Biosynthesis in Insects

Biosynthesis in Insects

Biosynthesis in Insects

E. David Morgan Chemical Ecology Group, Keele University, UK

RSeC advancing the chemical sciences

ISBN 0-85404-691-7

A catalogue record for this book is available from the British Library

0The Royal Society of Chemistry 2004 All rights reserved Apart from any fair dealing for the purpose of research or private study, or criticism or review as permitted under the terms of the UK Copyright, Designs and Patents Act, 1988, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page, Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 OWF, UK Registered Charity Number 207890 For further information see our web site at www.rsc.org Typeset by Refinecatch Ltd, Bungay, Suffolk, UK Printed and bound by TJ International Ltd., Padstow, Cornwall, UK

Preface “It is from the behaviour of simple molecules that we learn our most sign$cant lessons. Sir Frederick Gowland Hopkins, 1913

This book arose out of a course of lectures I was invited to give to young post-graduate students at Universidade Federal de Alagoas, Maceio, Brazil in 1999. Students working on practical aspects of insect pest control through the use of natural pesticides, pheromones and hormones need to know something about the origin of these substances in nature, if they are to use their skills fully in their work. There is not much information available to them in a clear and elementary form in the review literature or books, so I undertook to provide them with an introductory guide to biosynthetic pathways, with special emphasis on insects. I have subsequently expanded the notes prepared for them to make this small book, with the hope that it will prove useful and informative not only to them but to other students in science and technology, and perhaps attract more of them to this interesting and growing area of chemical ecology. Where biosynthetic processes differ between animals and plants or micro-organisms, I have concentrated on animal systems, but I have not confined myself strictly to the Insecta. The close interaction between plants and insects makes it necessary at times to look at plant substances and pathways, and I have, where important or interesting examples present themselves from other areas, strayed to other arthropods, and even man to complete the story. Because this is intended as a didactic work for young students, I have not used the apparatus of references as in a scholarly review. Experience has shown me that faced with many references to research literature, they may consult none of them. I have rather referred in the text to a few especially useful or interesting papers, and added a list of further reading at the end of each chapter. I have also included a few problems so that readers can make a self-assessment of their grasp of what has been discussed.

V

Acknowledgements I am grateful to my friends Neil Oldham, John Brand, Ralph Howard, Athula Attygalle, Graeme Jones and Desire Daloze for reading various parts or drafts and giving me many helpful suggestions, and to Jane Parker for giving me a student view on an early draft. The request for the original lectures came from my former student Ruth do Nascimento, to whom I am much indebted. I thank the British Council office in Recife, and CNPQ for the financial support that enabled me to give the lectures, and Professor Ant6nio Euzebio G. Sant’Ana for his hospitality. I am very grateful to Dr John Shanklin of Brookhaven National Laboratory and Dr Ed Cahoon of the Danforth Plant Sciences Center for kindly providing the diagrams of castor oil desaturase, Prof John Mann and Dr Rishuo Nishida for permission to use figures from their work. I am obliged to Dr Jonathan Banks in Australia and Dr Keith S. Brown in Brazil for helping me concerning the final position of aphinin research. Those of us laboratory-bound do not get to see a great variety of insects in their natural surroundings. I can happily thank various friends for contributing their photographs of (biosynthetically) interesting insects. Stefan0 Turillazzi, Universita di Firenze, for his picture of Polistes wasps, Athulla Attygalle and Maria Eisner for Epilachnis pupae, Steve McWilliam of rECOrd, Chester, for Coccinella septempunctata adults and Jim Klaisch of the Department of Entomology, University of Nebraska - Lincoln for the C. septempunctata larva, Dr Mike Quinn, Texas A&M University, Stephenville, for superb pictures of Hippodamia, Oncopeltus and Danaus, Tom Larsen for Scolopendra, Jens Christian Schou for Arctia caja, Warren E. Savary for Lytta magister, Markku Savela for Harpaphe haydeniana, Dr Hamish Robertson of the South African Museum for Ceroplastes, Dr Paul Choate, Department of Entomology and Nematology, University of Florida for Manduca sexta larva, Socikte Nouvelle des Editions Boubee for permission to reproduce the photo of a female silk moth taken by Jacques Six, Anthony Papadoupolos for the fire bug Pyrrhcorus apterus, the University of Oklahoma Veterinary School for Amblyomma americanum, and our own Terry Bolam for the Myrmica ant trail-following.The cover photo is one vii

...

Vlll

Acknowledgements

of the many beautiful insect photos taken by Ken Preston-Mafham of Premaphotos Wildlife. I have tried to find the owner of the picture of the cheese mite, Tyrophagus putrescentiae without success. I have sought to locate owners of all reproduced material not in my own possession. In a few cases I have been completely unsuccessful, but trust I have not inadvertently infringed any copyrights. Should I have done so I shall of course take appropriate action for any subsequent editions. I would also welcome comments and suggestions on this book. ([email protected])

Contents Chapter 1 Introduction 1.1 The Structures of Natural Products 1.2 Compounds and Function 1.3 Studying Biosynthetic Pathways 1.4 Plant Versus Insect Biosynthesis 1.5 Arthropods and Insects Background and Further Reading Questions Chapter 2 Enzymes and Coenzymes 2.1 The Chemical Reactivity of Enzymes 2.1.1 Lysozyme 2.1.2 Carboxypeptidase 2.1.3 Cytochromes 2.2 Coenzymes 2.2.1 Coenzyme A 2.2.2 Nicotinamide Adenine Dinucleotide 2.2.3 Flavin Adenine Dinucleotide 2.2.4 Thiamine Diphosphate 2.2.5 TetrahydrofolicAcid 2.2.6 S-Adenosylmethionine 2.2.7 Pyridoxal Phosphate 2.2.8 Vitamins 2.2.9 Biosynthesis of Formic Acid in Ants 2.3 Pyruvic Acid 2.4 Chirality 2.4.1 Asymmetric Induction Background and Further Reading Questions

10 10 10 12 13 13 14 15 14 17 19 19 19 21 21 22 23 24 26 26

Chapter 3 Fatty Acids and Derived Compounds 3.1 Fatty Acids 3.1.1 Biosynthesis

28

ix

28 29

Contents

X

3.1.2 Unsaturated Acids and Desaturase Enzymes 3.1.3 Eicosanoids 3.1.4 Branched Fatty Acids 3.2 Cuticular Hydrocarbons 3.2.1 Hydrocarbon Pheromones 3.3 Lepidopteran Sex Pheromones 3.4 Coleoptera 3.4.1 Coccinellines 3.4.2 Epilachnine 3.5 Cockroaches 3.6 Termites 3.7 Honeybees 3.8 Ants 3.9 Spiders 3. I0 Hemiptera 3.10.1 Green Leaf Volatiles 3.1 1 Lactones Background and Further Reading Questions

32 35 36 37 40 41 45 46 47 48 49 50 51 52 53 53 53 55 55

Chapter 4 Polyketides and Acetogenins 4.1 Acetogenins 4.2 Polyketide Derivatives 4.3 Volatile Pheromones 4.3.1 Cyclic Ketals 4.4 Defensive Secretions Background and Further Reading Questions

57

Chapter 5 Experimental Methods 5.1 Tracing Biosynthetic Pathways 5.1.1 Specific Incorporation 5.1.2 Locating the Site of Synthesis 5.2 Radio-isotope Labelling 5.2.1 Examples 5.3 Heavy Isotope Labelling 5.3.1 Examples 5.3.2 Carpophilus Beetle Pheromone 5.3.3 l3C-I3cCoupling 5.4 Isotope Effects 5.4.1 Kinetic Isotope Effects

69 69 70 71 72 73 75 75 80 80 81 81

57 51 61 65 66 67 67

Contents

xi

5.5 Analytical Aspects 5.6 Chirality Background and Further Reading Questions

82 82 83 84

Chapter 6 Terpenes 6.1 Monoterpene Biosynthesis 6.1.1 The Methylerythritol Phosphate Pathway 6.2 Monoterpene Pheromones 6.3 Monoterpene Defensive Compounds 6.3.1 lridoids 6.3.2 Degraded Terpenes 6.4 Sesquiterpenes 6.4.1 Sesquiterpene Pheromones 6.4.2 Cantharidin 6.4.3 Lac Insects 6.5 Homosesquiterpenes 6.6 Juvenile Hormone Background and Further Reading Questions

85

85 87 89 91 91 94 94 96 97 98 99 101 102 103

Chapter 7 Higher Terpenes and Steroids 7.1 Diterpenes 7.1.1 Termites 7.2 Sesterterpenes 7.3 Triterpenes and Steroids 7.3.1 Sterols in Insects 7.3.2 Saponins from Triterpenes 7.4 Insect Moulting Hormone - Ecdysteroids 7.5 Tetraterpenes Background and Further Reading Questions

104 104 105 107 108 111 113 114 116 119 119

Chapter 8 Aromatic Compounds 8.1 Aromatic Compounds in Nature 8.2 The Shikimic Acid Pathway 8.3 Phenyl-C, Compounds 8.3.1 Aromatic Pheromones 8.3.2 Compounds from Chorismic Acid 8.4 Aromatic Amines 8.4.1 Adrenaline Group 8.4.2 Serotonin Group

121 121 121 123 123 125 127 127 128

xii

Contents

8.5 Phenols 8.6 Quinones 8.7 Insect Pigments 8.7.1 Melanin 8.7.2 Quinones 8.7.3 Aphins 8.7.4 Pterins 8.7.5 Tetrapyrroles 8.7.6 Ommochromes and Ommins Background and Further Reading Questions

129 130 132 132 133 134 136 138 139 140 141

Chapter 9 Alkaloids and Substances of Mixed Biosynthetic Origin 9.1 Alkaloids 9.1.1 Alkaloid Precursors 9.1.2 Plant Alkaloid Biosynthesis 9.1.3 Insect Alkaloids 9.1.4 Other Examples 9.1.5 Alkylpyrazines 9.2 Compounds of Mixed Biosynthetic Origin 9.2.1 Luciferin 9.2.2 Volicitin Background and Further Reading Questions

143 143 144 145 145 151 152 154 157 158 159 159

Chapter 10 Plant Substances Stored, Changed or Unchanged, by Insects 10.1 Toxic Plant Substances in Insects 10.1.1 Cardiac Glycosides 10.1.2 Veratrum Alkaloids 10.1.3 Pyrrolizidine Alkaloids 10.1.4 Cyanogenic Glucosides 10.1.5 Glucosinolates 10.1.6 Coniferyl Alcohol 10.1.7 Other Types 10.1.8 A Parting Thought Background and Further Reading Questions The Bonus Question

161 161 162 163 164 166 169 171 171 174 175 175 176

Contents

...

Xlll

Answers to Questions Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 The Bonus Question

177 177 177 178 178 179 180 181 182 183 184 184

Appendix - Common Abbreviations Subject Index

185

187

CHAPTER 1

Introduction The materials in living plants and animals are divided by scientists into two groups: primary and secondary metabolites. Primary metabolites are the substances fundamental to all living matter: simple sugars, aminoacids, nucleotides and fatty acids, etc. Secondary metabolites are substances made by one or a group of species which are not generally vital to the life of the organism. Secondary metabolites may be structural materials, such as bone, chitin or hair, antibacterial or antifungal compounds, they may give protection from predators or foragers, they may be signalling substances (hormones or pheromones) or they may have, as yet, no known function in that organism. The range of secondary metabolites is enormous and presents a never-ending source of research and exploration. What is equally surprising is that this great array of substances are made from relatively few basic building blocks. Figure 1.1 attempts to summarize, very briefly, the way in which all these types of compounds found in nature are made. Notice that the carbon atoms of all substances, from plant or animal, are ultimately derived from carbon dioxide via photosynthesis. The figure shows that many groups of compounds are formed via relatively few biosynthetic paths. Biosynthesis is the building up of chemical compounds through the physiological processes that take place in living animals, plants and micro-organisms. There are by some estimates about one million insect species. They have colonized almost the entire terrestrial world, and are very varied in habitat and behaviour. They share some biochemical characteristics with all living organisms, others with all animals, but others are peculiar to insects alone, or to a few species or even a single caste of a single species. In the words of Jerrold Meinwald and Thomas Eisner, pioneers in insect chemical ecology, “The ability to synthesize or acquire an extremely diverse array of compounds for defence, ofence and communication appears to have contributed significantly to the dominant position that insects and other arthropods have attained. ” The kind of compounds the insects produce are therefore a challenge to our ability to understand their structures 1

2

Chapter 1

and functions. The groups of compounds that are of special interest to us in the study of insects are indicated in boxes in Figure 1.1. The great diversity of secondary metabolites indicated in Figure 1.1 are often spoken of by chemists as natural products. They are varied in their chemical structure, but they are all made by one of these few biosynthetic pathways (in some cases, a combination of more than one of them). By understanding their biosynthetic origins one can make some sense of this great diversity of natural products and group them according to their origin. Moreover, as we come to understand better these biosynthetic mechanisms, we gain greater insight into how we might regulate such reactions in pest species, as well as understanding how these pathways evolved. The general principles are considered first in each case and then their application to insects is discussed. In some cases the principles are discussed first in relation to micro-organisms or plants, because that is where they were first studied or where more is known of them. It should also impress upon the reader the unity and diversity of biosynthetic products.

1.1 THE STRUCTURES OF NATURAL PRODUCTS Knowing the probable biosynthetic origin of a new compound can help to decide what is its likely structure, and what is an improbable structure, and help us to arrive at its structural formula. It can be difficult to rule out a possible structure completely, because nature is full of surprises. This book should help the reader to decide which among some alternative structural possibilities is the more likely. In Figure 1.2, the compounds on the left are insect pheromones where the likely biosynthetic origin can be easily deduced from the structures, while it is very difficult to see how those on the right can be made by the routes we know, but both the structures on the right have been found in at least one insect. When structures like those on the right are proposed, it is particularly important to show that they are correct by synthesizing the structure proposed and comparing it with the natural compound.

