase at bioSAS concentration increasing up to their micelle−formation beginning. The temperature has a slight influence on the value of catalysis constants, that can be explained by practically zero activation energies and depend on activation entropy.

1

3а Naukova Str., Lviv–60, 79060, UKRAINE; e–mail: [email protected]. e–mail: [email protected]. 3 4 Kosygin Str., Moscow, 119991, RUSSIA; e-mail: [email protected]. 2

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INTRODUCTION One of the most problems in the fermentative catalysis is a problem of enzymes fermentative activity increasing depending on different physical−chemical factors. The aim of the presented work was to study the influence of concentration and nature of the biosurface−active substances (bioSAS) on kinetics of fermentative catalysis of sunflower−seed oil hydrolysis reaction by lipase. Kinetics was studied in stationary state accordingly to Michael’s−Menten conception. Sometimes in the fermentative kinetics biologists recognize as the criterion of fermentative activity the general rate of fermentative reaction that is not always true, since in accordance with the definition the catalysis of chemical reaction is determined by decreasing the value of reaction potential barrier, in comparison with non−catalytic reaction. That is why as the criterion of catalytic fermentative reaction should be accepted the catalytic constant which is the function of activation energy and preexponential multiplier. If such constant is changed via the process of fermentative reaction under the action of some medium factor, etc., and concentration of the reagents via the process does not change, then the change of the general rate of fermentative reaction can be used as a criterion of fermentative activity. If via the process some factor is changed (for example, concentration of the reagents), then the general rate does not characterize the change of fermentative activity. In this case it is necessary to use the catalysis constant as a criterion of fermentative activity.

EXPERIMENTAL The value of lipase activity was expressed in mCat/l [1], and the activity itself was determined by spectrophotometrically with the use of spectrophotometer of UV–visible diapason Uvmini–1240 (P/N 206–89175–92; P/N 206–89175–38; Shimadzu Corp., Kyoto, Japan) at the wave length λ = 410 nm. Hydrolysis of sunflower−seed oil by lipase was carried out in accordance with the technique described in [2]. Sunflower−seed oil was used with characteristic described in [3].

RESULTS AND DISCUSSIONS At studying the initial reaction rates (when the substrate consumption can be disregarded), we assume, that [S] ≈ [S0] and

υ=

k 2 [ E 0 ][ S 0 ] K S + [S 0 ]

(1)

where S is the concentration of substrate; υ is the rate of fermentative reaction, with proceeds in accordance with scheme:

Kinetics of the Fermentative Process in Stationary State for Sunflower-Seed Oil … 181 KS

k2 E + S ⇔ ES ⎯⎯→ E+P

The equation (1) permits experimentally to determine the constants k2 and KS. In a case when k2 and Ks are effective values (that is, they depend on рН medium and also side reactions take place and etc.), they are «catalytic constants kcat.» and imagine Michael’s constant Кimag. Then the eq. (1) will be as follow

υ=

k kat [ E 0 ][ S 0 ] K imag . + [ S 0 ]

(2)

The equation (2) is the Michael’s−Menten equation. It characterizes the hyperbolic dependence of fermentative reaction rate on the initial concentration of substrate and linear rate on concentration of ferment. Composition of kкаt [E0], which has the dimensionality of reaction rate is denominated as «maximal reaction rate (Vm)». Linearization of the experimental data in coordinates

1 1 , , which are denominated as υ S0

Lainuiver−Berck’s coordinates, permits to determinate the values υ max and Кimag. Kinetic regularities of catalysis under the conditions when the ferment «saturated» by substrate (that is, at S0 >> Ks), are some others. In this case the kinetics of fermentative process is determined by intermolecular chemical transformation of Michael’s complex ЕS in activated reaction complex [ES]#. The motive force of the catalysis is free energy of the interaction [ES]# in the transition state (but not into an intermediate complex ЕS). Thus, free activation energy is determined as a difference between free energy of activated complex [ES]# and free energy of Michael’s complex, that is, under such conditions the reaction of catalysis is «pseudomonomolecular». We can see from Tables 4.1−4.4, that Vmax. is increased till some concentration – 0,1 mgг/ml with the bioSAS concentration increasing. Following increase of the bioSAS concentration leads to the Vmax decreasing. This effect is explained by fact that the denoted range of bioSAS concentration is characterized by micelle−formation. Probably, the decreasing of surface aggregations from the micelles leads to Vmax. reducing. As we can see from Tables 4.1−4.4, the value of catalytic constant k2 is increased less in twice with bioSAS concentration increasing. These points out, some activation energy decreasing via the fermentative activity process. This process demands of separate detailed investigations; at the same time, it can be assumed the following scheme of reaction:

S + bioSAS ⇔ S ⋅ bioSAS

(3)

Е + bioSAS ⇔ Е ⋅ bioSAS

(4)

S ⋅ bioSAS + E ⋅ bioSAS ⇔ S ⋅ E ⋅ bioSAS →

(5)

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→ [S ⋅ E ⋅ bioSAS ] → E + P + bioSAS ≠

(6)

Activation energy is determined as the energies difference between activated complex [S E bioSAS]# and енергією Michael’s complex S E біоПАР. Probably, at bioSAS concentration increasing the Michael’s complex energy is rather increased, than the energy of activated complex that leads to decreasing the activation energy value and increase of the reaction rate constant. Increasing the energy of Michael’s complex can be explained at the expense of less thermodynamic probability of its formation. It’s not excepting that as a result of the fermentative reaction in the presence bioSAS it is important also the value of activation entropy ΔS#. In others words, generally the process should be characterized by value ΔF# of the reaction. It’s not excepting also is the process of the reagents concentration change under the action of bioSAS via fermentative reaction that is reflected on general rate of reaction and Vmax. This aspect also requires the additional experiments carrying out. We can see from Tables 4.1−4.4, that Vmax. and k2 in hydrolysis reaction are not much changed with variation of temperature. However, in the presence of enough great concentration of bioSAS it is observed some tendency to Vmax.і kкат.increasing. It is necessary to note, that the dependence of lgk2 on 1/Т is not rectilineal, in other words the Arrhenius’s equation is not fulfilled. The reason of this can be fact, that the constants are gross values. It’s not excepting, that at temperature increasing it is observed the reaction of protein (ferment) denaturation greatly. In such a case general rate of reaction will be consisted of V1 + V2, where V1 is the catalysis rate, and V2 is denaturation rate that leads to effective constants of hydrolysis. Table 4.1. Dependence of kinetic parameters of the fermentative catalysis on concentration of bioSAS at t = 20 0C Concentration RL, mgг/ml control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 2.35 3.07 4.00 5.00 6.66 5.71

k2, min.−1 0.94 1.22 1.60 2.00 2.66 2.28

Table 4.2. Dependence of kinetic parameters of the fermentative catalysis on the concentration of bioSAS at t = 40 0C Concentration RL, mgг/ml control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 2.55 2.95 4.12 5.40 6.52 5.90

k2, min.−1 1.02 1.18 1.64 2.16 2.60 2.36

Kinetics of the Fermentative Process in Stationary State for Sunflower-Seed Oil … 183 Table 4.3. Dependence of kinetic parameters of the fermentative catalysis on the concentration of bioSAS at t = 60 0C Concentration RL, mgг/ml control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 3.00 3.60 4.90 6.10 7.30 5.90

k2, min.−1 1.20 1.44 1.96 2.44 2.92 2.36

Table 4.4. Dependence of kinetic parameters of the fermentative catalysis on the concentration of bioSAS at t = 80 0C Concentration RL, mgг/ml control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 3.10 3.40 3.95 6.00 6.85 5.80

k2, min.−1 1.24 1.36 1.58 2.40 2.74 2.30

If assume, that at low temperatures the hydrolysis reaction is dominating (the range of temperatures 20−40 0С), then estimated activation energy of a process without bioSAS is equal to 1 ccal/mole. With taken into account the bioSAS in this range of temperatures, the activation energy is 0.1 ccal/mole (in other words, approximately in error limits). It can be considered that Еactiv. ≈ 0. The change of a rate at bioSAS concentration increasing proceeds at the expense of reagents concentration increasing; and kкат. increase takes place as a result of activation entropy increasing. As a result, the catalysis rate constant k2 is equal to preexponent, which weakly depends on temperature. Really,

kT k2 = e h

ΔS ≠ R

ΔH

e − RT

(7)

where k2 is constant of catalysis, k is Boltzman’s constant, h – is Plank’s constant, ΔS# is activation entropy of reaction, ΔН# is a heat of activation. When ΔН# ≈ 0 and ΔS≠ practically does not depend on temperature, we will obtained

kT k2 = e h

ΔS ≠ R

(8)

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184

In such a case the rate constants at the highest and the lowest temperatures will be

kT2 differed as multipliers ratio h

kT1 h

, or as T2

T1

; in other words the ratio is in ~ 1,2 times.

It is necessary to say some words as to Michael’s constants. Their values practically are not changed with the presence in reaction of bioSAS. Temperature has also an insignificant influence on their values. This fact is explained by fact that the ratio of elementary rate constants of fermentative catalysis of hydrolysis reaction both in absence of bioSAS, and also in their presence, are enough near. Michael’s constant

K imag .=

k1 + k 2 k1

(9)

or

K imag . = K eq. +

k2 k1

(10)

under condition, that k1 >> k-1

K imag .≈

k2 k1

(11)

In other words, knowing k2 and Кimag., it can be estimated k1. If for reaction k2 and Кimag. are near, then and k1 will be near. However, since k2 and Кimag. in the presence of bioSAS some are differed, respectively k1 also will be other. That is, reactive abilities of ferments and substrates will be in some manner differed.

REFERENCES [1] [2] [3]

Korolyuk M. A., Ivanova L. I., Mayorova I. G., Tokaryev V. E. Laboratornoye dielo, 1988, 1, 16-19. Becker G., Berger V., Domshke G. Organicum. Practicum on Organic Chemistry, 1979, 2, 447. Sunflower-seed oil, Unrefined "First-class", GOST Р 52465−2005.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 15

DYNAMIC TREND OF ENERGY EXCHANGE INTENSITY IN BRAIN UNDER CHRONIC STRESS Nana Koshoridze1 Iv. Javakhishvili Tbilisi State University2, Tbilisi, Georgia

ABSTRACT We studied dynamic trend of changes in the activity of creatinу kinase, aldolase and succinatdehydrogenase in brain cells under 30-day long stress induced by isolation and violated diurnal cycle. It was shown that these enzymes heterogeneously responded to 30-day long stress. Particular sensitivity was recorded by succinatedehydrogenase that showed the decline of activity in various sections of the brain at 60-80% on average. Unlike succinatedehydrogenase, aldolase activity increased on the 10th day of stress and then declined. Similar results were seen in relation to phosphokinase activity. It was observed that the change in the activity of the enzymes in question was accompanied by quantitative changes in nitric oxide-NO. In accordance with experimental data we suggested that the main signal molecule causing changes in enzyme activity should be NO.

INTRODUCTION Stress is a global problem closely linked with industrialization and globalization. An organism normally responds to stress as the demand to the individual comes to exceed the available personal and social resources [10]. Recent researches in the field have shown that chronic stress greatly affects biochemical indicators of an organism. According to reference materials, an organism under stress experiences failure of cell functions and becomes exposed for occurrence of various diseases such as atherosclerosis, amyotrophic lateral sclerosis, Parkinson’s Disease, Alzheimer’s Disease, Huntington’s Disease, etc. [1,2]. The fundamental reason for such diseases is the 1 E-mail: [email protected]. 2 1, Ilia Chavchavadze Ave., 0179 Tbilisi, Georgia.

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failure in the cell metabolism. In order to ensure a smooth flow of metabolic processes a cell requires energy generated by ATP. A stable available level of ATP is especially important for the cells with higher energy consumption, such as for those of the central nervous system and myocytes. Higher energy requirement – e.g. under convulsions, or reduced generation of energy makes the demand for ATP higher than the rate of its synthesis causing depletion of energy resources. Under such conditions quantitative composition of ATP is balanced by the creatine/creatinephosphokinase/phosphocreatine system. An efficient functionality of the system is regulated by the enzyme creatinephosphokinase (CK) that carries out transphosphorylation between creatine (CR) and phosphocreatine (PCr) [3]. There are 5 isoforms of the enzyme present in the cells. Three of them are cytosolic and the remaining two are mitochondrial. Mitochondrial phosphokinase transforms creatine into phosphocreatine at the expense of the ATP synthesized in redox processes. Under certain neurodegenerative diseases the damaged sections of the brain contain a reduced level of cytosolic creatinphosphokinase, which is related to the posttranslational modification of the enzyme [4,5,6,7]. Specifically, the enzyme molecule is characterized by an increased content of carbonyl groups, which speaks of the significant role of posttranslational modification as a factor in the change of enzyme activity. It was shown that mitochondrial creatinephosphokinase (MtCK) hinders generation of oxygen radical that induces certain processes, such as apoptosis, ischemic and neurodegenerative diseases [1,2]. The data obtained through the research indicate that reactive oxygen has a destructive impact that leads to neuropathy. Thus, modulation of the enzymes regulating oxidation stress is the mainline in the development of appropriate therapy. The suggested assumption is based on the protecting effect of creatine towards Huntington’s and Parkinson’s diseases and amyotrophic lateral sclerosis. It is noteworthy that reduction in ATP synthesis in neurons is characteristic of neurodegenerative diseases. This means that creatine’s protecting effect is conditioned by formation of phosphocreatine that facilitates higher rates of ATP generation [8,9,10]. MtCK content in the brain is fairly high. MtCK activity links oxidative phosphorylation and production of mitochondrial PCr, thus, transforming Cr into PCr at the expense of the ATP generated in mitochondria [8]. PCr is further exported to the cytosol while the produced ADP is pomped back to the mitochondria, which leads to more intensive phosphorylation. It was also shown that increase in the activity of MtCK facilitates reduction of mitochondrial permeability, which can lead to cell apoptosis and necrosis. It was the aim of the research to establish efficiency of the enzyme systems that have an active role in the synthesis and generation of energy cells, particularly in the glycolysis and Krebs cycle, as well as using creatine phosphokinase activity as an example. The research consisted in the study of 30-day stress induced by circadian cycle and isolation by standard methods.

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187

EXPERIMENTAL Materials and Methods Experiments were held on sexually mature male albino laboratory rats. The rats were kept in individual cages in the dark (dark-to-light ratio – 23.5/0.5 hrs) and were socially isolated. The animals were kept under the above-described conditions during 30 days. The control group consisted of animals kept in a common cage under natural conditions (10.00/14.00 hrs). The experimental animals were put to sleep by means of chloroform and decapitated. The brain was removed. Mitochondrial and cytosolic fractions were obtained through differential centrifugation in the sucrose gradient [11]. Creatinekinase activity was determined in the incubatory compound containing the suspension under study (0.1 ml) and 0.5 ml of creatine solution (1.9 mM). The suspension was prepared on glycine buffer with pH 9.7. The compound was added 0.07 mM of ATP and incubated 60 minutes at 37 0С. The enzymatic reaction was terminated by addition of trichloracetic acid (14%). The mixture was then centrifuged at 3000 g. The supernatant was added the mixture of ammonium vanadate and ammonium molybdate (1:1) and the intensity of blue coloration was measured by spectrophotometry at λ=400nm [12]. Succinatedehydrogenase activity in the mitochondria was determined by colorimetry by means of 3-(4.5-dimethylthyasol-2-il)-2.5-diphenyltetrazolium bromide (MTT). The mitochondria were washed twice in the incubatory solution prepared on HEPES buffer (140 mM NaCl, 5 mM NaHCO3, 1.1 mM MgCl2, 5.5 mM glucose and 20 mM HEPES, pH-7.4) and incubated for 45 minutes at 370С in the incubatory solution that contained MTT (0.5 mg/ml) and 3 mM of succinate. Afterwards the solution was removed and the blue formasane product was dissolved in 0.3 ml of 100% dimethyl sulfoxide. Intensity of blue coloring was measured by spectrophotometry at λ=540 nm [13]. Aldolase activity was determined by measuring the amount of soda-and-labile phosphorus of triphosphates. The incubatory compound contained 1 ml of glycine buffer (0.1 M, pH-9.0), 0.25 ml of fructose-1.6-diphosphate (2 mM) solution and 0.25 ml hydrazine solution (0.1 M). 20 mcl of the solution under study was added into the incubatory compound and incubated during 3 minutes at 370С. The reaction was terminated by means of 2 M of NaOH. The samples were suspended during 20 minutes at room temperature and inorganic phosphorus. Enzyme activity was determined by spectrophotometry at λ=340 nm [14]. The concentration of creatine in the samples was measured by means of diagnostic test systems (Dia Sys, Germany). Intensity of nitric oxide synthesis was assessed by the product of NaNO2 reaction. To this end, 0.2 ml of Griss reagent was added into 0.4 ml of the mixture under study and incubated during 15 minutes at room temperature whereupon optic density was measured by spectrophotometry at λ = 540 nm [15]. Proteins were determined by Lawry assay. The results were processed by Student.

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DISCUSSION OF RESULTS Formation of chronic stress under various factors is known to be a staged process and consists in modification of physiological and biochemical parameters. Taking this fact into account, we studied change in enzymatic activity resulting from 10-, 20- and 30-day stress (Figure 2). It was found that 10-day long stress does not affect CK activity. The figure shows that enzymatic activity is reduced only after 10 days of stress, particularly during a 20-day period of stress. Compared to the control group, enzymatic activity of mitochondrial and cytosolic isoforms is reduced by 20-25%, with the exception of cytosol in the cortex of cerebral hemispheres, where CK activity shows an insignificant drop when compared to the samples with 10-day stress. As for the enzymatic activity under 30-day stress, the data remain virtually the same as with 20-day long stress. The exception here was the mitochondrial fraction from the hippocampus, where a 15% reduction in enzymatic activity was recorded. 25

20

Control 10-day stress

15

20-day stress 30-day stress

10

5

0 Hc

Hm

Cc

Cm

Figure 1. Trend of Change in Creatine Phosphokinase Activity of White Rat Brain under Chronic Stress Ordinate: creatine phosphokinase activity in µmol Рi/mg protein-1 min-1. Hc: cytosolic fraction of hippocampus, Hm: mitochondrial fraction of hippocampus. Cc: cytosolic fraction of cerebral hemisphere cortex, Cm: mitochondrial fraction of cerebral hemisphere cortex. 1,2 1 0,8

Control 10-day stress

0,6

20-day stress 30-day stress

0,4 0,2 0 Hm

Cm

Figure 2. Trend of Change in Succinate Dehydrogenase Activity of White Rat Brain under Chronic Stress Ordinate: change in optic density at λ=540nm Hm: mitochondrial fraction of hippocampus, Cm: mitochondrial fraction of cerebral hemisphere cortex.

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189

It is known that CK activity is related with the process of oxidative phosphorylation. Enzymatic activity is presumably proportional to the intensity of mitochondrial processes [3, 7]. In order find the right answer to this issue we studied activity of succinate dehydrogenase – an enzyme from Krebs cycle in the cortex and hippocampus subjected to chronic stress. It was found that the enzyme activity in both sites of white rat brain, if compared to the control, is lower. It is necessary to note that high enzymatic activity is characteristic to the cortex of cerebral hemispheres. Meanwhile a 10-day chronic stress reduces the enzyme activity approximately by 70% (Figure 2). The same was much lower under 30-day stress. It is known that apart from oxidative phosphorylation there is a cytosolic, anaerobic process – glycolisis involved in the supply of a cell with ATP. Hence, we were also interested in establishing intensity of glycolisis under chronic stress. The process was studied on the example of aldolase activity. The results are presented in Figure 3. The data show that 10-day chronic stress causes an abrupt increase in aldolase activity (approximately by 200%). Thereafter the activity has a similarly acute drop (90%) compared to 10-day stress. These date indicate a critical reduction in the intensity of glycolisis. The data presented herein indicate that 30-day chronic stress caused by violation of circadian cycle and isolation is accompanied by both the reduction in creatine phosphokinase activity and ATP synthesis, which affects the production of energy-rich compounds in the sufficient quantity. The hippocampus is known to be in charge of very important processes, such as formation of emotions in animals, learning and memory. Failure in the energy balance under chronic stress must presumably have an impact on these processes. 35 30 Control

25

10-day stress 20-day stress

20

30-day stress

15 10 5 0 Hc

Cc

Figure 3. Change in Aldolase Activity of White Rat Brain under Chronic Stress. Ordinate: measurement of the amount of soda-and-labile phosphorus of triosophosphates, expressed in µmol Рi/mg protein1min-1. Hc: cytosolic fraction of hippocampus, Cc: cytosolic fraction of cerebral hemisphere cortex.

Therefore, it was our objective to reveal any intracellular signaling processes that provide for stability in the afore-mentioned metabolic processes. The most interesting results were obtained in relation with nitric oxide (NO). NO has a messenger function on the intracellular and intercellular levels and has an active part in the regulation of various metabolic processes, thus, providing for a successful performance of an organism. NO is in charge of regulating a number of enzymes, particularly components of the respiratory chain and enzymes present in

Nana Koshoridze

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glycolisis. Figure 4 shows that volume of NO under 30-day chronic stress is correlative of the changes in the activity of the enzymes under study. In particular, NO concentration was found to have risen. Reference data related to quantitative changes in NO under stress are heterogeneous [16,17]. If we take into account the results obtained through our research we can assume reduction in the activity of enzymes participating in mobilization and generation of cell energy may be caused by increased amount of nitric oxide. 1,4 1,2 1 Control 10-day stress

0,8

20-day stress

0,6

30-day stress

0,4 0,2 0 Hc

Hm

Cc

Cm

Figure 4. Trend of Change in NO Amount in White Rat Brain under Chronic Stress. Ordinate: amount of NO (mM). Hc: cytosolic fraction of hippocampus, Hm: mitochondrial fraction of hippocampus . Cc: cytosolic fraction of cerebral hemisphere cortex, Cm: mitochondrial fraction of cerebral hemisphere cortex.

90 80

Control 10-day stress 20-day stress

70 60

30-day stress 50 40 30 20 10 0 Hc

Hm

Cc

Cm

Figure 5. Quantitative Changes in Creatine Content in White Rat Brain under Chronic Stress. Ordinate: quantitative content of creatine (µmol ). Hc: cytosolic fraction of hippocampus, Hm: mitochondrial fraction of hippocampus. Cc: cytosolic fraction of cerebral hemisphere cortex, Cm: mitochondrial fraction of cerebral hemisphere cortex.

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NO is known to realize its effect in a cell in various manners, including its interrelation with creatine that is a substrate of creatine phosphokinase. It is also known that creatine has a protecting function in maintaining the redox status of a cell and any failure in the process leads to apoptosis or necrosis [18]. Therefore, we defined a trend in the concentration of creatine under 30-day chronic stress. The results are presented in Figure 5. The figure shows that the concentration of creatine changes after 10 days of stress. Although no significant changes were recorded on the 10th day, the indicator changed drastically on the 20th day and all the fractions under study had significantly lower creatine content compared to the control. Such results indicate that during the 30 days of chronic stress, functional changes in cell process occur, bringing about energy-related changes in the brain.

REFERENCES [1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

I. Cernak , V. Savic, J. Kotur, V. Prokic, B. Kuljic, D. Grbovic, M. Veljovic Alterations in magnesium and oxidative status during chronic emotional stress. Dev. of Research on Magnesium., 2000, 13, 29-36. H.Youn, I. Ji, H.P. Ji, W.R. Markesbery, Ji TH. Under-expression of Kalirin-7 Increases iNOS Activity in Cultured Cells and Correlates to Elevated iNOS Activity in Alzheimer's Disease Hippocampus., J. Alzheimers Dis., 2007, 12, 271-281. T. Kekelidze, I. Khait, A.Togliatti, J.M. Benzecry, B. Weiringa, D.Holtzman Altered brain phosphocreatine and ATP regulation when mitochondrial creatine kinase is absent. J. Neurosci. Res. 2001, 66, 866-872. M.Y. Aksenov, M.V. Aksenova, D.A. Butterfield, J.W. Geddes, W.R. Markesbery Protein oxidation in the brain in Alzheimer’s disease. Neuroscienc., 2001, 103, 373383. T.S.Bürklen, Hirschy A, Wallimann T. Brain-type creatine kinase BB-CK interacts with the Golgi Matrix Protein GM130 in early prophase. Mol. Cell. Biochem. 2007, 297, 5364. T.S. Bürklen, U. Schlattner, R. Homayouni, K. Gough, M. Rak, A. Szeghalmi., T. Wallimann. The Creatine Kinase/Creatine Connection to Alzheimer's disease: CKInactivation, APP-CK Complexes and Focal Creatine Deposits. J. Biomed Biotechnol. 2006, 3, 35936-35945. L.E. Meyer, L.B. Machado, A.P. Santiago, W.S. Da-Silva, et al., Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity. J. Biol. Chem.., 2006, 281, 37361-37371. N. Brustovetsky , R. LaFrance, K.J. Purl , et al. Age-dependent changes in the calcium sensitivity of striatal mitochondria in mouse models of Huntington's Disease. J. Neurochem. 2005, 93, 1361-1370. M. Dolder, B.Walzel, O. Speer, et al. Inhibition of the Mitochondrial Permeability Transition by Creatine Kinase Substrates. J. Biol. Chem.- 2003, 278, 17760-17766.

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[10] F.E. Pfeiffer, H.Homburger, T Yanagihara. Creatine kinase BB isoenzyme in CSF in neurologic diseases. Measurement by radioimmunoassay. Arch. Neurol., 1983, 40, 169172. [11] E. De Robertis Structural components of the synaptic region. Handbook of Neurochemistry, 1969, 2, 365-372. [12] I. Ueda, T. Wada Determination of inorganic phosphate by the molybdovanadate method in the presence of ATP and some interfering organic bases. Anal. Biochem., 1970, 37, 169-174. [13] K.Abe, A. Matsuki Measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and LDH release using MTT. J.Neurosci. Res., 1974, 38, 325–329. [14] A.Chappel, N.J. Hoogenraad, R.S.Holmts Purification and properties of the native form of rabbit liver aldolase. Biochem. J., 1975,175, 377-384. [15] K. Pahan, X. Liu, M.J. McKinney, C. Wood, F.G. Sheikh, J.R. Raymond Induction of nitric oxide synthaze and activation of nuclear factor-kB in primary astrocytes. J. Neurochem., 2000, 74, 2288-2295. [16] W.J. Welch, J.P. Suhan. Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J. Cell Biol., 1986, 103, 2035-2052. [17] M.W. Eysenck, M.T. Keane. Cognitive Psychology: A Student's Handbook (4th Ed.) 2000, Philadelphia: Psychology Press

PART 3. NEW COMPOUNDS FOR MEDICINE

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 16

SYNTHESIS AND TRANSFORMATION OF NEW ANEMIA-OPPOSITE ADAMANTANE DERIVATIVES OF FERROCENE Oliko Lekashvili1, Davit Zurabishvili, Levan Asatiani and Nodar Lekishvili Ivane Javakhishvili Tbilisi State University2, 1 Ilia Chavchavadze Ave., 0128 Tbilisi, Georgia

ABSTRACT Hydrazides are characterized with different pharmacological activity, amongst, compounds with inhibitory action of hydrophobia virus and human immunodeficiency virus. Therefore, we considered as perspective synthesis of adamantine containing hydrazides and anaemia opposite ferroocenes admixtures. Ferrocene-A(ferrocenyl-1phenyl-dioxy-1,4-butin-2) hes significant antitumour and antibacterial properties. The derivatives of adamantine have broad pharmacological activity, low toxicity, high membranotropic and antibacterial properties. The search new biologically active compounds, we have synthesized ferrocene- and adamantine-containing derivatives.

INTRODUCTION The derivatives of adamantine have significant pharmacological activity and they are used in medicine, especially against the viral and bacterial diseases. It is well known that these diseases are accompanied by the fall of hemoglobin, bat also it is known that derivatives of ferrocene effect against this process [1-6]. Adamantane containing compounds possess antivirus and wide spectrum of pharmacological properties, after long research we have established, that adamantine derivatives 1 E-mail: [email protected]. 2 1 Ilia Chavchavadze Ave., 0128 Tbilisi, Georgia.

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influence on reproduction of the stimulants of different microbial and virus infections and including anti activity of rabies and human immunodeficiency virus, also they have significant inhibitory activity and they are the most interesting objects for research against of those infection diseases [7-10], for which the medical preparation at the present time are not created. We have found that some derivatives of adamantine-1-carbon-acide hydrazide have antimicrobial and antivirus activity. Ferrocene and adamantane have unique structure conditioning their application ferrocene products are characterized with wide spectrum of biological activity; it is known the ferrocene containing polymers are applied as semiconductors. Products of ferrocene are characterized with the high heat stability, excellent dielectric properties. Alkyl derivatives of ferrocene are widely used in medicine, as preparations for treatment of disease caused by the iron deficiency in blood. Diatomic alcohols of ferrocene containing acetylene series are antioxidants and ant carcinogens. It should be noted that adamantanes as well are distinguished with unique pharmacological properties [7-9]. It was established that introduction of the adamantane lipophilic radical in the molecules of known biologically active compounds, frequently, causes reduction of the preparation toxicity and increase of the pharmacological action, that may be explained by immune and membrane-acting activity of the adamantane radical. Due to these properties, modification of preparations of different groups, amongst, antibiotics, glycosides, neuroleptics, steroids and others, by their adamantylation and adamantoilation reactions, with favorable results [9-10]. With the purpose of studying the biological activity, ferrocene containing dihydroxyacetylene compounds were synthesized [3-5]. Ant oxidation, antibacterial and ant carcinogenic action of certain representatives were established. It was revealed that 1ferrocenyl-1-phenyl-1,4-dihydroxy-2-butyne (Fc-A) displays ant carcinogenic action. In the moment of carcinogen application, existence of Fc-A in the blood prevents transformation of malignant tumor and at the same time effects the index of mitosis of human lymphocytes in the cell culture. Presumable mechanism of the ant carcinogenic action of the preparation was proposed [3]. Taking into account all the above mentioned, we have considered as perspective to perform synthesis of 1,1´-bis-(1-phenyl-1,4-dihydroxy-2-butynil)ferrocene, adamantane containing preparation Fc-A prototypes, adamantane and ferrocene containing hydroxylproducts of acetylene series and certain transformations, with the purpose of studying the comparative biological activity.

GENERAL RESULTS With the purpose of searching for the new biologically active organic and metal organic substances, synthesis and bioactivity of adamantane containing hydrazides and anaemia opposite ferrocenes admixtures end their transformation. Synthesized adamantine containing di and three acilhidrazides and ferrocene- and adamantane-containing oxy-derivatives of acetylene series.We have found antimicrobial and antivirus activety some of derivatives adamantine-1-carbonacide hydrazide.[7-12].

Synthesis and Transformation of New Anemia-Opposite Adamantane Derivatives… 197 We have synthesized N-adamantoyl-N/-arilidenhydrazides, N-adamantoyl-N/-acylhidrazides. Compound was obtained by boiling the adamantine-1-carbonacide with ethinilalcohol at the presence of sulfuric acid, and interaction the etilether adamantine-1-carbonacides with hidrazinhidrate.

COOH

HOC2H5 H2SO4

COO C2H5 H2N-NH2* H2O

CONHNH2

We have carried out acylation by corresponding anhydride and chloranhidride in abs. benzene and ether in the presence of triethilamine: Ac2O

CONH-NHCOCH3 +

COCH3

CONH-N III

II

COCH3

CONHNH2 ArCOCl TEA

I

CONH-NHCOAr Cl

O2N

Ar =

[IV],

Cl

Cl

[V],

Cl

[VI],

[VII].

HO

Scheme 1

Data of the IR, UV and NMR spectra confirm the structure of the synthesized compound (Fig 1 and Figure 2). Bioscrining of the azomethines obtained by us was performed for the antimicrobial activity by the method of diffusion in agar-agar. Compounds have selective action towards to the microorganisms, such as seracia, clebsiera and staphylococcus.

Figure 1. IR spectrra of compound V (Scheme 1).

Oliko Lekashvili, Davit Zurabishvili, Levan Asatiani et al.

198

Figure 2. 1H NMR spectrra of compound V (Scheme 1).

Ferrocene-A (ferrocenyl-1-phenyl-dioxy-1,4-butin-2) has significant antitumour and antibacterial properties. The derivatives of adamantane have broad pharmacological activity, low toxicity, high membranotropic and antibacterial properties. In order to search new biologically active compounds, we have synthesized ferroceneand adamantane-containing oxy-derivatives of acetylene series:

HO

HO

HO

HO C

C C R

C

,

R/

Fe

C

C

,

Fe

C

C CH3

R= R/= H, CH3,C6H5; R=CH3, R/=1-Ad, C6H5, Fc; RR/C=C10H14 These compounds were obtained by condensation of 2-hidroxy-2-ethyladamantane, ethynylbenzole, propargyl alcohol and lithium derivatives of ferrocene with corresponding ketones, in the medium of nucleophilic solvents (C2H5OC2H5, THF). The antimicrobial activity of synthesized compounds was studied. The correlation between antibacterial activity and chemical structure was estimated. Data of the IR spectra and elemental analysis confirm the structure and composition of the synthesized compound. In the IR spectra of the compound absorption bonds 3310-3300 cm-1 (≡C-H), 2100-2090 cm-1 (C≡C) are characteristic, and in the ketone spectra 1720-1660 cm-1 (>C=O). The said absorption bonds are not found in condensation products. In the IR spectra of the compound there are characteristic absorption bonds, cm-1:3600-3200 (OH); 3000-2830, 3120-3030 (C-H

Synthesis and Transformation of New Anemia-Opposite Adamantane Derivatives… 199 aromatic); 2970-2850, 1460, 1355, 1103, 770 (C-H adamantyl, alkyl); 2260-2220 (C≡Cdisubstituted). Table 1. Characteristic of Hydrazides (II-VII), (IR and IH NMR) figures #

M.p 0C

Rf meTanoli/ qloroform 1:12

IR speqtrebi, v,sm-1

I

II

161162

0.90

3240(N-H),3108(C-H ar), 2931,2854(C-H Ad),1697, 1650,1457(CONH),1373(C-N).

δ=9.6(s,1H),8.98(s,1H),2.04 (s,3H),1.84(s,9H),1.70(s,6H).

III

200

0.75

IV

228230

0.4

3425(N-H),2923,2854(C-H Ad), 1623(C=O),1589,1457 (CONH), 1218(CN).

δ=9.9(s,1H),9.5(s,1H),7.46(m,4H),1.9 9 (s,3H),1.87(m,6H),1.69(m,6H).

V

209210

0,64

3448,3386(N-H),3178(C-H ar.) 2931, 2854(C-H Ad), 1681,1643 (C=O),1373(C-N).

δ=10.08(s,1H),9.2(s,1H), 7.9(d,J=8.8Hz,2H),7.4(d,J=8.8Hz,2H), 2.05(s,3H),1.9(s,6H), 1.66(s,6H).

VI

110112

0,35

3448,3394 (N-H), 3093, 3030 (C-H ar.), 1681, 1643(C=O), 1357(C-N).

δ=10.54(s,1H),9.36(s,1H)8.2(d,J=8.8, 2.4Hz,1H),8.56(d,J=8.8Hz,1H),7.66(d ,J=2.4Hz,1H), 2.06 (s,3H)1.93(s,6H), 1.74(s,6H).

VII

140

0.52

3355, 3278(N-H), 2854, 2723 (C-H Ad),1689(C=O),1589,1457 (CONH), 1373(C-N).

δ=9.9(s,1H),9.4(s,1H)9.6(s,1H),7.64 (d,J=2.4Hz,1H),7.53(dd,J=8.8,2.4Hz,1 H),7.4(d,J=8.8Hz,H, 2.06(s,3H),1.96(s,6H),1.68 (s,6H).

H NMR, (DMSO -D6)

δ=11.4(s,1H),2.10,1.66(m,21H).

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

A.N. Nesmeyanov, N.S. Kochetkova. Progress of Chemistry 1974. TXLIII, pp. 15181523. G. Perevalova, M.D. Reshetova, K.N. Grandberg. Methods of Organic-Metallic Chemistry. Iron-Organic Compounds, Ferrocene. M. Nauka, 1983, 544 p. M. Pochkhidze, Z. Chitaishvili, L. Asatianin, M. Tsartsidze. Bull.Georg.cad.Sci.1999, 160, 1, 169-171. L.P. Asatiani, B.A. Lomsadze, C.Kh. Kiladze, S.Sh. Metskhvarishvili. Chem. Pharm. Journal. 1984-1985, 546-579. L.P. Asatianin, Z.Sh. Lomtatidze, et al. Organo-Metallic Bactericides. News of AS of Georgia. 1992, 146, 1, 66-69. L.P. Asatiani, Z.Sh. Lomtatidze, et al. News of AS of Georgia. 1989,133.3, 633-636. E.I. Bagri. Adamantanes. Obtaining, Properties, Application. Moscow."Nauka", 1989, 264 p. I.S. Morozov, V.I. Petrov, S.A. Sergeeva. Pharmacology of Adamantanes. Volgograd. Volgograd Medical Academy. 2001, 320 p.

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N.G. Artsimovich, T.S. Galushina, T.A. Fadeeva. Adamantanes - Medicines of the XXIst Century. Int.J.Immunorehabilitation. 2000, 2, 1, 55-60. [10] F. Szizicskai, I. Pelyvas, Z. Dinya, L. Sziligyl. Syntese und virushemmende in vitroWirkung neuerer 1 – Substituerter Adamantan – dwrivative. pharmazie, 1975, H 9, 571581. [11] G.I. Danilenko, E. A. Shablovskaya, L. A. Antonova, S. V. Guzhova, I. A. Lobanova, A.P. Diachenko, A.U. Tanasjuk, Synthensis and Protective Action of Phenyladamantane Derivatives with respect of Hydrophobia Virus. Chem. Pharm. Journal. 1988, 2 (Second edittion), 28-30. [12] G.I. Danilenko, S.P. Rybalko, Y. N. Maximov, V.F. Baklai, S.V. Guzhova. Chem. Pharm. Journal, 2000, 34, 1, 24-26.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 17

PHARMACOLOGICAL PREMISES OF THE CREATION OF NEW ANTITUMOR PREPARATIONS OF THE CLASS OF NITROSOALKYLUREA J. A. Djamanbaev1a, Ch. Kamchybekovaa, J. A. Abdurashitovaa, and G. E. Zaikovb* a Institute of Chemistry and Chemical Technology, Kyrgyz National Academy of Sciences2, Bishkek, Kyrgyzstan b N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences3, Moscow, Russia

ABSTRACT Perspectives in the field of creation of highly effective anticancerogenic preparations have been evaluated. For their creation is offered a new regio-selective method of glycosylation of alkylurea in conditions of nucleophilic catalysis with some following nitrosing of glycosyl carbamides of the D- and L-rows. This method opens principally new possibilities for modification of compounds by means of glycosylamides bond leading to preparations possessing small toxicity and high selectivity.