1.2 COMPOUNDS AND FUNCTION Many of the compounds from insects considered here are pheromones (Greek, phero = carry or convey), defensive or offensive substances (allomones, Greek, allos = other), or hormones (Greek, hormao = excite or impel). Pheromones can be considered as chemical communication between individuals of the species, while hormones are chemical communication within the individual. In evolutionary terms, it has been

In troduet ion CO2

+

3 hv

H20

GLUCOSE

(plus other 4 , 5 , 6 and 7 carbon sugars)

PHOTOSYNTHESIS Nucleic acids Polysaccharides Glycosides H

S

o Etythrose 4-phosphate Phosphoenol pyruvate

p o H 'OH

\ Shikimic acid

Aromatic amino-acids Aliphatic amino-acids CH~COSCOA

( in plants )

Acetyl coenzyme A

Citric acid cycle

C02

+

H20

+

+ co:!

glyceraldehyde 3-phosphate

ENERGY

+

Fatty acids

\ Acetogenins Hydrocarbons Hormones Pheromones Eicosanoids

CH~COSCOA

Steroids

CH~COCH~COSCOA Acetoacetyl coenzyme A

+ CH~COSCOA

Mevalonic acid

Figure 1.1 A summary of the chief biosynthetic routes to primary and secondary metabolites. Those ofparticular interest here are enclosed in boxes. Notice that glucose, phosphoenol pyruvate and erythrose-4-phosphate are the key intermediates for all these classes of compounds. P indicates a phosphate ester (Adapted from a figure in J. Mann Chemical Aspects of Biosynthesis 1994, by permission of Oxford University Press)

Chapter I

4

rather than

acoocH OH

a trail pheromone of ants

an alarm pheromone of ants

Figure 1.2 On the left are two structures with simple biosynthetic origins, while on the right are two for which a simple biosynthetic route cannot be given

suggested that pheromones were among the first communication chemicals affecting animal behaviour, and the pheromones of primitive singlecell organisms may have evolved into the hormones of multi-cellular animals. On the other hand, the types of compounds used as pheromones and allomones are so varied, they appear to have evolved many times, while the hormones are relatively conserved, and the same hormones serve many or all insects and can be common to many invertebrate classes. Chemicals for communication (semiochemicals, Greek semeion = sign or signal) between species and between plants and animals are called collectively allelochemicals, and are further sub-divided in a system depending upon whether they benefit the sender (allomones, as above), receiver (kairomones), or both (synomones), and other categories. Pheromones are the group of insect compounds that have found greatest application in agriculture and forestry. For example, a large number of lepidopteran species are important agricultural pests. They use sexual pheromones to attract males for mating. The pheromones can be used to aid control of pests in one of several ways. Traps baited with synthetic pheromone can be used to detect the arrival of a pest, or to assess the build-up of the species in a crop, so that insecticides can be used more sparingly and at the correct time. In a few cases, trapping alone can be effective in removing enough of the males to control the pest. Sometimes the pheromone is scattered throughout the crop so that males are unable to locate females (mating disruption). Sometimes a wrong isomer can completely inhibit the response to a pheromone, so a lure containing some of the inhibitor can disrupt mating. Both Coleoptera and Lepidoptera can be pests in forestry, and there too, pheromone traps have been found effective. Pests in stored products are particularly suitable for pheromone trapping, where use of insecticides is undesirable. Sales of pheromones world-wide still represent only a few percent of the total value of sales of insecticides, which are of the order of billions of US dollars, but pheromones sales are steadily growing.

Introduction

5

Insect defensive compounds are usually effective at short distance and their toxicity or repellency is not sufficient for them to have found any industrial application. Venoms can be powerful, but usually require injection. Of the insect hormones, ecdysteroids (Chapter 7) have not yet found practical application, but there are several examples, in special circumstances, of very effective use of juvenile hormone mimics. 1.3 STUDYING BIOSYNTHETIC PATHWAYS When considering the formation of naturally occurring substances, whether simple amino-acids, sugars, or complex proteins, alkaloids, polyketides, terpenes or steroids, it should be remembered that all the reactions involved follow the normal laws of chemistry. One of the fascinating areas of chemistry today is trying to understand how these biosynthetic reactions occur. How it is that reactions we find extremely difficult in the laboratory are accomplished efficiently and quickly at room temperature and near neutral pH inside cells? What kinds of organic chemical reactions can be used in living cells? The immediate answer to these questions is that nature has evolved efficient catalysts called enzymes that lower the energy of activation for these reactions, to make them proceed much more quickly. Enzymes became active catalysts through repeated accidental, evolutionary changes over time. Whatever the apparent “magic” effect of enzymes, the reactions must still obey the laws of thermodynamics, the reaction will still be explicable in terms of electron push and pull, of bond and charge movements, just as in the rest of organic chemistry. Enzymes cannot make reactions go forward if the energetics are unfavourable to formation of the product. To give a fuller explanation it is necessary to consider the nature and function of enzymes and some of the co-enzymes that often function with them. Emil Fischer, at the beginning of the 20th century had two enzymes called invertin and emulsin. Invertin hydrolysed only a-D-glucosides (sucrose is an example) while emulsin hydrolysed P-D-glucosides. From this and his knowledge of sugars he correctly deduced that these enzymes are asymetrically constructed molecules; in modern terms, they are chiral. Biochemical reactions take place on the chiral surface of an enzyme (Chapter 2), which makes an important distinction from solution chemistry. The first enzyme obtained in a pure, crystalline state was urease, in 1926. It soon became clear that it and other enzymes were proteins. The energetics and kinetics of these enzymic reactions are important to the biochemist, but are not essential to our understanding of what kinds of compounds are produced by insects.We should, however, bear in mind that these systems are not static. Schoenhemierand Rittenberg showed in 1936

6

Chapter I

that when an animal was allowed to drink heavy water (D,O) for a few days, its fatty acids became labelled with deuterium. When normal water then replaced heavy water, the dueterium disappeared from the fatty acids, showing that cells, and whole animals, are in a state of dynamic equilibrium. The kinds of organic chemical reactions that take place in living systems can be divided into five simple types, which are illustrated in Figure 1.3. Enzymes are known which catalyze all these types of reactions, but there may be several ways in which the reaction is catalyzed, particularly so in oxidation-reduction and hydrogenation-dehydrogenation reactions. a. Substitution

R-X

+

b. Addition

R-CH=CH-R H

R

c. Elimination

R++R R

X

d. Rearrangement

R-Y +

Y--

R' R+CH,-OH R

R

+

XY

-

xX R R w R R Y R +HX

- RmR R

R R+CH,-RI

R

OH

or

e. Oxidation-reduction

Figure 1.3 A summary of the types of organic biochemical reactions

1.4 PLANT VERSUS INSECT BIOSYNTHESIS Plants have the ability to make a much greater diversity of compounds than animals can show. Generations of natural product chemists have devoted their skills to solving the structures of plant compounds. For example, there are about 15,000 known terpenes (Chapter 6) made by plants. Above all, plants can use photosynthesis, splitting water in the light reaction (see Figures 1.4 and 5.6) and in the dark reaction creating carbohydrates from carbon dioxide and hydrogen. Plants (and microorganisms) have exclusive access to the shikimic acid pathway (Chapter 8), and the aromatic amino-acids, and to the methylerythritol pathway to terpenes. The case of the polyunsaturated acids (Chapter 3) may be unclear, since at least three insects have been shown able to make linoleic acid, but linolenic acid remains from plants only. The sterols (Chapter 7) can be made by plants and higher animals, but not by insects. The formation of carotenes (Chapter 7) by insects is doubtful. Compounds such as

7

Introduction Light reaction: H20

+

NADP+

ADP

+

H2P04-

hv

NADPH

+

H+

+

i02

ATP

Dark reaction:

Figure 1.4 A summary of the reactions of photosynthesis in green plants

chlorophyll, starch, cellulose, lignin, tannins, anthocyanins, flavones and triterpenes belong only to plants. On the other hand, all the biosynthetic methods, in their broad sense, used by insects, discussed in this book, are available to plants. That is, the formation of fatty acids and their derivatives, such as hydrocarbons; the acetogenins; and especially the terpenes and aromatic compounds are all used by plants. Acetogenins are not as prominent among plant products as the others, except in the formation of anthocyanins and flavones. Only special areas are left to insects alone. It is surprising, as more information accumulates, how insects and plants seem often to have found similar or the same way to biosynthesize certain compounds. Some authors call this parallel evolution. Plants and insects have been evolving together for about 300 million years. In that time plants have produced both physical (hairs, spines and thick waxy surfaces) and chemical (stinging trichomes, alkaloids, toxins and feeding deterrents) defences against insects, while insects have been evolving ways to overcome them. An interesting example of plant counter-attack are the phytoecdysteroids made by plants, which mimic the natural moulting hormone of insects and are stored in the leaves to disrupt normal development of the insect feeding on them (Chapter 7). There are plant anti-juvenile hormone compounds too. Nevertheless, there is probably not a single plant species without at least one insect that has found a way to overcome its defences. 1.5

ARTHROPODS AND INSECTS

The arthropods were the first organisms to emerge from the sea, and insects were the first invertebrates to fly. The arthropods consist of Crustacea (crabs, lobsters, shrimp, barnacles and woodlice), Chelicerata (spiders, ticks, mites, scorpions and others), Hexapoda or Insecta, and Myriapoda (millipedes, centipedes and other minor groups). These classes separated a long time ago, so they have developed quite differently, but it is interesting to discover parallel developments.

8

Chapter I

Spiders and millipedes have sometimes developed chemical defences or communication chemicals similar to those of insects. It is therefore useful occasionally to make comparisons. The insects are the largest single group of animals, with over 800,000 identified species, far more than all the other animals put together. New species are reported at the rate of about 5,000 per year, and total number estimates range from 1 to 10 million. It is estimated there are lo'* individuals alive at any time. They are divided into the Apterygota, primitive wingless insects (springtails and silverfish) which have as yet received little chemical study; and the Pterygota, or winged insects, which form the great majority. The latter in turn are divided into the Exopterygota or Hemimetabola, which hatch from eggs to nymphs which closely resemble their final adult form or imago (grasshoppers, cockroaches, termites, bugs, stick insects, etc.) (Figure 1S); and Endopterygota or Holometabola, which hatch from egg to larvae which may have a very different form and habitat from the adult. They then go into a resting form called the pupa, while the tissues are completely remodelled and from that emerges the adult form (Figure 14. The Holometabola include beetles, butterflies and moths, flies, fleas, bees, wasps and ants. Almost half of all the insect species are beetles. Potentially, the subject of this book is gigantic. The isolation of insect chemicals began slowly. Kermesic acid or venetian red, a pigment from beetles (Chapter 8) has been known and used from ancient times. Wray, in 1670, reported formic acid by distillation of formicine ants. It was not until the 1930s that it began to be recognized that some Lepidoptera males were chemically attracted to females, and

Figure 1.5 Representation of the lije cycles of a hemimetabolous and a holometabolous insect. The symbols J H and M H between stages indicate where the juvenile hormone (Chapter 6 ) and moulting hormone (Chapter 7), which regulate development, are produced Reprinted from ComprehensiveNatural Products Chemistry, Vol. 8, E. D. Morgan and I. D. Wilson. Insect hormones and insect chemical ecology, pp. 263-375. Copyright 1999, with permission from Elsevier.

Introduction

9

only in 1956 was the first sexual attractant (bombykol, from the silk moth Bombyx mori) isolated and identified. From that time onward, with the development of chromatographic and sensitive mass spectrometric techniques, the study of insect natural products has grown to be a major discipline of science.

BACKGROUND AND FURTHER READING

J. B. Harborne, Introduction to Ecological Biochemistry, 4th edition, Academic Press, London, 1993 (Chapters 5 & 8, plants and insects). E. Haslam, Metabolites and Metabolism, Oxford University Press, Oxford, 1985, pp. 161 (Chapter 6, secondary metabolites). H. Oldroyd, Insects and Their World, British Museum of Natural History, London, 1966, pp. 144 (general introduction to insects, out of print) . P. Howse, I. Stevens and 0. Jones, Insect Pheromones and Their Use in Pest Management, Chapman and Hall, London, 1998, pp. 369 (Chapters 1 , 2 & 3, introduction to pheromones). J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 7). H. H. Rees, Insect Biochemistry, Chapman and Hall, London, 1977,pp. 64. V. H. Resh and R. T. Card6 (Editors), Encyclopedia of Insects, Academic Press, San Diego, 2003, pp. 1266 (for reference at any point). K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapters 1 & 2) T. D. Wyatt, Pheromones and Animal Behaviour, Cambridge University Press, 2003, pp. 391 (Chapter 1 & 2). A. Zanetti, The World of Insects, Abeville Press, New York, 1985, pp. 256 (general introduction to insects, out of print). QUESTIONS 1. An ant has hydrocarbons on its surface cuticle to repel water and prevent desiccation. The ant feeds largely on honeydew secreted by aphids, which feed on a plant stem. With the aid of Figure 1.1, trace the source of the ant hydrocarbons from carbon dioxide. 2. A bee expends energy in flying from flower to flower collecting nectar. What, according to Figure 1.1 is the ultimate source of that energy? 3. Classify the following substances as primary or secondary metabolites: vitamin A, alanine, camphor, deoxyribose, glucose, penicillin. 4. When a flower produces a perfume to attract pollinating bees, is that an allomone, kairomone or synomone?