AIMS AND BACKGROUND In this work we tried to motivate searching and elaboration of the methods of the syntheses of the antitumor preparations among the derivatives of nitrosomethylurea with the purpose of obtaining new biologically active substances with high selective action, low toxicity by means of putting the carbamides fragment in to monosaccharides. 1 E-mail: [email protected]. 2 67 Chui Ave., 720 071 Bishkek, Kyrgyzstan. 3 4 Kosygin Street, 119 334 Moscow, Russia. E-mail: [email protected].

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These investigations are of great interest for the solution of practically important problems for physiologically active substances and medical preparations with known therapeutic action. The given direction provides working out of the recommendations on decrease of toxicity, change of water and lipid solubility preparations as well as obtaining derivatives with selective permeability through cell membranes [1]. A particular interest to nitrosoalkylurea, as potentially antitumor agents, was shown at the beginning of the 1960s, after revealing the high antileukemic activity of N-methyl-Nnitrozoguanidin and N-methyl-N-nitrosourea [2,3]. These compounds soon increased the essential range of antitumor preparations of alkylation action [4,5]. However, a number of side effects of the compounds of this type, first of all high myelotoxicity, restrained their introduction to medical practice. Modification of preparations by introduction to the molecular structure of different substituents has increased the range of potentially active compounds, but has not eliminated the undesirable influence of the preparations on an organism. The success reached in the pharmacology of nitrosoalkylureas is described in particular in Refs. 2 and 6. First natural carbohydrates analogue of N-nitrosomethylurea, the antibiotic ‘streptosotocin’, was gotten from cultural liquid—a streptomycin was found in the same years [4,7]. It was realised that ‘streptosotocin’ possesses a broad spectrum of actions—antidiabetic, diabetogenic, mutagenic and antitumor and presents by itself a carbohydrate derivative of nitrosomethylurea from secondary carbon atom of glucoses. The multiple pharmacological studies carried out showed that though ‘streptosotocin’ has the above-mentioned side effects, its general toxicity is much below the most citotoxic fragment, connected with atom C2 D-glucopiranosyd ring. These observations stimulated studies on syntheses and tests on antitumor activity of carbohydrate derivatives of N-methylN-nitrosourea. Repeatedly voiced suggestions that the carbohydrates molecules are the transport carrier, relieving carrying the citostatic groups in the tumors fabric promoted the development of this work. In the process of pharmacological investigations, it was ascertained that glycosylation leads to the sharp decrease of toxicity of medicinal preparations (LD50 falls, as a rule, by two orders). In clinical oncology, of great importance is not only the high antitumor activity of preparations of the group of nitrosourea, but also their ability to run through the hematoencephalitic barrier that opens real possibilities for chemotherapy of metastasis and primary tumors of the cerebrum. The majority of known preparations do not get through the hematoencephalitic barrier and so do not warn of a spreading metastasis in the cerebrum, and they are not practically efficient at defeat of the central nervous system. As to the selectivity of action, it is known that permeability of some cell membranes of animals regarding monosaccharides is different, and the degree of permeability correlates with the nucleophilic reaction ability of monosaccharides (Table 1) [8]. Thereof comes the principle possibility of processes regulation of the passing carbohydrates preparation through cell barriers by changing the nature of the carbohydrates’ carrier, as well as their biological activity. Attention must be given to the fact that a number of therapeutic characteristics greatly depend on the place of joining of a physiologically active fragment to the carbohydrates ring. It is especially revealed in the process of studying preparations of antitumor action.

Pharmacological Premises of the Creation of New Antitumor Preparations…

203

Table 1. The comparative dependence of the permeability of the monosaccharides from cell membranes from reaction ability of monosaccharides Monosaccharides Glucose Galactose Fructose Mannose Ksilose Arabinose Ribose Licsose

B1 1.0 1.1 0.4 0.2 0.15 0.09 -

B2 0 0.64 0.37 0.81 1.03 1.19 1.25

B1 – the comparative permeability of the monosaccharides. B2 - the comparative reaction ability of the monosaccharides.

Rather perspective for practical application, in our opinion, is the usage as ‘blending links’ of more acid-stable amide bonds. What is the value of the amide bond formation? What are the prospects of their use in applied aspects of the chemistry of carbohydrates? It is enough to point to the broad presence of urea fragments amongst natural materials—the derivatives of pyramidine of the row: nucleosides, riboflavin, teobromin, caffeine and others, and development of the direction of heterocycles syntheses, including analogues of the nucleosides on the basis of carbohydrates derivatives of urea. The development of the work on specified directions greatly depends on elaboration of technically acceptable and economically profitable methods of putting the carbamides fragment into mono-, oligo- and polysaccharides.

EXPERIMENTAL, RESULTS AND DISCUSSION, CONCLUSION As a result of searching conditions of the synthesis of N-nitroso derivatives of monosaccharides unprotected hydroxyl groups, providing the simplicity, effectivity of the process, it is perfected the general methods of getting N-alkyl-N-(β-D-glykopiranozyl)nitrosourea in which there were combined reactions: a) the interaction of monosaccharides with alkylurea under the condition of nucleophilic catalysis with addition of aryl amines and b) nitrozilation of N-alkylglycosylureas [9, 10, 11]. Methods of investigation: paper chromatography, element analysis, IR, PMRspectroscopy.

PREPARATION OF N-METHYL-N/-(Β-D-GLUCOSYL)N-NITROSOUREA A mixture of 1.98 g of D-glucose, 0.85 g of methylurea, 0.19 g of m-nitroaniline and 0.06 ml of a concentrated hydrochloric acid in 10 ml of methanol is heated at reflux for 20 minutes. The formed precipitate in the amount of 1.45 g is separated and added with 5 ml of

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glacial acetic acid, 1 .6 ml of distilled water, 0.83 g of sodium nitrite and stirred for 2 hours at the temperature of –2°C. The solution is evaporated; the residue is recrystallized from ethanol. The product yield is 1.44 g (90% of the theoretical). M.p. 180°C with decomposition, [α]D =-19° (water), Rf=0.56. Found, % N - 15.62. Calculated, % N -15.84. In this example and hereinafter the Rf is determined in the system: benzene-butanolpyridine-water 1:5:3:3.

PREPARATION OF N-METHYL-N/-(Β-D-GALACTOSYL)N-NITROSOUREA A mixture of 1.98 g of D-galactose, 0.85 g of methylurea, 0.19 g of m-nitroaniline and 0.07 ml of a concentrated hydrochloric acid in 10 ml of methanol is refuxed for 25 minutes. The resulting precipitate in the amount of 1.6 g is separated and added with 7.5 ml of glacial acetic acid, 1.5 ml of distilled water, 0.93 g of sodium nitrite and stirred for 2 hours at the temperature of –10C. The residue is filtered off, washed with an alcohol to give 1.26 g of the product (70% of the theoretical). M. p. 1210C., [α]D= +21.80 (water), Rf=0.53. Found, % N 15.50. Calculated, % N -15.84.

PREPARATION OF N-METHYL-N/-(Β-D-XYLOSYL)-N-NITROSOUREA A mixture of 1.5 g of D-xylose, 0.8 g of methylurea, 0.04 g of m-nitroaniline and 0.04 ml of a concentrated hydrochloric acid in 7 ml of ethanol is heated at reflux for 10 minutes. The resulting precipitate in the amount of 1.3 g is separated and added with 6 ml of glacial acetic acid, 1.2 ml of distilled water, 0.89 g of sodium nitrite and stirred at the temperature of –20C for 2 hours. The residue is filtered-off, recrystallized from an alcohol to give 0.9 g of the product (62% of the theoretical). M.p., 1090C (with the decomposition), [α]D=-21.90 (water), Rf=0.66. Found, % N - 17.62. Calculated, % N - 17.87. The study of the reaction with the participation of glycosyl bonds is important not only for the theory of the carbohydrates structure and reaction ability of carbohydrates. They present also a significant interest for solving a number of actual problems of bioorganic chemistry as the glycosyl bond is one of the most important structural elements of many biologically active compounds. Studying of the reactions of glycosyl centre has allowed the determination of some typical peculiarities of this type of nucleophilic catalysis. In this connection, the attention was concentrated on synthetic aspects of the homogeneous catalysis and elaboration of methods of directing synthesis of carbohydrates to physiologically active substances. They have much significance for the creation of medical preparations, in particular antitumor preparations, possessing low toxic activity, high solubility in water and high selectivity. Thus, a rather easy and mobile method of syntheses of a large group of carbohydrates derivatives of nitrosoalkylureas was worked out. On this basis it seems possible to have investigations of the biological activity of modified medical preparations with the help of the glycosylamides bond.

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REFERENCES [1]

N.N. Nikoliskiy, A.S. Troshin: The Transport Sugar through Cellular Membrane. Science, Leningrad, 1973. [2] N.M. Emanuel, D.B. Corman, L.A. Ostrovskaya, L.B. Gorbacheva, N.P. Dementieva: Nitrozoalkylureas - A New Class of Antitumor Preparations. Moscow, 1978, p. 290. [3] M.O. Grenn, J. Greenderg: The Activity of Nitrozoguanidines against Ascites Tumors in Mice. Cancer. Res., 1960, 20, 8, 1166. [4] U. Ross: Biological Alkylation Substances. Medicine, Moscow, 1964. [5] V.A. Chernov: Citostatic Substances in Chemotherapies of Oncomas. Medicine, Moscow, 1964. [6] N.N. Blohina, C.H. Zubroda: The System of the Creation of Antitumor Preparations in USSR and USA. Medicine, Moscow, 1977. [7] R.R. Herr, H.K. Jahnke, A.D. Argoudelis: The Structure of Streptozotocin. J. Am. Chem. Soc., 1967, 89, 4808, [8] V.A. Afanasiev, I.F. Strelicova, et al.: Construction and Reaction ability of Nglycosydes. Ilim, Frunze, 1976, p. 221. [9] J.A. Djamanbaev, V.A. Afanasiev: The Method of the Synthesis of N-alkyl-(N-aryl)-N(β-D-glykosyl)urea. A.C. № 772102 (USSR). [10] V.A. Afanaciev, J. A. Djamanbaev: Patent USA. 4: 656.259. 1987. [11] V.A. Afanasiev, J.A. Dzhamanbaev, G.E. Zaikov: The Derivatives of Carbohydrate with Carbamides Fragments. Successes in Chemistry, 1982, 51, 4, 661.

PART 4. BIOFIBERS

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 18

GRAFTING OF SOME BIOFIBRES WITH CARBOXYLIC ACIDS UNDER COLD PLASMA CONDITIONS C. Vasile11, M. Totolin1and M.C. Tibirna2 1

Romanian Academy, “P.Poni” Institute of Macromolecular Chemistry, Department of Physical Chemistry of Polymers2, Iasi, Romania 2 Laval University, Québec, Canada

ABSTRACT Physical and biochemical functionalisation of bast fibres are ways to improve thermo- and moisture regulation, anti-bacterial anti-allergies, hygiene, creating “smart” textile. Enhancing natural properties of vegetable fibres is an intermediary step in the obtaining of new products with special applications. The vegetable fibers are biodegradable, can be recycled, and in natural state are highly polar and hydrophilic. To improve the properties of the cellulosic fibers, the chemical and/or physico – chemical modifications were applied. The surface esterification of the natural polymer with acids can be carried out to obtain biodegradable materials, novel fibres with tailored functionalities for special applications. In this paper, starting from Spanish broom (Spartium junceum, syn. Genista juncea) fibers, under action of cold plasma discharges, and using different kinds of carboxylic acids, cellulose esters with short and long side chains have been synthesized. The new grafted polymers were characterized by FT – IR spectroscopy (ATR), XPS and SEM in order to assess the existence of incorporated functional groups. The thermal characterization of the obtained fibres reveals their particular behaviour.

INTRODUCTION The development of synthetic materials (e.g. plastics) at the beginning of the 20th century has caused the steady replacement of bio-based products. As a result of this change in raw 1 [email protected]. 2 41A Grigore Ghica Voda Alley, Ro 700487 Iasi, Romania.

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material utilization, combined with an enormous increase in energy and chemical demand, the world is now facing an ecological crisis. This crisis will greatly intensify with the expected growth in demand for industrial products in developing countries. It has been estimated that global industrial output will be five to ten times that of world production in 1987, when the world population stabilizes some time in the 21st century. Thus, the world community is facing a challenge of having to decrease pollution levels while at the same time, significantly increasing industrial output. Such predictions have led to a number of political initiatives, including support for enhanced industrial use of renewable resources (e.g. biomass) at the expense of non-renewable resources (plastic, glass fibres etc.). Plant fibres may therefore face a renaissance, not only for past uses, but also for the manufacture of three-dimensional products by hot-pressing of fibre mats or by extrusion or injection moulding of plant fibres in combination with plastic [1-4]. A classification of the plant fibres is given below – Scheme 1 [5].

Scheme 1. Classification of the plant fibres.

The applications of the plant fibres are very diverse such as: technical yarns, mechanical bonded nonwovens, various fields of application as reinforcing fibre, friction linings, paper production. Automotive components including natural fibres are currently being used by the following vehicle manufactures (as: Fiat, Ford, Mercedes Benz, Opel, Peugeot, Renault). Plant fibres have also found application in production of cement-based composites. A number of alternative crops have been identified particularly for southern Europe, while some progress has been made, as further production, processing and market development is required for large scale commercialization. A range of harvesting processes are being developed for each crop but harvesting cost and efficacy is still a limiting factor to economic production of high quality fibre. Much of the benefits of products derived from plant fibres are built on biodegradability. These benefits must be seen to be maintained in all new products. Spanish Broom a member of the Pea family (Leguminosae), and known botanically as Spartium junceum (, syn. Genista juncea), also known as Weaver's Broom, is a perennial, leguminous shrub. It is a native of the Mediterranean region and the Canary Islands, in southern Europe, southwest Asia and northwest Africa, where it is found in sunny sites, usually on dry, sandy soils and is often cultivated. Spanish broom is a handsome shrub with long switch-like green few-leaved or leafless branches and large yellow sweet-scented papilionaceous flowers. Spanish Broom typically grows to 2-4 m tall, rarely 5 m, with main

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 211 stems up to 5 cm thick, rarely 10 cm. It has thick, somewhat succulent grey-green rush-like shoots with very sparse small deciduous leaves 1-3 cm long and 2-4 mm broad. The whole plant, but especially the flower shoots and seeds have a bitter taste and tonic and diuretic properties, and were formerly used medicinally. The fibres of the young stems were used in making nets, carpets, mats, baskets, etc. The stem fibres are a hemp substitute being used mainly for coarse fabrics, cordage and paper. The stems are very pliable and can be used in basketry. It is also used for stuffing pillows etc., and for making paper. The smaller stems are used in basket making. The branches are often made into brooms. A yellow dye is obtained from the flowers. An essential oil is obtained from the flowers; it is used in perfumery [6]. During the last years, an increasing awareness of the public opinion about environmental and health problems pushed towards the utilization of natural raw materials, drawing the attention to industrial fibre crops. Industries all over the European Community are looking for raw material for replacing artificial fibres in composite materials to alleviate problems related with composite materials disposal at the end of the technical life. In Europe the use of natural fibres in the automotive industry in 1999 was about 21,300 tones and in 2000 about 28,300 tones. In 2005 the use of natural fibres was about 70,000 tones and in 2010 could increase to more than 100,000 tones of natural fibres for the effect of EU end-of-life vehicle directive than influence this development. The sources of raw material used in composites for automotive industry are mainly represented by flax, hemp, jute, kenaf, sisal and coconut fibres. Recently there has been a revival of interest in Spanish Broom as a possible source of natural fibre in automotive industry. Natural abundance, much higher strength per unit weight than most inorganic fillers, lower density and their biodegradable nature make natural fillers attractive as reinforcements of engineering polymer systems [7 – 9]. Spanish Broom cortical fibres are multiple elementary fibres (ultimates) arranged in bundles. The elementary fibres are bound together by lignin. A thick secondary cell wall indicates high cellulose content. The diameter of ultimates varies from 5-10 μm while the diameter of the whole bundle is about 50 μm. The values in tensile strength and elastic modulus are promising supporting the hypothesis that these fibres can be a potential replacement for man made fibres in composite materials. Spanish broom (Spartium junceum) fibres are new biofibres used as such or in composites [10]. The improvement of the properties is necessary in most purposes. Chemical processes of esterification with these acids are developed in special solvents for cellulose, in the presence of acid chlorides, pyridine and trifluoroacetic acid at very long reaction times. In these cases the pollution is high. [11] To avoid these drawbacks, a physical way of functionalization of vegetable fibre is proposed [12]. A highly hydrophobic product with retention of fibrous structure was obtained by the he reactions with higher saturated fatty acids (C10–C18) yielded lower DS values but still comparable hydrophobicity [13]. Possible ways of Spanish broom valorization are given in Scheme 2 [14, 15]. Enhanced fibre properties for improving the properties of new products and applications can be obtained by: new processing and production concepts include the development of environmentally friendly and energy-efficient processing and surface modification of fibres, yarns and fabrics. Creation of new natural polymer surfaces by plasma grafting and the deposition of thin film coatings by plasma assisted processes are applied in order to: improve: hydrophilisation, dyeing, reactive dyeing, printing, increase of the flame retardance and thermal insulation properties, increase abrasion resistance, to obtain higher conductivity, barrier layers to chemicals, UV protection, etc. use of various gases, mixtures of gases and monomers in vapor form and solutions, new softeners, to remove the impurities (reduction of chemical

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pretreatments) which are summarized in the Scheme 3. Special applications are also for: specific protein attachment [16], biomolecule (e.g. heparin) immobilization [17], improved cell attachment and spreading [18, 19] reduction of calcium carbonate nucleation [20] siloxane coating [21] to create smart clothes / wearable computing [22] elaboration of technologies for nano and micro coatings on textiles, etc.

Scheme 2. Possible ways of Spanish broom (Spartium junceum, syn. Genista juncea) valorization.

Scheme 3. Low temperature plasma: effects on textile.

Several low temperature plasma systems are known as : low pressure plasma systems (glow discharge) and atmospheric plasma systems (corona discharge, dielectric barrier discharge, glow discharge) The last one can be integrated into the production line manufacturing processes and also offers some advantages such as sample size is unlimited, are running at lower temperatures, [23, 24] and secondary reaction are avoided. Use of various gases, mixtures of gases and monomers in vapor form and solutions can be used [25 -

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 213 29]. Composites of natural fibers and thermo-plastics can be combined to form new enhanced materials. One of the problems involved in this type of composites is the formation of chemical bonds between the fibers and the polymers at the interface. Low energy glow discharge plasmas are used to functionalize cellulose fibers implanting polystyrene between the fibers and the matrix that improve the adhesion of both components, the adhesion in the fiber-matrix interface increasing with time in the first 4 min of treatment [30]. Methods of textile functionalization by plasma treatment include: a) plasma polymerization using different reactive gases, monomers or prepolymers, mixture of gases and monomers; b) plasma activation which mainly involves incorporation of new functional groups, which can be achieved by treatment with solutions and physical vapor deposition. Different procedures have been used to modify the cellulosic fibres from jute [31], wood fibres [32] cotton fabrics [33] for which the effect of corona discharge consists in the removal of impurities (reduction of chemical pretreatments), improvement of mercerisation process and dye uniformity in continuous dyeing, improvement of rubbing fastness of pigment printing and also it can influence the chemical finishing (softening, crease recovery, flame retardancy) and physical properties of linen fabrics and make raw cotton hydrophilic. The uniformity of dyeing is similar when the wetting agent in the recipe is replaced by a corona treatment. The authors claim that by corona treatment the wetting agents in different operations can be avoided, for example, in desizing, mercerization, bleaching and dyeing, higher number of barium is obtained in cotton mercerization, higher yield, penetration and rubbing fastness are obtained in pigment printing. In easy-care finishing, higher crease angle recovery, lower formaldehyde release, as well hydrophilisation is obtained. This paper deals with plasma grafting of Spanish broom (Spartium junceum, syn. Genista juncea) fibres with several acids in order to establish the optima conditions for their modification and obtaining of new improvements.

1. EXPERIMENTAL Materials and Methods The composition of the Spanish broom (Spartium junceum) fibres undergone to plasma treatment and those modified were globally characterized by FT-IR spectroscopy. The FT-IR spectra have been recorded by means of a DIGILAB Scimitar Series FT-IR spectrometer (USA) at 4 cm-1 resolution. Five recordings were performed for each sample and the evaluations were made on the average spectrum obtained from these five recordings. FTIR spectra are recorded in KBr pellets. Processing of the spectra was done by means of Grams/32 program (Galactic Industry Corp.). The FT-IR spectra of the sample under study are given in Figure 1 and the assignment of the bands is presented in Table 1.

C. Vasile, M. Totolin and M. C. Tibirna

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Absorbance, a.u.

0.3

0.2

0.1

0.0 4000

3500

3000

2000

1500

W avenumber, cm

-1

1000

Figure 1. FT-IR spectra of the fibres from Spanish broom (Spartium junceum) fibres.

Table 1. Assignment of the FT-IR bands from the spectrum of Spanish broom (Spartium junceum) fibres Band position (cm-1)

2913 s

Band assignment O(2)H…O(6) intramolecular H – bonds in cellulose, OH intermolecular, H – bond in the 10Ī plane O(6)H…O(3) intermolecular H bonds in cellulose Asymmetric CH valence vibration

2861 sh

Symmetric CH valence vibration

3418 vs 3277 sh

1723 sh 1639 s 1503 sh 1453 s 1429 s 1373 s 1320 s 1277 m 1239 m 1202 sh 1160 s 1114 s 1057 s 1031 s 896 w

2

2

C=O stretch in unconjugated ketone, carbonyl and in ester groups (frequently of carbohydrate origin) Protein impurity and water associated with lignin C=C stretching of the aromatic ring (G)CH deformation CH in pyran ring symmetric scissoring; OH in plane bending in cellulose I and cellulose 2

II H-O-C in plane bending of alcohol groups CH bending in cellulose I and cellulose II CH wagging in cellulose I and cellulose II 2

CH bending in cellulose I and cellulose II OH in plane deformation, also COOH OH in plane bending in cellulose I and cellulose II C – O – C asymmetric stretching in cellulose I and cellulose II Ring asymmetric stretching in cellulose I and cellulose II C – O valence vibration mainly from C(3) – O(3)H Stretching C – O in cellulose I and cellulose II Anomere C – groups, C – H deformation, ring valence vibration 1

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 215 In spectrum are identified all the bands of the main components mentioned in literature cellulose, lignin, pentosans, and extractives and also traces of amine groups [34]. A qualitative appreciation of the relative content of cellulose and lignin in these samples can be obtained from the ratios of the integral adsorption which are presented in Table 2. These ratios are proportional with lignin content which lied between 25 and 30 % [35, 36]. Table 2. Lignin/Carbohydrates ratio from FT-IR integral absorbances Sample

Relative intensities of aromatic skeletal vibration I /I I /I I /I 1505 1738

Spanish broom fibres

Height 0.88

1505 1375

Area 0.55

Height 0.38

1505 1158

Area 0.24

Height 0.44

Area 0.40

According to the literature data, the chemical composition of the whole stem outlined a high content of cellulose (67-76 %) while lignin (13-22%), pentosans (4-5%) and extractives (6-7%) were low [37]. The following carboxylic acids have been used: butyric acid, oleic acid, olive oil, sunflower oil, lactic and polylactic acids. Butyric acid was purchased from Merck, its purity is >99% being grade for synthesis; Lactic acid was also purchased from Merck, its purity was between 88 – 92 %; Olive oil contains between 55.0 - 83.0 oleic acid, 3.5 - 21.0 % linoleic acid and 7.5 - 20.0 palmitic acid, 0.3 - 3.5 % palmitoleic acid, 0.5 - 5.0 stearic acid and others in amounts less than 1%; Oleic acid from sunflower oil which belongs to the vegetable oils group with a high content in mono and polyunsaturated acids of about 94 ,90% ( 23.7 % oleic acid and 59.8 % linoleic acid) and saturated acids 11,30% (mainly palmitic and stearic). It contains 99,90 % lipids, trigliceride 99,20%, moisture content 0,1% [38]. Polylactic acid was laboratory synthesized and it has a number average molecular weight of Mn = 2000.

Procedure of Grafting by Cold Plasma The experimental set - up for cold plasma fibers’ grafting is presented in figure 2. In a typical experiment, after several washing cycles with inert gas (nitrogen) from the gas metallic reservoir (6), into the cylindrical shaped vacuum plasma reactor (1), the working pressure was established (0.3 mm Hg) and then the R.F. power was transferred to the reactor through the semi cylindrical, external, silver-coated electrodes (8). The R.F. power was dissipated to the electrodes from a R.F. generator (11) with the possibility of generating 50 300 W. The samples were deposited on special glass support for fibres exposure to grafting (12). The Spanish broom fibres have been treated in plasma at a P=300 W, frequency 13.56 MHz, pressure 0.3 mm Hg for 5 or 10 minutes. Before treatment the samples were impregnated with solutions of acids as: butyric acid, sunflower oil and olive oil (solutions 20 % in acetone) or with lactic and polylactic (Mn = 2000) acids (solution 4 % in ethanol).

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Figure 2: Experimental set-up for fibers’ grafting: 1 – vacuum plasma reactor; 2 – close glass vacuum system; 3 – vacuum gauge; 4 – vacuum pump; 5 and 7 – glass valves; 6 – monomer flask (only for distilled monomers in plasma medium, not for impregnated samples); 8 – semicylindrical external silver coated electrodes; 9 – glass support for samples; 10 – central monomer’s admission glass tube; 11 – HF generator (13.56 MHz); 12- special support for fibres exposure to grafting.

Unbounded acid from the treated samples was extracted for 6 h in a Soxhlet extractor. The treated fibres were dried and analysed. After plasma treatment all modified fibres become much softer comparatively with untreated fibre.

2. INVESTIGATION METHODS FT-IR spectroscopy – see above.

2.1. XPS Analysis of the Fibres Surface XPS spectra have been recorded by means of an Axis-Ultra de Kratos (UK) instrument equipped with an electrostatic analyzer with a great radius and a detection system having 8 channels. Two X-rays sources are used one double of Al-Mg without monochromator and one of Al with monochromator. The system is also provided with a source of electrons of low energy in order to neutralize the electrostatic charge which will appear on the samples when they are exposed to the monochromatic X-rays beam. The spectrometer operates in a high vacuum of 5·10-9 mm Hg. The XPS chamber of analysis is connected with a

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 217 transport/preparation chamber having multiple uses allowing rapid introduction of the samples in the position suitable for analysis. The samples in quartz crucibles have been introduced in XPS chamber and a pressure of 1-3·10-7 mm Hg was realized which is also necessary for the XDR source running. The XPS spectra were acquired with an Al source of 300 watts power. For the elemental composition determination the recording of the total spectra was done with an energy of analyzer of 160 eV, with step size of 1 eV, lens act in hybrid mode which assures the maximum sensitivity. The spectra of high resolution for chemical analysis have been recorded using an energy of 20 eV, step size having 50 meV (lens in hybrid mode). X-rays diffraction (XRD) analysis was done using a Bruker diffractometer equipped with a Kristalloflex 760 sealed-tube copper anode generator, operated at 40 kV and 40 mA, and a two-dimensional position-sensitive wire-grid detector (Bruker AXS) pressured with xenon gas. Collimation was effected by a graphite monochromator with a 0.8-mm pinhole sampleto-detector distance = 9 cm. Samples were placed in sealed Mark-Röhrchen glass capillaries (Charles Supper) of 1.0 mm inner diameter. Scans: 1200 s (1200 scans) SEM images have been taken with a scanning electron microscope TESLA BS 301 (Acceleration potential 10 kV). Differential scanning calorimetry (DSC).Thermal characterization was performed using a Mettler Toledo DSC 823e differential scanning calorimeter (DSC), calibrated with indium and flushed with nitrogen. The heating and cooling scans were performed at 10°C/min, on 5 to 15 mg of sample packed into standard aluminium pans. First-order transition temperatures are given by the peak values. Thermogravimetry. Thermogravimetric analysis (TGA) was carried out under constant nitrogen flow (200 ml/min) at a heating rate of 10 °C/min using a Mettler Toledo TGA/SDTA 851 balance. The heating scans were performed on 10 to 15 mg of sample. The kinetic parameters have been evaluated by integral and differential methods. The kinetic parameters have been evaluated by integral or differential methods using VERSATILE commercial program which gives kinetic parameters by various methods (see below).

3. RESULTS AND DISCUSSION 3.1. FT-IR Spectra Results FT-IR spectra of the studied samples are given in Figures 3. The spectra of the treated samples are different from that of untreated fibre and they are particular for each acid used for grafting. In all cases the evidence of the ester bonds in plasma grafted samples is clear at 1740 and 1610 cm-1. Bands shift and splits are also found at 2850 cm-1 and 2925 cm-1 (Figures 3 a and b). A very different spectrum shows the sample grafted with polylactic acid. Supplementary bands being present at: 3020 and 1500 cm-1 and the band at 1750 cm-1 found in other samples is shifted to 1725 cm-1. On the basis of these results it can appreciate that the grafting took place with a high yield.

C. Vasile, M. Totolin and M. C. Tibirna

218

a)

b)

c)

d)

Figure 3. FTIR spectra of Spanish broom (Spartium junceum) fibres untreated and treated with different carboxylic acids.

XPS Results The measurement of binding energy (BE) can be used to characterize materials. The observed BE depend on the specific environment where the functional groups are located, most changes being within ± 0.2 eV of variation [39, 40]. The C1s BE is observed to increase monotonically with the number of oxygen atoms bonded to carbon, that is C-C < C-O < C=O < O-C=O < O-(C=O)O- consistent with that the carbon becomes more positively charged with increasing number of oxygen atoms bonded to carbon. The XPS intensity (integrated area under the photoelectron peak) is proportional to the atom quantity in the detected volume, therefore by integrating the area under a given peak and correcting for its ionization cross section, quantitative elemental analysis of the material can be made [41, 42]. This requires an algorithm for peak fitting, which should include all characteristic elements for a photoelectron peak, i.e. the peak height, width, shape. The relatively simple shape of a photoelectron peak, due to the one electron process involved, allows in most cases the deconvolution of complex experimental peaks. To extract quantitative information from the XPS spectra, the area and the BE of each subpeak for a given orbital, e.g. C1s, must be determined. Typically, the spacing between subpeaks is similar or inferior to observed peak widths (∼1 eV). Thus, it is rare when individual subpeaks are completely separated in an experimental spectrum. This requires the use of a peak fitting

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 219 procedure. Quantities in such procedures, performed using appropriate software, include the background, peak shape (Gaussian, Lorentzian, asymmetric, or mixtures thereof), peak position, and peak height and peak width. In the XPS spectra of organic compounds, peaks corresponding to carbon atoms in different chemical environments every so often exhibit almost equal binding energies, and they cannot be correctly separated using a standard mathematical procedure, especially in substances with unknown chemical structures of the surface [43]. For example, in examining substances containing functional groups like those below: O ⎯ C ⎯ OH

⎯C⎯O⎯C

⎯C⎯O⎯C⎯

Peaks due to these groups in C1s spectra exhibit almost equal binding energies (about 286.5 eV) and, hence, cannot be separated using mathematical treatment. All the above is also true in the determination of functional groups by other XPS spectra such as O1s or N1s. This problem became more severe once those modification techniques were developed to tailor the polymer surface properties, the chemical derivatization in XPS being firstly used in the fundamental research of phenomena that occur in surface polymer layers under exposure to plasma and other modifying agents. XPS results for untreated and Spanish broom fibres grafted in cold plasma conditions are presented in Figures 4 and Tables 3 and 4. The main bands in XPS spectra – Figure 4a are as it is expected those of carbon and oxygen. Each sample contains in traces some impurities. The Si2p and Ca 2p bands are present in almost all spectra, this element being contained in raw material. Si might be from the holder of the samples. The unmodified fibres also contain traces of nitrogen and chlorine which are also find in plasma treated fibres with lactic acid and oleic acid. The Zn2p band was found in the fibres treated with butyric acid while the sample treated with polylactic acid contains manganese. Concerning the bands position, it can be observed a shift in C1s band for sample treated with lactic, oleic and olive oil, while O1s band is shifted to lower binding energy only in the spectra of sample treated with lactic acid – Table 3. The percentage area of the main elements takes values particular for each treated samples. The C1s area increases in the following order: untreated fibre < lactic acid < butyric acid < polylactic acid < oleic acid < olive oil cold plasma treated sample. The O1s area has an opposite variation. This is an evident proof that the grafting reaction took place. The most evident change in the fibre structure at least at their surfaces is in the number and type of carbon atoms, as it appears from deconvoluted carbon bands in Figures 4b. The untreated fibres and butyric acid treated sample have four types of carbon atoms, while the other treated samples have at least six types of carbon atoms. The oxygen deconvoluted bands – Figure 4c – are much simpler being evidenced two types of oxygen atoms in untreated fibre and butyric acid or lactic acid treated fibres, while the oleic acid-, olive oil- and polylactic acid grafted fibres exhibit three kinds of oxygen atoms.

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unmodified

Butyric acid 10 min

Oleic acid 10 min

Olive oil 10 min

Polylactic acid

Lactic acid

Figure 4a. XPS spectra of the studied samples.

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 221

Untreated sample

Butyric acid

Oleic acid 10 min

Olive oil

Lactic Acid

Polylactic acid

Figure 4b. Deconvoluted carbon bands of XPS spectra of the studied samples

222

C. Vasile, M. Totolin and M. C. Tibirna

Untreated sample

Butyric acid 5 min

Oleic acid 5 min

Olive oil

Lactic acid

Polylactic acid

Figure 4c. Deconvoluted Oxygen bands of the XPS spectra of the studied sample.

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 223 Table 3. Band position and percentage area of total area for untreated and cold plasma treated samples

Atom C1s O1s N1s Si2p Cl2p Ca2p Zn2p

Untreated Band position/% of total area 282.91/67.6 529.91/31.3 397.91/0.4 99.91/0.4 197.91/0.1 3442.9/0.2

Butyric acid Band position/% of total area 282.91/74.08 529.91/24.58 397.91/0.57 99.91 /0.42 194.91/0.13 344.91/0.21

Oleic acid Band position/% of total area 281.91/87.7 529.91/11.5 396.91/0.3 98.91/0.3 193.91/0.1

Olive oil Band position/% of total area 281.91/89.90 529.91/9.32 98.91/ 0.64 342.91/0.08 1020.91/0.06

Lactic acid Band position/% of total area 281.91/70.43 528.91/28.10 396.91/0.44 96.91/ 0.32 196/ 0.06 343/ 0.04

Polyactic acid Band position/% of total area 282.91/ 82.36 529.91 /16.80 99.91/ 0.32 344.91/0.29

Table 4. Results of C1s and O1s high resolution spectral fitting (%) Groups

Untreated

Butyric acid

Oleic acid

Carbon atom containing group Positions of the carbon atoms in deconvoluted curves 285.00 285.00 285.00 C-C (285.0eV) 285.88 286.63 286.55 C-O-C (286.6 eV) 286.78 287.99 287.97 O-C-O (288 eV)

Olive oil

Lactic acid

Polyactic acid

285.00 285.68 286.40 287.17 287.91 289.22 289.87 30.36 17.29 4.01 4.40

285.00 286.15 286.85

285.00 285.85 286.73

288.00 289.21 290.1 37.26 26.32 11.75 7.01

288.52 289.32 290.46 27.23 20.87 15.36 5.28

533.02 534.10 94.00 6.00 -

532.79 534.10 535.26 35.87 37.98 26.15

287.76 O-C=O (288.7 eV) 289.13 289.25 289.17 O-CO-O (290.4 290.43 eV) C1(C-C;C-H) 32.34 44.63 47.58 C2(C-O-) 51.57 41.62 17.73 C3 (O-C-O;C=O) 13.45 10.03 4.89 C4 (O-C=O) 2.65 3.72 4.42 Oxygen atom containing group Position of the oxygen containing groups in deconvoluted curves 532.61 532.81 532.93 532.89 533.82 535.55 533.55 533.79 535.02 534.16 O1 (OC=O) 78.41 90.04 45.18 39.36 O2 (C-O) 21.59 9.96 43.30 48.31 O3 11.52 12.33

The assignment of bonds which belongs these atoms is given in Table 4 and it was made according to the literature data [44 – 47]. • • • •

C1 is a carbon bonded only to another carbon (C-C) or a hydrogen atom (C-H); C2 – atom singly bonded to a oxygen atom (C-O-), other than a carbonyl atom; C3 – a carbon atom single bonded of two oxygen atoms (-O-C-O-) or to a single carbonyl atom (-C=O); C4 – a carbon atom single bonded to an oxygen atom and to a carbonyl oxygen atom (-O-C=O). C4 group (-O-C=O) is very low in untreated sample (2.65 %) and increases in treated sample as a result of ester bonds appearance. In the same time the

C. Vasile, M. Totolin and M. C. Tibirna

224

• • •

content of C2 group (C-O- from C-O-H) decreases from the same reason. The results are in good agreement with FTIR measurements. O1 is a oxygen atom linked to a carbon atom by a double bond, and also an oxygen single linked (O-C=O); O2 – an oxygen atom linked to a carbon atom by single bond (C-O- ); O3 – oxygen atom from supplementary oxidation processes.