CHAPTER 2

Enzymes and Co-enzymes 2.1 THE CHEMICAL REACTIVITY OF ENZYMES An enzyme contains one or more active sites, at which the reaction occurs. The substrate, the substance that is being altered, becomes attached to this site in some way. A co-enzyme, if one is involved, is also attached to, or held close to the active site. “An enzyme first binds its substrate in a particular orientation by using a variety of weak binding forces (hydrogen bonding, electrostatic attraction, dipole-dipole interaction, hydrophobic attraction, and so on), and then uses a variety of strategically placed functional groups and controlled conformational changes to induce reaction between them.” J. W. Cornforth, 1984, see Further Reading

Cornforth has done much of the work in understanding the stereochemistry of many biosynthetic reactions and was awarded the Nobel Prize for Chemistry (with V. Prelog) for this in 1975. For the biochemistry of enzymes the reader is directed to T. Palmer, Understanding enzymes 3rd edtion, 1991, Ellis Horwood, Chichester, and for a detailed treatment of enzyme and co-enzyme reaction mechanisms, T. Bugg, An introduction to enzyme and co-enzyme chemistry 1997, Blackwell Scientific, Oxford. 2.1.1 Lysozyme

Lysozyme has often been chosen as a simple example of how an enzyme works. It is said that Alexander Fleming (who later discovered penicillin) when he had a cold, at one time let the drips from his nose fall onto a bacterial colony on a Petri dish. Rather than throw it away, he kept it to see what would happen. He discovered that his nasal discharge inhibited the growth of the bacterium and this led to the discovery of the mildly antibiotic substance lysozyme in tears. He gave it this name because it is 10

Enzymes and Co-enzymes

11

an enzyme that caused bacterial Zysis. Later lysozyme was found in other body fluids, and elsewhere, but particularly in the white of egg. Lysozyme acts on a group of bacteria that have a cross-linked polysaccharide on their cell surfaces. Lysozyme cuts up the polysaccharide, making the bacterial cell wall very fragile. Lysozyme has a relatively small molecule for a protein, with 129 amino-acid residues linked in a single protein chain, and a molecular mass of 13,930. It was the first enzyme to have its total structure determined by X-ray analysis (1965), and to have its active site discovered. The protein chain of lysozyme is twisted and folded into a shape like a ball with a cleft down one side. The polysaccharide network on the surface of the bacteria fits into the cleft. The structure of the polysaccharide (the substrate) is shown in Figure 2.1 and the schematic structure of the enzyme molecule is shown in Figure 2.2 with the polysaccharide adsorbed on to the active site ready to be cleaved. Six rings of the

HOOC

HOOC

Figure 2.1

The polysaccharide molecule found in the walls of certain bacterial cells is the substrate broken by the lysozyme molecule. The polysaccharide consists of alternating residues of two kinds of amino sugar N-acetylglucosamine and N-acetylmuramic acid. In the portion of polysaccharide chain shown here A, C and E are N-acetylglucosamine residues; B, D and F are N-acetylmuramic acid residues. Ring D is distorted when adsorbed on the enzyme. The position attacked is indicated by the arrows

Figure 2.2

The amino-acid chain of lysozyme ( a ) is folded so it roughly forms a ball, with a cleft down one side, into which the polysaccharide chain of the bacterium fits. In ( b ) is shown how the aspartic acid 52 and glutamic acid 35 work together to break the sugar chain

12

Chapter 2

polysaccharide fit into the cleft of the lysozyme molecule, and are held firmly in position by hydrogen bonds and other interactions (see the quotation from Cornforth, above). Ring D is held in a flattened conformation, so the bond to ring E is strained and prepared for reaction. Adjacent to it are two carboxylic acid groups; that of aspartic acid 52 is in polar surroundings and is in the ionized form; that of glutamic acid 35 is in non-polar surroundings and is therefore in the un-ionized form. These two groups and a water molecule complete the reaction as shown in Figure 2.2.

2.1.2 Carboxypeptidase In the example of lysozyme, the catalytic effect is entirely due to the protein. In many enzymes there is a prosthetic group that often contains a metal ion bound to the protein, as in the example of carboxypeptidase, which contains a zinc atom in the active site (Figure 2.3), that takes part in the reaction. There are at least four carboxypeptidases which differ in the particular amino-acid linkages they are able

__--_

Figure 2.3 The carboxylic acid end of a protein sitting in the active site, a pocket in the enzyme carboxypeptidase, showing how it is held in place by bonding to the Zn2+atom and various amino acids. The probable mechanism, based on the hydrolysis of a known peptide, is shown, with the water molecule used for the hydrolysis shown in bold type. Once the amino-acid is cleaved, it can difuse away and the protein moves up into the pocket for the next aminoacid to be cleaved in the same way

Enzymes and Co-enzymes

13

to hydrolyze. Carboxypeptidase hydrolyzes amino-acids from the carboxyl end of proteins, one by one. Some 400 enzymes are known that contain zinc atoms. Other common metals in enzymes are Fe, Mg, Ca and Mn. A more advanced picture of enzyme action in biosynthesis is given in Chapter 3 when discussing desaturase enzymes.

2.1.3 Cytochromes There is in all living cells a family of enzymes known as cytochrome P,,, oxygenases. They contain a haem group (Figure 2.4) attached covalently, and have an absorption maximum at 450 nm in the violet region of the spectrum, and are coloured yellow, hence their names. They are important in the oxidizing of alkanes to alcohols, alkenes to epoxides, in the transformation of sterols (see Chapter 7) and for introducing an OH group into aromatic rings. They are also important for the detoxifying of many ingested substances, whether they be from plants or animals, or are synthetic substances, like pesticides or environmental pollutants. Cytochromes activate molecular oxygen, which attacks the substrate of whatever kind, as in Figure 2.5. Notice that the Fe3+atom must be reoxidized to Fe4+before the enzyme can be used again. It accepts another molecule of 02, splits off OH- and is restored to Fe4+-O-for re-use. Other substances that contain haem are haemoglobin, other cytochromes and catalase.

HOOC

Figure 2.4

-COOH

The structure of haem. The tetrapyrrole without the iron atom is known as protoporphyrin I X (see also Chapter 8 )

2.2 COENZYMES Working with the enzyme is frequently a co-factor, called a co-enzyme, a relatively small (compared with the enzyme) organic molecule which may itself be reversibly changed during the reaction. Remember that there are only five basic types of chemical reaction (see Figure 1.3) and all these types of reaction are encountered with enzymes and coenzymes.

Chapter 2

14 .Yenzyme

Efenzyrne

I

0’)

RLH

The overall reaction is: R-H

+ O2 i-2H+ +

-

2e-

R-0-H

R-OH

+

H20

Figure 2.5 Schematic drawing of the centre of a haem group of a cytochrome enzyme catalysing an oxidation of an organic molecule. This is a free radical reaction, as indicated by the arrows with a single barb

2.2.1 Coenzyme A Coenzyme A can be described as a “handle” for carboxylic acid groups. It picks up and drops acetyl groups, or acyl groups in general. It is particularly important in the degradation of fatty acids and in the first stages of terpene synthesis (Chapter 6). Its structure is shown in Figure 2.6. The essential part of the molecule is the thiol group, so it is usually represented as CoA-SH. The thiol reacts with, e.g., acetic acid, to give a thioester, coenzyme A thioacetate, or briefly CH,COS-CoA or AcS-CoA. Why a thioester? Esters are much more common in organic chemistry, but esters are less reactive than thioesters. Aldehydes and ketones have relatively reactive carbonyl groups, with reactivity slowed chiefly by bulky R groups. In esters, reactivity is decreased by the OR group through orbital overlap. There is no orbital overlap with the larger sulphur atom, so that reactivity of the thioester is more like that of a ketone. Notice the large increase in the acidity constant for the removal of an a-proton from a thioacetate compared with an acetate in Figure 2.7. The importance of this will become clear when considering the biosynthesis of fatty acids (Chapter 3) and terpenes (Chapter 6). pantothenic acid A

mercaptoethylamine

adenine

p-alanine

diphosphate

0I OH 0-7-0’

o’

ribose 3-phosphate

acetyl-CoA R-C, IP 0-H

Figure 2.6

+

H-S-COA

+

synthetase

~

R-c/t

+

H20

S-COA

Coenzyme A , showing its constituent parts, and the reaction between coenzyme A and an acid

Enzymes and Co-enzymes

15

2.2.2 Nicotinamide Adenine Dinucleotide Nicotinamide adenine dinucleotide (NAD' and NADP') are the reagents for alcohol to aldehyde, ketone or carboxylic acid oxidation-reductions. The equivalent reagents in the laboratory would be KMnO, or Na,Cr,O, for oxidations and NaBH, or LiAlH, for reductions. The complexity of the molecule should not hide its essential reactive part, the nicotinamide portion (Figure 2.8). The rest of the molecule is a polar handle to orient it correctly in the enzyme active site.

H3C-t C 0-R H&-6:

0

Z

@

+

HzC-C, 0 /p 0-R

@

+

HzC-C,

0 /P

K, = 10-'O

S-R

S-R

Figure 2.7

3x

K,

There is a large increase in the ease of dissociation of an a-proton in a thioester compared with a normal ester, which is important for biosynthetic condensation reactions

QcoNH22

?W

-

0+P,-O' 0

Ho

H

H

CONH2

e-

H+ NADH or NADPH

O-(H or phosphate)

Figure 2.8 Nicotinamide adenine dinucleotide NAD' (or N A D P with an extra phosphate on C-2 of ribose) oxidized and reducedforms. The sphere in the right-hand structure represents the remainder of the molecule

The reaction of a general alcohol with NAD' is illustrated in Figure 2.9. Note that when the alcohol is held on the enzyme surface it is possible to distinguish between the pro-chiral hydrogen atoms (for an explanation see the section on chirality at the end of this chapter). We know that the pro-R hydrogen atom is removed, and becomes attached to the upper side (as drawn) of the reduced nicotinamide molecule. When NADH is used for reduction, the same hydrogen, from the upper side of the molecule is transferred to the back of the carbonyl group (as drawn in Figure 2.9).

Chapter 2

16

The overall reaction is: RCH20H

Figure 2.9

+

-

alcohol dehydrogenase enzyme R-CHO

NAD+ Cf

+

NADH

+

H+ CI-

Oxidation of an alcohol with NAD+, and its reverse reaction, reduction of an aldehyde or ketone. B: represents some basic group on the enzyme. The subscripts R and S are a means of distinguishing between the two hydrogen atoms of the methylene group, see the section on chirality at the end of this chapter

This has been established by replacing either the pro-S or the pro-R hydrogen by deuterium and seeing whether the deuterium is retained by acetaldehyde or is taken up by NADH. NAD’ is used in metabolism or catabolism, for example, turning sugars into energy, NADP’ is used for anabolism or building up chemicals. Nature, in this way, keeps the two kinds of process separated. 2.2.3 Flavin Adenine Dinucleotide Flavin adenine dinucleotide (FAD) is also an oxidation-reduction reagent more specifically confined to reduction of C=C bonds and removing 2H from adjacent carbon atoms. The two hydrogen atoms added to the coenzyme and removed from the substrate are circled (Figure 2.10). FAD also oxidizes oxy-acids, amines and some aminoacids. The reduced form of the coenzyme has to be re-oxidized by molecular oxygen to FAD for use again. 0

FH2

YH‘OH

FAD

CH-OH

H2C-0,

,o\

FADH2

y.42

CH-OH

(ICJ

,o.

o ’ p \ o ~ o ~ ~ ~ oc w

OH

HO

-&A-I I

H H

The overall reaction is:

+

FAD

enzyme

‘c=c/ /

+ FADH~

\

Figure 2.10 Flavin adenine dinucleotide, FAD, oxidized and reducedforms. The two circled hydrogen atoms in FADH, are those removedfrom carbons

17

Enzyrn es and Co-enzyrn es

2.2.4 Thiamine Diphosphate Thiamine diphosphate is a particularly interesting substance because it illustrates a chemical evolution from lactic acid bacteria to yeasts and then to higher animals. It converts pyruvic acid, which is an a-keto-acid, into the equivalent of a 9-keto-acid (with C=N' instead of C=O), which can then lose CO, by decarboxylation. We know that the hydrogen atom next to nitrogen in the thiazole ring is acidic and easily removed because if we shake thiamine with D,O, we get rapid exchange of that atom (Figure 2.1 1). Some simple bacteria, such as those that produce yoghurt, reduce the pyruvic acid to lactic acid, and the reaction stops there, without much of

@ = phosphate

thiamine diphosphate

Figure 2.11 Thiamine diphosphate exchanges one labile hydrogen atom with D,O, indicating the reactive centre

0

+ OH

NADH

+

H+

YH C

H&'/,'9

0

+

NAD+

OH lactic acid

Figure 2.12 The reduction of pyruvic acid to lactic acid with the consumption of NADH formed earlier in the degradation of glucose

the energy of glucose being released (Figure 2.12). For each molecule of glucose that is broken down in metabolism, two molecules of NAD' are required (see Figure 2.20). Eventually two molecules of pyruvic acid are produced together with two molecules of NADH + H'. The NADH produced in catabolism of glucose is used by the lactic acid bacteria to reduce the pyruvic acid.

18

Chapter 2

+

c02

H+ H,

enzyme

H+

The overall reaction is: CH3COCOOH

-

CH3CHO + NADH

+

CH3CHO

H+-

+

C02 CH3CH20H

+ NAD+

Figure 2.13 The reaction of thiamine diphosphate with pyruvic acid in yeasts to release carbon dioxide and give acetaldehyde, and then ethanol. The structure marked A is used again in Figure 2.14

More advanced organisms, like yeasts, can use thiamine diphosphate with different enzymes to oxidize glucose to ethanol and carbon dioxide (Figure 2.13). The NADH produced in the glucose break-down is then used to reduce acetaldehyde to ethanol. Yeast is therefore used by the brewer for the alcohol produced and by the baker for the CO, to aerate the bread. Higher organisms have evolved a system to oxidize the pyruvic acid further, with a substance called lipoic acid as an intermediate, to acetic acid (as CoA thioester) and CO, (continuing the reaction from stage A in Figure 2.13). Approximately half the CO, we exhale is produced by decarboxylation with thiamine. The lipoic acid inserts itself after the decarboxylation step (Figure 2.14). The acetyl coenzyme A produced in the last step is either ultimately oxidized to CO, (through the citric acid or Krebs cycle) or it is the vital starting material for the biosynthesis of fatty acids, acetogenins and terpenes (Figure 1.1).

A

part of lipoic acid

tl

Figure 2.14 The$nal steps in the oxidation of pyruvic acid with thiamine diphosphate and lipoic acid to give acetyl coenzyme A

Enzymes and Co-enzymes

19

2.2.5 Tetrahydrofolic Acid The ultimate source of a one-carbon fragment is tetrahydrofolic acid, which obtains one carbon atom from formic acid, formaldehyde, or the amino-acids serine or glycine. The carbon atom from one of these sources becomes attached to N-5 of the folic acid and is reduced to methyl with the now familiar NADH (Figure 2.15).

tetrahydrofolic acid

Figure 2.15 The reduction of a one-carbon fragment attached to tetrahydrofolic acid to give the source of a methyl group. "C" represents one of several sources of a single carbon atom. This sequence of reactions can run in either direction, to give a methyl group from a formyl group, or to produce a formyl group from a methyl group as required

2.2.6 S-Adenosylmethionine The methyl group attached to N-5 of tetrahydrofolic acid becomes transferred to S-adenosylhomocysteine and the S-adenosylmethionine thus formed is the compound that transfers methyl groups (for methyl esters and ethers, and N-methyl groups) in nature (Figure 2.16).

S-adenosylhornocysteine

S-adenosylmethionine

S-adenosylhornocysteine

Figure 2.16 Reaction between 5-methyltetrahydrofolic acid and S-adenosylhomocysteine gives S-adenosylmethionine which can react with an alcohol, phenol, or carboxylic acid to give a methyl ether, a phenolic methyl ether or a methyl ester respectively, regenerating S-adenosylhomocysteine

2.2.7 Pyridoxal Phosphate Pyridoxal phosphate is the coenzyme that removes amine groups in metabolizing amino-acids (Figure 2.17). This is achieved by a series of reactions while the pyridoxal phosphate is bound to a transaminase

Chapter 2

20

The overall reaction is:

H

R-c-CoO-

+

pyridoxal

9

R-C-COOH

+

pyridoxamine

NH3+

Figure 2.17 The removal of ammonia from an amino-acid by pyridoxalphosphate ( P L P ) and u transaminuse enzyme. The PLP is held tightly to the enzyme by a lysine and ionic bonding of the phosphate group. B indicates some general base

enzyme by several interactions. Pyridoxal forms an imine with the amino-acid, and that is converted to an imine of pyridoxamine and a keto-acid. Hydrolysis of the imine gives an a-keto-acid and pyridoxamine which must be converted back to pyridoxal for re-use. In the metabolism of proteins the protein is first broken down to the individual aminoacids, which are de-aminated in this way. The carbon skeleton of the amino-acid (now as an a-keto-acid) is passed to the citric acid cycle (Figure 1.1) to be converted to energy. In fish the very toxic ammonia is usually excreted directly. In higher animals it is converted into harmless products, in insects uric acid is the important one. In mammals the amine group is transferred to the urea cycle and is excreted as urea. Pyridoxal is also used to move amino groups between amino-acids and, with different enzymes takes part in a number of other reactions involving amino-acids. An example of the steps by which an amino-acid

Enzymes and Co-enzy m es

21

Y

f

+ co2 l p

R-C N

H+

,

H+

0'

A

A

pyiiboxal

Figure 2.18 The sequence of steps by which an amino-acid attached to pyridoxal and a decarboxylating enzyme is converted to an amine and CO,

is decarboxylated are shown in Figure 2.18. Amines derived from aminoacids are discussed in Chapter 9.

2.2.8 Vitamins It should be noted in passing that higher animals have lost the ability to synthesize some of the coenzymes or parts of them. By definition, a vitamin is an essential substance that the body cannot make for itself and must acquire through its food. Humans cannot make pantothenic acid (vitamin B5)needed for coenzyme A, nicotinamide (vitamin B3) for NAD+,riboflavin (vitamin B2) for FAD, thiamine (vitamin BJ, folk acid (vitamin Bc or M) or pyridoxal (vitamin B6). We possess the ability to phosphorylate or otherwise convert the vitamins into active coenzymes. Some of these substances are also vitamins for insects, plus some others which higher animals can make themselves.

2.2.9 Biosynthesis of Formic Acid in Ants All ants of the subfamily Formicinae have lost the ability to sting but can spray a concentrated solution of formic acid (up to 65% in some species) from their venom glands. Blum and Hefetz (Science, 1978, 201, 545) studied the biosynthesis and showed by using radio-labelled compounds that the formic acid can be formed from serine (CH,OHCHNH,COOH) or glycine (CH2NH,COOH) with the help of tetrahydro folic acid (Figure 2.19). Apparently any compound capable of contributing a C , fragment can be a potential source of formic acid. The four enzymes necessary for these steps were all shown to be present in the venom gland. Other insects, including the larvae of the lepidopteran Schizura concinna and many beetles of the family Carabidae and some other arthropods also make and use formic acid in defensive or offensive secretions.

Chapter 2

22 H,

..-.C.

labelled serine

SOOH

M

M

' f E Y

I

ti

part of folate molecule

k

formic acid

Figure 2.19 Formation of formic acid in ants according to Blum and Hefetz. The black dot on carbon indicates the labelled atom which shows which atom of serine is used for formic acid

2.3 PYRUVIC ACID Pyruvic acid and its derivative phosphoenol pyruvate have already appeared in Figure 1.1, and pyruvic acid was required in discussion of the action of thiamine diphosphate (Figures 2.12 to 2.14). They are important intermediates and will appear again. It is worth looking briefly at the origins of these compounds now. When glucose is broken down in metabolism, the first steps are to convert glucose to glucose &phosphate, which is isomerized to fructose &phosphate and then converted to fructose 1,6-bisphosphate (Figure 2.20). This is cleaved by an aldol reaction running in reverse, and catalyzed by the enzyme aldolase. The products are dihydroxyacetone 1-phosphate and glyceraldehyde 3-phosphate, which are really equivalent compounds because they are interconverted through a common enol form. Glyceraldehyde 3-phosphate is phosphorylated again and oxidized to glyceric acid 1,3-bisphosphate, linked to the conversion of one molecule of NAD' to NADH. Glyceric acid bisphosphate loses one phosphate to ADP, forming ATP while the 3-phosphate is isomerized to 2-phosphate. Loss of water from this compound gives phosphoenol pyruvate and transfer of the phosphate to ADP gives another molecule of ATP, and conversion of enolpyruvate to the keto-form gives pyruvic acid. The summary in Figure 2.20 does not give all the stages, nor considers the mechanisms or the energetics of this important process. For that the reader is referred to a standard textbook of biochemistry. Under anaerobic conditions, the NADH produced during the oxidation of glyceraldehyde to glyceric acid is re-oxidized in the reduction of pyruvic acid to lactic acid (in bacteria), or in the reduction of acetaldehyde to ethanol (in yeast); and, under aerobic conditions, is oxidized (via

23

Enzymes and Co-enzymes

"

\

HO-CH2 Dihydroxyacetone phosphate

glucose 6-phosphate

H$-OH CH

fructose 1,6-bisphosphate phosphate

@ = phosphate

Q H,C-C-C,

,p

OH pyruvic acid

glyceric acid bisphosphate

-H20

c--

/ H H27-C-<

O

phosphoenol pyruvate

Figure 2.20 A summary of the steps by which pyruvic acid andphosphoenol pyruvate are obtained from glucose during metabolism. The key step is a retro-aldol reaction on fructose 1,6-bisphosphate

the citric acid cycle) to produce more energy in higher animals. Glycerol is produced by reduction of glyceraldehyde 3-phosphate. Some insects are cold-hardy, that is, they have evolved ways of surviving very low temperatures at which their blood might freeze and the icecrystals pierce their cell walls. One way is for insects to produce large quantities of glycerol which lowers the freezing point of their blood. Glycerol concentration can reach 2 M or more, representing 20% of the fresh body weight of the insect in winter months. It has been shown that the cold-hardy gall moth Epiblema scudderiana produces a special aldolase that converts fructose 1,6-bisphosphate into glycerol. The enzyme is more active at 5 "C than at 20 "C.

2.4 CHIRALITY The great majority of compounds in nature are chiral. A substance is chiral when it and its mirror images are not identical (the mirror image of the molecule cannot be superimposed upon it). Chirality in natural products is a direct consequence of being produced by enzymes, which are formed from chiral amino-acids and are themselves chiral. The two mirror-image forms, called enantiomers, are different substances. Although most of their chemical properties are identical, they can have very different biological properties. Since our own taste receptors (on our tongues) and odour receptors (in our nasal passage) are themselves chiral, we can detect some chiral differences. For example the amino-acid

24

Chapter 2

L-asparagine, first isolated from asparagus juice, has a bitter taste, while its enantiomer, the unnatural D-asparagine tastes sweet (Figure 2.21). Our odour receptors can distinguish between (8-(+)-carvone and (R)(-)-carvone (Figure 2.21), and we have a much lower threshold for detecting the first form. The different flavour of lemons and oranges is due to the enantiomeric forms of the monoterpene limonene (Figure 6.1). mirror

0

mirror

; Ho H'NH2 oC 0 nfNH2

N$

I

L-asparagine or (S)-(-)-asparagine bitter taste

I

D-asparagine or (R)-(+)-asparaghe sweet taste

(S)-(+)-carvone spearmint odour lower threshold

(/?)-(-)-cawone caraway odour higher threshold

Figure 2.21 Some examples of enantiomers which we are able to distinguish by taste and odour

The natural juvenile hormone of insects (+)-JH I (Chapter 6) is about 10,000 times more active than its enantiomer (-)-JH I. Cholesterol (Chapter 7) has nine asymmetric centres, yet exists naturally as only one enantiomer. The usual cause of chirality in organic molecules is an asymmetric carbon atom, one with four different groups attached (Figure 2.22). The rules for describing the arrangement of groups about an asymmetric carbon atom, the Cahn-Ingold-Prelog rules, can be found in any textbook of organic chemistry. The priority of atoms or groups are assigned in descending order of atomic mass (Figure 2.22).

(9-brornochlorofluoromethane

Figure 2.22

(R)-bromochlorofluoromethane

(9-(+)-alanine or L-alanine

(/?)-(-)-lacticacid

Some simple examples of chiral molecules. The numbers denote priority of groups according to the Cahn-Inglod-Prelog rules. (S) - (+) -ahnine and (R) (-)-lactic acid are naturally occurring compounds. Note there is no relation between the assignment of S and R to u chiral centre and optical rotation

2.4.1 Asymmetric Induction The formation of a chiral substance from an achiral starting material is called asymmetric induction. Glycerol has a symmetric molecule, but when it is held on the surface of an enzyme and one hydroxyl group is

25

Enzymes and Co-enzymes

+ ADP

glycerol-3-phosphate

Figure 2.23 Glycerol held on an imaginary enzyme surface, being selectively phosphorylated. The numbering of glycerol phosphate presents a problem, because ~-glycerol-3-phosphateis the same compound as D-glycerol-I phosphate. To avoid confusion, for glycerol, the pro-S CH, group is numbered 1 and the pro-R CH, numbered 3. Therefore the compound shown is the 3-phosphate

selectively phosphorylated, that symmetry is destroyed and chiral glycerol-3-phosphateis formed (Figure 2.23). If a molecule is not chiral, it is designated as prochiral if a single substitution will make it chiral. Ethanol is an achiral compound, its CH, group is described as prochiral. To distinguish between the two hydrogen atoms of this group, the Cahn-Ingold-Prelog rules are applied to describe the group, after each hydrogen in turn is notionally replaced by deuterium, and the priority rule applied (Figure 2.24). If the new chiral centre has the S configuration, then the hydrogen that has been replaced is pro-S, and, conversely, if the new centre with deuterium is R, then the hydrogen that was replaced is pro-R. The enzyme alcohol dehydrogenase a pro-chiral centre

kH

H3C

1 OH ___c

H

2 first, replace this

ethanol

hydrogen with deuterium

H f,)2 f H D 3 C ,

OH IH

then H3C

3 S configuration

AHs this hydrogen is pro-S

a pro-chiral centre

j )\HH

next, replace

/ this hydrogen

H3C

H

1

3

___)

OH ,I,,HR H3C H

this hydrogen is pro*

R configuration

Figure 2.24

Ethanol, a substance with no chiral centres, has a prochiral CH, group. The hydrogens of this group are assigned as pro-S and pro-R by imagining them replaced in turn by deuterium and applying the rules

Chapter 2

26

achiral

Figure 2.25

prochiral

chiral

Methanol is achirul, by substituting one hydrogen by deuterium it becomes prochiral. Replacing another hydrogen by tritium, the molecule is now chiral. A methyl group substituted with deuterium and tritium is often used in studying the mechanism of biosynthetic reactions

stereospecifically removes from ethanol one of the two hydrogen atoms attached to this carbon in the oxidation to acetaldehyde. It is the pro-R hydrogen that is removed (Figure 2.9). We have to imagine the ethanol molecule being held rigidly on the enzyme surface, much like glycerol in Figure 2.23, and being attacked by the co-enzyme from one side. Methanol is not prochiral. Two changes have to be made before it becomes chiral, as in Figure 2.25. Another example of chiral induction will be found in Chapter 6 in the biosynthesis of terpenes, where isopentenyl pyrophosphate is held on the enzyme surface and the pro-R hydrogen is selectively removed (Figure 6.2). The same pro-R hydrogen is removed in terpene chain extension, as, for example, in the formation of geranyl pyrophosphate (Figure 6.3). BACKGROUND AND FURTHER READING J. W. Cornforth, Stereochemistry of life, Interdisciplinary Science Reviews, 1984,9, 393-398. E. Haslam, Metabolites and Metabolism, Oxford University Press, Oxford, 1985, pp. 161. J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 2). H. H. Rees, Insect Biochemistry, Chapman and Hall, London, 1977, pp. 64. K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapters 3 & 7). QUESTIONS 1. Cytochromes P,,, are a group of oxidizing enzymes. What characteristic metal-co-ordinated group do they share? When acting on exogenous compounds (natural or synthetic compounds from outside

Enzymes and Co-enzymes

2. 3.

4.

5. 6.