Taking into account a content of hydroxyl groups in the superficial layers of the cellulose fibers (up to ~ 10 nm depth) of 51,57 % and also the variation of the C2 groups (C-O- from C-OH) which decrease from 51.57 % to 16.13 % and the increase of the C4 (-O-C=O) groups from 2.65 % to 7.01 % can be estimate a degree of grafting of superficial layers which depends on the type of carboxylic acid employed as it appears in Table 5. . Some errors in XPS results for non-smooth topographies samples as in our case (see SEM results) can appear, but they do not affect the find results, because the modifications are very clear. Table 5. Degree of grafting of the Spanish broom fibres with various carboxylic acids Fibre grafted with: Untreated Butyric acid Oleic acid Olive oil Lactic acid Polilactic acid

Degree of grafting (%) 0 19.3 53.6 45.8 68.8 62.8

O/C atomic ratio 0.463 0.332 0.131 0.104 0.399 0.204

The highest degree of grafting was achieved with lactic acid and this decreases in the order: lactic acid > polilactic acid > oleic acid (sunflower oil) > olive oil > butyric acid. In cold plasma conditions the grafting can take place both with unsaturated and saturated carboxylic acids, therefore the fibres grafted with sunflower and olive oil should have both chains with oleic acid, linoleic acid , palmitic and stearic. The formation of other kinds of bonds is also possible, taking in the view the diversity of the active species possible to be formed in plasma conditions. Comparing the values of the O/C atomic ratio it could appreciate that the grafting of the lactic, polylactic and butyric acids do not change significantly the surface composition while the other can be bonded by different kinds of bonds which lead to decrease of the oxygen percentage on the surface. The morphological aspects of the grafted samples have been studied by XRD and SEM.

XRD Results X-ray diffraction (XRD) techniques were used in the study of the effect of grafting on crystallinity. Generally XRD patterns of the cold plasma treated samples are similar with that of the untreated fibre – Figure 5.

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 225

Untreated

Butyric acid grafted Figure 5. Continued on next page.

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C. Vasile, M. Totolin and M. C. Tibirna

Oleic acid grafted

Olive oil grafted Figure 5. Continued on next page.

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 227

Lactic acid grafted

Polylactic acid grafted Figure 5. XDR patterns of the untreated and cold plasma treated Spanish broom (Spartium junceum, syn. Genista juncea) fibres.

The crystalline peaks are found in the untreated sample at 2 theta degree of: 3; 15.8; 22.5; 29 and 34 and they correspond to the Iα - cellulose crystalline structure [48, 49]. The same peaks characterize the crystalline fraction of all cold plasma grafted samples but they are shifted to higher 2 theta degree and they are much wider and the rings become much diffuse and for the fibres grafted with oleic acid, olive oil and lactic acid some rings can not be

C. Vasile, M. Totolin and M. C. Tibirna

228

distinguish. This observation is in accordance with the data obtained for other cellulose esters [50]. It was shown that X-ray diffraction analysis indicates distinct crystal patterns for these crystalline cellulose esters, and differential thermal analysis shows strong melting peaks. Xray diffraction analysis of secondary cellulose esters, that is, esters having a substantially lower degree of esterification, shows very diffuse patterns which are only slightly indicative of crystalline structure. On the other hand, differential thermal analysis, shows strong endothermic peaks which appear to indicate melting of crystalline material. A small peak appears at 38 2 theta degree in the samples treated with butyric acid, oleic acid, lactic acid or polylactic acid, probably because of some changes in crystallinity network. The crystallinity degree was evaluated as the ratio between the area under the crystalline peaks and the area under the amorphous halo which is a broad hump in the XRD pattern – Table 6. The decrease of the crystallinity degree after grafting with carboxylic acids is much important for the fibres grafted with oleic and lactic acid, therefore even the bulk morphology of the Spanish broom fibre is affected by grafting. Table 6. XDR data for untreated and grafted Spanish Broom fibres with carboxylic acids

Crystallinity index (%)

Spanish broom untreated

Treated butyric acid

Treated oleic acid

Treated olive oil

Treated lactic acid

Treated polylactic acid

55.8

45.53

39.52

42.41

32.86

40.54

SEM Results

Untreated sample X 810 (1 cm = 10 μm) Figure 6. Continued on next page.

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 229

Butyric acid grafted fibers (10 min) X 810 (1 cm = 10 μm)

Oleic acid grafted fibers (10 min) X 410 (1 cm = 20 μm) Figure 6. Continued on next page.

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C. Vasile, M. Totolin and M. C. Tibirna

Olive oil grafted fibers (10 min) X 430 (1 cm = 22 μm)

Acid polylactic grafted fibers (10 min) X 810 (1 cm = 10 μm) Figure 6. Continued on next page.

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 231

Lactic acid grafted fibers (10 min) X 430 ( 1 cm = 22 μm) Figure 6. SEM micrographs of the untreated and grafted Spanish broom (Spartium junceum, syn. Genista juncea) fibres under cold plasma conditions.

The existence of the graft layers on the surfaces of the Spanish broom fibers is clearly evidenced by scanning electron microscopy (SEM) (Figures 6). Important morphological differences can be observed between the support and the grafted derivatives in all cases. It can be noticed that the aspect of the carboxylic acids grafted fibers are totally different from the surface of the control fibers (support). On the surfaces of the fibres grafted with oleic and olive oil some irregularities looking as depositions on particles are observed, while the fibres grafted with butyric acid, lactic and polylactic acid show a uniform aspect, the fibres becomes voluminous and fibrils seems to be expanded and distinct.

Thermal Properties DSC Results Two important aspects can be followed by DSC study of the cellulosic materials: influence of the substituents on thermal characteristics and because of their hydrophilicity the interaction with absorbed water. Strong correlationship between the melting point and the length of substituents at the secondary hydroxyl groups at C2 and C3 positions, have been found for the cellulose fatty acid heteroesters (cellulose propanoate diacetate, cellulose butanoate diacetate, cellulose acetate dipropanoate, cellulose butanoate dipropanoate, cellulose acetate dibutanoate, and cellulose propanoate dibutanoate) [51]. Hatakeyama et al. found that vaporization peak is split into two peaks, one is at around 60°C and the other is at around 120°C. The high temperature vaporization peak is related with the structural change of amorphous chains of cellulose by desorption of bound water [52, 53]. Cieśla et al., established that the profiles of thermal effects depend on water content, time of conditioning, film pretreatment etc [54].

C. Vasile, M. Totolin and M. C. Tibirna

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Both heating – Figure 7 - and cooling – Figure 8 - cycles have been applied from -50 – 250 oC to the untreated and grafted fibres. The DSC curves recorded by heating show two or three peaks. The process with peak temperature around 100 oC can be due to release of the absorbed water. A large amount of non-freezable strongly bounded water was also detected. Cellulose absorbs water and the structural changes appear on ordering of polymer fraction. Due different strengths of water binding in the case of the grafted fibres the characteristic temperatures and the enthalpies vary with nature of grafts. Differences between interactions of particular cellulose fibres with water can be detected during the first, the second and the third heating. Because of water loss in the first run, in the next runs its quantity (and enthalpy) decreases and some temperatures are changed. Comparing the data of Table 7 of the grafted fibres with those of untreated one the following conclusions can be draw: the first peak is shifted to lower temperature after grafting but its enthalpy is generally higher than of untreated fibre. Some peaks at negative temperatures are present in the DSC curve of fibres grafted with oleic and olive oil. The biggest difference between peak temperatures of the peak of absorbed water release is found for samples grafted with olive oil, lactic and polylactic acid. This should means that water is stronger bonded in grafted fibres than in untreated one but in the lower amount as was found also by TG/DTG (see below).

a )

8

7 6

Exo

5

4

3

3 '

-5 0

0

5 0 T e m

1 0 0

p e r a tu r e

1 5 0

2 0 0

(° C )

Figure 7. DSC thermograms of the treated and untreated samples obtained by heating at 10°C/min [with 8 = untreated; 7 = treated with oleic acid; 6 = treated with butyric acid; 5 = treated with lactic acid; 4 = treated with polylactic acid; 3 = treated with olive oil (first heating); 3’ = treated with olive oil (second heating)].

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 233 Similar values have been obtained for cellulose esters with linear aliphatic acyl substituents ranging in size from C[12] (lauric acid) to C[20] (eicosanoic acid). A series of transitions that represented motion by both ester substituents and cellulosic main chain. Broad crystallization and melting transitions attributed to side-chain crystallinity were observed in the range between -19 and +55°C. Melting temperature values of 96°C and 107°C [55]. As concerns the variation of the temperatures of the second peak which is probably a decomposition/melting step expecting fibres grafted with oleic acid, all other grafted fibres exhibit lower decomposition/melting temperatures and also lower enthalpy of this process, the most important decrease being found for fibres grafted with olive oil and lactic acid (which acts probably as plasticizer). Table 7. Characteristic temperatures and enthalpies of the processes evidenced in DSC curves recorded by heating of the studied samples Sample

Untreated Butyric acid

Oleic acid

Olive oil Lactic acid Polylactic acid

Peak 1 Tonset, (oC) 94.3 -46.5 1.84 56.0

Tpeak, (oC) 149.6 -18.2 45.62 99.9

Tendset, (oC) 191.0 17.7 76.72 138.9

-59.63 -10.5 -3.48 -107.2

94.06 -32.8

130.9 -10.6

178.1 3.9

-93.75 -0.54

36.3

47.6

56.4

-1.14

99.05

132.8

172.4

-57.6

-41.47

2.08

15.82

-4.81

44.32 59.1 59.26

106.49 104.4 111.91

153.1 147.1 158.07

-68.07 -79.9 -92.76

ΔH(J/g)

Peak 2 Tonset, (oC) 179.5

Tpeak, (oC) 186.4

Tendset, (oC) 198.5

-32.85

144,7

164.4

172.7

-0.53

198.1

202.8

216.0

-1.32

121.7 154.2 163.58

139.7 160.1 169.47

173.8 168.1 179.68

-3.28 -1.57 -0.91

ΔH(J/g)

The DSC curves recorded by cooling are particular for each fibre studied and there are big differences in respect with the untreated sample. The DSC curves of the untreated sample and that of the fibres grafted with butyric acid and polylactic acid show two exothermic processes with peak temperature at 38 and 181 oC, the temperatures of the grafted fibre being close to those of the untreated sample, that should means that this grafts do not influence these crystallization processes. The crystallization process which occurs in the temperature range 50 – 200 oC is also present in the DSC curves of fibres grafted with oleic acid but it occurs at lower temperature. The fibres grafted with oleic acid, lactic acid and polylactic acid shows an exothermic process around 100 oC while the fibre grafted with olive oil and oleic acid show a crystallization peak at low temperatures at around -20 oC which could be due to the frozen bonded water.

C. Vasile, M. Totolin and M. C. Tibirna

234

a)

b)

3

Exo

8

7

-50

0

50

100

150

200

-50

0

50

100 150 200 250

Tem perature (°C)

Figure 8. DSC curves of the treated and untreated samples obtained by cooling at 10°C/min of a) untreated sample (8); treated with oleic acid (7) and b) treated with olive oil (3).

Table 8. Characteristic temperatures and enthalpies of the processes evidenced in DSC curves recorded by cooling of the studied samples Sample

Untreated Butyric acid Oleic acid

Olive oil Lactic acid Polylactic acid

Peak 1 Tonset, (oC) 48.39 55.1

Tpeak, (oC) 38.42 33.3

Tendset, (oC) 1.89 21.9

ΔH(J/g)

Tpeak, (oC) 181.23 193.6

Tendset, (oC) 171.1 182.9

ΔH(J/g)

0.77 0.54

Peak 2 Tonset, (oC) 190.6 198.4

-14.75

-24.12

27.6

1.46

202.3

195.2

184.8

0.32

39.5

29.3

11.5

0.35

116.4

110.97

98.7

0.43

-3.4 13.06 106.2 46.09 131.5

-18.4 10.7 100.06 35.1 117.9

-43.87 8.22 89.9 21.7 99.2

4.22 0.78

220.8 194.5

215.2 191.5

201.94 185.4

2.08 0.26

0.13 0.13

202.1

193.5

173.99

0.76

2.16 0.27

The sorbed water molecules are directly bound to the hydrophilic site to form nonfreezable water. Then, beyond a certain water content threshold, the sorbed water molecules become freezable, but with a melting point lower than 0°C, due to their location in the second hydration layer [56, 57]. The peak at high temperature seems to be due to of a reversible process because it appears both during heating and cooling at close temperature. This is very difficult to be

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 235 explained. It is possible as some substituents to act as plasticizer as was found by Aranishi et al., for a melt spun fiber comprising a thermoplastic cellulose mixed ester composition containing as plasticizer polylactic acid [58], and for cellulose oligomers with n>20: (cotton, wood, paper), a melting temperature > 250 oC is appreciated, but all time this process is accompanied by carbonization. For Eastman cellulose acetate a glass transition temperature is found at 180-186 oC [59] while the melting range lies between 230-250 oC. Therefore the first type of transition associated with other phenomena could be much probable in our case.

TG Results There is a great number of studies thermal degradation of cellulose and cellulose esters such as cellulose acetates, nitrate, cellulose phosphate [60]; cellulose benzoate, cellulose succinate and cellulose cinnamate [61] esters with fluorine-containing substituents [62] cellulose fibers partially esterified with some long chain organic acids such as: undecylenic acid; undecanoic acid; oleic acid and stearic acid [63] and a particular behaviour was found in each case. The TG/DTG curves of under study samples are given in Figure 9 and characteristics thermogravimetric data are summarized in Table 9.

Figure 9. TG/DTG curves of Spanish broom fibres untreated and grafted with different carboxylic acids in cold plasma conditions.

In the first thermogravimetric step, all characteristic temperatures for grafted fibres are superior those of untreated Spanish broom fibres and mass loss is lower. This process could attributed to water loss and should means that after grafting the bonding with water is stronger, but the absorbed amount is lower. In the second thermogravimetric step onset temperatures for all grafted fibres are higher than that of untreated one, while peak temperatures are almost unchanged. This step corresponds to decomposition and the shift of the onset temperatures to high temperature indicates an increase in thermal stability at least at the fibres surface. This process is very complex showing many inflexions which appear at low temperatures for grafted fibres, indicating some changes in reaction mechanism as was previously shown [64].

236

C. Vasile, M. Totolin and M. C. Tibirna

Table 9. Thermogravimetric data of Spanish broom fibres untreated and functionalized with different carboxylic acids Thermogravimetric Characteristic Peak I Tonset (oC) Tpeak (oC) Tendset (oC) Δw (%) Peak 2 Tonset (oC) Tpeak (oC) Tendset (oC) Δw (%)

Spanish broom untreated

Treated butyric acid

Treated oleic acid

Treated olive oil

Treated lactic acid

Treated polylactic acid

22,5 49,0 118,3 5,4

24.9 49.0 118.3 5.0

25.1 53.8 114.6 3.5

23.5 49.0 126.3 3.9

23.9 49.0 115.6 3

23.6 55.2 117.4 2.7

177,2 358,5 (i) 409,0 (i) 449,8 550,6 75,2

182.2 358.5 (i) 405.7 (i) 449.2 547.9 77.3

184.3 359.5 (i) 406.5 (i) 444.5 578.2 79.9

198.3 358.5 (i) 406.4

179.9 358.5 (i) 406.4 (i) 457.2 534.5 76.8

180.0 358.5 (i) 406.4 (i) 449.1 542.6 78.4

529.2 79.8

i – temperature at inflexion point of DTG curve.

To have many information about the involved processes the overall kinetic parameters have been evaluated – Table 10, using both differential and integral methods coupled in the commercial computing program VERSATILE. Table 10. Kinetic parameters (E - overall activation energy and n - reaction order) of thermal decomposition (second step) of Spanish broom fibres untreated and grafted with different carboxylic acids Sample

Methods of evaluation Coats – Redfern [65] Flynn-Wall [66] van Krevelen [67] Urbanovici-Segal [68] Achar [69] Freeman Caroll [70] Piloyan 71]

Spanish broom untreated E, kJ/mol; n 89.18; 1.7 94.38 ; 1.7 131.09; 2.0 90.53; 1.7 71.44; 1.3 75.54; 62.97 -

Treated butyric acid

Treated oleic acid

Treated olive oil

Treated lactic acid

E, kJ/mol; n 87.75; 1.6 93.02; 1.6 129.76; 1.9 89.13; 1.6 72.98; 1.3 70.01; 64.25; -

E, kJ/mol; n 85.52; 1.5 90.92; 1.5 121.88; 1.7 86.89; 1.5 67.75; 1.1 65.30; 66.25;

E, kJ/mol; n 106.95; 1.7 111.30; 1.7 156.50; 2.0 108.13; 1.7 91.46; 1.4 90.91; 83.98; -

E, kJ/mol; n 88.99; 1.7 91.06; 1.6 127.70; 1.9 90.25; 1.7 75.09; 1.4 66.98; 60.31; -

Treated polylactic acid E, kJ/mol; n 87.08; 1.6 92.40; 1.6 124.74; 1.8 88.44; 1.6 77.48; 1.4 64.44; 62.90; -

Significant changes in the kinetic parameters are obtained only for fibre grafted with olive oil which contains 55.0 - 83.0 oleic acid, 3.5 - 21.0 % linoleic acid. To explain the important changes in properties of the Spanish broom fibres after plasma treatment, the plasma action must be considered. Plasma aided synthesis (deposition and grafting reactions) involves fragmentation of plasma gases and reorganization of the resulting neutral and charged species, inside and outside the plasma area, into nonvolatile, highmolecular-weight structures. Recombination mechanisms developed in the absence of plasma

Grafting of Some Biofibres with Carboxylic Acids under Cold Plasma Conditions 237 (outside the plasma zone) usually lead to the incorporation of less fragmented building blocks into the nascent macromolecular structures [72-75]. Graft-polymerization reactions initiated from plasma-activated polymer surfaces involve two distinctive and consecutive processes: implantation onto polymer substrate of active sites, like free radicals or reactive functionalities followed by the initiation of conventional graftpolymerization reactions in situ or ex-situ conditions depending on the stability of the active sites. Supramolecular structure of polysaccharides controlled by intramolecular and intermolecular hydrogen-bonding (conformation of macromolecules), existence of crystalline and amorphous zones, exerts a significant influence on both their physical and chemical properties. Conventional modification techniques performed on lignocellulosics to make them compatible with other polymers or to enhance their surface properties by grafting with some additives (such fatty acids) alter significantly the supramolecular order of the macromolecular networks, diminishing their inherent physicochemical characteristics. Cold plasma environments offer a unique way for modifying these materials without altering the bulk structures and characteristics. Intrinsic properties can be preserved in this way, materials with advanced performances and tailored properties can be obtained. Peroxide functionalities are possible to be formed and they can strongly interact with the counterpart on surface. The interaction of the active species of a plasma with polymer surfaces involves electron mediated processes and positive ion-induced reactions. The last leads through neutralization reactions. To energy concentrations localized on macromolecular chains (electronically excited states) which can promote hemolytic bond cleavages leding to the formation of freeradical sites. These reactive centers lead to a large variety of functionnalization mechanisms depending on the reaction environments under in situ or ex situ plasma conditions. Several possible ways of grafting are given in Scheme 4. 6

* CH OH O

O

6CH OH 2 5C O

C 4 OH H 3C

H

Active sites at different carbon atoms

O

H C 1 H C 2

OH

5C

2

*

C* 4 OH H 3C* H

O

C*

O

OH

N2 CH

Fragmentation

O

2

5C

C 4 OH H 3C

H

C 4 OH H 3C

1

6

plasma

O

CH OOC-R 2 5C O

R

OH

+ R-COOH

or other different ways with various probabilities

OH O

O H C * 1 H C* 2

OH

O H C 1 H C 2

CH O

2

5C

C 4 OH R'COOH 3C

H

OH O

O H C (R) 1 CH H 2 (OOCR) OH

Scheme 4. Suggested reaction mechanism for nitrogen plasma induced molecular activation, fragmentation and grafting of the Spanish broom (Spartium junceum, syn. Genista juncea) fibres.

238

C. Vasile, M. Totolin and M. C. Tibirna

The trapped free radicals can “survive” in the polymer matrix and their intensities vary significantly with the nature of fibres and plasma gases. A good correlation has been found between free-radical concentrations and ex situ plasma surface oxidation reactions. As it was mentioned the characteristic cellulose peaks are: C-OH, C-O-C (286.6 eV) and O-C-O (288 eV) and also the existence of O-C=O (288.7 eV) and O-CO-O (290.4 eV) can be noticed. The formation of new functionalities (O-C=O and O-CO-O) are possible through the cleavage of C1-C2 bonds of the pyranosic ring. It was demonstrated that all possible four hydroxyalkyl radicals (1,2, 3) are generated as primary structures, through the hydrogen abstraction mechanism, but with a preference at C2 and C5 carbon atoms. Plasma irradiation produces preferentially the alkoxy-alkyl radicals at C1 of the glucose units.

CONCLUSIONS The research was concentrated to obtain fibres with new or significantly improved properties, with tailored functionalities for special applications starting from natural fibres based on concept of development of environmentally friendly and energy-efficient processing by surface modification of fibres, yarns and fabrics. New fibres with enhanced properties based on existing natural compounds have been obtained by plasma grafting of the Spanish broom (Spartium junceum, syn. Genista juncea) fibres with carboxylic acids. The heterogeneous esterification reaction with five different acids was realized in mild conditions with a high yield. The characterization carried out with X-ray photoelectron spectroscopy (XPS), XRD, SEM, differential scanning calorimetry and thermogravimetry showed that the individual fibers were covered with the corresponding esters by partial degrees of substitution of the cellulose and that the surface degree of substitution of the cellulose fiber was higher than for the bulk, showing that the esterification reaction was a surface phenomenon, mainly in cold plasma conditions. New biofibres obtained could be processed in products which should improve comfort performance and enhance micro-climate of bed rooms. Develop additional properties enhancing natural properties of vegetable fibres lead to promotion of fibers with lower ecological impact, promotion of integrated European production chain These fibers could be also useful as reinforcements in various composites (mainly containing polyolefins).

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In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 19

SPECIFIC PROPERTIES OF SOME BIOLOGICAL COMPOSITE MATERIALS N. Barbakadze, E. Gorb and S. Gorb Max Plank University, Shtutgart,Germany Georgian Technical University1,Tbilisi, Georgia

ABSTRACT Miniaturisation of technical systems creates the need for today’s science and engineering to assess the mechanical properties of small volumes of material. A specific feature of the structure and the combination of the desirable properties across several different length scales are fascinating by the many examples in biology. Determining the extraordinary properties of natural materials at the nanometer scale is regarded as a very attractive target for materials science. Mechanical behavior of various biological materials such as insect and plant cuticles was studied by applying experimental approaches of material science in order to explain their structural design and working principles. Experiments were performed on the head articulation cuticle of the beetle designed for friction minimisation and on the wax covered plant surfaces adapted for attachment prevention. Both insect and plant cuticles are multifunctional composite materials and have a multilayered structure. Gula cuticle of the beetle Pachnoda marginata is a part of the head articulation, which is a micromechanical system similar to a ball bearing. The surfaces in this system operate in contact and must be optimised against wear and friction and provide high mobility within the joint. The measurements on the gula cuticle were performed in order to understand structure and mechanical behavior of the material working for friction minimizing. The wax layer on the plant surfaces is a barrier for the attachment system of insects. Antiattachment function could be caused by contamination of attachment pads of insect with the wax crystalloids. Increase in roughness due to location of the wax crystals on the plant surface causes decrease in the real contact area between the plant surface and attachment pads of the insect. These are two of the hypotheses why insects cannot walk on the plant surfaces structured with wax. Nanoindentations on different plant surfaces were performed in order to understand the deformation behavior of the wax layer. This study is believed to 1

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N. Barbakadze, E. Gorb and S. Gorb be one of the first for mechanically testing insect cuticle and the very first for wax-coated plant surfaces in native condition.

MOTIVATION AND LITERATURE REVIEW “Why should engineers be interested in biological materials? One only has to look to the history of technology, starting from the first use of tools, to see that revolution and progress are driven by the new possibilities which new materials create.” Julian Vincent

Natural materials have been used in engineering over twelve millennia. The first houses and bridges were built from grass, wood and stone. The first clothes and shoes were made from animal skins. There are still no alternatives to wood and horsehair for many musical instruments (Wegst and Ashby, 2004). We use natural materials everyday. They are always around us and the interest in biological systems is still growing. The natural systems inspire the engineering sciences in the design of new materials. One example is the development of different replacement tissues from metal or plastic (Vincent, 1990). Most natural materials are structured fiber-matrix composites (Wainwright et al., 1976; Vincent, 1990). The components in them are quite similar. They consist mainly of natural polymer fibers embedded in a protein matrix. For example, cellulose, pectin and protein build a plant cell wall (Wegst and Ashby, 2004); insect cuticle is the composite of chitin and protein (Neville, 1975; Wainwright et al., 1976; Vincent, 1990; Vincent and Wegst, 2004). The biological materials exhibit a hierarchical structure. Natural systems such as skin, bone, cartilage and cuticle are layered composites with different single layer structures. Despite the limited constituents, the natural systems display a great variety of mechanical properties. Collagen and elastin are the main components in skin, bone and cartilage. However, depending on the amount of the constituents and the fiber orientation, each system has different mechanical properties (Wegst and Ashby, 2004). The structural and mechanical design of natural systems can be mimicked to provide new so-called “intelligent” materials with “smart” structures. Skin, for instance, shows compressive properties useful in the development of tactile sensors (Vincent, 1990). The man-made hydrophobic surfaces have been inspirited from the “lotus effect” of the plant leaf. The technique of the turn around of the beetle arouses the interest of robotics researchers to design a robot being able to cope with any difficulties in movement. In figures 1 and 2 (Vincent and Wegst, 2004) the wide spectrum of mechanical properties of natural materials is shown. Figure 1 presents Vickers hardness for a range of natural materials. For example, the arthropod (invertebrates) cuticle displays a great variation of hardness values depending on its function and condition (wet or dry). In figure 2, Young’s modulus is plotted versus density for different natural materials. Again arthropod cuticle shows different elastic modulus comparable with human skin, cork, wood and even aluminum. Determining the extraordinary properties of natural materials at the nanometer scale is regarded as a very attractive target of materials research. However, tribological systems in biology have not been studied much from a materials science point of view. The majority of

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work in tribology research has focused on the material properties and working mechanisms of human and animal joints (Fung 1993; Persson 1998). It was found that articular cartilage has a unique quality of lubrication and shock absorption that is mainly due to the multilayer structure (Fung, 1993). Being related to bone, cartilage has a very small coefficient of friction for relative motion between two articular pieces (Fung, 1993).

Figure 1. A compilation of hardness data for natural materials. (Figure adapted from Vincent and Wegst, 2004, created using the Natural Materials Selector).

There are considerable data in the literature about ultrastructural architecture and mechanical properties of the arthropod cuticle (Fraenkel and Rudal, 1940; Neville, 1975; Hepburn and Joffe, 1976; Andersen, 1979; Andersen et al., 1996; Vincent, 1980; 2002; Binnington and Retnakaran, 1991; Gorb, 1997; Arzt et al., 2002; Enders et al., 2004; Vincent and Wegst, 2004). It is the most widely distributed high-performance composite material after plant cell and wood (Vincent, 2002). Much work has been published on the correlation of microtribological properties of biological systems with its material structure (Scherge and Gorb 2000; Gorb and Scherege 2000; Gorb et al. 2001; Dai et al. 2002; Gorb and Perez Goodwyn 2003; Perez Goodwyn and Gorb 2004). Wax-coated plant surfaces were studied in relation to insect attachment systems over the last century (Kerner von Marilaun, 1898). Great work has been done on the structure and the chemical composition of the wax crystals (Hallam and Chambers, 1976; Bianchi et al., 1978; Haas and Schonherr, 1979; Baker, 1982; Avato et al., 1984; Jeffree, 1986; Baker and Gaskin, 1987; Walton, 1990; Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998; Gorb, 2001; Eigenbrode and Jetter, 2002; Gorb and Gorb, 2002). However, little is known about the mechanical properties of the wax layer. This study aims to explore mechanical performance of insect and plant cuticle using the material sciences approach. Head articulation cuticle of the beetle (Pachnoda marginata) designed for friction minimization and wax-covered pea (Pisum sativum) plant surfaces adapted for insect attachment prevention have been studied. Mechanical properties were determined by nanoindentation.

HEAD ARTICULATION OF THE BEETLE The body of insects is completely covered by cuticle, which is a multifunctional interface between the animal and the environment (Hepburn and Joffe, 1976; Gorb, 2001).

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Figure 2. Young’s modulus versus density chart for natural materials. (Figure adapted from Vincent and Wegst, 2004, created using the Natural Materials Selector).

It serves primarily as an exoskeleton that gives the body its shape and stability. The cuticle is also a main barrier against evaporation of water from the body and protects the insects against desiccation (Locke, 1964; Binnington and Retnakaran, 1992). Like most biological materials, cuticle is a fiber composite (Hepburn and Chandler, 1980). The fibers consist of chitin and the matrix is formed by proteins. Chitin is a natural polymer composed of 300 nm long and 3 nm thick nanofibrils (Vincent, 1980; 2002). Each nanofibril contains about 19 molecular chains (Fraenkel and Rudall, 1940; Vincent, 1980; 2002) running antiparallel to one another (i.e., alpha chitin) (Neville, 1975; Vincent, 1980). Chitin filaments are distributed in the protein matrix (Andersen et al., 1996) which stabilises the chitin. Water contained in the proteins (about 90% of the protein matrix) presumably functions to separate the two main components of the cuticle from each other (Vincent, 1980).

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Arthropod (invertebrates—animals without backbones) cuticle has a multilayer structure (Neville, 1975; Andersen, 1979) (figure 3). It typically consists of three main layers: epicuticle, exocuticle and endocuticle. The latter two layers form the procuticle. In some insects there is a mesocuticle between the endocuticle and the exocuticle (Neville, 1975; Andersen, 1979; Binnington and Retnakaran, 1992). The nonchitinous, tanned lipoproteinous epicuticle is the outer layer of the cuticle, which is very thin and has a relatively high tensile strength. The surface of the epicuticle is coated with wax and lipids. The hard and stiff solid part has a dense chitin-protein structure and forms the exocuticle. The endocuticle is the thickest region of the cuticle and has low chitin content (Locke 1964; Neville 1975, Binnington and Retnakaran 1992). From a mechanical point of view, there are two main types of insect cuticle: soft and hard. In larvae the cuticle is soft and colorless. A simultaneous hardening and darkening, called sclerotisation (Fraenkel and Rudall, 1940), forms a hard and colored integument in most adults. There is a great variation in kinds of the cuticle depending on the proportions of the main components and, accordingly, mechanical properties (Hepburn and Chandler, 1976). In various animal groups and on different body parts there are different types of cuticle from very hard and brittle to soft, ductile and also rubber-like (Jensen and Weis-Fogh, 1962).

Figure 3. Schematic of the multilayered structure in the insect cuticle. The shaded areas represent the single cuticle layers: outer surface layer, epicuticle which does not contain chitin fibers; procuticle with two layers: exocuticle and endocuticle. Last two cuticle layers contain chitin fibers oriented nearly always parallel to the surface. The endocuticle is connected with epidermis cell structure.

The wide spectrum of mechanical properties of the insect cuticle suggests that each body part is optimised to its function. Any movement involving contact between two surfaces or between a surface and a medium has to deal with the resistance of the surfaces or medium. This resistance is a friction phenomenon, believed to have a great influence on the design of biological structures (Gorb, 2001). Living microsystems have developed different ways to save muscular energy by the use of friction-optimised systems. Surface pairs in biological objects are designed to maximise or to minimise contact forces within joints. In biology they are defined as friction and anti-friction systems respectively. Working principles of friction and anti-friction systems are based on mechanical interlocking and a maximisation or a minimisation of the contact area (Gorb, 2001). Frictional systems require high friction to fixate body parts to one another (Gorb, 2001). The surface pair can be predefined as in the

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wing-locking mechanisms of beetles (Hammond, 1989; Gorb, 1997; Gorb, 1999b) and the head-arresting system of dragonflies (Gorb, 1999a; Gorb, 2001). Among various cases of contact pairs in biology, anti-friction systems always have a predefined pair of surfaces like the head articulation system in beetles (Arzt et al., 2002; Enders et al., 2004) or the hemelytra-hindwing locking mechanism in bugs (Perez Goodwyn and Gorb, 2004). An interesting example of anti-friction system is a head articulation of the beetle Pachnoda marginata. The surfaces in this system operate in contact and must be optimised against wear and friction to provide high mobility within the joint. A predefined, functionally corresponding pair of the contacting surfaces forms a tribological system with specific ultrastructural architecture and properties. However, each contacting piece has a different structure. The rather smooth surface of the hemispherical head-part or gula is believed to contribute to the friction minimisation in contact with its counterpart in the ventral part of the prothorax, covered by asymmetric outgrowths (Enders et al., 2004). In living conditions the organic substances on the cuticle surface, such as lipids and waxes are expected to serve as lubrication materials to reduce the friction in this biological microsystem. For this study a head part (gula) cuticle of the head articulation of the beetle Pachnoda marginata was selected. Both the structure and the mechanical properties of this material are largely unknown. The goal of these investigations was to obtain detailed information about the cuticle structure in the gula and to determine its mechanical properties. Assembly of the head articulation and structure of both contacting surfaces were studied by means of computer tomography and scanning electron microscopy (SEM). SEM images were also used for the evaluation of nanoindentation tests. Hardness and elastic modulus were measured using the nanoindentation technique. The samples were mechanically tested in the fresh, the dry and the chemically treated conditions in order to understand the influence of desiccation (fresh versus dry condition) and removal of an outer wax layer (chemically treated condition). The questions asked were: What are the structure and the local mechanical properties of the head articulation cuticle? How does the liquid content influence the mechanics of the joint material? How do surface waxes influence hardness and elastic modulus of the gula? Since the structure and the mechanical properties of the material are very important for tribological performance, this study aims to make a contribution to exploring the working principle of the gula cuticle.

MATERIALS AND SAMPLE PREPARATION Samples were prepared from the head articulation system of the beetle Pachnoda marginata. The beetles were anesthetized with CO2. Heads were dissected from the body and freed of soft tissue. To prevent the drying of the specimens, fresh gula cuticles were tested immediately (in 3-5 min after dissecting from the body). Dry samples were prepared from fresh cuticles by drying. To obtain the same degree of desiccation, fresh samples were dried in an oven for 24 h at 40°C. Chemically treated samples were prepared from the dry gula cuticles. In order to remove waxes from the surface, dry samples were treated in a solution of chloroform and methanol (2:1) for 50-60 min and air-dried.

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SHAPE AND STRUCTURE OF THE CUTICLE To observe the working position and the shape of the contacting surfaces, the anatomy of the head articulation system of the beetle Pachnoda marginata was explored with high resolution computer tomography (X-Ray µ-CT system, model 1072 tomograph by Sky Scan at the Institute für Kunststoffprüfung (IKP) at the University of Stuttgart, Germany). This method allows non-invasive imaging of the internal structure. The measurements were performed on dry samples. The images of the head articulation of the beetle were taken in two directions: transversal and sagittal (magnification × 30). Images showing cross sections of the contact parts (gula and prothorax) of the head articulation were taken in dry insects in two directions: transversal (figure 4 A) and sagittal (figure 4 B). The hemispherical convex shape of the gula cuticle can be seen in the cross section image in both transversal and sagittal direction. As can be distinguished in the figures, the head articulation system is an open joint. In the transversal image, the head articulation of the beetle is reminiscent of a technical ball bearing system.

Figure 4. X-Ray tomography (µ-CT system) images in transversal (A) and sagittal (B) planes of the head articulation of the beetle Pachnoda marginata.

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Figure 5 shows the surface structures of the gula plate (figure 5A and 5C) and its counterpart (prothorax) (figures 5B and 5D). The gula is rather smooth while the prothorax is covered by cuticular outgrowths. The arrows in the figures 5 A and 5 B show that these surfaces contact each other in the head articulation of the beetle. The surfaces of the contacting pair of the head articulation system were studied in the scanning electron microscopy (SEM). The samples were fixed in 2.5% glutaraldehyde in a phosphate buffer (pH = 7.3). The specimens were dehydrated in an ascending row of ethanol and then critical-point dried. Pieces of the dried material were fractured using a razor blade. The prepared samples were mounted on holders, sputter-coated with gold-palladium (10 nm thickness) and examined in a Hitachi S-800 scanning electron microscope (SEM) at 20 kV. SEM images in the figure 6 provide information on the shape of the gula cuticle and the ultrastructural architecture within a single cuticle layer. The gula has a hemispherical smooth surface (figure 6 A). Island-like places, occurring on the surface, are, presumably, dried secretory substances, delivered to the surface by well-defined pores, which run deep into the material and apparently are connected with epidermal cells (figure 6C and 6D). Cracks found on the surface are probably caused by material desiccation (figure 6D). The cross-section of the cuticle shows the layered structure of the fiber composite (figure 6B). The thickness of the gula cuticle is about 80 µm. A very thin (7-8 µm) and dense epicuticle can be distinguished. Procuticle is present with two layers: dense exocuticle, which is about 22 µm thick and the endocuticle, which is very thick (about 50 µm) and less dense compared to the exocuticle layer. As can be distinguished in figure 6 B, chitin fibres are oriented nearly perpendicular to the surface in the exocuticle layer and parallel to the surface in the endocuticle. The exocuticle structure of the gula differs from the layered pattern of regular cuticle, where the orientation of the chitin fibers is parallel to the surface (Neville, 1975; Wainwright et al., 1976; Vincent, 1990). The fibers in the exocuticle are oriented perpendicular or at some angles to the surface. A similar fiber orientation has previously been found in attachment pads of grasshopper (Gorb and Scherge, 2000). However, the advantage of such structures is not understood at present. Further investigations are required to clarify the reason for this perpendicular fiber orientation.

SURFACE ROUGHNESS The sample surface was studied using a white-light interferometer (Zygo New View 500, Zygo Corporation, USA). This technique can be used to obtain the average surface roughness (Ra), the average absolute value of ten-point height (Rz) or root mean square (rms) representing the height profile’s roughness. The results of surface roughness measurements are summarized in table 1 and shown in the figure 7. In the fresh condition, the surface of the hemispherical convex gula cuticle is extraordinarily smooth for a biological material (Ra = 0.033 ± 0.005 µm, rms = 0.038 ± 0.007 µm) (figure 7 A). After desiccation, the outer layer of the cuticle builds up high roughness (figure 7 B). The water loss is thought to cause the constriction of the structure in inner layers and folding of the outer layer. These structural changes could be the reason why dry samples exhibit a rougher surfaces (Ra = 0.161 ± 0.011 µm, rms = 0.197 ± 0.015 µm).