27

the body) do cytochromes convert them to more lipid-soluble, or water-soluble compounds? Write and balance the reaction between acetone and NADH and H'. Complete a balanced equation for the reaction between butyric acid and FAD. Show a scheme for the reaction between thiamine diphosphate, lipoic acid and a-keto-isovaleric acid (structure in Figure 3.12). What is the product of the reaction between hexanoic acid and S-adenosylmethionine? [2-3H]Acetic acid is prochiral. Label the pro-S and pro-R atoms on this molecule.

CHAPTER 3

Fatty Acids and Derived Compounds 3.1 FATTY ACIDS The fatty acids are primary metabolites, but are also the source of many secondary metabolites. They exist chiefly in nature as triglycerides (Figure 3. l), in which three molecules of fatty acids (usually a mixture of acids) are esterified to one molecule of glycerol. J. N. Collie (Journal of the Chemical Society, 1907, 91, 1806) noticed that the acids of natural fats always contained even numbers of carbon atoms, and suggested that they are made by head-to-tail linking of acetic acid units. Later when radioactive isotopes became available, his suggestion was shown to be correct. :

,o

o

H

..c--

0

z

H2C, 0

>

H

.

O

-

0

0

CH3(CH2)12COOH CH3(CH2)14COOH CH3(CH2)1BCOOH CH3 (CH,),CH=CH(CH2),COOH

palmytoylgroup

c-~ stearoyl group

-

Myristic acid Palmitic acid Stearic acid Oleic acid

oleyl group

C14:o C,6:o cj8:O c18:I

Figure 3.1 An example of a triglyceride or glycerol triester, (the structure shown is 1-palmitoyl-2-stearoyI-3-olein) and the formulae of the most common fatty acids, their common names and abbreviations

Fatty acids are indeed synthesized by head-to-tail condensation of two-carbon units, but the reality is a little more complex. In building up fatty acids, nature uses the Claisen condensation.to make the new carbonto-carbon bonds. In the laboratory we use a strong base (ethoxide ions) and anhydrous conditions to make this difficult reaction work between two molecules of ethyl acetate. Nature can perform the same in an 28

Fatty Acids and Derived Compounds

29

aqueous system and at neutral pH, but in order to make an acetate group sufficiently reactive, two activating effects are applied. We have already seen in Chapter 2 (Figure 2.7) that using a thioester increases reactivity. The second effect is converting the acetate into a malonate (Figure 3.2). The acidity value for the CH, group in a malonic thioester is not readily available, but it is evidently very high. In biosynthetic reactions the combined effects of thioester and additional carboxylate are used to activate the CH, portion of acetic acid for condensations.

@

4?

H3C-C,

0

+

0 /p

H2C-C,

OR

0

K, = 3~

OR K, = 1 x10-13

0 H3C-<

0

S-R

0 +

co2

0

&OH

indirectly

*

H2CkS-R

0

Figure 3.2 The removal of a proton from the CH, of an acetate derivative is a ditj’icult process, that is,for this reaction the acetate is a very weak acid. Conversion to a malonate derivative increases the acidity almost I0l2 times, use of a thiomalonate increases it still more

3.1.1 Biosynthesis The degradation and synthesis of fatty acids are very similar, so both synthesis and degradation are summarized together (Figure 3.3), and then the steps of the biosynthesis are examined in detail one by one. The enzymes of fatty acid biosynthesis in animals are held together in a complex known as fatty acid synthetase, and during all the stages of synthesis of the fatty acid, the growing chain remains attached to this complex, passing from one enzyme to the next. When the chain has grown to 16 or 18 carbon atoms, it becomes detached from the complex. It requires 64 individual steps from acetic acid to make a molecule of stearic acid. To make a malonyl group from an acetate (Figure 3.2), nature uses N-carboxybiotin as a carrier of CO,. From the biotin (another coenzyme and vitamin) the carboxyl group is transferred to acetyl coenzyme A, converting it to the much more reactive malonyl coenzyme A (Figure 3.4). In micro-organisms and plants the growing chain and a molecule of malonate are both attached to acyl carrier protein (ACP). In animals, including insects, the sequence is a little more complicated. An acetyl group is transferred from coenzyme A (Figure 3.5 Part A) to ACP and

Chapter 3

30 Synthesis -CH2-C\

(anabolism)

(catabolism)

/coo-

n

+ H2c\c',-o

/p S-ACP

ulvv

\

S-ACP

I p

Degradation

CH2- CH2-CH2-C\"" I S-COA

1

FAD_-FADH2 V

0

-CH2-d-CH2-C\'

, S-ACP

1 NADPH

-

-CH~-CH-CH-C\'

I

NADP+

S-COA

1

If --CHZ.C-CH~-C\'/

A

OH

0

S-COA

+I

NAD+-

NADH

+

H+

-cH~-cH-cH-c\'/ I S-ACP

1 NADPH-

I CoAS-H

NADP+

t

+ H3C-q' CH2. C\ S-COA S-COA

.vvvv.

Figure 3.3 The essential steps of fatty acid synthesis and degradation. The growing chain in synthesis is attached to acyl carrier protein ( A C P ) through a thioester. In degradation the shrinking molecule is attached to coenzyme A, also through a thioester. Note that while the p-hydroxyl is the (R)-enantiomer in the synthesis process, it is the (S)-enantiomer in degradation; and while NADPH is used to reduce the double bond, FAD is used to produce it

biotin N-carboxybiotin

Figure 3.4 Carbon dioxide, as HCO; is attached to biotin, and then transferred to acetyl coenzyme A forming malonyl coenzyme A for fatty acid synthesis

from there to P-ketosynthase. Then condensation between acetate and malonate occurs, followed by reduction of the keto-group, dehydration of the alcohol and reduction of the double bond (Figure 3.5 Part B). The butyryl group thus formed moves to the P-ketosynthase and is replaced by another molecule of malonate on ACP. Now condensation occurs again and the cycle continues (Figure 3.5 Part C). Note that the synthesis uses NADPH for reduction of a double bond, but the same

31

Fatty Acids and Derived Compounds

&

A. Transfer from coenzyme A to enzymes and condensation SH

1

SH ACYI carrier

C CH3COSCoA H~COSCOA

protein

I

synthase

,

L

'

-

protein

's

H 3 C - d0

p-ketoac

cki-\ I

synthase

acetyl coenzyme A Acyl carrier rnalonyl protein

malonyl coenzyme A

111 rnalonyl transferase

,

protein

J

6. Reduction of the keto-group, dehydration and reduction of double bond f

Y

H

33O

H3C--Cdc,<

W !( H+ acetoacstyl ACP

C. Repeat the cycle

Acyl carrier protein

fbhydroxybutyryl ACP

SH

- H20

H H3C'c*cyc\

/P

Acyl carrier protein

a[--f----l

crotonyl ACP

NADPH

+

/-. SH

protein

butyryl ACP

Figure 3.5

Thefirst and second round of steps in the synthesis of a fatty acid in animals. These stages are repeated until the chain reaches its full length of 16 or 18 carbon atoms of the fatty acids. K is a basic group on the enzyme

32

Chapter 3

bond is formed in degradation by the use of FAD, and that NADPH is used for the reduction of C=O to CHOH in synthesis, while NAD' (without the extra phosphate) is used in the oxidation of CHOH to C=O. In synthesis an (R)-OH group is formed by reduction, while in degradation an ($)-OH is the intermediate by hydration of the double bond (Figure 3.3). There is also spatial separation of these processes, synthesis occurs in the cytosol, the liquid portion of the cytoplasm, while degradation takes place in the mitochrondria. These are further examples of the way that degradation and synthesis are separated as chemical processes.

3.1.2 Unsaturated Acids and Desaturase Enzymes Double bonds, as in oleic acid (Figure 3.1), can be introduced in two ways, either a double bond is left in the growing chain (in anaerobic bacteria) or by removal of two hydrogen atoms from the complete molecule (aerobically, in plants and animals, including insects). This latter is a most remarkable reaction and worth a closer examination. It also illustrates the action of a type of biosynthetic enzyme that has received a great deal of investigation. Double bonds are introduced by desaturase enzymes. They are remarkable because they can remove hydrogen atoms from an unactivated alkyl chain, with great precision of position and stereochemistry, something that chemists have not yet learned to do. The C-H bond is very stable, it has a bond enthalpy of 413 kJ mol-' or 98.7 kcal mol-'. It requires the oxidative power of O2to break this bond. The byproduct is water. There are two types of desaturase; the first are soluble enzymes, found only in plants, and located in the plastids. Their substrate is a fatty acid attached to acyl carrier protein (acyl ACP). The second type is integrally bound to the membrane of the endoplasmic reticulum. These enzymes are found in animals generally, including insects, as well as in plants and fungi. Their substrate is the fatty acid bound to coenzyme A (acyl CoA). Soluble enzymes are much easier to study, so more is known of the first type, but from many studies with a variety of spectroscopic, X-ray and molecular biological techniques, it seems the mechanism of reaction is the same in both types. Although the full story of the enzymes is not yet known, the description here summarizes our present knowledge of membrane-bound fatty acid desaturases found in insects. The description is of a A9-desaturase, the most common type, which converts stearic acid to oleic acid. The location of the double bond is measured from the carboxylate end of the molecule. Palmitic acid with the same enzyme gives palmitoleic acid. If an unnatural C1, or C,9acid is supplied to the

Fatty Acids and Derived Compounds

33

desaturase enzyme, a A-9 acid is always formed. The double bonds introduced in this way in fatty acids always have a 2 or cis configuration. There are other enzymes that produce a double bond at different positions in the chain, and with different geometry. Desaturase enzymes of insects usually place double bonds at uneven positions in the fatty acids. The desaturase protein chain consists essentially of four a-helix coils, imbedded in the membrane, with a long narrow pocket into which the alkyl end of the fatty acid fits. The general appearance is shown for a soluble A9-desaturase from the castor oil plant in Figure 3.6 and Plate 1. Note the bent configuration that the molecule assumes at the C-9 atom (regardless of chain length), where the new double bond will be formed, and the two iron atoms within catalytic distance of this position. The iron atoms are buried deep inside the molecule, held by several histidine residues. When the substrate fits into the pocket, oxygen becomes attached to the di-iron cluster (Figure 3.7), which then attacks the hydrocarbon chain (Figure 3.8). The subsequent steps are still not known in great detail. Two further enzymes, both bound in the same membrane, and two coenzymes are required to complete the cycle. Cytochrome b, restores the desaturase iron to its reduced state, and the cytochrome in turn is reconverted to its reduced state by cytochrome bSreductase which uses FAD as coenzyme. The FAD is converted to FADH, (see Chapter 2) by NADPH. Small varj,ations in the enzyme structure give other desaturases, Region involved iiI regiospecificty\

A Diiron active site

1

ACP

Hydropho substrate cavity

specificity

Figure 3.6 A drawing of the A9-desaturase enzyme from the castor oil plant showing the monomer with the long, narrow pocket into which the fatty acid, shown as a curved line,fits. Note the di-iron cluster, and the region at the bottom of the cavity which determines how deep thefatty acid canfit, and consequently where the double bond will be inserted. The direction from which the electrons come from cytochrome which re-oxidize the iron cluster is also shown. See also Plate 1. Figure provided by J Shanklin and E. Cahoon

34

Chapter 3 H--0 I in--c..l\

Figure 3.7

The proposed arrangement of ligands (including histidine and glutamic acid residues) at the di-iron cluster in a desaturase enzyme enzyme

enzyme

1

.n

slow

H3C

Figure 3.8

S-CoA

H3C

S-COA

The removal of the pro-H, from C-9 and the formation of a double bond in a fatty acid, illustrated by the conversion of stearic acid (as a CoA thioester) to oleic acid by a A9-desaturase enzyme. It is a free radical reaction with the removal of two syn-oriented vicinyl hydrogen atoms

H

OH

-Figure 3.9 By variation of the protein structure, hydroxylase enzymes, similar to desaturases, are produced which can introduce hydroxyl groups into a fatty acid at the same position

hydroxylases, which introduce hydroxyl groups into the fatty acid at C-9 (Figure 3.9), and epoxidases. Variations produce A-7 or A-11 desaturases, and different conformations of the alkyl chain in the pocket of the enzyme can give trans double bonds as found in some of the lepidopteran pheromones (see later). Acetylenases, methyl oxidases (converting methane to methanol) and other enzymes have similar di-iron active sites.

35

Fatty Acids and Derived Compounds

Linoleic and linolenic acids (Figure 3.10) are made by plants by further desaturation of oleic acid. It is generally accepted that animals cannot make these acids, in other words, they are essential in their diets, but recent experiments have shown that a cricket and a cockroach, as well as a slug and a garden snail can make linoleic acid. It is possible then that all insects can make linoleic acid. There is no indication that linolenic acid can be made by animals. Arachidonic acid (5,8,11,14eicosatetraenoic acid) is made by animals only, by chain extension and desaturation from linoleic acid obtained in their food.

linolenic acid

OH

in animals 7 , COA-SH 2, desaturase

+

--d 0 arachidonic O acid H Figure 3.10

The biosynthetic relationship between oleic, linoleic, linolenic and arachidonic acids

3.1.3 Eicosanoids A series of compounds called collectively eicosanoids, important in human physiology, are made from arachidonic acid using cyclo-oxygenase enzymes (Figure 3.1 1). They include prostaglandins, leucotrienes, thromboxanes and lipoxins. They are widely distributed in various tissues and exert hormone-like effects, and are effective at very low concentrations. One of the most important effects of aspirin (0-acetylsalicylic acid) is to block the cyclo-oxygenase enzyme and prevent the formation of prostaglandins. The subject of human eicosanoids is well covered in many biochemical texts. What is important here is that prostaglandins and eicosanoids appear to be produced in all kinds of vertebrates and invertebrates. In insects they affect egg-laying, salt and water transport and cellular immune defenses. Prostaglandin GA, (PGA,) (Figure 3.1 1) seems to be important in insect immune response.

Chapter 3

36 Q

G

c

O

H

;

cyclo-oxygenase

O'7

1 +

Figure 3.11

G

O

H NH2 isoleucine

3P.