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Figure 5. SEM images of the gula (A, C) and prothorax (B, D) surfaces. On the gula surface pores (A, C) and dried organic substances (C) can be distinguished. The figures B and D show cuticlar outgrowths on the prothorax surfaces. The gula and the prothorax surfaces operate in contact (shown with arrows on the figures A and B) in the head articulation system of the beetle Pachnoda marginata.

Figure 6. SEM images of the gula surfaces. A: shows entire surface of the gule. B: cross section of the gula cuticle showing the single cuticle layer: EPI – epicuticle, EXO – exocuticle and ENDO – endocuticle. Perpendicular orientation of the chitin fibers can be distinguished in the exocuticle layer. The well-defined pores, dried organic substances and cracks can be seen on the cuticle surface (C and D).

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After the chemical treatment, the surface roughness becomes lower (Ra = 0.103 ± 0.008 µm, rms = 0.117 ± 0.011 µm) (figure 7 C). It could be caused by dissolving the dry organic substances or structure on cuticle surface. However, the roughness of the fresh samples remains lower than that of dry and chemically treated ones. Table 1. Summary of surface roughness parameters, Ra and rms, obtained by whitelight interference microscopy for fresh, dry and chemically treated gula cuticles. The surface roughness measurements were carried out on three samples for each condition (SD = standard deviation). Surface roughness parameter Ra (nm)

SD

rms (nm)

SD

fresh

33

±5

38

±7

dry

161

± 11

197

± 15

chemically treated

103

±8

117

± 11

Sample condition

Figure 7. Surface profiles of fresh (A), dry (B) and chemically treated (C) cuticles. The images were obtained by white light interference microscopy.

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MECHANICAL PROPERTIES Nanoindentation provides a fast and reliable technique for evaluating local mechanical properties, such as hardness and elastic modulus, of very small volumes of the material (Oliver and Pharr, 1992; Bhushan and Li, 2003). During the past decade, this method has become an important tool in materials characterization (details about this method see in Barbakadze 2005; Barbakadze et al., 2006). In figures 8 A and 8 B hardness and elastic modulus results are plotted vs. displacement (indentation depth into the sample surface). Each data point is the average value of approximately 150 indents (10 beetle heads with 15 indents on each, 150 indents in all). The measurements revealed a strong dependence of the mechanical behavior on the preparation conditions of biological samples. The significance of the differences due to the conditions was checked by means of statistical calculations. The experimental data were compared with a one-way ANOVA and a Tukey post tests (Origin 7 SR2). Hardness and elastic modulus values of the fresh, the dry and the chemically treated samples were compared with each other by different displacements of 250, 500, 1000 and 1500 nm. The results of the statistical calculations are summarised in table 2. Hardness and elastic modulus differ significantly for the fresh, dry and chemically treated conditions. Exceptions are the elastic modulus values of the dry and chemically treated samples at indentation depths of 1000 and 1500 nm. The effect of the chemical treatment on the sample stiffness at these depths can hardly be observed. Statistical calculations show no significant difference between elastic moduli of the dry and chemically treated samples at indentation depths higher than 1000 nm. The average hardness value (H=0.10±0.07 GPa) of the fresh samples is significantly lower than that of the dry (H=0.49±0.14 GPa) and the chemically treated (H=0.52±0.15 GPa) ones (figure 8B) (ANOVA p<0.0005, Tukey post test p<0.0005). The difference between the hardness results of the dry (H=0.49±0.14 GPa) and chemically treated samples (H=0.52±0.15 GPa) is lower but also significant (statistic: ANOVA p<0.0005), especially in the displacement range of 200-1000 nm. The same tendency was observed for the elastic modulus (figure 8C). The dry (E=7.50±1.80 GPa) and chemically treated (E=7.70±1.90 GPa) samples are significantly stiffer than fresh ones (E=1.50±0.80 GPa) (statistic: ANOVA p<0.0005, Tukey post test p<0.0005). In this case the chemical treatment causes a slight increase of the elastic modulus below 1000 nm indentation depth into the sample surface. At a depth higher than 1000 nm, the elastic modulus values of the dry and chemically treated samples are not significantly different. The maximum displacement for the fresh samples was 3000 nm, but for the dry and chemically treated samples only 2000 nm, because the maximum load of the instrument had been reached. High values of the hardness and elastic modulus in the first 50 nm of indentation are erroneous data caused by contact building between the indenter tip and sample due to the roughness and contamination of the sample’s surface. The hardness and elastic modulus for all samples slowly decreases with indentation depth. However, after 1700 nm, both parameters drop rapidly for the dry and chemically treated specimens.

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Figure 8. The results of hardness and elastic modulus from indentation tests on the gula-cuticle: hardness (A) and elastic modulus (B) are plotted versus displacement for fresh, dry and chemically treated samples. Hardness and elastic modulus were calculated by using the CSM technique from the projected contact area during indentation. Each data point corresponds to the mean value of approximately 150 measurements.

B

In general, soft and compliant cuticle contains more water than hard and stiff (Vincent and Wegst, 2004). Since the gula cuticle belongs to the hard and brown type of cuticle, it has a low content of water. But even so, the results show that desiccation has a great influence on the mechanical behavior of the cuticle tested. After drying, the gula cuticle becomes about 5 times harder and stiffer (H = 0.49 ± 0.14 GPa, E = 7.50 ± 1.80 GPa) than in the fresh state (H = 0.10 ± 0.07 GPa, E = 1.50 ± 0.80 GPa). Water content seems to be the crucial factor, which influences the mechanical properties such as hardness and elastic modulus. According literature data, humidity loss changes the mechanical behavior of biological materials significantly (Andersen et al., 1996; Arzt et al., 2002; Enders et al., 2004; Vincent and Wegst, 2004). In the fresh cuticle, chitin filaments are spaced within a protein-matrix containing about 90 % water (Andersen et al., 1996; Vincent, 1980). Therefore absence of water, one of the main components of the cuticle, is expected to lead to some changes of the structure. There are literature data on the structural changes depending on water reduction in the cuticle during sclerotisation (Andersen et al., 1996), but nothing is known about how drying alter the cuticle structure. However it is obvious that the water content makes cuticle soft and compliant. The increase in hardness and elastic modulus, especially of the cuticle layer near the surface up to 1700 nm, is thought to be caused by structural changes due to water loss. Indentation tests on various biological surfaces (insects and plants) display a considerable difference in the mechanical behavior between fresh/hydrated and dehydrated materials (Hillerton et al., 1982; Arzt et al., 2002; Enders et al., 2004; Vincent and Wegst, 2004).

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Table 2. Results of statistical calculations of the significance (One-Way-ANOVA and Tukey post tests) of differences between the hardness and elastic modulus for various indentation depths (250 nm, 500 nm, 1000 nm and 1500 nm) in fresh, dry and chemically treated conditions. + means that there is a significant difference between the values compared; - means that there is no significant difference between the values compared. There is no significant difference in the elastic modulus values between the dry and chemically treated samples an indentation depth of 1000 nm and 1500 nm. Hardness Elastic modulus + +

Fresh + +

Chem. treated

+ +

+ + + + + +

Fresh

Chem. treated

+ +

+ + + + + +

Fresh + +

Chem. treated

+ +

+ + + —

+ — + +

Fresh

1000

dry

Depth, nm

+ +

+ + 500

Dry

250

Dry

+ +

+ +

+ +

Chem. treated

+ +

+ —

Condition

Fresh

Dry

1500

+ —

Dry

— Chem. treated

Removal of surface waxes and lipids causes only small changes in the indentation results (slight increase in hardness and stiffness), especially at a depth below 1000 nm. Without water, the mechanical behavior of the gula cuticle is influenced only a little by the outer wax/lipid layer and is presumably determined mainly by proteins and chitin, which could not be removed by the chemical treatment used in this study. In order to understand how the two

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main components (proteins and chitin) contribute to the mechanical properties of the cuticle, further studies are necessary. SEM images were helpful to evaluate indentation data. The maximum indentation depth in our experiments was 3 µm. Therefore, measurements of the mechanical properties were done in the epicuticle (figure 6 B), which is about 7 µm thick. According to several models (Jönsson and Hogmark, 1984; Burnett and Rickerby, 1987; McGurck et al., 1994; Rother and Jehn, 1996; Korsunsky et al., 1998) concerning the hardness determination for thin metal films, the results obtained could also be influenced by layers of the exocuticle. After Bueckle (1965) and Bhushan (1996), hardness measured up to an indentation depth of 0.7 µm (displacement < 1/10 of the layer thickness) can be assumed to characterize the epicuticle. With larger displacements the hardness will be characteristic of the whole composite of the epicuticle/exocuticle layers. This suggestion is true only for hardness measurement. The elastic modulus measurements will be influenced by the whole composite since it is a longrange effect. The mechanical behavior of the gula cuticle can be regarded as that of a soft substrate coated by a hard film. A decrease in hardness and elastic modulus with increasing indentation depth is caused by the underlying layers. As already mentioned, deeper layers in cuticle are softer and more compliant than the outer ones. The influence of the underlying layers on the mechanical properties is stronger on dry and chemically treated samples than on fresh ones. Without water, surface layers appear to become harder and stiffer than the deeper layers showing a lower density and higher protein content (Locke 1964; Neville 1975, Binnington and Retnakaran 1992). Several studies of elastic modulus and hardness of insect cuticle using different methods can be found in the literature. Almost all experiments were performed on dry or rehydrated samples. There are only a few measurements of the mechanical properties of cuticle made by indentation. The average hardness (0.10–0.52 GPa) and elastic modulus (1.50–7.70 GPa) values obtained in this study are similar to those of various sclerotised cuticles (Vickers hardness 0.2–0.5 GPa and Young’s modulus 1–10 GPa (Vincent and Wegst, 2004)). However the hardness (0.10 GPa) of the fresh samples is below this range (0.2–0.5 GPa). It is believed that these are possibly the first measurements of hardness and elastic modulus on the insect cuticle in native condition. Comparable hardness values were obtained on other cuticle elements. For example, the dry wing membrane cuticle of the dragonfly Aeshna cyanea tested with a nanohardness-testmachine (Hysitron TriboScope) exhibited a hardness of 0.2 GPa (Kreuz et al., 1999). The different parts of the dehydrated locust cuticle measured by means of a Leitz Miniload hardness tester (using a Vickers diamond) (Hillerton et al., 1982) revealed H = 0.24–0.33 GPa. However the dry samples of the beetle head tested in this study (H = 0.49 GPa) were still harder than the cuticles mentioned above. The elastic modulus values (E = 7.50 ± 1.80 GPa) obtained for the dry beetle cuticle is very high when compared to the elastic modulus from tensile test of the dry wing membrane of the dragonfly Aeshna cyanea, where E = 1.5 ± 0.5 GPa (Kreuz et al., 1999). The reduced modulus for different body parts of the dehydrated dragonfly (Odonata, Anisoptera) were calculated from quasistatic nanoindentation experiments (Hysitron Inc., Minneapolis, MN) (Kempf, 2000). The mean values were lower (1.5–4.7 GPa) as well than elastic modulus obtained for dry samples in this study. Recent nanoscale study (quasistatic nanoindentation testing instrument, Hysitron Inc., Minneapolis, MN) of Drosophila melanogaster integument

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during development (Kohane et al., 2003) shows that the thickness and the development stage of the cuticle was very important for stiffness measurements. The average reduced elastic modulus (Er) of 0.41 MPa, 15.43 MPa and 4.37 MPa were determined by in vivo experiments for the cuticles of the larval, puparial and adult insects respectively. Thickness of the cuticles of Drosophila melanogaster was very low (2.3–17.7 µm) when compared to that of gula cuticle (about 80 µm). However, the mechanical properties of the cuticle are strongly influenced also by proportions of the main components (chitin and proteins) (Fraenkel and Rudal, 1940; Locke, 1964; Neville, 1975; Hepburn and Chandler, 1976; Binnington and Retnakaran, 1992). The nanoindentation experiments in this study were carried out in the epicuticle, which is a nonchitinous layer. It contains proteins and is covered with lipids and surface waxes (Neville, 1975; Binnington and Retnakaran, 1992). Surprisingly the elastic modulus of the fresh gula cuticle (E = 1.50 ± 0.80 GPa) is comparable to that of the chitin filament (1.97 ± 0.07 GPa) (Joffe and Hepburn, 1973). There are contradictory data in the literature about which component (chitin or protein) is responsible for the elastic modulus of the cuticle. According to Fraenkel and Rudall (1940), the mechanical properties of the cuticle are determined by chitin, whereas Vincent (1980; 2002) suggests that the protein matrix is decisive for its mechanical behavior. Elasticity which is expected to be a long-range effect is influenced by the underlying exocuticle layer consisting of a high amount of chitin in a protein matrix (Neville, 1975; Binnington and Retnakaran, 1992). Nevertheless, according to our experimental results, it can be concluded that the elastic modulus in fresh cuticle is not only influenced by only chitin or only the protein-matrix, but by both components. After an indentation test the surface profiles of the samples were investigated by atomic force microscopy (AFM) (DME, DualScope™ C-21 with scanner DS 45-40 BIO). Figure 9 shows the surface profile of the gula cuticle. Residual deformation of the cuticle surface after indentation seems similar to elastic-plastic contact, which is typical of most engineering materials (Barbakadze 2005). An elastic recovery on the indent-image can be distinguished. This is due to a visco-elastic relaxation expected for soft biological materials (Wainright et al., 1976; Vincent, 1990; Kohane et al., 2003).

WAX-COATED PLANT SURFACES The cuticle is an essential element of a plant. Being the outer surface layer, it is of fundamental functional and ecological importance for the interaction between plants and their environment (Barthlott, 1990; Bianchi, 1995; Gorb, 2001). The plant cuticles display different surfaces. They may be pigmented or colorless, smooth, textured or hairy, dry or covered with epicuticular secretions (Jeffree, 1986; Gorb, 2001). The extraordinary diversity of the plant surface structure is a reflection of their different environment (Jeffree, 1986). Preventing desiccation and allowing controlled exchange of gases are the important functions of epicuticular components (Baker, 1982; Jeffree, 1986; Barthlott, 1990). The plant cuticle is a multilayered composite consisting of a polymeric cutin matrix and the cuticular waxes (Eigenbrode, 2002). Figure 10 shows the generalized structure of the plant cuticle (Gorb, 2001). An outer wall of epidermal cells is covered by a pectinaceous cuticle membrane, which is protected by a layer made up of cellulose microfibrils. On the

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cuticle proper consisting of a lamellate layer, cutin and the epicuticular waxes are deposited (Bianchi, 1995; Gorb, 2001). Cutin is high molecular weight lipid polyester (Bianchi, 1995).

Figure 9. AFM images of the cuticle surface after indentation test showing the residual deformation. The image of the indent shows elastic recovery, which is characteristic for the elastic-plastic contact in most engineering materials. However such shape of the indent here is thought to be caused by viscoelastic relaxation expected for biological materials.

The epicuticle waxes are surface lipids (Bianchi, 1995). They display considerable ultrastructural and chemical diversity. The plant waxes are a complex mixture of very longchain aliphatics including different chemical compounds, such as hydro-carbons, wax esters, primary and secondary alcohols, fatty acids, aldehydes, ketones, ß-diketones, triterpenoids and flavonoids (Walton, 1990; Eigenbrode, 2002). A current classification of plant epicuticular waxes, based on the studies of 13 000 plant species using high resolution scanning electron microscopy (SEM), distinguishes 23 types of wax crystals (Barthlott, 1990; Barthlott et al., 1998). The most common types of crystal shapes are tubes, solid rodlets, filaments, plates, ribbons and granules (Baker, 1982; Jeffree, 1986; Bianchi, 1995; Barthlott et al., 1998). The wax crystalloid structure correlates with its chemical composition (Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998). For example, aldehydes are very important for the formation of wax filaments, which is characteristic of the pea Pisum sativum leave surface (Gorb, 2001). The eucalyptus epicuticular wax, containing ß-diketones and primary alcohols, exhibits mixed tubes and plates structure (Hallam and Chambers, 1976; Jeffree, 1986).

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Figure 10. The generalised structure of a plant cuticle: W - epicuticular wax; CP - cuticle proper; CL cuticular layer of reticulate region traversed by cellulose micro fibrils; P - pectinaceous cuticle membrane and middle lamella; CW - cell wall; PL - plasma lemma (taken from Gorb, 2001).

All plant surfaces carry a partial or a continuous layer of the amorphous wax as the obligatory final layer of the cuticle (Baker, 1982, Barthlott et al., 1998). However some plants consist of even two amorphous wax layers. Upon the amorphous layer, wax crystals of different shapes emerge and grow. The wax crystal layer varies widely in thickness from 1 to 20 µm. The thickness of the wax crystalloids lies in the nanometer range (Gorb, 2001; Enders et al., 2004). Wax morphology varies with the environmental conditions. The thickness of the layer, the size, the orientation and the density of wax crystals can be significantly modified by temperature and humidity (Baker, 1982). The epicuticular waxes occur in plants primarily on the surface of leaves, stems and fruits (Bianchi et al., 1978; Avato et al., 1984; Baker and Gaskin, 1987; Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998; Gorb, 2001). On the surfaces of many plant species, such as Eucalyptus leaves, the wax is continuously regenerated. In this way the surface is protected and kept in a fully functional state. However, the epicuticular wax cannot be replaced after having been removed by abrasion from some plant surfaces (Bianchi, 1995). Although no study on mechanical properties of the wax layer has been carried out, it is believed in biology that the plant wax and the underlying layers display different mechanical properties. A cuticularisation of the thick outer epidermal walls contributes to mechanical stability of the surface. The wax covering is mechanically very unstable and in biology known as “soft film” (Barthlott, 1990, Barthlott et al., 1998). Being fragile, the waxes are easily removed by rubbing (Barber, 1955; Jeffree, 1986). The wax crystalloids increase the roughness. Together with the wax chemistry, the roughness makes the plant surface hydrophobic and decreases its wettability. The hydrophobic constituents of the wax and surface microroughness reduce the adhesion of the dust and other particles on the outer cell wall. This “anticontamination function” called “lotus effect” is thought to be the most

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important ecological function of the water-repellent plants (Baker, 1982; Barthlott, 1990; Barthlott and Neinhuis, 1997). An interaction between the plant surfaces and their environment also involves the complex and very important relation between plants and insects (Juniper and Southwood, 1986). Insects possess the attachment devices allowing the hold and the movement anywhere and on nearly any kind of surfaces (Gorb, 2001). The structures of attachment pads adapted for the natural, mainly plant surfaces enable the insects to move freely on any technical surfaces as well. However, some surfaces even in nature raise difficulties for their visitors. The wax layer on the plant is a barrier for the attachment systems of insects. Walking on the plant surface structured with the wax crystals was found to be impossible for insects (Eigenbrode, 1996; Gaume et al., 2002; Gorb and Gorb, 2002). Even though the interaction between the wax crystalloid layer and the insect has been of great interest to researchers for about a century (Kerner von Marilaun, 1898; Haberlandt, 1909; Knoll, 1914), the function of such epicuticular layer preventing adhesion remains largely unknown. There are only a few experimental studies reporting some hypotheses, why the insects cannot attach on the wax coated plant surfaces (Eigenbrode, 1996; Gorb and Gorb, 2002; Gaume et al., 2002): (1) the location of wax crystals on the plant cuticle may be responsible for an increase in the surface microroughness; this can be the reason for reduced real contact area between the plant surface and the attachment system of insects (Stork, 1980); (2) the wax crystals are mechanically unstable and fragile; attachment can be prevented by the contamination of the attachment system of insects by the adhesion of the wax crystals to its pads (Juniper and Burras, 1962); (3) the possible reaction between the wax layer and the secretion liquid of the attachment system of insects (Gaume et al., 2002). The interaction between the insect attachment systems and the plant anti-attachment surfaces motivated this work. The goal was to study the mechanical behavior of plant surfaces coated with wax crystals to understand the anti-attachment mechanism of the wax and to clarify the first two hypotheses. The leaves of two pea (Pisum sativum) mutants were selected for this study. The wild type pea leaves are strongly covered with wax crystals (filaments). In “glossy” mutation, the amount of waxy bloom is greatly reduced (Eigenbrode and Espelie, 1995; Eigenbrode and Jetter, 2002). The samples in fresh and dry conditions were mechanically tested using the Nano indenter® SA2. The structure of plant surfaces was explored by means of scanning electron microscopy (SEM). This study is believed to be the first attempt to characterize the mechanical properties of the wax crystals of plant surfaces.

MATERIALS AND SAMPLE PREPARATION The experiments were performed with the pea Pisum sativum. The wild and glossy mutants of the pea plant were planted and grown in the laboratory. The samples were prepared from the pea leaves of both mutants: the wild type with normal wax layer and the glossy type with reduced waxy bloom. The top and bottom sides of the leaves were tested in the untreated condition. The nanoindentations were performed on the specimens in the fresh and dry conditions. To prepare dry samples, the fresh cuts of leaves were air dried at room temperature for 48 h.

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STRUCTURE OF WAX LAYER The surfaces of the pea leaves were observed using scanning electron microscopy (SEM). The samples, 7×7 mm in size, were cut from the leaves and mounted on an aluminum holder. The surface structure of the wild and the glossy plants were tested at 5 kV. The observations of top and bottom surfaces of both pea mutants were performed in the untreated, fresh condition. Figures 11 and 12 show the surface structure of the wild and glossy pea leaves respectively. The images are taken on the top and the bottom side of the leaf for each plant type. The cell structure of the plant surfaces can be distinguished in figures 11 A, B and 12 A, B. The openings on the figures 11 A, B and 12 A, B are stomata for gaseous exchange. There is a great difference in the leaf surface between the wild and glossy pea. The wild pea samples are completely covered with wax crystals (figures 11 C, D, E, F), while the glossy pea surface only shows single wax crystals (figures 12 C, D, E, F). The comparison of SEM images of the two plant mutants (figures 11 C, D and 12 C, D) leads to the estimate that the surface of the glossy pea leaf bears only about one third of wax crystals. There is a small difference in the wax covering of the top and bottom side of the same plant species as well. The bottom side of the wild pea sample shows a denser wax crystal structure than the top side (figure 11 C, D). The single wax crystals appear to be larger on the top surface of the glossy pea leaf than on the bottom side (figure 12 C, D). The single wax crystals can be estimated to be 1 – 2 µm long with a diameter of 100 - 200 nm (figures 11 E, F and 12 E, F). As can be seen in the figures 11 E, F and 12 E, F, the wax crystals are oriented at different angles to the surface.

Figure 11. SEM images of the wax crystalloid layer on the wild pea leaf surface. A and C show the surface of the top side; B and D show the surface of the bottom side. Images A and B show cell structure of the plant leaf. On the C and D images wax crystal covering the leaf can be distinguished. The bottom side (D) of the pea leaf appears to have more dense waxy bloom than the top side (C). E and F show the top side of the wild pea leaf.

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Figure 12. SEM images of the wax crystalloid layer on the glossy pea leaf surface. A and C show the surface of the top side; B and D show the surface of the bottom side. Images A and B show cell structure of the plant leaf. On the C and D images single wax crystals on the leaf can be distinguished. The top side (C) of the glossy pea leaf appears to have more wax crystals than the bottom side (D). E and F show the top side of the glossy pea leaf.

MECHANICAL PROPERTIES Table 3 and 4 summarise the results of mechanical properties, hardness and elastic modulus. The mechanical behaviors of the sample surfaces differ presumably depending on the amount of wax crystalloids in the dry, but not in the fresh condition. It is surprising that there is no difference in the mechanical properties (hardness and elastic modulus) between the fresh samples with normal and with reduced wax layer. The maximum load corresponding to the maximum displacement (3 µm) is about 3.5 mN for all fresh samples. The wax layer appears to have an influence on the mechanical behavior of the dry plants. The top and bottom surface of the same plant differ due to the different density and amount of the wax crystals as well. In general, the surface with more wax shows lower hardness and elastic modulus values (figure 13 and 14). The wax layer is believed to make the plant cuticle surface soft and compliant. In the figure 13 and 14, hardness and elastic modulus (mean values of approximately 100 measurements are shown) are plotted versus displacement for all fresh (figure 13 A and 14 A) and all dry (figure 13 B and 14 B) samples. By looking at the curves showing hardness values at the beginning of the indentation, a little influence of the wax layer could be observed only at the first 200 nm of indentation depth. In this range the fresh samples display a slightly different behavior depending on the presence of the wax layer.

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Table 3. Summary of mechanical properties obtained at the displacement of 100 and 500 nm for fresh plant samples Hardness (GPa)

Elastic modulus (GPa)

by indentation depth of

by indentation depth of

100 nm

500 nm

100 nm

500 nm

wild top

0.7 ± 0.5

0.7 ± 0.2

3.2 ± 0.8

1.1 ± 0.2

wild bottom

0.7 ± 0.4

0.6 ± 0.4

2.7 ± 0.3

1.0 ± 0.3

glossy top

0.5 ± 0.5

0.5 ± 0.3

2.2 ± 0.5

1.0 ± 0.2

glossy bottom

0.4 ± 0.5

0.6 ± 0.2

2.6 ± 0.7

1.1 ± 0.1

Plant species

Table 4. Summary of mechanical properties obtained at the displacement of 100 and 500 nm for dry plant samples Hardness (MPa)

Elastic modulus (MPa)

by indentation depth of

by indentation depth of

100 nm

500 nm

100 nm

500 nm

wild top

5.2 ± 2.8

2.3 ± 0.9

285.2 ± 153.1

238.3 ± 194.2

wild bottom

1.2 ± 0.8

0.3 ± 0.1

168.2 ± 90.1

96.4 ± 59.3

glossy top

50.7 ± 41.1

23.7 ± 19.3

799.3 ± 569.1

570.4 ± 381.2

glossy bottom

7.5 ± 6.1

5.7 ± 3.9

526.8 ± 382.7

403.1 ± 291.1

Plant species

Fresh plant samples with normal wax layer tend to be harder only at the first 200 nm of indentation depth than those with reduced wax In the first 200 nm the indenter tip obviously contacts single wax crystals. This is thought to be caused by high mechanical stability of single wax crystalloids that build a very unstable and soft wax layer (Barthlott, 1990). However, a difference in the elastic modulus values between the fresh samples is not noticeable.

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Figure 13. Hardness versus displacement curves of all fresh (A) and all dry (B) pea leaves. The mean results of approximately 100 measurements are shown up to 1 µm.

a)

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b)

Figure 14. Elastic modulus versus displacement curves of all fresh (A) and all dry (B) pea leaves. The mean results of approximately 100 measurements are shown up to 1 µm.

All fresh samples exhibit the same mechanical behavior at larger displacements apparently due to the influence of the underlying layers. The plant cuticle has a multilayered structure (Eigenbrode and Jetter, 2002), which consists of the mechanically stable cuticularised epidermis covered by unstable and soft wax layer (Barthlott, 1990; Barthlott et al., 1998). The hardness and the elastic modulus values corresponding to the fresh samples are expected to be strongly influenced by cell walls and pressure of the cell fluid. However, the cell wall itself appears to be soft and compliant (dry samples are softer and more compliant than fresh ones). The cell structure, filled by fluid, appears to be influenced by internal pressure of the cell fluid and therefore, is hard and stiff. This is why the effect of the wax layer cannot be detected. A schematic of the cross section of the plant sample during the indentation is shown in figure 15. In the dry condition, the influence of the cell fluid is minimized or removed and there is a great difference in the mechanical behavior between the plant surfaces with normal and with reduced wax layer. The dry samples with normal wax layer are softer and more compliant than the dry ones with reduced wax layer. In addition, the same mechanical behavior was observed in the case of pitcher plant Nepenthes alata, eucalyptus Eucalyptus guinnii and red cabbage Brasica (Barbakadze, unpublished results). The desiccation greatly influences the mechanical behavior of biological materials (Gorb, 2001; Vincent and Wegst, 2004). However the effect of water loss is different for insect and plant cuticles. Fresh insect cuticles are softer and more compliant than dry ones. By contrast, the hardness and the elastic modulus values of fresh plant cuticle surfaces are higher than that of dry ones.

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Figure 15. Schematic of the cross section of the plant during indentation experiment. The arrows show pressure of the cell fluid under the indenter.

WAX COVERINGS AND INSECT ATTACHMENT PADS The attachment abilities of insects were recently tested on the plant surfaces investigated in this study. The leaves of the wild pea (Pisum sativum) (Eigenbrode and Jetter, 2002) were difficult for insects to cope with. While walking on these surfaces, the insects often stopped and cleaned their feet, whereas they had less difficulty on the surface of the glossy mutant with reduced waxy bloom. To elucidate the hypotheses of reduced contact area and contamination effect of the wax crystals, let us consider the results obtained in connection with the blowfly Calliphora vicina (Niederegger et al., 2002), which is the model for most studies of the structure and the attachment behavior of pads. What would the hardness and elastic modulus values obtained in this study mean for this insect? The blowfly C. vicina weighs about 4 mN and possesses attachment pads structured with a few thousand (4000–6000) flexible (1 N/m spring constant) cuticular outgrowths called seta. A single spatula (tip of the seta) has an area of 1 µm2 (Niederegger et al., 2002). Thickness of the wax crystals lie in a few hundred nanometer range. By walking, three feet of the insect with one third of the whole amount of setae come in contact. However, the area of a spatula increases in contact by about 35% (Niederegger et al., 2002). An estimation of the load exerted by one single seta in contact with the plant surface gives values ranging between 134 and 400 nN.

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In the nanoindentation experiments, the hardness and elastic modulus values corresponding to this load range were obtained at displacements of 20–40 nm: for the wild pea, H = 50–340 MPa and E = 2.50 8.00 GPa (figure 16). With these considerations, one can assume that wax crystals remain stable under insect pads and contribute to a decrease of the contact area between the insect attachment pad and the plant surface (hypothesis 1). However, the wax crystals are not oriented perpendicularly to the surface but at different angles. The insects do not only push down the plant surface but also use lateral forces while walking. These considerations make the estimation more difficult. Regarding all these factors, contamination of the insect pads (hypothesis 2) due to the breaking and adhesion of wax crystals to the pad surface cannot be excluded. Further measurements on the isolated cuticle (without cell structure) surface are necessary.

Figure 16. Hardness and elastic modulus of the wild pea samples (top side of the leaf) in the displacement range of 0–80 nm. Grey area shows the displacement area of 20 to 40 nm, corresponding to the load range of 134–400 nN. This load range is estimated to act during the walking of the blowfly C. vicina on the plant surface.

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Jeffree, C. E. (1986) The cuticle, epicuticular waxes and trichomes of plant, with references to their structure, functions and evolution. In: Insects and the plant surface. Juniper, B. E., Southwood, T. R. E. (eds.) Edward Arnold: London, pp. 23-64. Jensen, M. and Weis-Fogh, T. (1962) Strength and elasticity of locust cuticle. In: Biology and Physics of locust flight. Philosophical Transactions of the Royal Society, B 245 pp. 137-169. Joffe, I. and Hepburn, H. R. (1973) Observations on regenerated chitin films. Journal of Materials Science, 8: 1751–1754. Jönsson, B. and Hogmark, S. (1984) Hardness measurements of thin films. Thin Solid Films, 114: 257-269. Juniper, B.E. and Burras, J.K. (1962) How pitcher plants trap insects. New Scientist. 13: 75-77. Juniper, B. E. and Southwood, R. (1986) Insects and the Plant Surfaces. London, UK: Edward Arnold. Kempf, M. (2000) Biological materials, determination of Young’s moduli of the insect cuticle (dragonflies; Anisoptera). Application note, Hysitron Inc, www.hysitron.com. Kerner von Marilaun, A. (1898) Pflanzenleben 2. Bibliographisches Institut, Leipzig/Wien. Knoll, F. (1914) Über die Ursache des Ausgleitens der Insektenbeine an wachsbedekten Pflanzendteilen. Jahrbuch für Wissenschaftliche Botanik, 54: 448-497. Kohane, M., Daugela, A., Kutomi, H., Charlson, L., Wyrobek, A. and Wyrobek, J. (2003) Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development. Wiley Periodicals, Inc.: 633-642. Korsunsky, A. M., McGurk, M. r., Bull, S. J. and Page, T. F. (1998) On the hardness of coated systems. Surface Coatings Technology, 99: 171-183. Kreuz, P., Kesel, A., Kempf, M., Göken, M., Vehoff, H. and Nachtigall, W. (1999) Mechanische Eigenschaften biologischer Materialien am Beispiel Insektenflügel. BIONA report, 14: 201-202. Locke, M. (1964) The structure and formation of the integument in insects. In: The physiology of Insecta. Rockstein M. (ed.), Academic Press, New York, pp. 123-213. McGurk, M. R., Chandler, H. W., Twigg, P. C. and Page, T. F. (1994) Modelling the hardness response of coated systems: the plate bending approach. Surface Coatings Technology, 68/69: 576-581. Neville, A. C. (1975) Biology of the arthropod cuticle. Springer Verlag, Berlin, Germany. Niederegger, S., Gorb, S. and Jiao, Y. (2002) Contact behaviour of tenent setae in attachment pads of the blowfly Calliphora vicina (Diptera, Calliphoridae). Journal of Comparative Physiology, A 187: 961-970. Oliver, W. C. and Pharr, G. M. (1992) An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. Journal of Materials Research, 7 (6): 1564-1583. Perez Goodwyn, P. J. and Gorb, S. N. (2004) Anti-frictional properties of contacting surfaces in the hemelytra-hindwing locking mechanism in the bug Coreus Marginatus (Heteroptera, Coreidae). Journal of Morphology (in press). Persson, B. N. J. (1998) Sliding friction. Springer: Berlin, Heidelberg, New York. Rother, B. and Jehn, H. A. (1996) Coating and interface characterization by depth-sensing indentation experiments. Surface and Coatings Technology, 85: 183-188.

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Scherge, M. and Gorb, S. N. (2000) Microtribology of biological materials. Tribology Letters, 8: 1-7. Stork, N. E. (1980) Role of wax blooms in preventing attachment to brassicas by the mustard beetle, Phaedon cochleariae. Entomologia Experimentalis et Applicata, 28: 100-107. Vincent, J. F. V. (1980) Insect cuticle - a paradigm for natural composites. In: The mechanical properties of biological materials. Symposium of the Society Experimental Biology, 34: 181-210. Vincent, J. F. V. (1990) Structural Biomaterials. The University Press, Princeton. Vincent, J. F. V. (2002) Arthropod cuticle –a natural composite shell system. Composite Part A - Applied Science and Manufacturing, 33 (10): 1311-1315. Vincent, J. F. V. and Wegst, U.G. K. (2004) Design and mechanical properties of insect cuticle. Arthropod Structure and Development, 33: 187-199. Wainwright, S. A., Biggs, W. D., Currey, J. D. and Gosline, J. M. (1976) Mechanical Design in Organisms. Princeton University Press. Walton, T. J. (1990) Waxes, cutin and suberin. In: Lipids, membranes and aspects of photobiology, Dey, P. M., Harborne, J. B. (eds.), pp. 105-158. Wegst, U. G. K. and Ashby, M. F. (2004) The mechanical efficiency of natural materials. Philosophical Magazine, 84, 21: 2167-2181.

PART 5. COMPOUNDS FOR ANTIBIOCORROSIVE COVERS AND PROTECTORS

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 20

ANTIBIOCORROSIVE COVERS AND CONSERVATORS BASED ON NEW CARBOFUNCTIONAL OLIGOSILOXANES AND BIOLOGICALLY ACTIVE COMPOUNDS Nodar Lekishvili, Khatuna Barbakadze, David Zurabishvili, Tea Lobzhanidze, Shorena Samakashvili, Zurab Pachulia, and Zurab Lomtatidze Ivane Javakhishvili Tbilisi State University, Georgia

ABSTRACT New carbofunctional oligosiloxanes containing trifluorinepropyl and methacrylic groups at silicon atoms have been synthesized and studied. Biological active nitroanilides with spatial adamantane-containing groups and cadmium complex compounds based on them have been obtained. By using the data of IR and NMR spectral analyses the composition and the structure of synthesized compounds have been established. New composite materials of multifunctional application for individual and environmental protection, based on the obtained silicon-organic carbofunctional oligomers and complex compounds, have been created. It was shown that the created composites could be used as: a) protective covers (film materials and impregnating compositions) stable to biocorrosion; b) materials with antimycotic properties for prophylaxis and treatment of mycosis; c) biologically active polymer materials for protection of archaelogical and museum exhibits; d) for human protection during their contact with microorganisms. Preliminary investigations have shown that the synthesized compounds have also a real perspective to be utilized as accessible antioxidants towards the cancer.

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INTRODUCTION At the first decade of the 21st century a wide assortment of synthetic and natural polymeric materials has been produced. At the same time, there appeared various aggressive microorganisms, which can destruct these materials [1, 2]. The actions of microorganisms on polymers are influenced by two different processes: a) deterioration and degradation of polymers, which serve as a native substance for growth of the microorganisms (direct action); b) influence of metabolic products of the microorganisms. Losses caused by destruction of natural and synthetic materials with micromycets reach enormous amounts and constitute annually milliards of dollars (indirect action) [1, 2]. One of the ways to protect the synthetic materials from the action of microrganisms is a creation of novel polymer covers with high bioactivity by modification of various polyfunctional adhesive polymer matrixes with biologically active compounds [3, 4]. Use of natural and synthetic biological active compounds as modificating additives unable to firm fixation in polymer matrix. Such polymers are characterized not only by contact [fungistatic] action, as the first ones, but could dosilly extract biological active compounds to environment. The latter is an important factor for guaranteed human protection during its long stay in a closed space [4]. In many regions of the world some diseases of agricultural plants, caused by various phytopathogenic microorganisms, are widely circulated. For example tumors, halles and nodes are formed as a result of intensive division of affected cells of meristem plant tissues. Roots’ and fruit-trees cancers are provoked by - А. tunefacicus; cancer of root crops, beets is provoked by X. campestris pv. beticols etc. These diseases distractively damage plants and significantly decrease harvesting efficiency. They also deteriorate the quality of a grape, water-melons, melons and other agricultural plants [4]. Therefore, synthesis of new compounds as plants’ protectors with high biologically activities, as well as conservators and compounds for antibiocorrosive covers of various natural, synthetic and artificial materials is extremely significant and requires further developments [5].