'0-0

arachidonic acid

H

The probable formation of PGA, in insects, based upon the known reactions in vertebrates. Thefirst step in the series is inhibited by aspirin. The prostaglandins PGG, and PGF,, are intermediates in the sequence

-

G

O0

r

A

o

H0

0

* + +

2-methylbutyric acid

(R)-14-methylhexadecanoicacid an anteiso acid

OH

0

OCHs methyl (R,Z)-l4-rnethyl-8-hexadecenoate

OH (R,Z)-l4-rnethyl-8-hexadecenol

Figure 3.12 The origin of branched iso- and anteiso-fatty acids illustrated for the C,, and C,,acids respectively. Iso-acids have an even number of carbon atoms, anteiso-acids have an odd number. The two unsaturated anteiso-compounds are part of the sex pheromone of some Trogoderma species

3.1.4 Branched Fatty Acids Branched fatty acids, known as iso-acids and anteiso-acids, occur normally in small quantities in fats. Their synthesis begins with the aminoacids valine and isoleucine (Figure 3.12). This has been demonstrated with radio-labelled isotopes, by radio-active monitoring and with stable isotopes by 13Cnuclear magnetic resonance spectroscopy or mass spectrometry. Both isobutyric acid and 2-methylbutyric acid are common defensive compounds among insects. Note that a chiral centre is introduced in 2-methylbutyric acid and anteiso acids.

Fatty Acids and Derived Compounds

37

The kapra beetle Trogoderma granarium and other species of Trogoderma use derivatives of the anteiso-unsaturated acid (R, 2)-14methyl-8-hexadecenoic acid, such as the methyl ester and the corresponding alcohol (Figure 3.12), the aldehyde and derivatives with the E-double bond, as part of the female-produced sex attractant. L-Valine has been shown to be incorporated efficiently into methacrylic and isobutyric acids in the defensive secretion from the pygidial glands of the carabid beetle Scarites subterraneus (Attygalle, Meinwald and Eisner, Tetrahedron Letters, 1991, 32,4849). The work was done by replacing all the hydrogens on carbon of valine with deuterium (Figure 3.13), and analyzing the methacrylic and isobutyric acids by mass spectrometry. Females of the hemipteran bug AZydus eurinus release 2-methylbutyl butyrate and (E)-2-methylbutenyl butyrate as an attractant pheromone (Figure 3.14). Adults, when disturbed, also produce a defensive secretion of butyric and hexanoic acids from metathoracic glands. Small amounts of odd-numbered fatty acids, e.g. CIS,CI7,and CI9are encountered in insects, as well as in vertebrate fat. These are biosynthesized starting from a propionic acid group to which are added acetate units in the usual way.

deuterated valine

deuterated isobutyric acid

deuterated methacrylic acid

Figure 3.13 The biosynthesis of isobutyric acid and methacrylic acid in a beetle demonstrated by deuterium labelling

Figure 3.14 Small-molecule esters that form the sex pheromone of the bug Alydus eurinus

3.2 CUTICULAR HYDROCARBONS The outer covering of insects consists of a layer of water repellent lipids, frequently made up of alkanes, methyl-branched alkanes and alkenes. This lipid layer is important to prevent dehydration and to repel rain; and in social insects (bees, wasps, ants and termites), the mixture is characteristic of the group, and the available evidence suggests the mixture helps individuals to distinguish between nestmates and individuals from

38

Chapter 3

another colony. Cuticular hydrocarbons have from 17 to 49 carbon atoms in the chain, and they have been shown to be derived from fatty acids through chain lengthening and decarboxylation. The carbon chain is extended with more acetate groups (converted to malonate for the synthesis) to make straight chain hydrocarbons (Figure 3.15). The long chain acid is then reduced to an aldehyde, and, with the aid of oxygen and a cytochrome P,,, the carbonyl group is lost as CO,. As the fatty acids have an even number of carbon atoms, the hydrocarbons have an odd number of carbon atoms. It has been shown by labelling that the hydrogen of aldehyde becomes attached to the end of the hydrocarbon chain. The cuticular hydrocarbons appear to be synthesized in the oenocyte cells of the epidermis or the fat body, and if the latter, are transported through the haemolymph (insect blood) by a protein called lipophorin. 0

C, stearic acid 0

1 reduction, NADPH i

C26 hexacosanoic acid OH

0

h

hexacosanal

decarboxylation, cytochrome P450rNADPH, O2

h ?

+c02 pentacosane

Figure 3.15 Outline of the synthesis of a C,, hydrocarbon by chain-lengthening from stearic acid and decarboxylation. The dot on the hydrogen atom indicates that a labelled hydrogen atom in this position is retained on the terminal carbon atom of the alkane

Methyl-branched hydrocarbons are produced through the intervention of propionic acid (converted to methylmalonate) as shown in Figure 3.16. The methyl-branched fatty acids and the microsomal synthetase for making them have been isolated. The system is capable of selecting between malonyl CoA and methylmalonyl CoA and adding the correct intermediate at each step in the chain lengthening. While common fatty acids have no chiral centres, introduction of a propionate group creates a chiral centre, and branched hydrocarbons are chiral, although not much is known about their chirality as yet. Beyond hexadecane (m.p. 18 "C), all straight-chain alkanes are waxlike solids. Pentacosane melts at 50 "C, triacontane at 60 "C and tetracontane (C40H82) at 80 "C. To keep the cuticular-hydrocarbon surface soft and flexible, where more long-chain alkanes are present in the cuticle, more alkenes and methyl-branched alkanes are added. Introducing an internal double bond reduces the melting point -50 "C compared with

Fatty Acids and Derived Compounds

39 0 C1 stearic acid

OH

C20 eicosanoic acid

0 Me-CZ2 acid

OH

0 OH

Me-CZ4 acid

decarboxylation

+ co* 3-methyl tricosane

I

Figure 3.16 An illustration of the synthesis of a methyl-branched hydrocarbon. Conversion of the acid to hydrocarbon occurs as a concerted step. Note that a chiral centre is introduced

the alkane, and a methyl branch (depending upon where in the chain) reduces melting point by -30 "C.The melting temperature range of the complex mixture on most insect cuticle will be low and very broad. Decarboxylation of isoalkanoic and anteisoalkanoic acids (Figure 3.12), with or without chain lengthening, gives 2-methylalkanes and 3methylalkanes respectively. But notice also that 3-methylalkanes can be formed in two possible ways (Figure 3.17), although the second alternative is more likely. Only labelling experiments will permit a decision between the two alternatives. Pupae of a number of lepidopterans make very long-chain methylbranched alcohols and acetates, like those shown for the southern armyworm Spodoptera eridania in Figure 3.18. From studies with

+ co.2

n CH3COOH -L--

anteisopalmitic acid

OH

then decarbox. a 3-methylalkane n-2 CH,COOH

HO (

r\

U

OH

palmitic acid

a 3-methylalkane

Figure 3.17

The formation of 2-methylalkanes and 3-methylalkanes. The latter can be formed in two alternative ways, starting from an anteiso-acid, or from a straight chain acid by insertion of a propionic acid unit later

40

Chapter 3 OH 22,26-dirnethyloctatriacontanol

OH 24,28dirnethyloctatriacontanoI

OH 26,30-dimethyldotetracontanol

Figure 3.18 Very long chain alcohols synthesized by the pupae of Spodoptera eridania. Such compounds appear to be characteristic of lepidopteran pupae

incorporation of [3H]acetateand [ 1-'4C]propionate it is concluded these are produced from the alkyl end and terminate at the alcohol end, so the methyl branches are added early in the synthesis. Terminal double bonds are not usually found in cuticle alkenes but do occur in other insect secretions. W. Boland's group have shown, by deuterium labelling in several places in the chain that terminal alkenes are formed by an anti elimination of the carboxyl group and the pro-S hydrogen (see Figure 2.25) on the second carbon atom of a fatty acid, which is held firmly in one configuration on the enzyme (Figure 3.19). The same mechanism applies to this reaction in plants.

Figure 3.19

The appearance of a 2-deuterio-fatty acid in the configuration in which it must be held on the enzyme during the elimination of the carboxyl group to give a I-alkene. Had it been a syn elimination then the deuterium would have been cis to the alkyl group as in the third formula

3.2.1 Hydrocarbon Pheromones

A number of hydrocarbon pheromones are known, chiefly in the Diptera, but also among Lepidoptera (see later). Sometimes hydrocarbons are part of the defensive secretion, as in the confused flour beetle, Tribolium confusum, which uses terminal alkenes e.g., 1-pentadecene, 1-hexadecene and 1-heptadecene, formed as described above. They are frequently found accompanying terpenes or oxygenated compounds in the secretions of many social insects, and C9to CI5alkanes frequently accompany formic acid in defensive secretions, where they are thought to act as spreading agents. The major sex pheromone of the female housefly Musca domestica is (Z)-9-tricosene. Incubating a mixture of ( 9 - 15-[1-14C]- and ( 9 - 15-

Fatty Acids and Derived Compounds

41

[15,l6-3H,]tetracosenoic acid with microsomes from houseflies gave equal amounts of tritiated (Z)-9-tricosene and 14C-labelledCO,. Moreover, by adding hydroxylamine as a trapping reagent for aldehydes, it was possible to capture the intermediate aldehyde as its oxime (Figure 3.20). Both the cuticular hydrocarbons and the tricosene pheromone are synthesized in the integument but the integument is closely associated with oenocyte cells. The tricosene is converted by a cytochrome to the other two components of the pheromone, the epoxide (major) and the unsaturated ketone (minor) (Figure 3.20). 3

T

G

T

O

0

H

I

NH20H-

trapped as oxime

1 4 ~ 0 ~

tritiated (Z)-9-tricosene

9.10-eDoxvtricosane

(Z)-lCtricosen-lO-one

Figure 3.20 Demonstrating the formation of the sex pheromone of the common housefly (Z)-9-tricosene from a C,, fatty acid through the aldehyde. T indicates tritium. Note that using a mixture of tritiated and14C-labelledcompoundsfor this purpose is the same as having all the labels in the same molecules. The epoxytricosene is another major component of the pheromone and the tricosenone is a minor component

3.3 LEPIDOPTERAN SEX PHEROMONES Lepidopteran pheromones have been intensively studied for three decades, largely because of the economic importance of lepidopteran pests, but also because many of these species are easy to rear, and because the biosynthesis of their pheromones was easy to relate to fatty acids. Females of very many species of Lepidoptera release volatile chemicals to attract males for mating. Most of these compounds have the following characteristics:

1) They are made of straight chains of 12, 14, 16, or 18 carbon atoms; 2) They usually have a primary alcohol, ester, aldehyde or acetate at one end of the chain; 3) They have up to three double bonds with an E or 2 configuration.

42

Chapter 3

Some of the questions to be answered were: 1) Were these pheromone compounds made from existing fatty acids or directly synthesized from acetic acid units in the gland? 2) Were the short chains made by stopping the synthesis at an earlier stage, or were the longer chains of fatty acids shortened? 3) At which stage were the double bonds introduced? Labelling experiments with radio-labelled and heavy-atom-labelled acetic acid and labelled fatty acid molecules have answered these questions. We know now that these pheromones are made from 16 and 18-carbon fatty acids circulating in the haemolymph as triglycerides. The fatty acids are converted to pheromones in abdominal glands when required, released, and carried by the wind to the males, which fly upwind when excited by the pheromone plume. The exact sequence of reactions that occur in the pheromone-producing gland varies with different insect groups, but Figure 3.21 shows the biosynthetic reactions for the pheromone of Argyrotaenia velutinana (the red-banded leafroller, an important agricultural pest), one of the first studied (Bjostad and Roelofs, Journal of Biological Chemistry, 1981, 256, 7936). The precise ratio of the two final products, (3and (Q-11-tetradecen-1-yl acetate (92:8), depends on the relative activity of several enzymes in the sequence. The ratio of (2)to (A)-1 1-tetradecenoic acids is 40:60. The configuration of the double bond can have a profound effect on reception by the male insect. Sometimes a wrong isomer can completely inhibit the response to a pheromone. For example, the moth Eupoecilia ambiguella produces (3-9-dodecenyl acetate. Males are inhibited by as \ many steps ASCoA

1

acetyl~o~ 1

6

i

s

C

o

*

~

i

o

:

4

~

S

C

o

palmitic acid

11

i

reduction to alcohol

OH 11

A

0

H Oacetylation

0 -

-11 -11

c 1

SCoA

Figure 3.21 Biosynthesis of the sex pheromone of A. velutinana. It is a mixture of ( Z ) and (E)-11-tetradecenyl acetate in an isomer ratio of 92.8

A

43

Fatty Acids and Derived Compounds Fatty acid synthesis

-

C1~ - C O A

A1l-desaturase

A1l-Ci &oA

A11-desaturase c1e-COA

1 4 2

* A1 1-Ci6-COA

Ag-C16-COA

Figure 3.22 A scheme showing how by chain shortening by one acetate unit at a time and the action of a unique A1 1-desaturase enzyme, many of the lepidopteran pheromone chains are made, from which speclJic blends of alcohols, aldehydes and acetates are created, as in the example of Trichoplusia ni

little as 0.1% of the (E)-isomer. A pheromone lure containing some of this @‘)-isomer can be used as a practical means to disrupt mating for this species. The intensive study of lepidopteran sex pheromones and their biosynthesis has made possible a scheme showing how many of them are produced by a small number of reactions, summarized in Figure 3.22. While the A9-desaturase enzyme for making unsaturated acids is common to plants and animals the A1 1-desaturase used here is unique to insects. The optimum chain lengths are apparently 12 or 14 carbon atoms, less than this is too volatile. There are fewer identified pheromone compounds with 16 carbons and still fewer with 18. With still larger molecules volatility is perhaps too low (except for hydrocarbons) for efficient detection by the males. There are more species in the world than there are suitable compounds for lepidopteran pheromones, therefore species use blends. Within a blend of any species it is usually found that the components vary by one or more of five possible differences: The chain length may vary by one acetate unit; The functional group may be altered, e.g., from alcohol to aldehyde or acetate; There may be one more or less double bonds; The double bond may move two positions along the chain in unsaturated alcohols; A 2 double bond may be converted to E and vice versa. For example the cabbage looper moth Trichoplusia ni uses a blend of six acetates, all of which are considered essential to the pheromone blend. They are dodecyl acetate, 11-dodecenyl acetate, (9-7-dodecenyl acetate, (Z)-5-dodecenyl acetate, (9-9-tetradecenyl acetate, and (27-7tetradecenyl acetate. The most frequently encountered compounds are

44

Chapter 3

(2)-9-tetradecenyl acetate, which is used in the blends of 223 lepidopteran species and (2)-11-tetradecenyl acetate (Figure 3.23) used by 214 species. Some further examples of lepidopteran pheromones are shown in Figure 3.23. Bombykol, the sex pheromone of the silkworm moth Bombyx mori (Plate 2) was the first pheromone isolated and identified, in an effort that took many years of work (Butenandt, Beckmann, Stamm and Hecker, Zeitschrift fur Naturforschung, 1959, 14b, 283). It is not easy to see how bombykol could be biosynthesized by use of a A1 1-desaturase. Indeed it is made in the insect from palmitic acid, which is converted to ( 3 - 11-hexadecenoic acid, and then an unusual 10,12desaturase converts that into (1OE,122)- 10,12-hexadecadienoicacid. Some species employ hydrocarbons as pheromones. That of Estigmene acrea (the salt marsh caterpillar) has been shown to be made from linolenic acid (Figure 3.24), probably obtained in its food, since most insects and higher animals are unable to make this acid. Chain lengthening and decarboxylation occur as described in the hydrocarbon section above. Similar chain lengthening and decarboxylation would give the pheromone of Phragmatobia fuliginosa. /OH Bombykol, pheromone of Bombyx mori, the silk worm moth

-0

CHO C H -O

-

+

Choristoneura fumiferana, the spruce bud worm, a mixture of €and Zisomers (96:4)

(4-114etradecenyl acetate used by many species -

L

-

+

O

\

0

the pink bollworm, Pectinophora gossypiella, a mixture of isomers 65:35 (top:bottom) (il)-6-heneicosen-l1 -one

0 H

1

(minor compornent)

Heiicoverpa zea, the corn earworm

0 0

(Z)-l,6-heneicosadien-l1-one 0

(E,Z)-3,6-heneicosadien-ll -one three compounds from Orgya pseudotsuga

Figure 3.23 Further examples of lepidopteran sex pheromones. These substances are all produced by adult females to attract males. In many cases the eflective pheromone consists of a mixture of compounds, often isomers or homologues. Disparlure, the pheromone of the gypsy moth Lymantria dispar has a branched chain which starts with an isobutyric acid unit and is extended with acetic acid units. The three C,, unsaturated ketones are the pheromone of the Douglas-fir tussock moth Orgya pseudotsuga

45

Fatty Acids and Derived Compounds

Phragmatobia fuliginosa

+OH

-

-

0

-

1, 2 x CH,COOH D 2, decarboxylation

3, epoxidation

linolenic acid

0

4 Estigmene acrea

(5S,SS)-dimethylheptadecane

Figure 3.24 The hydrocarbon pheromone of Phragmatobia fuliginosa, and the known route to the sex pheromone of Estigmene acrea, the saltmarsh moth. The only example at present of a methylalkane pheromone with known chirality is the dimethylheptadecane from the leaf miner moth Leucoptera scitella

At present, the only example where the chirality of a methyl-branched alkane pheromone has been determined is in the sex pheromone of the leaf miner moth Leucoptera scitella where (SS,9S)-dimethylheptadecane (Figure 3.24) has been shown to be highly attractive to males.

3.4 COLEOPTERA The Coleoptera (beetles) contains about 300,000 species and are very varied in form, diet and habitat. Equally they have very different kinds of pheromones and defensive secretions, many of them derived from fatty acids. The bean weevil Acanthoscelides obtectus uses an unusual allenic methyl ester (Figure 3.25). Its biosynthesis has not yet been investigated. Allenes are an unusual example of chirality. Anomala cuprea, a chafer grub produces two lactone sex attractants, derived from the unsaturated acids oleic and palmitoleic acids (Figure 3.25). Both are shortened by the loss of two acetic acid units, then oxidized (stereospecifically) at the allylic position and cyclized to the pheromone. =C b C O O C H 3

,

) ,

(-)-methyl (R,2€)-2,4,54etradecatfienoate

0

O

'

>

, -' oleic acid

-4SzAOH

-

J

O

0

H 0

-

OH

OH

0 OH

palrnitoleic acid

Figure 3.25 The unusual allenic ester pheromone of the bean weevil Acanthoscelides obtectus, and formation of the two-component sex pheromone of Anomala cuprea from a mixture of oleic and palmitoleic acids

46

Chapter 3

3.4.1 Coccinellines Coccinellid beetles, commonly called ladybirds, which prey on aphids, make a range of defensive compounds. They are produced in the fat body and stored in the haemolymph. When a ladybird is disturbed, it exudes some of this toxic haemolymph (this is called reflex bleeding) at its leg joints. One group of such toxic compounds are the coccinellines, tricyclic amines and their N-oxides (Figure 3.26). The coccinellines were first shown to be made from acetate units using feeding experiments by radiolabelling with sodium [ l-14C]acetateand [2-I4C]acetatein 1975. Recently the experiments were repeated with in vitro studies using excised body parts of ladybirds that gave much higher incorporation of the radio-label (Laurent, Braekman, Daloze and Pasteels, Insect Biochemistry and Molecular Biology, 2002, 32, 1017). The fatty acid route was demonstrated because the formation of coccinelline was inhibited by 2-octynoic acid, a known inhibitor of fatty acid synthesis. If the compound is derived from palmitic or stearic acid, it would require loss of one or two acetate units by P-oxidation, followed by further oxidations (Figure 3.26) at appropriate carbon atoms. This was supported by experiments that showed coccinelline formation was inhibited when the experiment was performed in a nitrogen atmosphere. The origin of the nitrogen atom in coccinelline was shown to be glutamine. This was demonstrated by

EH3 coccinelline

+ other isomers

hippodahne

convergine

arnidotransferase *

-k

NH2 glutamine

Figure 3.26

(NHd

NH2 glutamic acid

The probable transformation of a fatty acid by chain shortening, oxidation and cyclization to precoccinellines and coccinellines. The preferred source of the nitrogen atom is glutamine through the action of an amidotransferase. Ammonia is shown in brackets since it is unlikely to exist in the free state

47

Futty Acids and Derived Compounds

supplying various amino-acids to a tissue culture that could synthesize the coccinelline in the presence of oxygen. Precoccinelline (Figure 3.26) is basic, the final stage gives the weakly basic N-oxide. A number of isomers are possible by altering the stereochemistry at the ring junctions. The isomers most frequently met are coccinelline itself, found, for example in the common ladybird Coccinella septempunctata (Plate 3) which contains coccinelline and precoccinelline, and Hippodamia convergens (Plate 7 ) which contains hippodamine and convergine, the N-oxide of hippodamine. The true shape and difference between these isomers is shown more clearly in Figure 3.27. H

precoccinelline

hippodamine

Figure 3.27 Stereodiagrams of precoccinelline and hippodamine to show their diflerent shapes

Over 50 alkaloids (see Chapter 9) of several structural types have been isolated and identified from ladybirds. Two further compounds, now known to have very similar origin to the coccinellines are (-)-adaline and (-)-adalinine (Figure 3.28), from Adalia beetles. The intermediate piperideine (A in Figure 3.26) undergoes further oxidation and a Mannich reaction to give adaline (Figure 3.28). Feeding Adalia bipunctata with (-)-adaline deuterated in the side chain caused them to make deuterated adalinine, but when fed (+)-adaline, similarly deuterated, there was no labelled adalinine produced, only unlabelled compound. From this it is concluded that adalinine is formed from adaline by a reversal of the Mannich reaction on the other side of the ring. Addition of water and a further oxidation gives adalinine. In the Australian ladybird Cryptolaemus montrouzieri is found two compounds, the first might be described as 6-methylpelleterine. Pelleterine is itself found in the root bark of the pomegranate tree and is long used as an anthelmintic. The second compound, clearly related to the first, can also be seen as a lower homologue of adaline (Figure 3.28).

3.4.2 Epilachnine Epilachnine is an example of a quite different substance made by a coccinellid beetle and derived from a fatty acid, although that origin might not be immediately obvious. It is the defensive secretion of the pupae of the Mexican bean beetle Epilachna varivestis. The pupae carry the toxin

Chapter 3

48

=I&[

&+---@O

H

03

adalinine

adaline

0 (-)-adaline

adalinine

n

(+)-adaline

w v H

0

from Cryptolaemus montrouzieri

Figure 3.28 Outline of the formation of adaline and adalinine,fatty acid-derived defensive compounds of Adalia beetles, The structure A is the same as in Figure 3.26. The compoundsfrom Cryptolaemusmontrouzieri are evidently ofrelated origin

in droplets at the ends of hairs covering the body (Plate 4). It has been shown by labelling experiments to be made from oleic acid, which loses four carbon atoms from the carboxylate end, and the amino-acid serine (Attygalle, Blankespoor, Eisner and Meinwald, Proceedings of the National Academy of Sciences USA, 1994,91, 12790). The route to epilachnine has been studied in great detail with polydeuterated precursors. For example, fully deuterated stearic acid fed to larvae gave compound A isolated from pupae (Figure 3.29). Another intermediate, B, without deuterium at C-15 was incorporated into epilachnine without loss of deuterium. Therefore the chain is specifically oxidized and cyclized at this carbon atom as shown.

3.5 COCKROACHES Cockroaches belong to the family Blattodea, and have received special attention because they are such a nuisance in buildings. The female German cockroach Blatta germanica produces 3,ll -dimethylnonacosan2-one as a contact pheromone. From its structure it might not be seen as close to the fatty acids, but it follows easily from the consideration of branched-chain hydrocarbons. Condensation of an acetate unit and a propionate unit gives the first branching point. Three more acetate units are added before another propionate, and the chain is completed with nine more acetates. The sequence is completed by decarboxylation and

Fatty Acids and Derived Compounds

49

-"-;?,

0

H

I

epilachnine

CD3

A

$OH -----)

B

Figure 3.29 Outline ofthe way in which epilachnine is biosynthesizedfrom oleic acid and the amino-acid serine. Totally deuterated stearic acid and partially deuterated oleic acid were incorporated into deuterated epilachnine as shown. A t the bottom is shown the sequence ofreactions at the C-15 of oleic acid that completes the ring

oxidation of a CH, group in two stages to a ketone (Figure 3.30). This is an interesting example because the biosynthetic route is not the most obvious one expected. Usually the methyl branches are added late in the sequence, after the long straight chain has been formed, here they are known to be added at the beginning. It has been shown that this pheromone is produced in the fat body and transported by the protein lipophorin to the abdominal surface. 3.6 TERMITES

Not many trail pheromones of termites have been identified, but one group is worth mentioning here. In the group Macrotermitinae, for some species (Z)-3-dodecan-l-01 is the trail pheromone, while for another (2,2)-3,6-dodecadien-l-o1is. In both cases the sternal gland is the source. It is suggested that these alcohols are formed from oleic acid and linoleic acids, respectively, by loss of three acetate units and reduction to the alcohol (Figure 3.31).

Chapter 3

50 CH3COS-COA

GH3 + CHz-COS-CoA

J\COS-C~A 3 x CH~COSCOA then CH3CH2COSCoA

\

-COS-CoA

I OH

0

COS-COA decarboxylation oxidat ion

I Oxidation 3,11-dimethylnonacosan-2-one

Figure 3.30 Outline of the biosynthesis of the contact pheromone of the German cockroach

Figure 3.31

The suggested conversion of oleic and linoleic acids to the termite trail pheromones dodecenol and dodecadienol. This dodecadienol also acts as sex attractant (at higher concentration) in Ancistrotermes pakistanicus

What is called chemical parsimony (stinginess) is applied among termite pheromones. One compound serves as pheromone for a number of species. Moreover, dodecadienol, produced by females at higher concentration, acts as sex pheromone for males (about 0.1 pg cm-’ for trails and about 1 ng as sex pheromone). This dual use also applies to (Z,Z,E)-3,6, 8-dodecatrien-1-01, which in some Rhinotermitidae acts as trail pheromone, at low concentration, and sex pheromone at higher concentration.

3.7 HONEYBEES Several C,, acids are important in honeybees. (25)-9-Oxodecenoic acid is the sex attractant of the virgin queen. After mating the fertile queen produces a mixture of this and four other substances (Figure 3.32), each weakly active alone, as components of the ‘queen substance’ which prevents queen cell construction by workers and a number of other actions which signal the presence of a dominant reproductive queen. Both enantiomers of 9-hydroxydecenoic acid have to be present for highest activity, a rare example of synergism between enantiomers in insect

Fatty Acids and Derived Compounds

51

semiochemicals. The workers make (2E)-10-hydroxydec-2-enoic acid (known as royal jelly acid, because it is part of the food given to queen acid. Both 9-hydroxy- and 10larvae) and (2E)-9-carboxynon-2-enoic hydroxy-compounds are made preferentially from stearic acid. The stearic acid is oxidized at the 17- or 18-position, and then the chain shortened from the carboxyl end by loss of four acetate units by P-oxidation (Figure 3.33). This can be done by both queens and workers, so presumably, the 17-oxidation route operates in the queen preferentially and the 18oxidation route in the workers. The actions are catalyzed by P,,, oxidases.