EXPERIMENTAL Synthesis of adamantane-containing anilides: to the benzene solutions of hydrochlorides of initial amines [6] and basic agents (triethylamine, NaHCO3 or NaOH) was added dropwise benzene solution of the chloroanhydride of various carbonic acids (adamantane-1carboxylic acid, acetic acid, benzoic acid or phenylacetic acid, correspondingly). The mixture was heated and stirred during 1.5-3 hrs. The precipitated crystals were separated by filtration and washed with H2O; they were dried and physical constants of obtained compounds were determined. Synthesis of adamantane-containing nitroanilides: to the solution of adamantanecontaining anilides in acetic anhydride and acetic acid was added drop-wise 56-58% HNO3. The mixture was cooled on 5-100C and stirred during 0.5-2 hrs. The reaction mixture was dismissing (by pour out on ice water). The precipitated crystals were separated by filtration

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and washed with H2O; they were dried and physical constants of obtained compounds were determined.

Method of Analysis Spectral analysis: IR spectra were obtained from KBr pellets, using UR-20 (Karl Zeis®) spectrophotometers and a Nicollet Nexus 470 machine with MCTB detector [7]. NMR spectra were obtained with an AM-360 (Brucker®) instrument at an operating frequency of 360 MHz NMR spectra were obtained with an AM-400 (Brucker®) and Talsa BS-467 instrument at an operating frequency of 400 MHz using CDCl3 as a solvent and tetramethylsilane as an internal standard [8]. Elemental analysis of the obtained compounds was carried out according to the classic methods of microelemental analysis [9]. The quantitative determination of functional groups was performed by using procedures described in ref. 8. The content of the active hydrogen in Si–H was determined according to the ref. 10. Quantum-chemical calculations were performed on PC with AMD processor with the built-in coprocessor by using Mopac2000 and CS Chem3D Ultra, v8. We gave the following key-words to guide each computation: EF GNORM=0.100 MMOK GEO-OK AM1 MULLIK LET DDMIN=0.0 GNORM=0.1 GEO-OK. Thermogravimetric and differential-thermal analysis (TGA and DTA) was performed on a derivatograph (Paulic, Paulic and Erdey) at the speed of the heating 10Kmin-1. Chromatography analysis of original reagents and the reaction products were performed by using the device LKhM-80 (Russia), type 2 (the column 3000 x 4 mm, the head – “Chromosorb W, the phase-5 mass % SE-30, and gas-carrier-helium). Wide-angle X-ray diffractograms have been obtained by DRON-2 instrument (“Burevestnik”, Petersburg, Russia). Cu Kα was measured without a filter; the motor angular velocity was ω ≈ 2 deg. min-1.

RESULTS AND DISCUSSION Antibiocorrosive covers contain two components – biologically active compound and polymer matrix, where the biologically active compound is dropped [11]. Some polyfunctional hetero-chained organic polymers, such are polyurethane elastomers, polyurethaneacrylates, ionomers, etc., has been successfully used as a matrix for creation of antibiocorrosive covers [12]. The polymer matrix for the antibiocorrosive covers may be also obtained based on polyepoxide resins in mass in presence of the active diluents [13]. The use of the organic polyepoxide resins “ED-20” and “ED-26” for creation of the matrix for antibiocorrosive covers did not give the satisfactory results. The obtained covers cracked during the exploitation and turn yellow. To modify the aforementioned coating material we used silicon-organic oligomers with fluorinealkyl radicals at silicon atoms. These oligomers we obtained by hydrosilylation oligoorganohydridesiloxane (MF-1)[14] with perfluorinealkylacrylate (12FA) [15] by formula CH2=CH–C(O)OCH2(CF2)5CF2H in presence of Speier’s catalyst (0.1 mole solution of H2PtCl6 in iso-propanole):

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nMe3SiO[i-(MeHSi)a(Me2SiO)b(MePhSiO)c]mSiMe3+ mb CH2 = CHC(O)OCH2(CF2)5CF2H Me3SiO[MeSiO)a(MeSiO)b(MePhSiO)]mSiMe3 CH2CH2C(O)OCH2(CF2)5CF2H where: a = 0.3, b = 0.4, c = 0.3; m = 10 Scheme 1.

In the IR spectra of synthesized compounds together with absorption bands related to SiOSi, SiCH3, SiC6H5, CH3, C6H5 groups(1040-1090 cm-1, 1425 cm-1, 1440 cm-1, 1330 cm-1, 2970 cm-1, 1605 cm-1, 3080 cm-1) there were found absorption bands related to H2C=C (in acrylic groups), C=O (in ester groups) and C–F group (in CF2 groups) (1640cm-1, 1720cm1, 1330cm-1)15. In IR spectra there was also observed a week maximum of absorption related to Si–H group confirmed non-complete (100%) conversion of Si–H groups. The addition of the fluorine-containing oligomers to the composites based on “ED-20” and “ED-26” results the diluent effect, improvement of hydrophobic properties and thermalstability but the hardening of the aforementioned oligomers, in conditions of their application, is difficult. To make easier of the hardening process of silicon-organic modifiers we have synthesized the new fluorine-containing carbofunctional oligoorganosiloxane with methacrylic groups at silicon atoms (MF-1-AMA-F3) by two stages according to the following scheme:

I. Me3SiO[(Me2SiO)a(SiMeO)b(SiMePhO)c]mSiMe3 H + mb CH2 = CH

CH2

O(O)C

C = CH2 CH3

Me3SiO[(Me2SiO)a(SiMeO)b(SiMePhO)c]mSiMe3 (CH2)3O(O)C

C = CH2 CH3

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II. Me3SiO[(Me2SiO)a(SiMeO)b(SiMePhO)c]mSiMe3 (CH2)3O(O)C + nF3

C = CH2 CH3

Me Me3SiO[(Me2SiO)a SiO (SiMeO)b(SiMePhO)c]mSiMe3 R 3n(CH2)3

O(O)C

C = CH2 , CH3

where

R = CH2CH2CF3

a=0.3, b=0.4, c=0.3; m≈10 Scheme 2. General reaction scheme of obtaining of the oligomer MF-1-AMA-F3

On the first stage we synthesized the comb-type oligosiloxane with side allylmethacrylate fragment (Scheme 2) by the hydrosilylation of the MFA-1 with AMA. The process was controlled by determination of the content of active Si–H groups’ in oligoorganohydridesiloxane in time by the method described in ref. 10. The corresponding kinetic curves were created (Figure 1). Based on the obtained kinetic curves the reaction rate constant have been determined (k=0.13•10- l.mol-1c-1, T=70°C). The total reaction order equals to 2. In Figure 1a is shown that hydrosilylation reaction proceeds rapidly during the 1-1.5 hours and then slows down. The reaction proceeds at 70°C with conversion about 74-%. The arising of the reaction temperature on 20°C the conversion of the active Si–H groups increased till 80% (Figure 1). The synthesized oligomer is viscous liquids soluble in acetone, dioxane, benzene and toluene. The structure and composition of the synthesized oligomer were established by elemental analysis, FTIR and NMR spectral data. In the FTIR spectrum (Figure 2) together with the absorption bands (1040-1080 cm-1, 1440 cm-1, 1450 cm-1, 2930 cm-1, 2970 cm-1, 1605 cm-1) related to Si−O−Si, Si−CH3, Si−C6H5, CH2, CH3 and C6H5 groups, correspondingly, the absorption bands related to C=O, C−O−C and H2C=C groups (1735 cm-1, 1160 cm-1, 1650 cm-1) have been observed. In the IR spectrum was not observed an absorption band for Si–H group.

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Figure 1. Dependence of the conversion of Si−H group (a) and reciprocal value of the concentration (b) on time (1.- 700C; 2.- 800C; 3.- 900C) during the reaction of MF-1 with AMA.

Figure 2. IR spectrum of adduct obtained from the interaction of MF-1 and AMA.

In the 1H NMR spectrum of the hydrosilylation product (Figure 5a) there were identified the resonance signals with chemical shifts 1.92 ppm, 5.40 ppm and 6.99 ppm, related to the protons of the following group:

5.40 ppm H 6.00 ppm

H

C

C

CH3 1.92 ppm

In the spectrum one can also observe the resonance signals of phenyl protons with chemical shifts in the range of 7.0-7.6 ppm. There were also observed resonance signals with chemical shifts 1.2 ppm of the methylene groups, 1.1 ppm of the methyl groups and multiplet resonance signal in the range of 4-1.8 ppm related to the methine group. These data confirm a

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formation of the fragment CH2−CH2 (α) and CH3−CH (β) of feasible derivation of Markovnikov and anti-Markovnikov addition products (Figure 3a). In the 13C NMR spectrum (Figure 3b) of the same sample one can observe the presence of the resonance signal with the chemical shift 65.75 ppm related to the protons of the OCH2 group and the resonance signal with the chemical shift 17.71 ppm related to the fragment of CH3 that indicates formation of both (Markovnikov and anti-Markovnikov) products:

21.61 ppm Si

CH2 12.63 ppm

CH2 CH2

O ,

18.60 ppm CH3

65.75 ppm

28.95 ppm CH CH2 O 65.75 ppm Si

In the 13C NMR spectrum of the obtained oligomer we have identified four type Si–CH3contained groups, this is once again proves the presence of α and β adducts (Markovnikov and anti-Markovnikov) [16, 17].

0.82 ppm CH3 CH3

Si CH3

,

0.51 ppm CH3 Si CH2

,

0.15 ppm

1.33 ppm CH3

CH3 Si

,

CH

CH3

Si C 6H 5

By the ratio of integral intensity of correspond resonance signals we determined the ratio of α and β adducts (39.13 : 60.87).

Figure 3a. 1H NMR spectrum of adduct of oligomethyl-phenylhydridsiloxane and allylmetacrylate.

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Figure 3b. 13C NMR spectrum of adduct of oligomethylphenylhydridsiloxane and allylmetacrylate.

We have performed calculations by the semiempirical AM1 method for modeling reaction between of oligomethylphenylhydrosiloxane (MF-1) to AMA using software Chem3D [15, 18, 19]. Such calculations for polymethylhydrosiloxane and AMA are not doable since the software does not produce reliable results for systems with more than 100 atoms. Necessarily, numerical values for the model reaction will be different than for the polymers studied experimentally, but they will provide better understanding of the experimental results (Scheme 2) [20]. We consider the hydrosilylation of (CH3)3SiOSi(CH3)(H)SiOSi(C6H5)(CH3)2 with AMA in view of the anti-Markovnikov and Markovnikov rules. According to the model reactions compounds I and II will be obtained (Scheme 3). The hydrosilylation reaction is considered through the following model reaction: (CH3)3SiOSi(CH3)(H)SiOSi(C6H5)(CH3)2 + + CH2=CH–CH2–O–C(O)–C(CH3)=CH2 → β (I) and α (II) adducts

CH3 CH3

C6H 5

Si

O

CH3

CH3

CH3

Si

Si CH3

O

CH3 H

+ H2C

CH

CH2 O

C O

C

CH2

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CH3 CH3 I

Si

O CH3

CH3

Si

CH3 C6H5

Si

CH3 (CH2)3 O

C

C

CH2

O

O

CH3 CH3 CH3

Si

O CH3

CH3

II

Si

CH3 C6H5

Si

O

CH3 CH CH3

CH2 O

C

C

CH2

O

CH3 Scheme 3. Model system for the calculation of ΔΔΗ# and ΔΔΗ for products of hydrosilylation of MF-1 with AMA.

The activation energy of α-adduct is ΔΔΗ# = 124.8 kJ/mole (RSiC = 2.30 Å), and for β adduct is ΔΔΗ#=114.4 kJ/mole (RSiC = 2.25 Å). In the both cases the combination process is exothermic (ΔΔΗ = -199.3 kJ/mole and ΔΔΗ = -191.2 kJ/mole respectively). The low value of activation energy of β-product indicates the superiority of performing the reaction in this direction. We compare ∆Hf values for compounds of addition taking into account Figures 4 and 5. Clearly, hydrosilylation reaction of (CH3)3SiOSi(CH3)(H)SiOSi(C6H5)(CH3)2 to AMA is energetically more favorable according to the anti-Markovnikov rule behind to Markovnikov rule. This result is in good agreement with NMR spectral data.

Figure 4. Dependence of enthalpy (ΔΗ) on the reaction coordinate (RSiC) for α-adduct.

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Figure 5. Dependence of enthalpy (ΔΗ) on the reaction coordinate (RSiC) for β-adduct.

On the second stage we have carried out the catalytic cooligomerization reaction of obtained methacrylate with trimethyltri(trifluorinepropylene)cyclotrisiloxane (in toluene, at 80°C), in the presence of sulfocationit “CU 23“(copolymer of divinylbenzene with styrene – 1.5-2.0 mass %) and hydroquinone (inhibitor, 1.0 mass %). Investigation of the corresponding model reaction (Scheme 4) by CLC method18 has shown that it is characterized by the establishment of the equilibrium at the room temperature (25°C) during 8 hours. The conversion of the hexametyldisiloxane (HMDS) reaches 50% [21]. By an increase of the temperature till 70°C, the time of the establishment of the equilibrium decrease till 3 hrs.

[CH2=C(CH3)-COO(CH2)3 Si(CH3)2] 2O + [(CH3)2Si]2O 2 CH2=C(CH3)-COO(CH2)3 Si(CH3)2-O-Si(CH3)3 Scheme 4. General scheme of the model reaction.

The value of the specific viscosity of the product of co-oligomerization depends on the molar ratio of MF-1–AMA and cyclosiloxane (Tible 1) and on the reaction temperature. The corves of dependence of the value of specific viscosity ηsp on the time (Figure 6) have an extreme character: in the beginning, during 2.5-3 hours, the ηsp reaches the maximal value (conversion of the organosiloxane – 80-85%); then it decreases (during 3.0-3.5 hrs) until a certain constant value (Figure 6, Table 1).

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Figure 6. Dependence of the value of specific viscosity of co-oligomerization products of MF-1-AMA with F3 (1) at the 800C.

It must be noted that the value of the specific viscosity of the product of co-oligomerization, in comparison to analogical systems [15], is increasing slowly, which may be connected with the increasing of the spatial (steric) factor at the silicon atoms in MF-1AMA. Table 1. The reaction conditions of co-oligomerization of MF-AMA and F3, and some characteristics of reaction products Initial substances molar ratio #

T,°C

Duration of the reaction, hr

ηspec.

Mη*

MF-AMA

F3

1

1

4

80

6

0.035

2523

2

1

6

80

6

0.028

1784

. *Mη = [η 5000]

1,515

The obtained co-polymers are colorless transparent or white viscous products soluble in ordinary organic solvents (toluene, dimethylformamide). The composition and structure of synthesized co-oligomers we established by elemental analysis and IR spectra. In the IR spectrum we abserved the absorption bands (1040-1080 cm-1, 1440 cm-1, 1450 cm-1, 1720 cm-1, 1645 cm-1, 2970 cm-1, 1600 cm-1) belonging to Si–O–Si, Si–CH3, Si–C6H5, C=O, C=C, CH3, C6H5 groups. The absorption bands (1170 cm-1, 1270 cm-1) related to C-F(CF3) groups were also observed [7]. For the synthesized oligomers, we have carried out wide-angle X-ray scattering (WAXS) analysis. Figure 7 shows that the oligomers are amorphous one-phase systems. Diffraction patterns display two maxima. First 2Θ0≈10.5 corresponds to the maximum of the inter-chain distance d1≈8.85Ǻ, while the second (2Θ0≈21) corresponds to d2≈4.33Ǻ, which characterizes both intra-molecular and inter-chain interactions [19].

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Figure 7. Wide-angle X-ray patterns of synthesized oligomers: MF-AMA/F3 (1:6), 2. MF-AMA (1:1), 3. MF-1-13FA (1:1).

By differential-scanning calorimetric (DSC) studies we determined that synthesized oligomers are amorphous one-phase systems (Figure 8). From analysis of DCS curves it is shown that the incorporation of perfluoromethacrylic radical in the chain of oligomethylphenylsiloxane (MF-1) modified with allylmethacrylate results to the rise of the transition temperature (Tg) on 19°C. We studied the thermal-oxidative stability of the synthesized organosiloxane by DTA and TGA analysis methods. We established that their destruction is starting at 280-290°C. The intensive destruction process proceeds above 450°C. We manufactured antibiocorrosive covers based on the synthesized bioactive compounds, dropped into the aforementioned polymer matrix, for goods from different materials.

Figure 8. DSC curves of oligomers: 1. MF-AMA (1:1); 2. MF-1- AMA-F3 (1:1).

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As biologically active compounds for antibiocorrosive polymer covers and protectors we have synthesized adamantane-containing nitroanilides (Scheme 5) and new cadmium complex compounds based on them (Scheme 6) [22, 23].

Scheme 5. Some of synthesized adamantane-containing nitroanilides.

While selecting nitroanilides and complex-compounds we foresaw their biologically activity and able to form dipole-dipole and hydride bonds with the polymeric matrix [6, 22, 24]. In order to select of these compounds we also considered the availability of their synthesis and possibility of their perspective wide commercialization. The composition and structures of synthesized compounds (Schemes 5 and 6) were established by using IR and NMR spectral data In the FTIR spectra of obtained compounds we have observed the absorption bands for the next groups: νas N–H (3430-3130cm-1), νas C–H of aromatic rings (3120-3030 cm-1), νas C–H of adamantyl group (2910-2830 cm-1), νas>C=O (1670-1640 cm-1), δ N–H, C–N (15401500 and 1360-1330 cm-1) and NO2 (1330-1350 cm-1), also νas C–O–C (1270-1230 cm-1). In 1H NMR spectra [24] of the synthesized compounds (I-II, Schemes 5) one can observe singlet signals with chemical shifts within the range 9.01-10.07 ppm for the protons in the NH groups. In spectra we could also observe quartet signals with chemical shifts at 7.30-7.94 ppm, which correspondes to the four protons in phenyl groups. Multiple signals with chemical shifts at 1.57-2.01 ppm correspond to the 15 protons in adamantyl groups. Singlet signals with chemical shifts within the range 3.71-3.83 ppm correspondes to the three protons in the methyl groups (VII, XIV). In 1H NMR spectra of the synthesized adamantane-containing nitroanilides (I-II) we can observe dublet signals with chemical shifts within the range 7.46-7.51 ppm for the protons C(3)H; the protons C(2)H and C(5)H one can observe in the form of two dublet signals with chemical shifts within the range 7.29-7.65 ppm. Constant of spin-spin interaction J = 2.8 what confirms the substitution of nitro-group in 2-position. In 13C NMR spectra one can observe a signal with a four chemical shifts at 27.6-40.7 ppm typical for adamantyl group the signals with chemical shifts within the range 165.17175.57 ppm are corresponded to the carbon atom of C=O groups; the signals with chemical shifts within the range 113.37-155.73 ppm related to the carbon atoms of phenyl groups. The synthesized compounds are stable on air white powders insoluble in water and in nhexane. They are dissolved in chloroform, DMFA, acetone and ethanol [24]. The synthesized nitroanilides we used as the ligands for manufacturing of the cadmium complex compounds [24]:

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Scheme 6. General reaction scheme of the synthesis of Cd complex compounds with adamantanecontaining nitroanilide ligands.

The composition and structures of synthesized compounds were established based on data of elemental analysis and FTIR spectra [7]. In the FTIR spectra together with the following absorption bands – 3448, 3371 (NH), 3090 (CH, Ar), 2928, 2850 (CH, Ad), 1689 (C=O), 1342 (NO), 1265, 1242 (C-O-C), we have observed the absorption bands related to bromate ion (νas 779-810 cm-1) and iodat-ion (νas 745-750 cm-1). By using of differential-thermal (DTA) and TGA analysis (Figure 9) methods we have established that starting destruction process of the obtained complex compounds (Scheme 6, VI, Scheme 7) proceeds in interval 165-400oC, with 58.4% loss of mass (organic fragments; theoreticaly: 58.7%). The thermolysis of inorganic fragments of the complex proceeds difficultly. Particulary, in the temperatural interval 400-460°C mass loss is 16.6% what corresponds to remuving of the I2O5 (theoreticaly: 16.4%). The endothermic peak at 580°C corresponds to the obtaining of the CdO with the mass loss 9.5% (theoreticaly: 10.0 %).

Figure 9. Corves of DTA (a) and TGA (b) analysis of the complex compound based on diiodatocadmiat of the 4-ethoxy-2-nitro-N-(1-adamantoyl)anilide.

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The composition and structures of synthesized compounds were established based on the data of elemental analysis and FTIR spectra 7In the FTIR spectra together with the following absorption bands – 3448, 3371 (NH), 3090 (CH, Ar), 2928, 2850 (CH, Ad), 1689 (C=O), 1342 (NO), 1265, 1242 (C-O-C), we have observed the absorption bands related to bromate ion (νas 779-810 cm-1) and iodat-ion (νas 745-750 cm-1). In the infrared spectra of the synthesized complex compounds the characteristic frequency υas for nitro groups (1342 cm-1) is splitting on two absorption bands: 1504 cm-1 and 1311 cm-1. The characteristic frequency υas of C=O groups shifts on 10-40 cm-1 (Figure 11). That confirms a combination of nitro and C=O groups with inorganic (metal-containing) fragments 25. These data are in good correspondence with the results of the quantum-chemical calculations (semiempirical method AM1) of structure and complexformation of adamantanecontaining nitroanilides 26. By using of differential-thermal (DTA) and TGA analysis (Figure 12) methods we have established that starting destruction process of the obtained complex compounds (Scheme 9, XVII) proceeds in interval 165-400oC. The thermolysis of inorganic fragments of the complex proceeds difficultly. Particularly, in the temperature interval 400-460°C mass loss is 16.6% what corresponds to remuving of the I2O5. The endothermic peak at 580°C corresponds to the obtaining of the CdO24.

Figure 11. dibromatokadmiate of 4-methoxi-N-(1-adamantoil)-2-nitroanilide.

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Figure 12. Corves of DTA (a) and TGA (b) analysis of the complex compound based on diiodatocadmiat of the 4-ethoxy-2-nitro-N-(1-adamantoyl)anilide.

According the aforementioned process we can suppose that the thermolysis of the investigated samples proceeds from the following summary general scheme [24]: Cd(IO3)2 → CdO + I2O5 I2O5 → 2 IO2 + 1/2 O2 2 IO2 → I2 + 2 O2 Scheme 8. General scheme the end stage of the thermolysis of Cd-complexes based on adamantanecontaining nitroanilides.

We have tested the bactericidal and fungicidal activity of synthesized nitroanilides (Scheme 5, I and II) and complex compounds IV, V and VI (Scheme 6). To this target we have applied the test-microorganisms – Pectobacterium aroideae, Fusarium arenaceum, Autinomyces Griseus and Fusarium proliferate. Bactericidal and fungicidal properties were determined according to the method described in ref. 24. The test results showed that synthesized compounds IV and V (Scheme 6) have revealed selected bactericidal properties and have suppressed the development of research cultures. The compounds IV and V have a relatively high activity towards the bacterium Pectobacterium aroideae, which strikes the melons and gourds and provoke rot. In a case of compound IV at concentration 0.01 g/l, the zone of inhibition was 0.4 cm, correspondingly, whereas for compound V, at concentration 0.01 g/l, the zone of inhibition was 0.7 cm. The compound VI is inactive taword to aforementioned microorganisms, although have reveal weak antifungicidal activity with respect to Fusarium arenaceum, which destroys some

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synthetic carbo-chain polymers. For this compound, at concentration 0.1 g/l, zone of inhibition was 0.2 cm. For the compound IV, at concentrations 1, 0.1 and 0.01 g/l, zone of inhibition toward a same bacterium zone of inhibition was 0.1 cm. Compounds IV and V, at concentration 0.01 g/l, have a sufficiently good activity (zone of inhibition were 0.1 and 0.3 cm, correspondingly) towards Pectobacterium aroideae and Act. Griseus. It must note that the bioactivity of the initial nitroanilides is less than bioactivity of their complex compounds. For example, in case of Fusarium arenaceum, zone of inhibition for 4(p-chlorophenoxy)-2-nitro-3-chloro-N-(1-adamantoyl)anilide at concentration 0.1 g/l (Scheme 5, II) was 0.1 cm. The antibiocorrosive covers prepared by the following way: to bioactive substances (≤ 3 %) and oligoorganosiloxane MF-AMA (modifier for matrix compound ≤ 10 %) in composition with the “ED-26” (matrix) have been added the hardening agent – hexamethylenediamine (≤ 5 %). The mixture was stirred till obtaining homogenous mass. Later, the produced thin layers on the surface of the selected material (lead, plastic, etc.) were holding on the air for 24-48 hours at room temperature. After hardening, there was produced homogenous, smooth, thick mechanically stable protective layer. It has to be noted that the produced compositions are available and their production is technologically simple. The basic properties of protective layers (homogeneity, viscosity, compatibility of polymer matrix components with bioactive compounds), physical and chemical characteristics (water absorption ≤ 0.2 %), and adhesion strength ≥ 4.0 MPa) are in compliance with the compositions of protective layers of objective types [4, 5, 25]. The created composites may be recommended as: a) protective covers with multivectorial application (film materials and impregnating compositions) stable to biocorrosion; b) materials with antimycotic properties for prophylaxis and treatment of mycosis; c) c) biologically active polymer materials for protection of archaelogical and museum exhibits; and d) for human protection during its contact with microorganisms. Preliminary investigations have shown that the synthesized compounds have also a real perspective to be utilized as accessible antioxidants towards the cancer.

CONCLUSIONS New composite materials based on carbofunctional fluorinecontaining siliconorganic polymers with side methacrylic groups and bioactive adamantane-containing compounds (nitroanilides and Cd-complex compounds based on them) for antibiocorrosive covers having various applications for individual and environmental protection have been obtained and studied. The created composites may be recommended as: a) protective covers (film materials and impregnating compositions) stable to biocorrosion; b) materials with antimycotic properties for prophylaxis and treatment of mycosis; c) c) biologically active polymer materials for protection archaelogic and museum exhibits and d) for human protection during their contact with microorganisms [25]. The preliminary investigations have shown that synthesized compounds have also a real perspective to be utilized as accessible antioxidants towards the cancer.

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REFERENCES [1] [2] [3] [4] [5]

[6]

[7] [8] [9]

[10] [11] [12]

[13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

Gu Ji-Dong. Int. Bioterior. and Biodegr, 2003, 1, 52, 69-91. G.T. Howard. Int. Bioterior. and Biodegr. 2002, 4, 49, 242-252. J. Hazziza-Laskar, et al. Journal of Applied Polymer Science, 1995, 1, 58, 77-84. E.Z. Koval and L.P. Sidorenko. Mycodestructores of the industrial articles. Kiev: Naukova Dumka, 1989, 192 (Russ.). 41st IUPAC World Chemistry Congress. Chemistry Protection Health, Natural Environment and Cultural Heritage. Programme and abstracts. Turin (Italy), August 511, 2007. Kh. Barbakadze, Sh. Chipashvili, L. Nanuashvili, K. Revia, D. Zurabishvili. Proceedings of Ivane Javakhishvili Tbilisi State University, Chemistry (Editor Nodar Lekishvili), 2006, 361, 35-39. K.Nakamoto, IR and Combination scattering spectrum of inorganic and coordination compounds, MIR, 1991. H. Friebulin. ‘Bask One- and two-dimensional NMR Spectroscopy’, VCH, Germany, 1991, 218. A.P. Kreshkov, V.A. Bork, E.A. Bondarevskaya, L.V. Myshlyaeva, S.V. Syavtsillo and V.T. Shemyatenkova, "Manuel for Analysis of Organosilica Compounds", M. Goschimizdat, 1962 (Rus.). T. Iwahara, M. Kusakabe, M. Chiba and K. Yonezawa, J. Polym. Sci. 1993, A 31, 2617 Pat. 14952А Ukraine. A.P.Grekov, V.Ya.Veselov et.al. - Appl. 04.03.97 (Ukr.). Yu.V.Savelyev, L.A. Markovs’ka, О.А. Savelyeva et al. Pat. 33837 Ukraine. Method of making polyurethane foams with biocompatibility and bactericidy. - Appl. 17.03.2003 (Ukr.). Yu. Savelyev, E. Akhranovitch, P. Pissis, Polymer, 15, 39, 3425-3429 (1998). Sh. Samakashvili. Diss...Cand.Chem. Sci. Ivane Javakhishvili Tbilisi State University, 2006. G..Goldin and M. Baturin. Zhurnal Prikladnoi Khimii (Zhurnal Applied Chemistry). 1985, 58, 1379 (Rus.). N. Lekishvili, V. Kopilov, D. Murachashvil, I. Sokol'skaya and M. Kezherashvili. Oxid. Commun., 2009 (in press). N. Lekishvili, Sh. Samakashvili, G. Lekishvili. Polymers and Polymer Composites, 2008, 1, 16, 35-45. Michael J. S. Dewar, Eve G. Zoebisch, Eamonn F. Healy and James J. P. Stewart. J. Am. Chem. Soc. 1985, 107, 3902-3909. N.L. Allinger. J. Am. Chem. Soc. 1977, 99, 8127-8134. O. Mukbaniani, G. Titvinidze, T. Tatrishvili, N. Mukbaniani, W. Brostow and D. 1183 Pietkievicz. J. Appl. Polym. Sci., 2007, 104, 1176. V.P. Zubov, S.D. Stavova, I.P. Chikhacheva, S.V. Budris, V.M. Kopylov, M.V. Zheneva and M.M. Obiedkova. Plasticheskie Massi., 1993, 5, 3-5. Kh. Barbakadze, T. Lobzhanidze, N. Lekishvili, D. Zurabishvili, M. Chubinidze. International Scientific Conference: “Modern Technologies and Materials”. Kutaisi, Georgia, May 14-16, 2008.

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[23] T. Lobzhanidze, R.Gigauri. Bulletin of the Georgian Academy of Sciences. 2003, 2, 168, 265-268. [24] Kh. Barbakadze, D. Zurabishvili, M. Lomidze, I. Sadaterashvili, T. Lobzhanidze, [25] N. Lekishvili. Proceedings of the Academy of Sciences of Georgia. Series of chemistry. 2008 , 45, 1 (Geo.- Abstract in English). [26] E. Markarashvili, N. Lekishvili, Z. Lomtatidze, Sh. Samakashvili, Izvestia VUZOV. Khimia i Technologia (Rus.), 2006, 48, 5, 117 -119.

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 21

SYNTHESIS, BIOCIDE PROPERTIES AND STRUCTURES OF SOME ARSONIUM POLYIODIDES FOR ANTIBIOCORROSIVE COVERS L. Arabuli1, N. Lekishvili and M. Rusia Ivane Javakhishvili Tbilisi State University2 Tbilisi, Georgia

ABSTRACT Quaternary arsonium triiodides [(Ph3AsCH2I]I3 and [(i-Bu)3AsCH2I]I3 have been synthesized and studied. The x-ray structures of [(Ph3AsCH2I]I3 and [(i-Bu)3AsCH2I]I3 have been determined. Crystals belong to the monoclinic (comp.1) system, space group P 21/n (No. 14) with a = 10.97 (1)Å, b=13.152 (1)Å, c=16.882 (1)Å , β=93.01 (1)o and to the triclinic system (comp.2), space group P-1 (No. 2) with a=8.413 (1)Å, b=9.109 (1)Å, c=15.876 (1)Å, α = 76.24 (1)o , β=75.60 (1)o, γ=75.26 (1)o. The structures were refined to an R value of 0.063 from 4082 (comp. I) and 0.091 from 4475 (comp.2) observed reflections. The As atom is coordinated tetrahedraly to the substituents and the anion has a linear structure. The synthesis of [R2(R')AsCH2I]I3 (where R= R' or R≠ R') are described. The possibility of the perspective application of synthesized compounds has been shown.

INTRODUCTION Inter-halogen compounds reacts with metal halides to form the XXn1- ions, as well as "mixed" anions such as I2Cl-, I4Cl-, ICl3F- etc. [1]. The Lewis acidity is expressed for diiodine toward electron pair donor molecules. The Lewis acidity is also evident in the interaction of halogen molecules with ion donors to give the range of ions known as 1 E-mail: [email protected]. 2 3, I. Chavchavadze Ave., 0128, Tbilisi, Georgia, e-mail: [email protected].

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polyhalides. I3- and I5- polyiodides are Lewis acid-base complexes, in which I- and I3- act as the bases and I2 acts as the acid. The Lewis structure of I3- has three equatorial lone pairs on the central I atom and two axial bonding pairs in a trigonal bipyramidal arrangement. As I3- ion can interact with an additional I2 molecule to yield larger mononegative polyiodides of composition [(I2)n(I-)]. In combination with a large cation, such as [N(CH3)4]+ [2] and [As(C6H5)4]+ [3], the symmetrical and linear counterion [I3]- is observed with a longer I-I bond than in I2. However, the structure of the triiodide ion is highly sensitive to the identity of the counter ion. For example, Cs+, which is smaller than the tetramethylammonium ion, distorts the I3- ion and produces one long and one short I-I bond [4, p. 556]: [I—2.82—I—3.10—I]- (I3 in CsI3) A more exam example of sensitivity to the cation is provided by NaI3, which can be formed in aqueous solution but decomposes when the water is evaporated: Na+(aq) + I3-(aq) → NaI(s) + I2(s) This behavior is another example of the instability of large anions in combination cations. Thus, the large cations are necessary to stabilize them in the solid state. Structures of arsenic-organic compounds are studied less, though about 6000 arsenic inorganic and organic compounds have been synthesized to present [4]. Thus, the identification and characterization of arsenic-containing compounds attract much interests due to their theoretical and practical importance, taking into account as anticancer drugs [5], biological active substances [6], additives in protective covers and polymers with specific properties [7], as auxiliaries in asymmetric synthesis [8,9], as catalysts [10], etc. Based on the movable anion of quaternary arsonium iodides [R3AsCH2I]I [11,12], like potassium iodide, we have attempted to obtain and study analogical type polyiodides. The Lewis basicity arises from the lone pair on the central atom of tertiary arsine (:AsR3), moreover, the central atom may exist in oxidation states +3 or +5 in AsR3 and AsR4+, respectively. Compounds containing lone pairs can be considered rich with electron and trialkyl(aryl)arsines (:AsR3) act as nucleophilic toward haloid-alkanes to produce tetraalkyl(aryl)arsonium salts (AsR4+), which contain As(V) [2, p.448]. Chemically active (with high toxicity) tertiary arsines can be stabilized by forming fourth bond with electrophilic substituents and 4-coordinated arsenic compounds are chemically stable and less toxic. Products of alkylation- arsonium salts are successfully used in synthetic inorganic chemistry as bulky cations to stabilize bulky anions [13]. Thus, tertiary arsines react with metlyleneiodide to produce “adducts” as follows [11]: R2As(R') + CH2I2 → [R2(R')AsCH2I]I , where R= R' =Pr, i-Pr, Bu, i-Bu, Am and Ph; R' ≠ R= Bu and Ph.

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EXPERIMENTAL Crystals of [(Ph3AsCH2I]I3 and [(i-Bu)3AsCH2I]I3) suitable for x-ray analysis were prepared by treatment of the arsonium iodides alcohol solutions with an excess (about 5%) of I2 alcohol solution. Dark-red solution was stayed during 2-3 weeks, after filtration the product was washed with deionized water, alcohol and dried in vacuum (with CaCl2). The crystals were characterized by x-ray analysis, IR spectroscopy and chemical (elemantal) analysis. Arsenic was analyzed by Evins' (iodometric) method [14], iodine – by mercurimetric titration method[15]. Materials. Crystalloographic data for the structures reported in this paper have been deposited on Data Centre as supplementary publication No. CCDC-661921 and CCDC661922. Copies of the data can be obtained free of charge an application to CCDC, 12Union Road, Cambridge CB2 1EZ, UK. [Fax: int. code +44(1223)336-033; Email: [email protected]]. IR spectra were obtained from KBr pellets, using Thermo Nicollet Avatar 470 spectrometer with MCTB detector.

GENERAL RESULTS Synthesis To prepare arsonium polyiodides, we have attempted interaction between iodmethylenetrialkyl(aryl)arsonium iodides and diiodmethane in various molar ratio such as: 1:1 (arsonium iodide: haloid alkane), 1:2 and 1:4. As analyses results have shown, one molecule of iodine combine to the cationic complex in all cases according to the reaction: [R2(R')AsCH2I]I + I2 → [R2(R')AsCH2I][ (I-)(I2)] The [R2(R')AsCH2I][I3] formulas for synthesized compounds were determined by means of the analytical procedures described in experimental section. Results of chemical analyses and loading of starting materials and yields of reaction products are given in tables 1 and 2. Table 1. Results of chemical analyses No. Formula 1. Pr3AsCH2I4 2. (i-Pr)3AsCH2I4 3. Bu3AsCH2I4 4. (i-Bu)3AsCH2I4 5. Bu2PhAsCH2I4 6. Am2PhAsCH2I4 7. Am3AsCH2I4 8. Ph3AsCH2I4

Analyses: calc.%/found% As 10.34/10.02 10.34/10.57 9.77/10.11 9.77/9.45 9.52/9.27 9.26/9.69 9.20/8.77 9.06/9.48

I 69.96/70.32 69.96/69.54 66.13/66.39 66.13/65.82 64.45/64.03 62.70/62.36 62.24/62.49 61.33/61.03

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Table 2. Loading of starting materials and yields of reaction products No. 1. 2. 3. 4. 5. 6. 7. 8.

arsonium g 1.17 1.20 1.80 1.65 1.50 1.44 1.35 1.86

iodide mole 0.0025 0.0025 0.0035 0.0032 0.0028 0.0026 0.0024 0.0032

iodine g 0.66 0.68 0.93 0.86 0.75 0.69 0.64 0.86

mole 0.0026 0.0027 0.0037 0.0034 0.0029 0.0027 0.0025 0.0034

yield g 1.64 1.73 2.37 2.12 1.85 1.72 1.62 2.33

mole 0.0023 0.0023 0.0031 0.0028 0.0023 0.0021 0.0020 0.0028

% 91.3 93.4 88.3 86.2 83.5 82.1 82.7 86.9

IR spectroscopic study: The IR spectrum of [(Ph3AsCH2I]I3 is shown in Figure1. The aromatic C-H stretching bands appear in the 3047 cm-1 and the aliphatic C–H - in the 2877 (asymm.) and 2946 (symm.) cm-1 regions. Skeletal vibrations, representing aromatic C=C absorb in the 1581-1434cm-1 range. The C–Har.bending bands appear in the regions 12411025 cm-1 (in plane bending) and 833-686 cm-1(out-of plane bending) [16]. The As-C stretching bands appear at the 462 cm-1 (As–Car.) and 655 cm-1 (As-Caliph.), characterized for As–C4 bonds in tetrahedral position [17].

Figure 1. IR spectrum of Iodmethylenedibuthyl(phenyl)arsonium triiodide

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STRUCTURAL ANALYSIS The crystallographic data and structure refinement are given in table 3, bond lengths [Å] and angles [o] –in table 4. By x-ray analysis have been confirmed the conclusions of our previous work [10], in which we indicated that CH2I+ group of methylene iodide (CH2I2) in the interaction of tertiary arsines (R3As or R2(R')As), is bonded to the arsenic atom and therefore exists in internal sphere of complexes but lone atom of iodine takes place in external sphere as counterion.

CRYSTAL STRUCTURE OF [PH3ASCH2I]+I3[Ph3AsCH2I]+I3- crystallises in the monoclinic, space group P21/n (No. 14) with a=10.197 (1)Å, b=13.152 (1)Å, c=16.882 (1)Å , β=93.01 (1)o and four formula units per unit cell. The crystal structure was solved via the Patterson method. For refinement full-matrix least-squares methods were applied. The cation has a tetrahedral shape and the anion has a linear structure. The structures of iodmethylenetriphenylarsonium cation and triiodide anion are shown in Figure 2. In the cation arsenic atom is bonded to four carbon atoms,- three of them assigned to the phenyl groups but fourth carbon is of alkylating group (CH2I). The As–C interatomic distances range in the 1.906-1.924 Å and C-As-C angles of the cation - 105.3- 109.8o. The series of tri- and four-coordinated arsenic compounds were studied [18 and ref. citated therein, 19], for example, in trimethylarsine (CH3)3As, As-C bond lengths are equal 1.98 Å and C-As-C angles– around 96o, the geometry is pyramidal, respectively. For tetracoordinate arsenic derivatives (sulphides As=S, oxides As=O and arsonium compounds (=As=)+X-) a tetrahedral configuration dominates and by authors [20] have been suggested that pπ-dπ and p, π conjugation is characteristic for the corresponding compounds. In the tetraphenylarsonium large cation As-C distances range 1.910-1.921 Å and angles at arsenic - 106.1-110.7o [21,22]. Moreover, as authors [23,24] indicate, in the arsonium salts As-C bond lengths range 1.8891.949 Å and C-As-C angles 106.9-112.5o characterized for a tetrahedral geometry. Carbon atoms of phenyl groups are in sp2 hybridization position and the values of bond angles range 117-120o. The fourth carbon atom (of CH2I group) is also in tetrahedral position like central atom. The anion I3- has asymmetric linear structure [I—2.86—I—3.00—I]-, this fact may be caused by structure and nature of substituents of the synthesized compound, the same explanation is proposed also by authors [25-27]. The angle of counterion I(21) –I(20) –I(22) is 179.38o.

Crystal Structure of [(I-Bu)3asch2i]+I3[(i-Bu)3AsCH2I]+I3- crystallizes in the triclinic, space group space group P-1 (No. 2) with a = 8.413 (1)Å, b = 9.109 (1)Å, c = 15.876 (1)Å , α= 76.24(1)o, β= 75.60 (1)o and γ= 75.26(1)o and four formula units per unit cell. The crystal structure was solved via the Patterson method.

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Figure 2. Strusture of iodmethylenetriphenylarsonium triioidide [Ph3AsCH2I]I3.

The cation has the shape of a tetrahedron and the anion has a symmetric linear structure. The structures of iodmethylenetri-izo-buthylarsonium cation and triiodide anion are shown in Figure 3. In the cation arsenic atom is bonded to four carbon atoms,- three of them assigned to the izo-buthyl groups and fourth carbon is of alkylating group (CH2I). The As–C interatomic distances range in the 1.921-1.953 Å and C-As-C angles of the cation - 106.8- 114.7o. The As-C bond lengths of Ph-substituted compound is shorter than izo-Bu-substituted compound. The anion (in contrast of comp. 1) has symmetric linear structure: [I—2.91—I— + 2.91—I] , as it is in [As(C6H5)4] [I—2.90—I—2.90—I] [3]. The bond lengths of the anion I3 are 2.9131 and 2.9243 Å but bond angles I–I–I = 180.0o. This is completely symmetric linear structure.

Figure 3. Structure of iodmethylenetri-izo-buthylarsonium triioidide [(izo-Bu)3AsCH2I]I3.

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Table 3. Crystal data and structure refinement for the compound 1 – [(C6H5)3AsCH2I]I3 and the compound 2 - [(i-C4H9)3AsCH2I]I3

BIOACTIVITY OF SOME SYNTHESIZED COMPOUNDS The aim of this work has been to study biocide properties of the above mentioned arsenic- containing compounds. Toxicity of several homologes of arsonium salts has been investigated. Various type of inorganic, natural and synthetic compounds with bacterial and fungicide properties successfully are used to a number of pathogen microorganisms participating in biodegradation. Some of them may be applied for obtaining of biostable polymers, paints and also against phitogen bacteria [28-30]. To this target we have studied antibacterial properties of iodmethylenetriphenyl-arsonium triiodide (Table 5, comp. 1), iodmethylenetri-izo-butilarsonium triiodide (comp. 2) and iodmethylenediamylphenylarsonium triiodide (comp. 3). We have applied test-microorganisms obtained from collections of cultures of Microbiology Institute (Georgia), namely, from phitogen gram-positive - Staphylococus aurous, from gut's group bacteria phitogen gram-negative - Esherichia coli, spore-forming bacteria – gram-positive Bacillus subtilis and gram-positive (from radiate mushrooms) Autinomyces griseus. In the experiment we have applied the following concentrations of substances: (g/l) 1.0; 0.1; 0.01. Biocide properties have been determined by the method described in ref. 19, for cultivation of microorganisms there have been used food areas – Krasilnikov' synthetic area for actinomicet and dry TPA – for bacteria.

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Results of the tests are presented in Table 5. Relatively high toxicity is characterized for iodmethylenediamylphenylarsonium triiodide (comp. 3) which inhibits growing of Esherichia coli, Staphylococus aureus and Bacillus subtilis with minimal concentrations. Inhibirability of studying substances rises with increasing of concentrations (0.1 g/l), though only iodmethylenediamylphenylarsonium triiodide with this concentrations reacts to the Autinomyces griseus as inhibiter. 1 g/l concentration of the substances has been toxic for all testmicroorganisms. Thus, according to the test results of the analysis have proved that iodmethylenediamylphenylarsonium triiodide is characterized with high toxicity, which besides arsenic and iodine, contains phenyl (C6H5), methylene (CH2) and amyl (C5H11) radicals and inhibits both – gram-positive and gram-negative bacteria. Iodmethylenetriizobutylarsonium triiodide is less toxic (comp. 2). Table 5. Antibacterial spectrum of Arsonium triiodides Test-culture

Esherichia coli Staphylococus auveus Bacillus subtilis Actinomyces griseus

Compound 2 1 (i-Bu)3AsCH2I4 Ph3AsCH2I4 Concentration of substance (g/l) 1.0 0.1 0.01 1.0 0.1 0.0 1 A zone of inhibition of test-culture (mm) 0 2.5 1.5 1.0 4.0 2.0 0 2.0 1.0 0 3.0 1.0 0 2.0 2.0 0 2.0 1.0 0 1.0 1.0 0 1.0 0

3 Am2PhAsCH2I4 1.0

0.1

0.01

6.0 5.0 5.0 1.0

3.0 2.0 3.0 1.0

1.0 1.0 1.0 0

Preliminary researches showed that on the basis of the synthetic compounds, new biologically active materials can be develope for various applications, such as: 1. Polyfunctional covers stable to bio-corrosion induced by microorganisms (Figure 4); 2. Materials with antimicosic properties - appretive and adhesive compositions as aqueous dispersions for manufacturing of skins, shoes and textiles; 3. Biologically active polymeric materials for covering agriculture plants from illness, caused by incomplete mushrooms and some microorganisms, also, in the time of contact of people with them.

Figure. 4. Antibiocorrosive covers based on arsonium triiodides and siliconorganic matrix for wood and plastic.

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L. Arabuli, N. Lekishvili and M. Rusia

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]

B. Duglas, D. MeDaniel, J. Alexander, Concepts and Models of Inorganic Chemistry, 2nd Edition, New York, John Wiley and Sons, Inc., 1983, 589. D. Shriver, P. Atkins, C. Langford, Inorganic Chemistry. Second Edition, New York, W.H. Freeman and Company, 1994, 555. A. Drozdov, V. Zlomanov, Chemistry of Main Group Elements of Periodic System. Halogens. Moscow, MGU, 1998. N. Evstaphev, C. Arephev, M. Pronin. Self-Occuring Arsenic. Chemistry and Life. 1991, 5, 27-30. G. Salem et al. J. Chem. Soc., Dalton Trans., 2000, 3603. V.A. Valiulina, V. I. Gavrilov, Vestnic Kazannskovo Technologicheskovo Universiteta. 1988, 1, 28-38. M. Caeter, N. Baker, R. Bunford, Journal of Applied Polymer Science. 2003, 58, 11, 2039-2046. 8] http://www.rsc.anu.edu.au/Annual/Report/Report2002/AH-report.html A. Kojina, Ch. D. Boden and M. Shibasaki, Tetrahedron Letters. 1997, 38, 19, 34593460. V.K. Aggarwal, M. Patel and J. Studly, Chem.Comm. 2002, 1514-1515. R] Gigauri, L.Arabuli, Z. Mazhaide, M. Rusia, Russian Journal of General Chemistry. 2005, 10, 2, 268-271. R. Gigauri, L. Arabuli, et al. Russian Journal of General Chemistry. 2006, 75(10), 1076-1079. I.S. Butler, J.F. Harrod, Inorganic Chemistry. Principles and Applications. California, The Benhamin/Cummings Publishing Company, Inc., 1989, 650. E. Evins, J. Chem. Soc. 1916, 109, 1355. R. Gigauri, L. Arabuli, M. Rusia, M. Kikalishvili, Georgia Chemical Journal. 2002, 29, 3, 195. B.H. Stuart, Infrared Spectroscopy: Fundamentals and Applications. Sydney, John Wiley and Sons, Ltd., 2004, 74. W.R. Cullen, G.B. Deacon, J. U. S. Green. Canadian Journal of Chemistry. 1965, 43, 3193-3200. [18] F. D. Yambushev and V. I. Savin. Russ. Chem. Rev. 48(6), 1979, 582-595 [19] D. B. Sowerby, in the book “The chemistry of Organic Arsenic, Antimony and Bismuth Compounds”, Edited by Saul Patai, John Wiley and Sons, Ltd.,1994, 27 M. Shindo, Y. Matsumara and R. Okawara, J. Organometal. Chem., 1968, 11, 299. U. Muller and H. D. Dorner, Z. Naturforsch., Teil B, 1982, 37, 198. M. P. Bogaard, J. Petersson and A. D. Rae, Acta Crystallogr., Sect. B, 1981, 37, 1357. D. G. Allen, C. L. Raston, B. W. Skelton, A. H. White and S. B. Wild, Aust. J. Chem., 1984, 37, 1171. A. Kostick, A.S. Secco, M. Billinghurst, D. Abrams and S. Cantor, Acta Crystallogr., Sect. C, 1989, 45, 1306. С.T. Mortimer and H. A. Skinner, J. Chem. Soc., 1952, 4331. J. Trotter, Canad. J. Chem., 1962, 40, 1590. J. Trotter, Canad. J. Chem., 1963, 41, 191.

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[28] Ilichev V.D., Bocharov B.V., Gorlenko M.V. Ecologicheskie Ocnovi Zashchiti ot Biopovrezhdeni. M.: Nauka, 1985, 264 pp. [29] Valiulina V.A. Biopovrezhdenia y Zashchita Materialov Biocidami. M.: IEMEZh, 1988, 63. [30] N. Lekishvili, L Arabuli, T. Beruashvili, T. Lobzhanidze, Kh. Barbakadze, M. Kezherashvili. 1st International Caucasian Symposium on Polymers and Advanced Materials. 11-14 September, 2007, Tbilisi, Georgia. [31] L. Arabuli, N. Lekishvili, M. Kadagidze, N. Kebuladze, R. Gigauri. The 41st IUPAC World Chemistry Congress, Book of Abstracts. Italy, 2007, 62.

PART 6. ENVIRONMENTAL CHEMISTRY

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 22

ANTIMUTAGENI AND ANTICYTOTOXIC ACTIVITY OF BIOENERGOACTIVATORS Ramaz Gakhokidze1 and Amiran Pirtskhelani Iv. Jvakhishvili Tbilisi State University, Georgia

ABSTRACT Pollution of the environment is caused by human industrial and agricultural activity. It poses a harmful factor to the genetic apparatus of organisms to which are connected hereditary diseases, premature aging, cardiovascular problems, etc. Numerous experimental investigations have shown that many chemical factors are characterized by a mutagenic influence. Following these discoveries, our laboratory elaborated many prophylactic measures, directed at prevention of genetic damage from the influence of harmful mutagenic agents on the organism. In the laboratory, the antimutagenic and anticytotoxic effect of bioenergoactivators (biorag, ragozan, ematon and ragil) on mice have been studied during mutation and cytotoxicity induced with the fertilizer ammonium nitrate and the pesticides (phosphamide, trichlorfon and celtan). The cytologic and genetic methods of investigation have been applied in the study. Experiments showed that tested bioenergoactivators exerted highly effective automutagenic and anticytotoxic action. Environmental pollution, which basically is caused by agricultural and industrial activity of human beings, comes back to them as factors harmful to organisms and their genetic apparatus, resulting in not only hereditary illness, malignant tumors and premature senescence, but also illnesses such as cardiovascular and digestive system disease, neural, allergy, and others. In connection with an annual increase of chemical pollutants, science stands in the front of genetic danger. Among those problems whose solutions are first and foremost, the protection of organisms and their progenies from chemical mutagens existeing in the environment is the most actual one. Wide application of chemical preparations in medicine, agriculture, industry and everyday life, as well as the existence of a great amount of chemical pollutants in the soil, water and atmosphere, allow us to talk about a sharp alteration of the ecologic situation [1].

1 E-mail: [email protected].

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Ramaz Gakhokidze and Amiran Pirtskhelani Many clinical and experimental research studies have shown that many chemical factors are characterized by mutagenic activity. Following from that, it is necessary to elaborate the prophylactic measures directed toward prevention of a genetic influence on the organisms. Such measures are: 1) revelation of dangerous mutagens, their withdrawal from usage and changing them by not having dangerous analogues; 2) determination of activation of a biologically safe dose of these preparations, overdose of which is characterized by genetic activity; 3) revelation and application of effective antimutagens, which will decrease natural and induced mutations’ frequencies until they are at a minimum.. Investigations about antimutagens first were conducted by A. Novick and L. Szilard [2] over 50 years of the past century, which showed that purinic ribonucleozides in the intestine bacillus (E. Coli) decrease the spontaneous and induced mutations. These compounds which showed up with similar properties in the next were being called by an anti mutagenic term [3]. The fundamental investigations conducted in this branch showed that there had been revealed some ten anti-mutagens, owing to this, there arose the problem of elaborating a classification of those compounds. By T.Kada classification [4] the antimutagens are divided into four groups: 1) dismutagens, which cause the inactivation of mutagens; 2)substances, which influence on the mutagens’ metabolism and transport; 3) substances, which reduce the replication and reparation errors; 4) anti- mutagens, which operate by an unknown mechanism. Complete classification of antimutagens with the foresight of their operation mechanisms was suggested S. De flora and C. Ramel [3]. Numerous research studies about anti mutagens were carried out by many scientists [5,6,7,8,9,10,11]. Extracts of the following plants: cabbage, bitter pepper, guinea squash, apple, pineapple, mint, garlic, tea, caffeine and others revealed high antimutagenic properties [12]. Vitamins have anti mutagenic properties, as well [5,6,8,9,11]. Interferons are characterized by high antimutagenic activity also [9]. Since 1983 we have been actively following the work for the discovery of different antimutagens (interferons, different vitamins, their complexes and carbohydrates) and usage of them to decrease the cytotoxic and mutagenic activity of different biologic and chemical factors of environment pollutants [6,9-11]. Now, we had investigated on the background of mineral fertilizer—ammonium nitrate—and pesticides (phosphamide, trichlorfon, keltan) the antimutagenic and anticytotoxic influence of bioenergoactivators (ragozan, ematon, ragil, biorag ) on the laboratory mice by calculation of chromosomal anomalies and pathologic mitoses. In the white mice, we introduced the following pesticides orally one by one: phosphamide, trichlorfon, keltan, also fertilizer ammonium nitrate. The mentioned substances in the enimals were introduced with the following doses: 1/2 LD50 and 1/5 LD50. At the investigation of the chromosomal anomalies, results of which are given in Table 1, we could see that the mentioned substances cause structural disturbances of chromosomes (singular and plural fragments, lysis of chromosomes), and also genomic mutations (triploids, tetra- ploids). The common number of anomalous metaphases at the influence of phisphoamide (dose 1/2 LD50) consists 10.4%, with trichlorfon 8.7%, with keton 7.1%, at the influence of ammonium nitrate this index was being reached to 8.8% (control 1.0%). There was expressed well the dose effect. At the usage of 1/5 LD50 dose this index was reduced positively (P<0,01).

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Table 1. Frequency of Chromosomal Anomalies in the White Mice Cells at the Influence of some Pesticides and Fertilizers (dose 1/2 and 1/5 LD50) Pesticides and fertilizers

Dose (mg/kg)

The number of investigated metaphases

Phosphamide

140 28 350 140 215 85 175 70

500 450 500 450 500 400 419 400

Trichlorfon Keltan Ammonium Nitrate Control

Structural disturbances of chromosome s (%±) 7,4 6,5 5,5 4,2 5,4 3,2 6,4 3,1

Genomic mutation s (%±)

General number of normal metaphases

P

3,0 2,3 3,28 2,8 1,7 1,5 2,7 2,6

10,4 ±1,3 8,8 ±1,1 8,78 ±1,2 7,0 ±1,2 7,1±1,1 4,3±0,8 8,8 ±1,5 5,7 ±1,07

<0,001 <0,001 <0,001 <0,001 <0,001 <0,001 <0,001 <0,001

0,8

0,2

1,0 ±0,2

-

It must be noted that from structural disturbances of chromosomes there most of all was being noted lysis of chromosomes then singular and plural fragments, but from genomic mutations—triploid cells then tetraploid ones. Therefore, the abovementioned pesticides and ammonium saltpeter in the sharp experiments conducted on the laboratory mice are characterized by expressed mutagenic influence. Citogenetic analysis showed also that the mentioned pesticides and nitrate cause with more high frequency the pathologic mitoses or they are characterized with cytotoxic and genotoxic influence, because of elimination of cells comprising pathologic mitoses. In table 2 is represented the disturbances frequency of pathologic mitoses and interphase nuclei in the white mice cells at the influence of pesticides (phosphamide, trichlorfon, keltan) and ammonium nitrate, from which is shown that mentioned substances in comparison with control one cause the trustworthy increase of porous metaphases, K-metaphases and chromosomes gluing frequency. (P<0,001). From pesticides the phosphimid is characterized with most high cytotoxic effect (21,6%), then come trichlorfon (14,65%) AND KELTAN (7.4%) (dose 1/2 LD50). In the case of ammonium saltpeter, effect reaches to 21.4%. So as at the mutagenic influence there was revealed the dose-effect. In comparison with 1/2 LD50 at the use of 1/5 dose the cyto toxic effect was positively low (P<0,oo1). If we compare the chromosomes structural disturbances and genomic mutations of mentioned substances on the one hand and cytotoxic effect or origination frequency of pathologic mitoses on the other hand, we see that they are in correlation with each other. There, where is high chromosomal anomalies, there is also a high cytotoxic effect and vice versa. As a result of the abovementioned xenobiotics influence in the animals the inter phase disturbances of nuclei are strongly increased (porous nuclei). At the influence of phosphamid (dose 1/2 LD50) origination frequency of porous nuclei reaches 5.6%, triclorfon 4.2%, keltan 2.2% and in the case of saltpeter this index reaches 4.5% (control 1.1).

Ramaz Gakhokidze and Amiran Pirtskhelani

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Table 2. Frequency of pathologic mitoses at the influence of pesticides and ammonium Nitrate (at every level 5 animals are used) Agrochemicals

Phosphamid Trichlorfon

Keltan Ammonium nitrate Control

Investigated metaphases number

General Number of Pathologic mitoses (%±)

Pathologic mitoses Porous metaphas es

Kmetap hase

Gluing of chromos omes

140 28 350 140

500 500 450 450

21,6±1,8 21,6±1,9 14,6±1,6 11,5±1,5

10,2 7,5 8,2 6,2

2,1 1,6 2,0 1,4

215 85 175 70

520 500 419 400

9,6±1,3 4,9±0,9 13,4±1,0 8,3±1,3

3,8 2,6 6,8 3,5

1,8

500

2,7±10

1,2

Dose (mg/kg)

Porous Nuclei

P

8,7 3,5 4,2 3,9

5,6±0,7 4,7±0,6 4,2±0,5 2,4±0,5

<0,001 <0,001 <0,001 <0,001

2,0 1,9

4,3 2,3 4,6 2,9

2,2±0,4 1,4±0,3 4,5±0,18 2,6±0,2

<0,001 <0,001 <0,001 <0,001

0,2

1,3

1,1±0,3

In contrast to normal nuclei, in disturbed ones the chromatin is located on the periphery but in the centre is a vacuum. Such nuclei are called porous ones. Microscopic analysis of such nuclei are conducted on those preparations being studied for mutagenic and cytotoxic activity, i.e., there is no need to use other testing. It is easy to calculate disturbed interphase nuclei, which takes 10 times less time then calculation of chromosomal anomalies. For preliminary estimation of general toxicity and cytogenetic effect of investigated substances, it is acceptable to analyze interphase nuclei, as an ascertainment of the effect is very easy; at the same time, as we noted above, it is in correlation to the frequency of origination of chromosomal anomalies and pathologic mitoses. Literature and our data [6,9,11] showed that pesticides and fertilizers stipulate the destruction of genetic apparatus of different organisms and human beings to which is connected the beginnings and development of very many illnesses in the human body. This circumstance stipulates the elaboration of such measures, the result of which will be the protection of the genetic apparatus of man. Ascertainment of the effective, antimutagenic substances and their usage, which will reduce to a minimum the spontaneous mutation frequency, so induced ones and cytotoxic influence can be considered as one of the basic means. For this purpose there was revealed and used by us some saccharides (biorag, ragozan, ematon, ragill), which were being introduced in advance one by one in the white mice during a five-day period; at the fifth day, there were introduced at the same time the pesticide (dose 1/2, 1/5, LD50). Cytogenetic analysis showed that at separate introduction of phosphamide, the frequency of chromosomal aberration and genomic mutation was 8.8%, pathologic mitoses 12.6%, disturbances of interphase nuclei 4.7%, but on the background of ragosin the mentioned indexes accordingly were decreased till 2.7%, 3.8%, and 2.1%. To reduce the cytogenetic effect, we used antimutagenic ematon discovered by us. It turned out that if there would have place separate influence of trichlorfon in the animal cells, the number of chromosomal anomalies reached 8.7%, pathologic mitoses 14.6%, disturbances

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of inter phase nuclei 4.2%; on the background of ematon, these indexes, accordingly, were decreased till 2.9%, 5.0% and 1.6% (control 1.0%, 3.7%, 1.1%). Antimutagenic effect was revealed also to bioenergoactivator ragil. Table 3 shows that in the cells of white mice the number of chromosomal disturbances and genomic mutations at the influence of celtan (dose 1/2 LD50 ) reaches 7.1%, pathologic mutases 9.6%, and inter phase disturbances 2.2%. On the background of ragil those indexes were accordingly decreased till 2.3%, 3.4%, 0.8%. Also investigated were mutagenic and cytotoxic effects of the widely used fertilizer ammonium nitrate, separately and also on the background of biorag. It turned out that the mutagenic effect of nitrate (dose 1/2 LD50) in the animals reaches 8.8%, cytotoxic effect 25.5% (pathologic mitoses, disturbances of inter phase nuclei). On the background of biorag, those indexes were decreased till 3.0% and 17.9%. There was a clearly revealed dose effect. At the application of dose 1/5 LD50 the mutagenic effect of ammonium nitrate in combination together with biorag decreased till 1.0%, but cytotoxic effect—till 5.1%. (Influenced only with nitrate, effects, accordingly, reached till 5.7 and 17.9%). Consequently, the bioenargoactivators (ragozan, ematon, ragil) revealed by us on average a three times decrease of mutagenic and cytotoxix effect, caused by pesticides (phosphamide, triclorfon, keltan), but the effect caused by ammonium nitrate under the influence of biorag is decreased four times. All series of nitrozocompounds—nitrogen oxide, nitrates, nitrozoammine and ammonium nitrate—represent very dangerous pollutants. The basic source of nitrates and nitrites in plant and animal organisms is the ammonium nitrate which is widely used in agriculture as a fertilizer. The great amount of nitrates and nitrites in the stomach is a risk factor for developing cancer [12,13,14]. Carcinogenic activity of nitrocompounds is revealed on plants, insects and mammalian animals [15]. Vegetables and grain-crops obtained from soil cultivated by ammonium nitrate contain a great amount of nitrates and nitrites. Meat foods of domestic animals, nourished by grass germinated from such soil are saturated with nitrates and nitrites, which provokes the beginning and development of many illnesses and malignant tumors between them [16,17,18,19]. In vivo and in vitro conducted experiments show that the nitrates can be converted into nitrites in mammalian animals as a result of metabolism [20,21]. Due to adsorption, the nitrates from the intestine come into the blood. The nitrates are strong oxidants and they have a connectionability to hemoglobin (Hb), resulting in normal Hb being converted into metahemoglobin (metHb) and there develops methemoglobinemia. In the normal case, a quantity of metHb does not exceed 2%, but with the nitrate influence, this index is increased till 10%.The high consistence of nitrates and nitrites in the food for babies represent a risk of gastrointestinal infection [22,23]. Nitrites have also penetration ability in the placenta resulting in methemoglobinemia and death [24]. As we noted the nitrates also produce in the organism the nitrozo-compounds which are characterized by a carcinogenic influence, which was confirmed by experiments conducted on animals [25,26].

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Table 3.Anti mutagenic and antitoxic influence of biorag, ragozan, ematon and ragil at the beginning of cytogenetic disturbances by pesticide and ammonium nitrate (on the separate experiment there is taken on average five animals, dose 1/2 LD50; 1/5 LD50)

Factors

Dose (mg/kg)

Number of metaphases

Structural disturbances and genomic mutations of chromosomes (%±st.ct).

Frequency of pathologic mitoses. (%±st.ct).

Frequency of porous inter phase nuclei. (%±st.ct).

Ammonium nitrate Nitrate+biorag

175

419

8,8±1,5

21,0±2,0

4,5±1

157+30

450

3,0±0,3

8,6±1,8

1,5±0,2

Nitrate

70

400

5,7±1,0

15,8±1,7

2,6±0,2

Nitrate+biorag

70+30

450

1,4±0,2

4,0±1,1

1,1±0,3

Phosphamide

2,8 1/2 LD50

500

8,8±1,4

12,6±1,3

4,7±0,6

Ragozan

50

500

0,7±0.04

1,0±0,04

__

Ragozan+ phosphamide trichlorfon

50+28

500

2,7±0,7

3,8±0,8

2,1

350 1/2 LD50

400

8,7±1,2

14,6±1,6

4,2±0,5

Ematon+ trichlorfon Ematon

50+350

500

2,9±0,6

5,0±0,9

1,6±0,4

50

300

0,5

1,2±1,04

0,4

Keltan

215 1/2 LD50

520

7,1±1,1

9,6±1,3

2,2±0,4

Keltan+ ragil control

215+30

400

2,3±0,7

3,4±0,8

0,8

-------

500

1,0±0,2

3,7±6,8

1,1±0,9

P

<0,001

<0,001

<0,001

<0,001

<0,001

The soil of Georgia and most countries is polluted by different mineral fertilizers and pesticides, which is one of the basic reasons for different illnesses and especially the rising frequency of malignant tumors. It should be noted that in the Svaneti region of Georgia, where the people do not use mineral fertilizers and pesticides, (for fertilization of the soil they use only organic fertilizermanure) the number of persons afflicted with a malignant tumor is very small.

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REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26]

N.P. Dubinin, I.V. Poshin. In book: Mutagenez. Moscow, Nauka, 1978. A. Novick, L. Szilard. Nature 1952, 170, 4335, 926-927. S. De Flora, C.Ramel. Mutat. Res., 1988, 202, 285-306. T. Kada. Mechanisms and genetic implications of environmental antimutagens. 1992, p. 355-259. U.K. Alekperov. Antimutagenez. Moscow, Nauka, 1984, p.13. N.B. Bichikashvili. Dissertation, Tbilisi, 2003. G.I. Goncharova, Antimutagenez. Minssk, Nauka,1974. S.B. Durnev, S.B. Seredinin. Mutagenes. Moscow, Medicina, 1998. A.G. Pirtskhelani. Dissertation, Tbilisi, 1994. A.G. Pirtskelani. N.A. Pirtskhelani, R.A Gakhokidze et al. Biomedicina, 2008, 1, 4446. R.Gakhokidze, A.Pirtskhelani. Bulletin of the Georgian Academy of Sciences, 2000, 161, 1,121-123, G. Montes, C. Guella et al. Cancer Lett., 1979, 7, 6, 307-312. J. Dolby et al. Scandinavion Journal of Gastroenterology. 1995, p.105-110. E. Colbers et al. Science, 1995, 134, 134. Y.Pasternak. Arzneimitel forsh, 1974, 147, 802. A. Bonnel. Nitrate concentrations in Vegetables. Council of Europe Press, 1994, Strasburg, p. 11-20. W. Corrn, T. Breimer. Literature Survey. 1993, No39. W.E. PhillipsPublic Health Implications. Canad. Inst. Food Technol, 1988, 1, 98-103. W. Rudell. et al. Lancet z., 1976, 1037-1039, S. Gangolli et al. Exp.cell. Res.1963, p. 320-326. C. Walters, P. Smith. Food Chemistry and Toxicology, 1981, 16, 297-302. H. Muller. Adverse health effects of nitrate and its metabolites: epidemiological studies in human. Strasbourg, Council of Europe Press. 1994, p. 255-268. L. Schudeboom. A survey of the exposure to nitrate and nitrite in foods. Council of Europe. Strasburg Press. 1994, p. 41-74. C.Jaffa. Clinical hematology. 1981, 10, 99-122. W.E. Phillips. Nitrate contents of foods. Public Health Implications. Canad. Inst. Food Technol, 1988, 1, p. 98-103. W. Rudell. et al. Lancet z.,1976, p. 1037-1039.

PART 7. PERSONALLY

In: Advanced Biologically Active Polyfunctional Compounds… ISBN 978-1-60876-114-2 © 2010 Nova Science Publishers, Inc. Ed: N. Lekishvili, G. Zaikov and B. Howell

Chapter 23

THE SCIENTIST WHO HAS OUTSTRIPPED HIS TIME Revaz Skhiladze, Tengiz Tsivtsivadze and Bachana Pichkhaia1 Georgian Technical University * Iv. Jvakhishvili Tbilisi State University, Georgia

ABSTRACT This article is dedicated to the 100 years anniversary of birth of Georgian prominent scientist, Professor Akaki Gakhokidze. He is one of outstanding representatives of Georgian chemist’s school. High theoretical preparation, mastery of experiment conduction, unusual scientific flair and intuition allow him to leave the great and light footstep for the posterity on the way of scientific research and pedagogical activity. Fundamental investigation of A.Gakhokidze won the international recognition. His woks was published and broadly considered in the special literature, monographs and manuals of chemistry. There are created the specific paragraphs as “Synthesis of Gakhokidze”, “method of Danilow- Gakhokidze” etc.

A.Gakhokidze was born on 14 August 1909 in the village Zeda Khuntsi, Martvili district of Georgia. Martvili belongs to the region of Mingrelia which is a native land of many remarkable persons in the past. They are pride of Georgian people today. One of them on which we want to pay your attention is a bright representative of scientific elite of Georgia Akaki Gakhokidze, the person world renowned, scientist of international scale, which has brought the huge contribution in the development of a chemical science. Initial education he had received at home, in his village. At first he attended the Khuntsi elementary school and then proceeded to the seven year school. At duration of school study he was very diligent and industrious pupil. From 1924 A. Gakhokidze is continuing studies in Tbilisi Melikishvili college, which he graduated with high academic success after what he was sent for practice passing in Baku in 1927 where A Gakhokidze begin work in the oil purification plant. There turned out that only theoretical preparation without practice is quite insufficient for him. Lately he remembered:

1 E-mail: [email protected].

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Revaz Skhiladze, Tengiz Tsivtsivadze and Bachana Pichkhaia

“I thought that I know very well oil technology but soon I have understood that engineer without practice is like to the unarmed warrior. There I have started to study everything from all, beginning from the simple worker, finishing the engineer. It is clear that all became easy for me and I have soon connected the theory with practice”. In 1928 A.Gakhokidze graduated the college very successfully and he begin work in the soap plant (next the chemical industrial complex) of Tbilisi. At the same time he begin preparation for examinations of higher school. On August of 1928 he entered in the polytechnic faculty of Tbilisi University. Parallel studying A.Gakhokidze interested the problem of sugar production in Georgia. In this time there were functioned two institute in the former USSR – in Kiev (one – educational, second - scientific research) where the sugar production problems were being solved. Student A.Gakhokidze was their frequent guest. For A.Gakhokidze the task to build the sugar production plant in Georgia was the one of great importance. He elaborated in Kiev the sugar production plant for Georgia. There is the fragment of recollection of A.Gakhokidze: “I composed and wrote the plant project right away, made up 30 design drawing, the explanatory letter took 400 page, in Tbilisi it was not required corrections. After it consideration professor I.Burjanadze signed the project on the it public presentation. This project drawing up gave me important knowledge”. When sugar production plant director in Kiev acquainted this project, he told to project author with admiration: “the building of sugar plant in Georgia must be realized by your project.” In June 1929 A.Gakhokidze successfully defended the sugar plant project and he became an engineer technologist. Since 1932 year A.Gakhokidze continued post-graduated study in Leningrad (St.Peterburg). He works there in the field of carbohydrate chemistry under direction of professor Danilow. S.Danilow was the student of Russian outstanding chemist A.Favorski, whose scientific activity goes back to the founder of organic chemistry Butlerow. A.Favorski was the head of Leningrad Chemical Society where in general meeting two times in month was being made the report about new discovery in chemistry. At 18 December 1935 professor Danilow in the next meeting made the brief information about Gakhokidze’s work. He noted that like simple aldehydes the sugars are proved analogous isomerization and there are obtained saccharinic acids. “One of our students represented dissertation about this problem”, told Danilow. Favorski asked him who was this young scientist and the last pointed to Gakhokidze. Later one jester chemist explained to Gakhokidze the reason of Favorski interest: “you are now his grandchild and he wanted to see you.” The joker meant following sequence: Zinin → Butlerow → Favorski → Danilow → Gakhokidze. At November of 1935 A.Gakhokidze successfully defends dissertation “Isomerization of glucose in saccharinic acid”. Scientific council by a solid vote appropriates to A.Gakhokidze the candidate‫ۥ‬s degree. In the same year was published his large experimental work in the German journal “Berichte der Deutschen Chemischen Gasellschaft”. This work had large comments between chemists. Method elaborated in it was included in the manual for students of higher school as the “Danilow-Gakhokidze method”.

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In 1939 A.Gakhokidze comes back in Georgia and begins work in the Institute of processing of a wood material as a docent. Since 1937 he works in Tbilisi Pedagogical State Institute as Dean and next as a Head of faculty of chemistry till his death. In 1934 A.Gakhokidze was sent again in Leningrad by recommendation of Georgian Science Academy as a person working for doctor's degree. Doctor dissertation was defended in 1948 very successfully. In this time he was a first doctor chemist in organic chemistry in Georgia. 1940-1947 years A.Gakhokidze worked (parallel of pedagogical activity) in the institute of chemistry of Georgia Science Academy and then in 1947-1950 – in the Scientific-Research Chemical- Pharmaceutical Institute in Tbilisi, where he founded the department of medicine technology. Professor A. Gakhokidse is most outstanding representative of Georgian Chemists School. His life credo is very many-sided. He was a kind and tactful person. There were not for him large or small problems. He with identical attention concerned them. All students for him were young men which should be become not only good specialists but also worthy citizens of our country. The fundamental investigations of prof. A.Gakhokidze have an international recognition. The goal of this article is to touch more detail to activity of prof. A.Gakhokidze in carbohydrate chemistry. He has elaborated original method of disaccharides synthesis by which there appeared a possibility to receive unknown (until then) type of disaccharides synthesis. This type compound caused a great interest because of foreigner scientists working on the same problem possibility of existence of new type (1,2- and 1,3- bond consisted) disaccharides except of trehalose, maltose and gentiobiose type (1,1-, 1,4- and 1,6-bond consisted) disaccharides. For example we can bring the schemes of synthesis of glucosyl- 2glucose (sophorose) and glucosyl-3-glucose (laminaribiose). From 2,3,4,6,-tetra-0-acetyl-αD-glucopyranosyl bromide(I) and 1,3,4,6–tetra-0-acetyl-β-D-gluco-pyranose (II) he obtained 2-0-β-glucopyranosyl-D-glucopyranose (sophorose, III): CH2OAc

CH2OAc O OAc

O +

OAc AcO

Br

OAc

ZnCl2, P2O5

OAc

AcO

AcO

OAc I

CH2OAc O OAc

OH

CH2OAc

II

O

Ac = COCH3

OAc AcO OAc

O

CH3ONa

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CH2OH O OH OH HO CH2OH

O

O OH HO OH III

Structure of new disaccharide were determined by author by following way: by action of hydroxylamine on the disaccharide was obtained corresponding oxyme (IV) and by interaction of last with acetic anhydride is obtained acetylated nitrile of glucosyl -2-gluconic acid (V). Author transferred this substance into glucosyl-1-arabinose, via of saponification and nitrile group splitting, which does not act with phenylhydrasine:

CH2OH

CH2OAc

OH OH

OAc CH

NOH

OAc

HO III

CH2OH

AcO CH2OAc

O

O

O

O

OH

OAc

HO

AcO OH

OAc IV

V

C N

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O HO HO CH 2 OH

OH

O

O

OH HO OH VI By oxidation of disaccharide (III) (with brome water) the author received glucosil-2glucone acid (VII) and it’s derivatives, but by hydrolyze of acid – glucose(VIII) and glucone acid (IX):

CH2OH OH OH

COOH

III

CH2OH O

COOH

CH2OH

HO

O O

OH OH

OH

+ HO OH

HO

OH

OH

OH

HO

CH2OH VIII

OH VII

IX

324

Revaz Skhiladze, Tengiz Tsivtsivadze and Bachana Pichkhaia

Analogously, disaccharides with different monosaccharide residues (glucosyl-2galactose, galactosyl-2-galactose, galactosyl-2glucose, manosyl-2glucose, manosyl-2manose) has been obtained. From 2,3,4,6,-tetra-0-acetyl-α-D-glucopyranose (X) and 1,2-0-isopropyliden-4,6-0benziliden-α-D-glucopyranose (XI) A.Gakhokidze obtained 3-0-β-D-glucopyranosyl-Dglucopyranose (laminaribiose,XII): By similar procedures, A.Gakhokidze synthesized disaccharides with different monosaccharide residues.After some tens years there were corroborated once again the structure of substances obtained by A. Gakhokidze by several scientists with application of modern physical and chemical research methods. Lately, it was proved that new type compounds discovered by A. Gakhokidze are widespread in nature and they have high physiologic (immunologic, antitumoral and others) activity. Methods of synthesis of those class carbohydrates in the world scientific literature are known under name of “Gakhokidze’s syntheses”. The central problems of A. Gakhokidze’s scientific researches were the investigation of physiological active compounds, their study and application. The theory of A. Gakhokidze was the foundation of application of carbohydrate as a transporter of medicine preparations.

The principle of glycosylation of medicine means elaborated by A.Gakhokidze, which is based on the active transport of carbohydrate fragments in the cell membranes, represents a new approach of a creation of medicine preparations of task-oriented action.

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Application of these preparations in clinical pharmacology which are not solved in water has very important disadvantage which consists that their obtaining is possible only internal way or with external influence. Mentioned circumstance obviously restricts to the possibility of their usage in medical practice. A.Gakhokidze provided for great importance of conversion of medicine means in water soluble form in which was being given the possibility to introduce parenterally these medicines. In result there would be changed not only absorption but accelerated physiologic effectiveness and sharply decreased the preparation toxicity. All above spoken pointed out to actuality of synthesis development of water soluble medical preparations. It was the question of very great significance solution of which had a great importance in the provision of population by medical preparations. Clinical medicine in this period widely used sulfa drugs which not being solved in water. A. Gakhokidze envisaging the actuality of problem laid down the aim to converse just sulfanilamide in solved form in water. After studying the question in detail, A.Gakhokidze solved the problem by following way. He realized the sulfanilamide condensation with glucose for obtaining of soluble one and determined the conditions of reaction. In the solution of diluted ethanol at the interaction of streptocide with glucose, at presence of calcium chloride gives monoglucosulfanilamide (XIII), but if repeat this reaction in the absolute ethanol mediom, we obtain diglucosulfanilamide (XIV). What about the question of obtaining of soluble aspirin, A.Gakhokidze obtained chlorine anhydride of aspirin by interaction of sodium salt of aspirin and thionyl chloride, then he realized the condensation of glucose and last component (with presence of quinoline) and partially saponification by sodium acetate of obtained pentaaspiringlucose (XV) he prepared pentasalicylglucose (XVI). Obtained product is the water soluble preparation what gives the possibility to prepare injection form of medicine for parenteral application.

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Prof. A.Gakhokidze obtained also water soluble monoaspiringlucose (XVII) by following scheme: OAc

OAc OH

O OAc Br

AcO

+

O OCOC 6 H 4 OH

Pb

H4C6 COO

OAc

OAc AcO OAc

2

OH

OH

O OCOC 6 H 4 OH

O OCOC 6 H 4 OAc

OH

OH

HO

HO

OH

OH

XVII

As a conducted researches showed insoluble preparations solve in the water by “fastening” of carbohydrate molecules and absorb easy by organism. There is significantly increased the physiologic effectiveness of medical preparations and decreased their toxicity. Clinical medicine today widely use such approach “to ennoble” the preparations for treatment of malignant tumors. For example interaction product of glucose and nitrogen yperit is used successfully in oncology: Cl OH O O

CO

CH2

OH

N

n

HO OH

Cl

n=1,2,3

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In comparison of alive cells with neoplasm ones the tumor cells are distinguished by intensive glycolise. This reason is considered by scientists unusual penetration of glucose in the shell of neoplasm cells. Therefore in such compound the carbohydrate residue is the carrier, which provides more selected concentration in the neoplasm cells. The same idea has laid down for obtaining of chlorethylamides of aldaric and aldonic acids. It is known that natural compounds are easy subjected to decomposition, therefore they can not perform the role of ideal medicines. This noted circumstance force the researchers find the way of natural compounds modification, for obtaining of stable medicine means of long duration pharmacologic influence. The light confirmation of this is the elaboration of carbohydrate condensation methods with ascorbic acid and other physiologic active substances. In today world the searching of new medical means is most expensive, long and less effective process. This was mentioned on the one of meetings of National Academe of USA in New York. Medicine service market today need not increase of new, synthesis means potential but the correction of known, approved by clinical medicine drugs i.e. increase of their specificity, acceleration of their participation in the pathologic region, reversal of toxic influence from organism what cardinally changes the pharmacokinetics and pharmacodynamics of preparations. Prof. A.Gakhokidze obtained also the soluble starch. There has a great importance of A.Gakhokidze‫ۥ‬s achievement in veterinary practice, in the obtaining of helminthologic means. There must be noted the obtaining new preparations between which substances consisted manganese and arsenic obtained by mining of natural deposits of Chiatura and Racha. One of basic purposes of A.Gakhokidze was the study and application of natural resources. By him was stated the structure of some glucosides and pigments and elaborated the methods of their synthesis. For example he isolated new flavonides – akrammerin (XVIII), olmelin (XIX) and 3-D-glycosylepicatechin (XX) with different acids and glycosides from plant gleditschia triacanthos to which the clinicians appropriate an important value in phitotherapy as a means of cardiovascular and malignant diseases. OH OH

O

O

OH HO OH

HO

OH H 3 CO

O

O

XVIII akramerini

XIX olmelini

OCH 3

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OH HO

O

OH

OH O O

OH HO

OH OH

XX 3-D-glukozilepikateqini

Prof. A.Gakhokidze synthesized akrammerin and olmelin according to the following scheme: OH HO

OCH 2 C 6 H 5

HNO3

OH

C6H5CHCl2 H 5 C 6 H 2 CO

OCH 2 C 6 H 5

OH

OCH 2 C 6 H 5 OH

OCH 2 C 6 H 5

OCH 3

OCH 3

(CH3)2SO4 H 5 C 6 H 2 CO

H 5 C 6 H 2 CO

COCH 3

H 5 C 6 H 2 CO

OCH 2 C 6 H 5

H 5 C 6 H 2 CO

OCH 2 C 6 H 5 OCH 3

OCH 3

OCH 3 H 3 CO

OCH 3

OCH 3

H 3 CO

OH OCH 3

O

COOK

OCH 3

OH

O

HI

HO

OH

OH HO

H 3 CO

O

OH

O

Demethylated akrammerin dimeTilirebuli akramerini

OCH 3 H 3 CO

O

OCH 3 OCH 3

H 3 CO H 3 CO

O

meTilirebuli Demethylated akrammerin Methylated akrammerin akramerini

The Scientist who Has Outstripped His Time HO

OH

HO

HCl

NC

+

329

OH

OCH 3

H2O + NH2

OH

-

Cl

OH

fluroglucini Phloroglucinol OCH 3 HO

OH O

HCOOC2H5

O

HO

OH OH

OCH 3

O

OCH 3

Prof. A.Gakhokidze elaborated method of hydroxicarbonic acids synthesis which is based on the condensation of ketones and esters of carbonic acids: R

R''

R

COOR'''

R'

R'' C(OH) CH

CO + CH2 R'

COOR''' R''

R C(OH) CH R'

COOH

On the base of these investigations by A.Gakhokidze was arisen an original theory of organic acid formation in the plants. In accordance with this theory, for example, citric acid is formed from glucose according to the following scheme: CHO H HO

OH H

COOH H HO

OH H

H

OH

H

OH

H

OH

H

OH

CH2OH

CH2OH

H

COOH OH

HO

H

H

OH CO CH2OH

COOH H

OH

HO

H

H

OH COOH

COOH CH2 CO CH2 COOH

COOH

HCOOH

CH2 C(OH)COOH CH2 COOH

+

HCOOH

This scheme was proved as chemical so biochemical synthesis. Splitting between 4 and 5 carbon atoms of 5-keto-gluconic acid gives rise to other organic acids. A.Gakhokidze paid a great attention to the application of agricultural wastes. He obtained from maize waste xylotrihydroxyglutaric acid. It can change the tartaric acid in the techniques. Yield of this acid from maize waste consisted 6%. A.Gakhokidze separated citric acid from tobacco waste, from different region of Georgia.There was stated that waste from Lagodekhi consisted 6.37% of citric acid, from Gagra region – 4.71%.

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By processing of shell of tung-tree A.Gakhokidze separated the dye and stated it‫ۥ‬s empirical formula. Prof. A. Gakhokidze has founded chemical investigation of the oil in Georgia, which is of great theoretical and practical meanings. He studied oil deposits of Supsa, Mirzaani and Shirakhi. In 1942 A. Gakhokidze came out with an original hypothesis about oil genesis. He showed that transformation of carbohydrates in nature besides biochemical conversion is possible also by geologic metamorphosis. For proving his opinion, he obtained 2methylheptane (isooctane) from glucose by method elaborated by him (Gakhokidze was the first investigator who used metalorganic synthesis in carbohydrate chemistry): CH2OH O

CH2OH Br2, CaCO3

OH HO

O O

OH

OH

(CH3CO)2O

HO

OH

H3C

COH

HOCH

HI, P

1. CH3MgI 2. H2O

OAc CH3

CH3

HCOH

O

OAc

AcO OH

H3C

CH2OAc O

CH3

CI CH2

H3 C Mg

CH CH2

CH2

CH2

HCOH

CH2

CH2

HCOH

CH2

CH2

CH3

CH3

CH2OH

This research is very important for power engineering in future. A.Gakhokidze‫ۥ‬s consideration were proved after 40 years by Canadian scientists. There were discovered microorganisms which transform the carbohydrates into oil (oil fermentation). Prof. A.Gakhokidze wrote many courses of chemistry for students; there were published course of organic chemistry, manual of organic and biological chemistry, inorganic chemistry and many other books. The life of prof. A.Gakhokidze interrupted very unexpectedly at the age of 55 years in 1964. He could not realize completely many new scientific ideas, new scientific beginnings, but what he has created that remains forever in a treasury of a world science.

INDEX A Aβ, xiii, 169, 170, 171, 175, 195 absorption, 41, 77, 135, 138, 139, 140, 168, 169, 198, 239, 245, 278, 279, 285, 287, 288, 289, 291, 325 absorption spectra, 135, 168, 169 academic success, 319 acceptor, 120, 174 accuracy, 136 acetate, 132, 156, 231, 235, 325 acetic acid, 36, 37, 70, 89, 168, 204, 276 acetone, 156, 215, 279, 287 acetonitrile, 2, 3, 4 acetylcholine, ix, x, 11, 12, 19, 21, 32 acetylene, 196, 198 achievement, 327 acidic, 130, 140, 239 acidification, 27 acidity, 138, 295 actin, x, 12, 16, 17, 21 activation, xiii, 2, 3, 5, 6, 7, 8, 15, 27, 30, 46, 88, 91, 92, 108, 109, 110, 111, 114, 115, 119, 122, 160, 161, 162, 164, 179, 180, 181, 182, 183, 192, 213, 236, 237, 283, 310 activation energy, 3, 46, 161, 164, 180, 181, 182, 183, 236, 283 activation entropy, xiii, 122, 162, 179, 182, 183 activation parameters, 6, 7, 8, 119 activation volumes, 109, 115 activators, 20, 21, 158 active centers, 17 active site, 237 active transport, 324 actuation, 16 acylation, 38, 95, 197 adamantane, x, xv, 35, 36, 37, 38, 54, 55, 57, 196, 198, 200, 275, 276, 287, 288, 290, 291

adaptation, 18, 98 additives, xii, 103, 105, 116, 126, 237, 276, 296 adducts, 281, 282, 296 adenine, 63, 65, 66 adhesion, 213, 259, 260, 267, 291 adhesion strength, 291 adiabatic, xii, 103, 104, 107, 108, 109, 110, 113, 114, 115, 116, 119, 120, 121, 124, 125 administration, 32, 89, 90, 91, 92, 94, 95, 96, 141, 142 ADP, 186, 191 adrenal gland, 94 adrenaline, 19, 20, 21 Adrenaline, 19 adsorption, 131, 139, 215, 313 adult, 26, 257 adults, 247 AFM, 257, 258 Ag, 106 agar, 57, 197 age, 15, 89, 268, 330 agent, 16, 19, 20, 130, 148, 213, 291 agents, xv, 16, 36, 38, 130, 145, 202, 213, 219, 276, 309 aggregates, 139 aggregation, 137, 140, 160, 164 aggregation process, 137, 140 aging, xv, 81, 85, 100, 309 aging process, 85 agonist, 20, 54, 55 agricultural, xv, 36, 57, 276, 309, 329 agriculture, xvi, 303, 309, 313 aid, 27, 30 aiding, 143 air, 69, 86, 168, 248, 260, 287, 291, 295 air-dried, 248 alanine, 83, 85

332 alcohol, 30, 198, 204, 214, 297 alcohols, 196, 258 aldehydes, 258, 320 aldolase, xiii, 185, 189, 192 algal, 21 algorithm, 218 aliphatic amines, 82 alkaline, xii, 82, 83, 129, 140 alkane, 297 alkanes, 296 alkylation, 202, 296 allergy, xv, 309 alpha, 246 alternative, 105, 210 alternatives, 244 aluminum, 148, 217, 244, 261 amide, 203 amine, 82, 83, 215 amines, ix, x, xi, 11, 12, 19, 21, 36, 81, 82, 84, 203, 276 amino, xi, 14, 73, 81, 82, 83, 84, 85, 86 amino acid, 14, 73, 81, 82, 83, 86 amino acids, 14, 73, 81, 82, 83 aminopeptidase, 54, 55 ammonium, ix, xv, 1, 2, 5, 6, 8, 71, 89, 187, 309, 310, 311, 312, 313, 314 ammonium salts, ix, 1, 5 amorphous, 228, 231, 237, 241, 259, 285, 286 amplitude, 14, 118 amyotrophic lateral sclerosis, 185, 186 anaemia, xiii, 195, 196 anaerobic, 189 analog, 30 anatomy, 249 anemia, 100, 195 angular velocity, 277 aniline, xi, 58, 81, 82 animal tissues, 88, 89 animals, 12, 23, 26, 36, 55, 56, 88, 89, 90, 92, 93, 94, 95, 96, 97, 187, 189, 202, 247, 311, 312, 313, 314 anisotropy, 269 annealing, 137, 140 anode, 217 anomalous, 117, 310 ANOVA, 253, 255 antagonist, x, 20, 25, 27, 28, 32, 54 antagonistic, x, 12 antagonists, x, 25, 26, 31, 88 antibacterial, xiii, 195, 196, 198, 209, 301 antibacterial properties, xiii, 195, 198, 301 antibiotic, 202 antibiotics, 196 anticancer, 130, 141, 296

Index anticancer drug, 141, 296 antidiabetic, 202 antihypertensive agents, 130 antioxidant, 89, 90, 95, 96, 100, 191 antioxidative, 100 antioxidative activity, 93, 96 antisense, 145 antisense oligonucleotides, 145 antitumor, 201, 202, 204 antitumor agent, 202 APA, 70, 72 apoptosis, 186, 191 APP, 191 application, xii, xv, xvi, 16, 20, 21, 37, 58, 70, 78, 87, 91, 92, 93, 95, 96, 126, 129, 131, 132, 136, 196, 203, 210, 275, 278, 291, 295, 297, 309, 310, 313, 324, 325, 327, 329 aqueous solution, xii, 103, 105, 116, 296 aqueous solutions, 116 arachidonic acid, 92 argon, 2, 105 arithmetic, 71, 90 aromatic rings, 287 arrest, xii, 104, 125 arsenic, 296, 299, 300, 301, 303, 327 arthropods, 269 articular cartilage, 245 articulation, xiv, 243, 245, 248, 249, 250, 251, 268 ascorbic, 71, 89, 96, 327 ascorbic acid, 71, 89, 96, 327 aspirin, 325 assignment, 213, 214, 223 assumptions, 164 astrocytes, 192 asymmetric synthesis, 296 atactic moieties, 152, 153 atherosclerosis, 81, 185 atmosphere, xvi, 2, 138, 309 atomic force, 257 atomic force microscopy (AFM), 257 atoms, x, xv, 3, 35, 43, 45, 47, 48, 63, 65, 66, 118, 218, 219, 223, 238, 275, 277, 278, 282, 285, 287, 299, 300, 329 ATP, 15, 17, 27, 28, 30, 31, 186, 187, 189, 191, 192 ATPase, 26, 27 atropine, 19 attachment, xiv, 17, 212, 243, 245, 250, 260, 266, 267, 269, 270, 271 autocorrelation, 49 autosomal dominant, 26 availability, 37, 88, 287 awareness, 211

Index

B babies, 313 bacillus, 310 Bacillus, 156, 301, 303 Bacillus subtilis, 301, 303 bacteria, 156, 301 bacterial, 195, 301 bacterium, 290 bandwidth, 120 barium, 213 barrier, xiv, 2, 104, 109, 159, 180, 202, 211, 212, 240, 243, 246, 260 barriers, 130, 141, 202 BAS, 105, 106 basicity, xiii, 167, 296 beetles, 248, 269 behavior, xiv, 104, 112, 116, 138, 142, 242, 243, 253, 254, 255, 256, 257, 260, 262, 265, 266, 296 behaviours, 143 bending, 214, 270, 298 benefits, 210 benzene, 2, 5, 36, 37, 38, 45, 46, 168, 197, 204, 276, 279 Bernoullian statistics, xiii, 147, 150, 151, 152 binding, 65, 66, 218, 219, 232 binding energies, 66, 219 binding energy, 65, 218, 219 bioactive compounds, 286, 291 bioantioxidants, 100 bioavailability, 59, 130 biocatalysis, 159, 160, 163, 164 biocatalyst, 164 biochemistry, 78, 156 biocompatibility, 131, 142, 292 biodegradability, 131, 210 biodegradable, xiv, 136, 141, 142, 143, 156, 209, 211, 239 biodegradable materials, xiv, 209 biodegradation, 301 biogenic amines, ix, x, 11, 12, 19, 21 biological activity, xi, 53, 55, 58, 87, 88, 94, 96, 196, 202, 204 biological consequences, 100 biological systems, 132, 244, 245 biologically active compounds, ix, xiv, 36, 96, 195, 196, 198, 204, 276, 287 biomass, 210 biomolecule, 212 biomolecules, 105, 117 biopolymer, 16 biopolymers, 127 biotechnology, 156

333

birth, xvi, 319 Blay and Dyer method, 70 bleaching, 213 blood, xi, 87, 89, 90, 92, 93, 94, 95, 96, 97, 100, 196, 313 blood plasma, xi, 87, 89, 94 body weight, 89 boiling, 138, 197 bolus, 56 bonding, 63, 235, 237, 296 bonds, x, 3, 6, 35, 63, 65, 66, 139, 168, 176, 198, 203, 204, 214, 223, 224, 238, 287, 298 brain, xiii, 26, 185, 186, 187, 189, 191 broad spectrum, 202 broadband, 118, 125 buffer, 82, 83, 84, 85, 105, 106, 111, 138, 140, 156, 187, 250 building blocks, 237 burning, 89 butyric, 215, 219, 224, 228, 231, 232, 233, 236

C Ca2+, v, x, 15, 16, 17, 21, 25, 26, 27, 28, 29, 30, 31, 32 cabbage, 265, 310 cadmium, xv, 275, 287 caffeine, x, 25, 26, 27, 28, 29, 30, 31, 32, 203, 310 calcium, 15, 16, 17, 21, 26, 32, 191, 212, 325 calcium carbonate, 212 calculus, 65 calibration, 28, 71 calmodulin, 21 calorimetry, 139 cancer, xv, 36, 54, 275, 276, 291, 313 candidates, 59, 131 capillary, 139 caprolactone, 145 carbohydrate, 202, 214, 320, 321, 324, 326, 327, 330 carbohydrates, 202, 203, 204, 310, 324, 330 carbon, 41, 45, 47, 48, 118, 119, 120, 121, 122, 124, 125, 196, 202, 218, 219, 221, 223, 224, 238, 287, 299, 300, 329 carbon atoms, 45, 47, 118, 219, 223, 238, 287, 299, 300, 329 carbonic acids, 36, 57, 276, 329 carbonization, 235 carbonyl groups, 186 carboxylic, xiv, 36, 38, 209, 215, 218, 224, 228, 231, 235, 236, 238, 276 carboxylic acids, xiv, 209, 215, 218, 224, 228, 231, 235, 236, 238 carcinogen, 196

334 carcinogenic, 62, 196, 313 carcinogens, 196 cardiac muscle, 26 carpets, 211 carrier, 202, 277, 327 cartilage, 244, 245 catabolism, x, 12, 21, 88 catalase, xi, xiii, 87, 89, 94, 155, 156, 157, 159, 160, 163, 164, 165 catalysis, xiii, xiv, 156, 158, 160, 179, 180, 181, 182, 183, 184, 201, 203, 204 catalyst, 5, 6, 8, 277 catalytic activity, 159 catecholamines, x, 12, 19, 20 cation, 2, 3, 6, 8, 63, 296, 299, 300 Caucasian, 305 C-C, 168, 218, 223 cell culture, 196 cell death, 88 cell membranes, 99, 202, 203, 324 cell metabolism, 88, 186 cellulose, xiv, 209, 211, 213, 214, 215, 224, 227, 231, 232, 233, 235, 238, 239, 240, 241, 242, 244, 257, 259 cellulose crystallites, 241 cellulose derivatives, 241, 242 cellulose fibre, 232, 241, 242 cellulosic, xiv, 209, 213, 231, 233, 240, 241 cement, 210 central nervous system, 26, 186, 202 cerebral hemisphere, 188, 189, 190 cerebrum, 202 CH3COOH, 38 channels, 216 Chara cells, 15 chemical bonds, 213 chemical composition, 148, 215, 245, 258 chemical energy, 17 chemical kinetics, xiii, 167 chemical pretreatments, 212, 213 chemical properties, 49, 54, 62, 105, 130, 156, 237, 269 chemical structures, 219 chemicals, 105, 211 chemokine, 54 chemotherapy, 202 chitin, 244, 246, 247, 250, 251, 254, 255, 257, 270 chitosan, 131, 132, 133, 134, 135, 137, 138, 141, 145 chloride, xiii, 147, 148, 239, 325 chlorine, 219, 325 chloroanhydrides, 38 chloroform, 70, 89, 168, 187, 248, 287 chloroplast, 16

Index cholesterol, 88, 92, 142 chromatin, 312 chromatograms, 71 chromatography, 37, 70, 89, 156, 157, 203 chromium, 167, 168, 170, 171 chromosomes, 310, 311, 312, 314 chronic stress, 185, 188, 189, 190, 191 circadian, 186, 189 citizens, 321 citotoxic, 202 classical, 112, 122 classification, 210, 258, 269, 310 cleavage, 3, 6, 238 cleavages, 237 clinical oncology, 202 clusters, 51 CMV, ix, x, 11, 12 C-N, 168, 199 CNS, 98 Co, 62, 105, 132, 156 coatings, 211, 268 coconut, 211 cognition, 32 Coleoptera, 268, 269 collateral, 36 commercialization, 37, 210, 287 community, 210 compatibility, 291 compilation, 245 compliance, 291 components, 2, 62, 70, 84, 100, 110, 114, 116, 118, 189, 192, 210, 213, 215, 244, 246, 247, 254, 256, 257, 277, 291 composites, xv, 210, 211, 213, 238, 244, 271, 275, 278, 291 comprehension, ix, 126 computation, 37, 277 computing, 212, 236 conception, 180 concrete, x, 12 condensation, 198, 325, 327, 329 conditioning, 196, 231 conduction, xvi, 124, 319 conductivity, 2, 14, 105, 211 confidence, 115 configuration, 62, 63, 122, 174, 299 conformity, 63 conjugation, 299 constant rate, 161 construction, 16, 109, 134, 139 consumption, 180, 186 contamination, xiv, 243, 253, 260, 266, 267, 268

Index control, 29, 30, 31, 76, 88, 89, 90, 92, 94, 95, 104, 108, 109, 110, 113, 115, 117, 122, 125, 159, 160, 163, 164, 182, 183, 187, 188, 189, 191, 231, 310, 311, 313, 314 control group, 95, 187, 188 conversion, 5, 17, 126, 278, 279, 280, 284, 325, 330 convex, 249, 250 cooligomerization, 284, 285 cooling, 139, 217, 232, 233, 234 copolymer, 131, 142, 143, 284 copolymers, 130, 143 copper, 217 COR, 123 corona discharge, 212, 240 correlation, ix, x, 1, 7, 12, 51, 132, 173, 198, 238, 245, 311, 312 correlation coefficient, 51, 173 corrosion, 303 cortex, 188, 189, 190 cotton, 213, 235 coupling, 26, 104, 105, 108, 126 covering, 131, 259, 261, 303 Cp, xii, 103, 104, 105, 118, 119, 120, 123, 124, 125, 126 cracking, 134, 138, 139 CRC, 79, 98 creatine, 186, 187, 188, 189, 190, 191 creatine kinase, 191 creatine phosphokinase, 186, 188, 189, 191 crops, 36, 210, 211, 276, 313 cross-validation, x, 35, 51 crust, 62 crystal structure, 241, 261, 299 crystal structures, 241 crystalline, 227, 228, 237 crystallinity, 224, 228, 233 crystallization, 233 crystals, xiv, 37, 243, 245, 258, 259, 260, 261, 262, 263, 266, 267, 276, 297 CSF, 192 C-terminal, 32 cultivation, 301 cultural heritage, ix, 36 culture, 156, 303 cuticle, xiv, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 255, 256, 257, 258, 259, 260, 262, 265, 267, 268, 269, 270, 271 cutin, 257, 271 cycles, 215, 232 cyclic voltammetry, xii, 103, 105, 126 cycling, 191 cyclohexanone, 2 cyclosis, ix, 11, 12, 14, 15, 16, 17, 18, 19, 21

335

Cyclosporine A, 142 cytochrome, xii, 103, 104, 105, 106 cytoplasm, ix, x, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 88 cytosine, 63, 65, 66 cytoskeleton, 17 cytosol, 186, 188 cytosolic, 186, 187, 188, 189, 190 cytotoxic, 143, 310, 311, 312, 313 cytotoxicity, xv, 144, 309

D danger, xvi, 309 data analysis, 124 data processing, xii, 103, 105, 126 data set, 49, 111 death, 313, 321 decay, xii, 104, 111, 124 deciduous, 211 decomposition, ix, xiii, 1, 2, 3, 4, 5, 6, 7, 8, 155, 156, 158, 161, 163, 204, 233, 235, 236, 327 deconvolution, 119, 150, 151, 218 deduction, 120 defence, 90 defense, 96 deficiency, 196 definition, 108, 180 deformation, xiv, 214, 243, 257, 258 degradable polymers, 143 degradation, 88, 90, 131, 143, 235, 242, 276 degradation rate, 90 degrees of freedom, 117, 118, 122, 124, 126 dehydrogenase, 189 delivery, 130, 131, 140, 141, 142, 143, 144, 145 denaturation, 161, 163, 164, 182 dendrimers, 131 density, 3, 63, 94, 108, 125, 133, 187, 188, 211, 242, 244, 246, 256, 259, 262 density fluctuations, 125 depolarization, 13, 17 deposition, 211, 213, 236 deposits, 327, 330 deregulation, 26 derivatives, xiii, 36, 131, 195, 196, 198, 201, 202, 203, 204, 231, 241, 242, 299, 323 desiccation, 246, 248, 250, 254, 257, 265 desorption, 231 destruction, 134, 140, 276, 286, 288, 289, 312 detection, 50, 51, 54, 216 deviation, 115, 117 diabetes, 81, 85 diabetes mellitus, 85

336 dialysis, 82, 85 dielectric constant, 109, 110 differential scanning calorimeter, 217 differential scanning calorimetry, 238 diffraction, 217, 228 diffusion, 92, 106, 113, 114, 120, 124, 131, 197 digestion, 71 diluent, 278 dimensionality, 181 dimethylformamide, 285 dipole, 287 direct action, 21, 276 direct measure, 150 direct observation, 17 discharges, xiv, 209 disclosure, 16, 118, 124, 125 discrimination, xii, 103, 105 diseases, 36, 81, 185, 186, 195, 196, 276 disorder, 26 displacement, 13, 17, 18, 20, 131, 153, 171, 176, 253, 254, 256, 262, 263, 264, 265, 267, 270 dissociation, 139, 158 distilled water, 82, 85, 89, 156, 204 distribution, xii, 21, 83, 118, 124, 132, 144, 147, 148, 150, 151 disulfide, 111 diuretic, 211 divergence, 119, 151 diversity, 224, 257, 258 division, 36, 276 DMFA, 287 DNA, v, xi, 61, 62, 66, 131, 132, 143, 144, 145 donor, 120, 295 donors, 295 drinking water, 89 drug delivery, 130, 141, 142, 143 drug delivery systems, 142 drug discovery, 58 drug release, 140, 142 drugs, xii, 32, 129, 130, 131, 327 dry matter, 71 drying, 70, 71, 133, 142, 248, 254 DSC, 139, 217, 231, 232, 233, 234, 241, 286 dualism, 96 durability, 15 duration, xi, 14, 82, 85, 87, 96, 319, 327 dust, 259 dyeing, 211, 213 dynamic control, 111 dysregulation, 32

Index

E earth, 62 ecological, 210, 238, 257, 260 eicosanoid, 92 elaboration, 201, 203, 204, 212, 312, 327 elasticity, 270 elastin, 244 elastomers, 277 electrical conductivity, 2 electrodes, xii, 103, 105, 106, 107, 111, 112, 113, 114, 115, 118, 120, 215, 216 electrolyte, 13, 126 electron, xii, 3, 63, 103, 107, 108, 112, 113, 114, 115, 117, 126, 217, 218, 237, 250, 268, 295, 296 electron density, 3, 63 electron microscopy, 231, 248, 250, 258, 260, 261, 268 electronic integrator, 149 electronic structure, 62, 63 electrons, 37, 63, 216 elementary school, 319 elongation, 3 elytra, 269 embryo, 26, 36 emulsifier, 141 encapsulated, 130, 131 encoding, 26 endocrine, 62, 144 endocrine glands, 62 endoplasm, 16 endoplasmic reticulum, 26 endothermic, 171, 174, 176, 228, 288, 289 energy, 3, 5, 6, 17, 37, 46, 63, 65, 91, 108, 110, 114, 117, 126, 140, 160, 174, 181, 182, 183, 186, 189, 190, 191, 210, 211, 213, 216, 217, 237, 238, 247, 283 energy consumption, 186 engraftment, 143 environment, ix, xii, xv, xvi, 14, 17, 36, 103, 104, 115, 116, 126, 131, 218, 245, 257, 260, 276, 309, 310 environmental conditions, 130, 132, 140, 259 environmental protection, ix, xv, 275, 291 enzymatic, 70, 72, 81, 95, 156, 158, 187, 188, 189 enzymatic activity, 95, 156, 188, 189 enzymes, x, xiii, 12, 21, 70, 88, 90, 91, 92, 93, 130, 180, 185, 186, 189 epidermal cells, 250, 257 epidermis, 247, 265, 268 epithelial cells, 141 equality, 13, 161

Index equilibrium, 6, 107, 109, 110, 119, 120, 132, 140, 144, 153, 169, 171, 172, 173, 174, 175, 176, 177, 284 erosion, 143 erythrocyte, 88, 92, 93, 94, 95 erythrocyte membranes, 88, 92, 93 erythrocytes, xi, 87, 89, 92, 93, 94, 97, 98 essential fatty acids, 69 ester, 214, 217, 223, 233, 235, 278 ester bonds, 217, 223 esterification, xiv, 209, 211, 228, 238, 239 esters, xiv, 209, 228, 233, 235, 238, 239, 241, 242, 258, 329 ethanol, 38, 105, 137, 140, 204, 215, 250, 287, 325 ethanolamine, 71, 89, 97 ethyl acetate, 132 ethylene glycol, 25 eucalyptus, 258, 265 evaporation, 131, 132, 141, 246 evolution, xiii, 147, 151, 152, 176, 269, 270 examinations, 320 excitation, 12, 13, 15, 16, 26 exoskeleton, 246 experimental condition, ix, 3, 119 exploitation, 277 exposure, 82, 92, 215, 216, 219, 315 extraction, 120, 141 extrapolation, 121 extrusion, 210

F fabrication, 141 failure, 185, 191 FAO, 239 fat, 70, 72 fatigue, 32 fatty acid, 69, 71, 92, 211, 231, 237, 239, 258 fatty acids, 71, 237, 239, 258 FDA, 131 feet, 26, 266 females, 89, 91 fermentation, 330 ferrocenyl, xiii, 195, 196, 198 fertilization, 314 fertilizer, xv, 309, 310, 313, 314 fertilizers, 311, 312, 314 fiber, 14, 213, 235, 238, 241, 244, 246, 250 fibers, xiv, 209, 213, 215, 216, 224, 229, 230, 231, 235, 238, 240, 242, 244, 246, 247, 250, 251 fibrils, 16, 231, 259 filament, 16, 257 fillers, 240

337

film, xv, 57, 70, 231, 256, 259, 275, 291 films, 104, 106, 118, 120, 256, 270 filtration, 37, 105, 276, 297 fixation, 2, 142, 276 flame, 211, 213 flavonoids, 258 flight, 270 flora, 310 flow, 132, 149, 186, 217 flow rate, 149 fluctuations, 116, 118 fluid, 117, 265, 266 fluoride, 25 fluorine, 235, 242, 278 folding, 250, 269 food, 26, 32, 70, 301, 313 force constants, xi, 61, 65, 66 formaldehyde, 213 fragmentation, 236, 237 free activation energy, 181 free energy, 6, 108, 117, 118, 158, 181 free radical, 3, 237, 238 free radicals, 3, 237, 238 free volume, 148 freedom, 117, 118, 122, 124, 126 free-radical, 237, 238 freezing, 117, 126 friction, xiv, 105, 113, 114, 115, 116, 119, 122, 210, 243, 245, 247, 248, 268, 269, 270 fructose, 187 fruits, 259 FTIR, 218, 224, 240, 241, 279, 287, 288, 289 FT-IR, 213, 214, 215, 216, 217 functional approach, 268 functional changes, 191 functionalization, 211, 213 fungi, ix, 14, 57 fungicidal, 57, 290 fungicide, 301 fungus, 15 Fusarium, 57, 290, 291

G gas, 4, 215, 217, 277 gas phase, 4 gases, 211, 212, 236, 238, 257 gastrointestinal, 313 gauge, 216 Gaussian, 119, 120, 121, 122, 219 gel, 70, 89 gene, 26, 32, 131, 141, 143, 144, 145, 268 gene expression, 144

Index

338 gene therapy, 141 gene transfer, 141, 143, 144 generation, 13, 17, 186, 190, 191 genes, 130 genomic, 94, 310, 311, 312, 313, 314 genotoxic, 311 gland, 94 glass, 2, 70, 89, 134, 140, 210, 215, 216, 217, 235 glass transition, 140, 235 glass transition temperature, 140, 235 globalization, 185 glow discharge, 212 glucose, xi, 81, 82, 83, 84, 85, 86, 105, 111, 187, 203, 238, 320, 321, 323, 325, 326, 327, 329, 330 glutaraldehyde, 250 glycation, 85 glycerol, 27, 104, 116 glycine, 83, 85, 86, 187 glycol, 144 glycolysis, 186 glycosides, 82, 196, 327 glycosyl, xiv, 201, 204 glycosylamides, xiv, 201, 204 glycosylation, xiv, 85, 201, 202, 324 gold, 106, 118, 250 government, iv GPCR, 53 G-protein, x, 12, 21 grafting, 211, 213, 215, 216, 217, 219, 224, 228, 232, 235, 236, 237, 238 grafting reaction, 219, 236 grafts, 232, 233 grain, 78, 313 gram-negative bacteria, 303 grants, 177 granules, 258 graph, 49 graphite, 217 grass, 244, 313 growth, 14, 62, 72, 91, 92, 94, 95, 174, 210, 276 guanine, 62, 63, 65, 66 gut, 301

H H1, 3 H2, 3, 46 halogen, 295 hanging, 133, 140 hardening process, 278 hardness, 244, 245, 248, 253, 254, 255, 256, 262, 265, 266, 267, 268, 269, 270 harvesting, 36, 210, 276

health, ix, 211, 315 health effects, 315 health problems, 211 heart, 56, 105 heat, 7, 8, 45, 46, 132, 133, 134, 139, 140, 183, 196 heat capacity, 133, 134, 139, 140 heating, xii, 129, 133, 134, 135, 136, 137, 138, 139, 140, 217, 232, 233, 234, 277 heating rate, xii, 129, 133, 135, 136, 140, 217 height, 218, 250 helium, 277 hemagglutinin, 143 hematology, 315 heme, 126 hemisphere, 188, 190 hemoglobin, 195, 313 hemoglobin (Hb), 313 hemp, 211 Heparin, 239 hepatocytes, 143 herbivores, 268 heterocycles, 203 heterogeneity, 72, 119 heterogeneous, 105, 107, 109, 111, 112, 113, 114, 117, 118, 119, 144, 190, 238 heterogeneous systems, 105 Heteroptera, 269, 270 hexane, 287 high pressure, xii, 103, 105, 106, 114 high resolution, 217, 223, 249, 258 high temperature, 139, 163, 231, 234, 235 high-frequency, 109 hippocampus, 188, 189, 190, 191 histidine, 27, 63 HIV, 144 HIV-1, 144 Hm, 188, 190 homeostasis, 26, 32, 88, 91 homogeneity, 291 homogenized, 132 homogenous, 291 homolytic, 3 hormone, 142 hormones, 94, 130 horse, 156 human, ix, xiii, xv, 61, 145, 195, 196, 244, 245, 275, 276, 291, 309, 312, 315 human immunodeficiency virus, xiii, 195, 196 humidity, 254, 259 hybrid, 217 hybridization, 45, 299 hydration, 63, 234 hydrazine, 187

Index hydride, 287 hydro, xiv, 209, 213, 234, 241, 258 hydrocarbon, 2, 71 hydrochloric acid, 203, 204 hydrogen, xi, xiii, 3, 37, 48, 61, 65, 66, 89, 155, 156, 159, 160, 163, 164, 167, 168, 169, 175, 223, 237, 238, 277 hydrogen abstraction, 238 hydrogen atoms, 3, 48 hydrogen bonds, xi, 3, 61, 65, 66, 163 hydrogen peroxide, xiii, 89, 155, 156, 163, 164 hydrogen sulfide, xiii, 167, 168, 169, 175 hydrolysis, xi, 30, 69, 70, 71, 72, 180, 182, 183, 184 hydrolyzed, 27, 28, 70, 72, 73, 77, 241 hydroperoxides, 2, 3 hydrophilic, xiv, 209, 213, 234, 241 hydrophilicity, 131, 231 hydrophobia, xiii, 195 hydrophobic, 134, 137, 139, 140, 163, 211, 240, 244, 259, 278 Hydrophobic, 240 hydrophobic groups, 134, 139 hydrophobic interactions, 163 hydrophobic properties, 278 hydrophobicity, 137, 140, 211 hydroquinone, 284 hydrosilylation, 277, 279, 280, 282, 283 hydrostatic pressure, 105, 111, 114, 115 hydroxide, 3, 82 hydroxyl, 196, 203, 224, 231 hydroxyl groups, 224, 231 hygiene, xiv, 209 hyperbolic, 181 hypertension, 99 hyperthermia, 26 hypothesis, 20, 88, 211, 267, 330

I ice, 37, 82, 89, 276 id, 55, 139, 140 identification, 16, 109, 296 identity, 296 images, 217, 248, 249, 250, 251, 252, 256, 258, 261, 262 imaging, 249 immersion, 106 immobilization, 212 immunity, 144 immunization, 144 impregnation, 14 impurities, 211, 213, 219 in situ, 237

339

in transition, 140 in vitro, 100, 131, 142, 143, 144, 145, 200, 313 in vivo, 100, 131, 141, 142, 144, 145, 257, 270 indium, 217 industrial, xii, xv, 58, 147, 148, 210, 211, 292, 309, 320 industrialization, 185 industry, xvi, 78, 211, 239, 309 infection, 196, 313 infections, 196 infrared, 289 inhibition, 16, 20, 92, 290, 291, 303 inhibitor, 53, 54, 55, 158, 284 inhibitory, xiii, 195, 196 initiation, 237 injection, 15, 100, 141, 210, 325 injection moulding, 210 inorganic, 14, 58, 71, 89, 105, 187, 192, 211, 288, 289, 292, 296, 301, 330 inorganic fillers, 211 inorganic salts, 14 iNOS, 191 inositol, 71, 89 insects, xiv, 243, 245, 246, 247, 249, 254, 257, 260, 266, 267, 270, 313 instability, 131, 296 instruments, 244 insulation, 211 insulin, 62 integration, 18 integrity, 54, 55, 70 integument, 247, 256, 269, 270 interaction, ix, x, xi, 11, 12, 16, 17, 19, 20, 21, 26, 27, 41, 61, 63, 65, 69, 81, 82, 83, 85, 116, 131, 132, 137, 145, 159, 174, 181, 197, 203, 231, 237, 257, 260, 280, 287, 295, 297, 299, 322, 325, 326 interactions, 63, 148, 163, 232, 285 intercalation, 144 interface, 126, 213, 245, 270 interfacial layer, 118, 125 interference, 252 interferons, 310 intermolecular, 3, 6, 158, 181, 214, 237 interphase, 311, 312 interval, xii, 19, 129, 134, 136, 138, 139, 140, 164, 288, 289 intestine, 310, 313 intracellular signaling, 189 intrinsic, xii, 103, 105, 107, 109, 113, 114, 115, 116, 117, 118, 119, 122, 126 intrinsic viscosity, 113, 116 intuition, xvi, 319 invariants, 49, 51

Index

340 invasive, 62 invertebrates, 244, 247 iodine, 71, 295, 297, 298, 299, 303 ion transport, 92 ionic, xii, 103, 105, 106, 117, 118, 126 ionic liquids, 105, 117 ionization, 218 ions, xi, xii, 3, 6, 16, 17, 21, 28, 30, 31, 43, 61, 103, 104, 119, 130, 295 IR spectra, 37, 39, 41, 176, 198, 213, 214, 217, 277, 278, 285, 297 IR spectroscopy, xiv, 209, 213, 216, 297 iron, 196 iron deficiency, 196 irradiation, 238 irritability, 12, 13 irritation, 12, 13, 20 ischemic, 186 isoforms, 26, 32, 186, 188 isolation, xiii, 27, 185, 186, 189 isomerization, 320

J joints, 245, 247 judge, 71

K K+, 17, 91 Kates modification, 70, 89 KBr, 168, 213, 277, 297 Keltan, 311, 312, 314 ketones, 30, 198, 258, 329 kinase, xiii, 185, 191, 192 Kinase, 53, 54, 191 kinase activity, 191 kinetic constants, 106 kinetic curves, 5, 27, 28, 29, 30, 31, 279 kinetic model, 86 kinetic parameters, xiii, 8, 123, 155, 182, 183, 217, 236 kinetic studies, 109, 113 kinetics, xiii, 118, 155, 156, 157, 159, 160, 163, 164, 167, 170, 180, 181, 242 Krebs cycle, 186, 189 Kyrgyzstan, 201

L lactic acid, 130, 142, 219, 224, 227, 228, 232, 233, 236

lactose, xi, 81 land, 319 large-scale, 113, 118, 124 larvae, 247 larval, 257 law, 161 LDH, 192 learning, 189 lecithin, 70 Leguminosae, 210 lens, 217 lesions, 88 Lewis acidity, 295 Lidocaine, 143 ligand, xiii, 53, 54, 62, 63, 167, 168, 169, 171, 172, 174, 175, 176 ligands, xiii, 62, 64, 167, 170, 174, 176, 177, 287, 288 light scattering, 124 lignin, 211, 214, 215 limitation, 131 linear, xv, 5, 6, 172, 176, 181, 233, 295, 296, 299, 300 linear dependence, 172, 176 linen, 213 links, 186, 203 linoleic acid, 215, 224, 236 lipase, 180 lipid, xi, 69, 70, 71, 72, 74, 77, 78, 87, 88, 89, 90, 91, 92, 93, 95, 142, 143, 144, 202, 255, 258 Lipid, v, 54, 55, 69, 98, 100 lipid oxidation, 72, 74 lipid peroxidation, xi, 71, 87, 88 lipids, 69, 70, 71, 72, 74, 75, 76, 77, 78, 88, 91, 92, 93, 95, 131, 143, 215, 247, 248, 255, 257, 258, 268 lipophilic, 36, 131, 196 liposome, 144 liposomes, 131, 144 liquid crystals, 117 liquid phase, 2 liquids, 105, 110, 114, 117, 118, 120, 279 lithium, 82, 148, 198, 239 lithium hydride, 148 liver, xi, 62, 87, 89, 90, 91, 92, 93, 94, 192 living conditions, 248 L-lactide, 142, 143 localization, 16 logarithmic coordinates, 120 London, 86, 177, 268, 269, 270 low molecular weight, 143, 144 low temperatures, 161, 163, 183, 233, 235 LPO, xi, 71, 73, 87, 88, 89, 90, 92, 94, 95, 96

Index LPO intensity, 71, 90, 94 lubrication, 245, 248 lumen, 26, 27, 28 luminal, 26 luminescence, 15 lymphocytes, 196 lysine, 141 lysis, 310, 311

M M.O., 23, 205 macromolecular chains, 237 macromolecular networks, 237 macromolecules, 130, 237 magnesium, 191 magnetic, 132, 268 Maillard reaction, xi, 81, 82, 85, 86 maize, 329 males, 89, 90, 91, 92 malignant, xv, 26, 62, 196, 309, 313, 314, 326, 327 malignant hyperthermia, 26 malignant tumors, xv, 309, 313, 314, 326 maltose, xi, 81, 321 mammalian cells, 192 manganese, 219, 327 man-made, 244 manners, 191 manufacturing, 142, 212, 287, 303 manure, 314 marine mammals, 78 market, 210, 239, 327 Markov, 150, 151 Markovian, xiii, 147, 150, 151 Markovnikov rule, 282, 283 MAS, 240 mass loss, 235, 288, 289 mastery, xvi, 319 material sciences, 245 materials science, xiv, 148, 243, 244 matrix, 108, 130, 131, 143, 213, 238, 244, 246, 254, 257, 276, 277, 286, 287, 291, 299, 303 meanings, 330 measurement, ix, 11, 134, 150, 189, 218, 256 measures, xv, 139, 309, 310, 312 mechanical behavior, xiv, 243, 253, 254, 255, 256, 257, 260, 262, 265 mechanical properties, xiv, 239, 243, 244, 245, 247, 248, 253, 254, 256, 257, 259, 260, 262, 263, 268, 271 media, 14, 38, 46, 105, 117 mediators, 94

341

medicine, xii, xvi, 129, 195, 196, 309, 321, 324, 325, 326, 327 Mediterranean, 210 melons, 36, 276, 290 melt, 235 melting, 51, 136, 140, 228, 231, 233, 234, 235 melting temperature, 233, 235 membranes, 13, 21, 26, 78, 88, 93, 94, 99, 144, 269, 271 memory, 189 men, 321 mercury, 82, 134 meristem, 36, 276 messengers, x, 12, 21, 94 metabolic, 15, 186, 189, 276 metabolism, 13, 15, 54, 55, 88, 94, 186, 310, 313 metabolites, 90, 315 metal ions, 61, 130 metalloporphyrins, 167, 174, 177 metals, 62, 63 metamorphosis, 330 metaphase, 312 metastasis, 202 methanol, 70, 89, 137, 148, 203, 204, 248 methemoglobinemia, 313 methine group, 280 methyl group, x, 25, 27, 30, 32, 41, 280, 287 methyl groups, x, 25, 27, 30, 41, 280, 287 methyl methacrylate, xii, 147, 148 methylene, 41, 111, 112, 124, 280, 299, 303 methylene group, 41, 280 Mg2+, 16 mice, xi, xv, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 141, 309, 310, 311, 312, 313 micelles, 160, 164, 181 microbial, 196 microcalorimetry, 132, 145 micro-climate, 238 microemulsion, 145 microfilaments, 16 microorganisms, ix, xi, xv, 35, 36, 55, 57, 197, 275, 276, 290, 291, 301, 303, 330 microparticles, 142, 143, 144 microscope, 217, 239, 250 microscopy, 26, 252, 257, 268 microspheres, 131, 141, 142, 143, 145 microstructures, 148 microsystem, 248 microviscosity, 92 middle lamella, 259 mitochondria, 26, 88, 91, 186, 187, 191 mitochondrial, 186, 188, 189, 190, 191 mitosis, 196

342 mobility, xiv, 92, 132, 148, 243, 248 model system, 45, 100, 111 modeling, x, xi, 8, 35, 49, 51, 61, 282 models, x, xiii, 5, 21, 35, 37, 49, 118, 120, 122, 147, 191, 256 modulation, 94, 186 modulus, 211, 244, 246, 248, 253, 254, 255, 256, 257, 262, 263, 265, 266, 267, 270 moieties, xiii, 147, 148, 152, 153 moisture, xiv, 70, 209, 215 moisture content, 215 molar ratio, 82, 83, 84, 85, 168, 284, 285, 297 molar volume, 109 mole, 45, 46, 65, 162, 164, 183, 277, 283, 298 molecular biology, 268 molecular mass, 16 molecular oxygen, 2 molecular structure, x, 25, 49, 202 molecular weight, 81, 82, 83, 85, 86, 131, 142, 143, 144, 149, 215, 258 molecular weight distribution, 149 molecules, x, xii, 2, 12, 17, 20, 26, 27, 28, 30, 32, 49, 62, 63, 71, 92, 103, 134, 173, 196, 202, 234, 295, 326 molybdenum, 167, 168, 174, 175 monochromator, 216, 217 monolayer, 104, 106 monomer, 216 monomers, 2, 211, 212, 216 monosaccharide, 324 monosaccharides, 201, 202, 203 morphological, 224, 231 morphology, 228, 239, 259, 268 motion, 16, 108, 119, 124, 233, 245 moulding, 210 mouse, 191 mouse model, 191 movement, ix, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 244, 247, 260 multi-component systems, 110 multilayered structure, xiv, 243, 247, 265 multiplier, 180 muscle, x, 25, 26 muscle cells, 26 muscle contraction, 26 muscles, 16 mushrooms, 301, 303 mutagen, 36 mutagenesis, 54 mutagenic, xv, 202, 309, 310, 311, 312, 313, 314 mutant, 111, 266 mutants, 260, 261 mutation, xv, 32, 65, 66, 260, 309, 312

Index mutations, xi, 26, 32, 61, 310, 311, 313, 314 myocytes, 186 myoglobin, 116 myopathies, 32 myosin, 16, 17, 21

N Na+, 91, 296 NaCl, 27, 156, 187 nanoindentation, 245, 248, 256, 257, 267, 269 nanometer, xiv, 243, 244, 259, 266 nanometer scale, xiv, 243, 244 nanoparticles, xii, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 nanoparticulate, 131, 142 nanotechnology, 130 National Academy of Sciences, 1, 58, 201 natural, xiv, 15, 36, 62, 88, 96, 100, 130, 131, 187, 202, 203, 209, 210, 211, 213, 238, 239, 243, 244, 245, 246, 260, 269, 271, 276, 301, 310, 327 natural fillers, 211 natural resources, 327 necrosis, 186, 191 neoplasm, 327 nerve, 14 network, 86, 228, 239 neurodegenerative diseases, 186 neuroleptics, 196 neurons, 186 neuropathy, 186 neutralization, 57, 237 Ni, 62, 63, 65, 66, 205 nickel, 38, 62, 63, 65 nitrate, xv, 235, 309, 310, 311, 312, 313, 314, 315 nitrates, 313 nitric oxide (NO), NO, xiii, 41, 45, 46, 48, 185, 187, 189, 190, 191, 192, 287, 288, 289 nitrobenzene, 37 nitrogen, 48, 63, 148, 167, 170, 215, 217, 219, 237, 313, 326 NMR, xii, xv, 37, 38, 39, 41, 42, 43, 46, 58, 118, 147, 148, 149, 150, 151, 153, 197, 198, 199, 240, 241, 275, 277, 279, 280, 281, 282, 283, 287, 292 N-N, 203, 204 nodes, 36, 276 non-biological, 104 nonequilibrium, 140 non-invasive, 249 non-renewable, 210 non-renewable resources, 210 nontoxic, 144 noradrenaline, 19, 20, 21

Index norfloxacin, 142 normal, 13, 32, 65, 66, 92, 117, 260, 262, 263, 265, 311, 312, 313 normalization, 94, 96 N-terminal, 143 nuclear, 5, 108, 192 nucleation, 212 nuclei, 311, 312, 313, 314 nucleosides, xi, 61, 62, 63, 203 nucleotides, 62, 63 nutrition, 26

O observations, 202, 261 oil, 180, 184, 211, 215, 219, 220, 221, 222, 223, 224, 226, 227, 228, 230, 231, 232, 233, 234, 236, 239, 319, 330 oligomer, 279, 281 oligomers, xv, 17, 21, 235, 275, 277, 278, 285, 286 oligonucleotides, 131, 144, 145 olive oil, 215, 219, 224, 227, 228, 231, 232, 233, 234, 236 omission, 161 oncology, 202, 326 optical, 71, 82, 169, 170 optical density, 71, 82 oral, 32, 59, 140, 142 organ, 13 organelles, 88 organic, xii, xv, 2, 37, 49, 66, 103, 105, 131, 132, 192, 196, 219, 235, 242, 248, 251, 252, 275, 277, 285, 288, 296, 314, 320, 321, 329, 330 organic C, 66 organic compounds, 219, 296 organic polymers, 277 organic solvent, 105, 131, 132, 285 organic solvents, 105, 131 organism, xv, 19, 26, 81, 88, 90, 91, 94, 96, 98, 185, 189, 202, 309, 313, 326, 327 orientation, 244, 250, 251, 259 osmotic, 93 osteotomies, 142 outliers, x, 35, 49 oxalate, 27 oxidants, 196, 313 oxidation, 2, 62, 69, 70, 72, 73, 74, 76, 88, 186, 191, 196, 224, 238, 296, 323 oxidation products, 73 oxidation rate, 88 oxidative, 70, 75, 88, 89, 96, 186, 189, 191, 286 oxide, xiii, 185, 187, 190, 192, 313 oxides, 299

343

oxidizability, 71, 92, 95 oxygen, x, 2, 25, 27, 30, 32, 48, 69, 167, 170, 176, 186, 218, 219, 222, 223, 224, 242

P paclitaxel, 141, 142 paints, 301 palladium, 250 pancreas, 94 parabolic, 108 parameter, 72, 74, 75, 77, 90, 91, 94, 104, 108, 117, 118, 120, 124, 131, 133, 140, 252 parasite, 15 parenchyma, 14 parenteral, 325 Parkinson, 185, 186 particles, x, xii, 12, 17, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 231, 259 partition, 53 pathology, 98, 99 pathophysiological, 99 pathways, 131 PCA, x, 35, 37, 49, 50, 51 pectin, 244 pedagogical, xvi, 319, 321 penetrability, ix, 11, 88, 92 pentads, xiii, 147, 149, 150, 151, 152 peptide, 144 peptides, 130, 131, 142, 143 perception, 12, 117 permeability, 58, 186, 202, 203 peroxidation, xi, 87 peroxide, 2, 3, 5, 6, 8, 72, 74, 89, 159, 160, 161 peroxides, 2, 74 pesticide, 312, 314 pesticides, xv, 309, 310, 311, 312, 313, 314 petroleum, 9 PGA, xii, 129, 130, 140 pH, 27, 82, 83, 84, 105, 106, 111, 130, 131, 132, 133, 135, 138, 140, 156, 187, 250 pharmaceutical, 54, 98, 130 pharmaceuticals, 57 pharmacodynamics, 327 pharmacokinetic, 144 pharmacokinetics, 327 pharmacological, xiii, 26, 27, 30, 32, 195, 196, 198, 202 pharmacology, 36, 202, 325 phase space, 117 phenol, xi, 81, 84, 168 phosphate, 71, 82, 83, 84, 85, 89, 105, 107, 192, 235, 250

344 phosphatidic acid, 71, 90 phosphatidylethanolamine, 144 phosphocreatine, 186, 191 phosphodiesterase, 21 phospholipids, xi, 70, 71, 73, 87, 89, 91, 92, 93, 94, 96 phosphorus, 71, 187, 189 phosphorylation, 88, 186, 189 photoelectron spectroscopy, 241 photon, 132 phylogeny, 269 physical properties, x, 35, 118, 142, 148, 150, 152, 213 physicochemical, xi, 87, 88, 90, 96, 144, 237 physicochemical properties, 144 physico-chemical properties, 105, 130 physiological, xii, 12, 15, 129, 188, 192, 324 physiology, 270 pigments, 327 pinhole, 217 Pisum sativum, 13, 245, 258, 260, 266 placenta, 313 planar, xi, 61, 62, 63, 65, 66 plants, 13, 14, 15, 16, 21, 36, 57, 88, 239, 254, 257, 259, 260, 261, 262, 268, 270, 276, 303, 310, 313, 329 plasma, xi, xiv, 87, 88, 89, 94, 95, 209, 211, 212, 213, 215, 216, 217, 219, 223, 224, 227, 231, 235, 236, 237, 238, 240, 242, 259 plasma membrane, 88 plasmid, 143, 144 plasmolysis, 19 plastic, 210, 244, 257, 258, 291, 303 plasticizer, 233, 235 plastics, 209, 213 platinum, 106, 139 play, 17, 94, 126, 137 PLGA, vi, xii, 129, 130, 131, 132, 133, 135, 136, 137, 138, 140, 141, 142, 145 PLS, x, 35, 37, 51 PMMA, xii, 147, 148, 149, 150, 151, 152, 153 PMSF, 25, 27 point mutation, xi, 61, 65, 66 poisonous, 55 poisons, 99 polar groups, 116 polarity, 131, 137, 140 polarization, 122 pollen tube, 14 pollutants, xvi, 309, 310, 313 pollution, xv, 210, 211, 309 poly(lactic-co-glycolic acid), 142 poly(vinyl chloride), 148

Index poly(vinylchloride), 150 polyester, 258 polyesters, 143 polyethylene, 70, 144, 242 polyethylenimine, 144 polymer, xiv, xv, 131, 139, 142, 143, 148, 209, 211, 219, 232, 237, 238, 244, 246, 275, 276, 277, 286, 287, 291 polymer materials, xv, 148, 275, 291 polymer matrix, 131, 143, 238, 276, 277, 286, 291 polymer systems, 211 polymeric materials, 240, 276, 303 polymerization, 2, 148, 213, 237 polymers, xiv, 2, 57, 131, 141, 142, 143, 148, 196, 209, 213, 237, 239, 241, 276, 277, 282, 285, 291, 296, 301 polynucleotide, 63 polyolefins, 238 polypeptides, 143 polypropylene, 148, 150 polysaccharides, 203, 237, 242 polystyrene, 213 polyurethane, 277, 292 polyurethane foam, 292 polyvinyl alcohol, 142 pools, 92 population, 36, 49, 210, 325 pores, 250, 251 porous, 311, 312, 314 porphyrins, 167 potassium, 17, 71, 296 powders, 287 power, 51, 62, 104, 111, 115, 215, 217, 330 power-law, 104 precipitation, 156 prediction, 51, 58, 117, 122 predictors, 51 preference, 238 pressure, xii, 103, 105, 106, 107, 109, 110, 111, 113, 114, 115, 118, 126, 138, 212, 215, 217, 265, 266 prevention, xiv, xv, 243, 245, 309, 310 primary products, 72 primary tumor, 202 printing, 211, 213 private, 21 probability, xi, 46, 54, 61, 65, 108, 150, 151, 182 probe, 118, 119, 120, 126 producers, 98 production, 99, 140, 186, 189, 210, 211, 212, 238, 291, 320 prognosis, 54 program, 53, 54, 65, 141, 169, 213, 217, 236 pro-oxidant, 95, 96

Index property, 12, 113, 147, 152, 153 prophase, 191 prophylactic, xv, 309, 310 prophylaxis, xi, xv, 36, 57, 275, 291 protection, ix, xv, xvi, 131, 211, 275, 276, 291, 309, 312 protein, x, xii, 12, 15, 16, 17, 21, 26, 27, 70, 72, 74, 75, 86, 89, 95, 103, 104, 105, 109, 110, 113, 114, 115, 116, 126, 156, 157, 161, 182, 188, 189, 212, 244, 246, 254, 256, 257 protein structure, 247 proteins, x, xii, 12, 16, 21, 81, 82, 85, 88, 92, 103, 104, 105, 109, 113, 114, 116, 130, 145, 156, 187, 246, 255, 257 proteolytic enzyme, 70 prothorax, 248, 249, 250, 251 protons, 41, 280, 281, 287 protoplasm, 17 PSA, 53 public opinion, 211 pulse, 149 pumping, 27 pupil, 319 purification, 27, 156, 157, 319 purines, 63 PVA, 132, 140 PVC, xiii, 147, 148, 150, 152, 153 pyramidal, 299 pyrimidine, 63 pyrrole, 168

Q QSAR, 49 quantum-chemical calculations, x, 35, 45, 289 quartz, 217 quaternary ammonium, ix, 1, 2 quaternary ammonium salts, ix, 1 Quercus, 241

R radiation, 98, 99 radical formation, 3 radius, 63, 216 rain, 191 rat, 189 rats, 55, 56, 100, 141, 187 raw material, 210, 211, 219 raw materials, 211 reactant, xii, 103, 107, 111, 126 reactants, 126

345

reaction center, 45 reaction mechanism, 109, 235, 237 reaction medium, xi, 81, 82, 114, 118, 122 reaction order, 3, 172, 173, 176, 236, 279 reaction rate, 5, 158, 159, 160, 162, 180, 181, 182, 279 reaction temperature, 279, 284 reaction time, 82, 83, 84, 211 reaction zone, 108 reactive oxygen, 186, 191 reactive oxygen species, 191 reactivity, ix, xi, 1, 2, 3, 6, 8, 81, 82, 85 reading, 106 reagent, 63, 187 reagents, 159, 175, 180, 182, 183, 277 reality, 15 reception, 12 receptors, ix, 11, 19, 20, 21, 92 recognition, xvi, 319, 321 recollection, 320 recovery, 192, 213, 257, 258 recrystallization, 241 recrystallized, 2, 204 red blood cell, 92 redox, xii, 103, 104, 105, 109, 111, 114, 115, 118, 119, 120, 126, 186, 191 redox-active, xii, 103, 104, 105, 115, 126 reducing sugars, 81 reflection, 15, 257 refractive index, 149 regenerated cellulose, 241 regeneration, 6 regular, 61, 250 regulation, ix, x, xiv, 11, 12, 88, 90, 92, 96, 189, 191, 202, 209 regulators, 156 relationship, xi, xiii, 6, 7, 49, 75, 81, 86, 147, 171 relationships, x, 35, 51, 141, 148 relaxation, 108, 113, 116, 117, 118, 124, 257, 258 relaxation process, 108 relaxation time, 116, 125 relaxation times, 125 renewable resource, 210 reparation, 310 replication, 65, 66, 310 reproduction, 196 reservoir, 215 residues, 92, 131, 324 resins, 277 resistance, 93, 106, 211, 247 resolution, 132, 213, 217, 223, 249, 258 resources, 185, 186, 210, 327 respiratory, 189

Index

346 retention, 51, 211 reticulum, x, 25, 26, 31, 32 returns, 13 reverse reactions, 176 rhamnolipids, 156 riboflavin, 203 ribose, 32 rigidity, 91, 92, 95 rings, 106, 227 risk, 313 RNA, 145 robotics, 244 ROOH, 3, 4, 5, 6, 7, 8 room temperature, xii, 70, 103, 105, 113, 114, 126, 132, 139, 187, 260, 284, 291 room-temperature, 118 roughness, xiv, 243, 250, 252, 253, 259 roughness measurements, 250, 252 rubber, 247 Russian Academy of Sciences, 1, 25, 69, 87, 96, 99, 155, 167, 179, 201 ruthenium, 27, 28, 29, 30, 31

S sacred, 268 safety, 69, 131 salt, 2, 3, 5, 6, 8, 325 salts, ix, 1, 5, 6, 14, 61, 132, 140, 296, 299, 301 sample, xiii, 70, 73, 75, 77, 139, 147, 149, 150, 151, 152, 153, 212, 213, 217, 219, 221, 222, 223, 227, 228, 233, 234, 250, 253, 261, 262, 265, 281 SAS, 156, 163, 164 saturated fat, 71, 92, 211 saturated fatty acids, 71, 211 scaling, 119 scanning calorimetry, 217 scanning electron, 268 scanning electron microscopy, 231, 248, 250, 258, 260, 261 scattering, 58, 124, 135, 139, 285, 292 school, xvi, 79, 319, 320 search, xiv, 118, 195, 198 searching, 196, 201, 203, 327 seaweed, ix, 11, 12, 14, 15, 16, 17, 19 secretion, 260 seed, 180, 184 seeds, 211 selecting, 287 selectivity, xiv, 201, 202, 204 SEM, xiv, 209, 217, 224, 228, 231, 238, 248, 250, 251, 256, 258, 260, 261, 262 SEM micrographs, 231

semen, 14 semiconductors, 196 senescence, xv, 309 sensing, 270 sensitivity, xiii, 20, 88, 91, 111, 132, 185, 191, 217, 296 sensors, 244 separation, 106, 110, 112, 120, 269 serine, 71, 89 serotonin, 19 serum, 144 seta, 266 sex, xii, 87, 91, 93, 96 shape, 62, 93, 108, 112, 126, 132, 138, 139, 218, 246, 249, 250, 258, 299, 300 shares, 76 sheep, 57 shock, 245 short-term, 94 side effects, 202 sign, x, 11, 20, 21, 96 signal transduction, 26 signaling, 88, 92 signals, xiii, 41, 147, 150, 151, 152, 280, 281, 287 silica, 70, 89, 143 silicon, xv, 275, 277, 278, 285 siloxane, 212 silver, 215, 216 similarity, 20, 27, 32, 65, 113 simulations, 118 singular, 310, 311 siRNA, 131 sites, 171, 189, 210, 237 skeletal muscle, x, 25, 26 skeleton, 16 skills, 57 skin, 62, 145, 244 sleep, 187 social resources, 185 sodium, 27, 89, 204, 325 soft substrate, 256 software, 106, 219, 282 soil, xvi, 309, 313, 314 soils, 210 solar, 126 solar energy, 126 solid state, 296 solid surfaces, 268 solubility, 58, 130, 202, 204 solvation, 116, 117, 118 solvent, 2, 3, 37, 38, 70, 104, 109, 110, 116, 117, 118, 119, 122, 125, 131, 132, 133, 134, 137, 138, 140, 141, 142, 148, 149, 167, 168, 277

Index solvent fluctuation, 116 solvents, 109, 110, 122, 198, 211 sorghum, 268 Soxhlet extractor, 216 soybean, xi, 69, 70, 72, 73, 74, 75, 76, 77 spatial, xv, 62, 63, 275, 285 species, 88, 104, 107, 111, 115, 175, 191, 224, 236, 237, 258, 259, 261, 263, 269 specific adsorption, 111 specific heat, 132, 133, 134, 135, 136, 137, 138, 139 specificity, 26, 27, 88, 327 spectrophotometric, xiii, 132, 139, 167, 168, 169, 170 spectrophotometry, 187 spectroscopy, xii, 58, 118, 132, 139, 147, 148, 149, 203 spectrum, 36, 41, 42, 43, 44, 135, 139, 168, 195, 196, 202, 213, 214, 215, 217, 218, 244, 247, 279, 280, 281, 282, 285, 292, 298, 303 speed, 12, 15, 18, 19, 20, 136, 140, 277 sphingolipids, 94 spin, 41, 287 spleen, xi, 87, 94 sponges, 143 spore, 301 stability, xii, xiii, 36, 65, 70, 72, 105, 129, 131, 132, 136, 138, 140, 143, 144, 145, 167, 169, 174, 175, 176, 177, 189, 196, 235, 237, 246, 259, 263, 278, 286 stabilization, 65, 163 stabilize, 63, 296 stages, 3, 4, 37, 77, 156, 157, 171, 175, 278 standard deviation, 252 staphylococcus, 197 starch, 327 statistics, xii, 90, 147, 148, 150, 151, 152 stereosequences, 148, 152, 153 steric, 6, 63, 285 steroids, 196 sterols, 71, 72, 77 stiffness, 253, 255, 257, 270 stimulus, 12, 20 stoichiometry, 171 stoma, 15 stomach, 140, 313 storage, xi, 69, 70, 72, 73, 74, 75, 76, 77, 78, 131, 136, 137, 268 strain, 156 strength, 106, 140, 143, 174, 176, 211, 241, 247, 291 streptosotocin, 202 stress, xiii, 115, 185, 186, 188, 189, 190, 191, 192 stretching, 214, 298 structural changes, 232, 250, 254

347

structural characteristics, ix, 1 styrene, 284 suberin, 271 substances, x, xii, 2, 12, 14, 25, 27, 28, 32, 70, 71, 72, 78, 89, 92, 94, 95, 100, 120, 129, 156, 180, 196, 201, 202, 204, 219, 248, 250, 251, 252, 285, 291, 296, 301, 303, 310, 311, 312, 324, 327 substitution, 41, 63, 109, 238, 287 substrates, 88, 105, 184 sucrose, 27, 187 sugar, 14, 81, 116, 320 sugars, 81, 83, 104, 320 sulfa drugs, 325 sulfur, 174 sunflower, 180, 215, 224 superiority, 283 supernatant, 70, 71, 187 supply, 189 supramolecular, 237 surface area, 53, 58 surface diffusion, 113 surface layer, 247, 256, 257 surface modification, 131, 211, 238 surface properties, 219, 237 surface roughness, 250, 252 surface structure, 250, 257, 260, 261 surfactant, 142 suspensions, 136 swelling, 131, 133, 139 symbols, 121 symmetry, 62 syndiotactic sequences, 148, 152 synthesis, x, xiii, xv, 12, 21, 36, 37, 49, 88, 186, 187, 189, 195, 196, 203, 204, 215, 236, 276, 287, 288, 295, 296, 321, 324, 325, 327, 329, 330

T tachycardia, 26 tacticity, xii, 147, 148, 149, 150, 151, 152 task performance, 26 taste, 69, 81, 211 Taxol, 141, 142 taxonomy, 268 T-cell, 143 tea, 310 temperature dependence, 140, 171 tensile, 211, 247, 256 tensile strength, 211, 247 teratogenesis, 54 tetrahydrofuran, 148 textile, xiv, 209, 212, 213, 240 textiles, 212, 303

348 TGA, 217, 277, 286, 288, 289, 290 therapeutic agents, 130 therapy, 36, 141, 142, 186 thermal analysis, 228, 241, 242, 277 thermal decomposition, 5, 236 thermal degradation, 235, 242 thermal expansion, 133 thermal stability, 235 thermal treatment, 83, 86 thermodynamic, xiii, 131, 132, 133, 136, 138, 167, 169, 171, 174, 182 thermodynamic parameters, 136, 138, 171 thermodynamics, 167 thermograms, 232 thermogravimetric, 235 thermogravimetry, 238 thermolysis, 2, 5, 288, 289, 290 thermoplastic, 235 theta, 227, 228 thin film, 211, 268, 270 thin films, 268, 270 thiobarbituric acid, 70, 89 thoracic, 269 threatened, 20 three-dimensional, 210 threshold, 13, 234 thymine, 65, 66 thyroid, 94 thyroid gland, 94 tissue, 13, 91, 94, 95, 141, 143, 248 tissue engineering, 143 titration, xiii, 2, 70, 167, 168, 169, 170, 297 tobacco, 329 toluene, xiii, 38, 167, 168, 169, 170, 171, 173, 174, 175, 176, 279, 284, 285 tonic, 88, 211 toxic, 131, 156, 204, 296, 303, 311, 327 toxic effect, 311 toxicity, xiii, xiv, 36, 90, 105, 141, 195, 196, 198, 201, 202, 296, 303, 312, 325, 326 TPA, 89, 95, 301 training, 50, 51 transfection, 131, 143, 144, 145 transfer, xii, 3, 103, 104, 108, 117, 118, 120, 126, 140, 141, 143, 144 transferrin, 144 transformation, 62, 65, 66, 158, 172, 181, 196, 330 transformations, 174, 196 transition, 6, 15, 62, 111, 134, 136, 137, 138, 140, 159, 174, 181, 217, 235, 286 transition metal, 62 transition temperature, 134, 136, 137, 138, 140, 217, 235, 286

Index transitions, 163, 233 translational, 122 translocation, 14, 143 transmembrane, 32, 92 transmembrane region, 32 transmission, 12, 21, 92, 93, 162 transparency, 134 transparent, 70, 285 transport, 21, 29, 30, 31, 32, 58, 88, 92, 106, 114, 120, 145, 202, 217, 310, 324 transportation, 130 trees, 36, 276 Triads, 151, 152 tribological, 244, 248 tribology, 245 trifluoroacetic acid, 211 triploid, 311 tumor, 196, 314, 327 tumor cells, 327 tumors, xv, 202, 276, 309, 313, 314, 326 tungsten, 167, 168 tunneling, 104, 110 turnover, 107, 112, 113 two-dimensional, 58, 217, 292

U ultrastructure, 269 uniform, 231 urea, 203, 205 UV, 37, 82, 156, 168, 180, 197, 211, 240

V vacuole, 14 vacuum, 148, 168, 215, 216, 297, 312 valence, x, 35, 45, 62, 63, 214 validation, 51 validity, 120 van der Waals, 65 vapor, 105, 211, 212 variability, 13, 72, 77, 95 variables, 51, 105 variation, xii, 103, 104, 105, 111, 112, 114, 116, 117, 118, 125, 126, 161, 182, 218, 219, 224, 233, 244, 247 vector, 144 vegetable oil, 215 velocity, ix, 11, 15, 18, 19, 30, 277 ventricular tachycardia, 26 vibration, 214, 215 Vickers hardness, 244, 256

Index vinyl monomers, 2 virus, xiii, 141, 143, 195, 196 virus infection, 196 viruses, ix viscose, 111 viscosity, xii, 88, 92, 103, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 118, 124, 125, 140, 284, 285, 291 visible, 88, 156 vitamins, 69, 78, 310 voltammetric, 112, 120, 121

W walking, 266, 267 water absorption, 291 water-soluble, 57 water-soluble polymers, 57 wavelengths, 169 waxes, 248, 255, 257, 258, 259, 268, 269, 270 WAXS, 285 weak interaction, 116 wear, xiv, 243, 248, 268 web, 37, 243 wells, 108 wettability, 259 wetting, 213, 240 wheat, 70

349

wood, 213, 235, 240, 244, 245, 303, 321

X xenobiotics, 311 xenon, 217 X-ray analysis, 64 X-ray diffraction (XRD), 224, 228, 241 X-ray photoelectron spectroscopy (XPS), xiv, 209, 216, 217, 218, 219, 220, 221, 222, 224, 228, 238, 241 X-rays, 216, 217 xylene, 38

Y yeast, 70 yield, 61, 83, 112, 167, 168, 204, 213, 217, 238, 296, 298 young men, 321

Z Zea mays, 13 zinc, 111