Figure 3.32 The substances that make up the queen substance of Apis mellifera. The quantity given under each structure is the amount found in a single queen bee O

C

;

in queens

1

b

,

H

O

b

C

O

H

t.

in workers

I

O

t

OH V

ICOOH

\ H O -

/

C

O

O H OH part of "queen substance"

COOH

"royal jelly acid"

Figure 3.33 The formation of bee substances from stearic acid. Both queens and workers are capable of using both pathways but those shown actually operate preferentially. 9-Hydroxydec-2-enoic acid is also used by the queen when swarming to hold the workers in the swarm

3.8 ANTS Among the many substances identified in ants as pheromones or allomones, one group of offensive compounds is worth mentioning. Crematogaster ants are a large, aggressive, world-wide genus. They have the unusual ability to bring their abdomens forward over their heads and place a toxic secretion on other insects with their flattened sting. Some of the European species store long-chain unsaturated keto-acetates, of the form shown in Figure 3.34, in their Dufour glands (part of the poison apparatus). These are ejected with enzymes from the poison gland (an acetate esterase and an alcohol oxidase) to hydrolyze the acetates and

52

Chapter 3

0 A

U

0 0-c, CH3

0 0-c, CH3

Figure 3.34 Examples of the substances stored in the Dufour gland by Crematogaster scutellaris ants. On ejection they are hydrolyzed to alcohols and then oxidized to aldehydes, the true oflensive compounds, by enzymesfrom the poison glands

oxidize the alcohols to aldehydes. These unsaturated keto-aldehydes are strong electrophiles, reacting with OH, SH and NH, groups, and are strongly toxic to other ants. The biosynthesis of the acetates has not yet been studied but would appear to be from oleic and palmitic acids with chain extension by an unknown C5fragment.

3.9 SPIDERS Spiders are generally solitary animals, but they still have to find mates. A few sex attractants of spiders have been discovered. That of Linyphia triangularis, female produced, is a mixture of (R)-3-hydroxybutyric acid and its dimer (R)3- [(R)-3-hydroxybutyryloxy]-butyric acid (Figure 3.35). Another female attractant, from Cupiennius salei, is (81,l’-dimethyl citrate (Figure 3.35). This pheromone is placed on the silk thread. On this small sample it has been suggested that spider pheromones may be adapted from primary metabolites. On the other hand, a desert spider Agelenupsis aperta uses 8-methyl-2-nonanone, which is more like a hymenopteran pheromone, to attract males. It is suggested this is probably made from leucine, via isovaleric acid, followed by chain extension and decarboxylation. 6-Methyl-3-heptanone was present with it in the spider but not pheromonally active.

Figure 3.35 Threefemale-produced sex pheromones of spiders. Thefirst is the dimer of the simple metabolite 3-hydroxybutyric acid and the second isfrom the primary metabolite citric acid. The third, 8-methyl-2-nonanone, is rather more like some insect pheromones

Fatty Acids and Derived Compounds

53

3.10 HEMIPTERA The defensive secretions of a large number of hemipteran bugs have been examined chemically. They are typified by hexanal, and (3-2-hexena1, often accompanied by 2-octenal and 2-decenal and their corresponding acetates, but they have not been studied from the biosynthetic aspect. 3.10.1 Green Leaf Volatiles An interesting by-product of plant metabolism is the production of 'green-leaf volatiles' by oxidation of unsaturated fatty acids (Figure 3.36). and (Q-3-hexenols and -3These volatile compounds, which include (3hexenals, (Q-2-hexena1, (Q-2-hexen- 1-01 and 1-hexanol are released by some plants naturally, and especially when damaged by insects, and apparently are then sufficiently noticeable to attract parasitic wasps to the damaging insects. They are also attractive to many insects and are used by others as pheromones (aggregation in bugs) and defensive secretions (of stink bugs). A millipede secretes 1-octen-3-01, the characteristic odour of mushrooms, but what its origin is, or what protective effect it offers the millipede are not known.

3.11 LACTONES Five-membered-ring lactones of beetles, and the special case of epilachnine, a large-ring lactone with a nitrogen in the ring, have been described. Large-ring lactones or macrolides are relatively common in insects. The Amitermes group of termites use lactones in their defensive secretions. 15-Pentadecanolide, 2 1-heneicosanolide, 22-docosanolide and

COOH

lipoxygenase

- H.

H H linolenic acid

- C

\

-O

COOH

I

0-0. H I

*

O

H

I:"-"* 1

-

COOH

A

reductase OH OH (Z)-3-hexenol

isomerase

*OH (€)-3-hexenol

+

/

CHO (€)-Phexenal

1-0cten-3-ol

Figure 3.36 Theformation of some of the 'green leaf volatiles' by plants and used by some insects. The biosynthesis in insects may not be by the same route. 1-octen-3-01 isfrom a millipede but of unknown biosynthetic origin

54

Chapter 3

c15

C X 2 = J 0

0

Figure 3.37 Defensive secretion macrocyclic lactones of Amitermes termites. Note the odd number of carbon atoms in two compounds

B

A

E

D

C

Figure 3.38 Macrocyclic lactones from beetle aggregation pheromones. Compound A is found in Cryptolestes pusillus, theflut grain beetle, compounds B and E in C . surinamensis, a mixture of enantiomers of C in C. turcicus, compound D and the R-enantiomer of E in C . ferrugineus, the rusty grain beetle

24-tetracosanolide have all been identified along with the a-hydroxy- and P-hydroxytetracosanolides shown in Figure 3.37. Males of some stored grain beetles use macrolides for aggregation pheromones, making them from fatty acids by oxidation at the terminal or next-to-terminal carbon atom (Figure 3.38). The Argentine ant Linepithema humile which has invaded Australia and Mediterranean Europe produces a macrolide evidently made from a fatty acid where synthesis begins with 2-methylbutyricacid (Figure 3.39). The Dufour glands of halictine and colletine bees frequently contain mixtures containing macrocyclic lactones. Colletes bees, for example,

18-octadecanolide 13-(1 '-methylpropy1)t ridecanolide

20-eicosanolide

eo 0

Linepithema humile

18-octadec-9-enolide

0

:

"

o-hydroxystearic acid

Figure 3.39 Further examples of insect macrolides, from the ant Linepithema humile, and cell-lining material from the glands of Colletes bees

Fatty Acids and Derived Compounds

55

contain mixtures of 18-octadecanolide, 20-eicosanolide and 18-octadec9-enolide (Figure 3.39) as well as o-hydroxy-fatty acids (which gives the clue to their biosynthesis) and hydrocarbons. These solitary bees line their brood cells with a waxy polymer made from these components. There are many more pheromone structures derived from fatty acids, but they all arise by chain extension or shortening, double bond introductions, oxidations and reductions as outlined in these examples. BACKGROUND AND FURTHER READING

M. S. Blum, Biosynthesis of arthropod exocrine compounds, Annual Review of Entomology, 1987,32,381 4 13. M. S. Blum, Biochemical defenses of insects, in M. Rockstein, editor, Biochemistry of Insects, Academic Press, New York and London, 1978, pp. 465-5 13. W. Francke and S. Schulz, Pheromones, in K. Mori, editor, Comprehensive Natural Products Chemistry, Vol. 8, Pergamon Press, Oxford, 1999, pp. 197-261. P. Howse, I. Stevens and 0. Jones, Insect Pheromones and Their Use in Pest Management, Chapman and Hall, London, 1998, pp. 369 (Chapters 5 & 6, isolation and structure of pheromones). J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 2). H. H. Rees, Insect Biochemistry, Chapman and Hall, London, 1977, pp. 64. J. A. Tilman, S. J. Seybold, R. A. Jurenka and G. J. Blomquist, Insect pheromones - an overview of biosynthesis and endocrine regulation, Insect Biochemistry and Molecular Biology, 1999,29,481-5 14. www-pherolist.slu.se for a comprehensive list of lepidopteran sex pheromones, species and pictures. QUESTIONS 1. (Z)-9-Tricosene is a pheromone on the surface of female Musca domestica the common housefly. Outline its probable biosynthesis from acetic acid units. 2. Outline the biosynthetic route to 5-methyltricosane. 3 . Suggest a probable biosynthetic route to (9E,l 119-9,ll-tetradecadienyl acetate, the sex pheromone of the apple moth Epiphytas postvittana, from palmitic acid. 4. [18,18,18-2H,]Stearicacid was used in the study of the formation of 9-hydroxy- and 10-hydroxy-dec-2-enoic acids (see Figure 3.32) in

56

Chapter 3

queen and worker honeybees, using mass spectrometry. How many of the deuterium atoms per molecule would you expect to find in 9-hydroxydec-2-enoic acid, and how many in 1 0-hydroxydec-Zenoic acid? 5. When 2-fluorostearic acid was used in the study in Question 4, none of the 9-hydroxy- or 10-hydroxy-decenoicacids was formed. Why was that? 6. If all the hydrogen atoms on carbon in isoleucine were replaced by deuterium, show where and how many deuterium atoms would be present in 14-methylhexadecanoicacid (Figure 3.12) if it is made from the labelled isoleucine. 7. Bruchin A is a substance made by certain strains of the larvae of the pea weevil Bruchus pisorum. It causes the formation of callus tissue around the larva inside the pea. Its structure is specific; propionic, lactic or 3-hydroxybutyric acids or no acid esterified onto the long chain diol all give inactive compounds. Predict the biosynthetic origin of bruchin A.

OH

HO bruchin A, stimulates neoplastic growth in peas

CHAPTER 4

Polyketides and Acetogenins 4.1 ACETOGENINS J. N. Collie, who predicted that fatty acids were made from acetic acid units (see Chapter 3), and one of his students went further and suggested that many other substances were made from acetic acid molecules joined head to tail. His speculations were largely forgotten, but the idea was resurrected by Sir Robert Robinson in the 1940s and extended by A. J. Birch. They showed that many complex natural compounds could be conceived as made of chains of acetate units linked together, first to form a polyketide (Figure 4. l), and then by suitable folding, condensation reactions (elimination of water) and an occasional further oxidation or reduction, to give the structure of the natural compound. Subsequently, using radioactively-labelled acetic acid, these predictions have been proved largely correct (Chapters 1 and 2 in E. Haslam Metabolites and Metabolism see Further Reading). We now recognize that a wide range of naturally occurring substances are derived through these linear polyketides. The polyketides shown in figures here may or may not exist as intermediates, but the structures provide a convenient way to show how the final products are obtained. The final products are called acetogenins because they are derived from acetate units. With a little experience, many compounds can be seen to be acetogenins by a quick examination of their molecular structure. Most of the known acetogenins come from micro-organisms. A very simple example is orsellinic acid, a common metabolite of many micro-organisms (Figure 4.1).

4.2

POLYKETIDE DERIVATIVES

Methyl 6-methylsalicylate is the trail pheromone of some ants, it is also found in carabid beetle, 6-methylsalicylaldehyde in the defensive secretion of a cerambycid beetle, and 6-methylsalicylic acid is made by some Penicillium moulds. Orsellinic acid and 6-methylsalicylic acid are 57

58

Chapter 4

4 CH3COOH

condense, - H20

HO

OH

orsellinic acid

Figure 4.1

The condensation of four acetic acid units to give an intermediatepolyketide which by further reaction gives an acetogenin. The polyketide is a formal intermediate, not always having a real existence CH3

condense

OH reduce to OH

enolize

6-methylsalicylic acid

Figure 4.2 Formation of the acetogenin 6-methylsalicylic acid

2-hydroxy-6-methylacetophenone

2-acetow-6-methylacetophenone

CH3 HO mellein 3,4-dihydro-8-hydroxy3-methylisocoumarin

/

CH3 Orcinol

CH3 3-methyl-2-cyclohexenone

Figure 4.3 Some further examples of acetogenins from insects. Mellein occurs in the mandibular glands and metapleural glands of ants. 3-Methyl-2-cyclohexenone comes from the pygidial glands of the ant Rhytidoponera chalybaea

examples of very simple acetogenins, made by folding a polyketide composed of four units of acetic acid (Figure 4.2). It is now possible to see why the structure on the left in Figure 1.2 was predictable, while it is impossible to see how any simple polyketide can be folded to give 5-methy lsalicylic acid. The Australian ant Rhytidoponera aciculata produces 2-hydroxy6-methyl-acetophenonein its pygidial glands. Notice the similarity to 6methylsalicylic acid. Other related acetogenins from insects are shown in Figure 4.3. Given the structure of a substance we can usually work back to the polyketide from which it must be derived. When side chains occur in acetogenins, these are always derived from the ‘tail’ of the polyketide, never the carboxylic acid ‘head’. The carbonyl oxygen nearest to the

59

Po lyke t ides and Ace t ogenins methyl "tail" of the polyketide

\

this P-keto-group is usually retained

Figure 4.4 The formation of an isocoumarin (produced by an Aspergillus mould), indicating the 'head' and 'tail' of the intermediate pentaketide tail

endocrocin, a red pigment of Aspergillus fungus

alternariol, a phenolic compound from the fungus Alternaria tenuis

griseofulvin, antibiotic used

for treating fungal infections

Figure 4.5 Further examples of products derived from polyketides. The head and tail of the polyketide are indicated

carboxyl group in the polyketide is usually retained and is presumably necessary to activate the a-CH, group for the cyclization (Figure 4.4). Some examples are given in Figure 4.5. There are two types of polyketide synthase (known as PKS) enzymes. In type I1 polyketide synthases, the whole polyketide chain is first formed and then cyclized, oxidized or reduced as necessary, as in the examples above. Aromatic compounds like orsellinic acid, 6-methylsalicylic acid and isocoumarin are produced with type I1 synthases. In type I polyketides, each acetate unit is altered (reduced, dehydrated, etc.) as it is added, as happens in fatty acid synthesis (Figure 3.3). One of the difficulties in studying this type is the near absence of isolatable intermediates, since the growing chain remains attached to the polyketide synthase enzyme complex. The genes for the necessary enzymes are clustered together on one chromosome, and the process of synthesis is carried out on large complexes of enzymes. It is sometimes difficult to decide from looking at a structure whether it is derived from a fatty acid or from a type I1 polyketide. In the future it is possible this may be most easily decided by looking at the cluster of genes that code for the necessary enzymes. 2-Heptanone, a pheromone used by honeybees and many ants is a simple example of a type I polyketide. We can think of the compounds as being composed of a chain of acetate units as shown in Figure 4.6, which are joined together and then decarboxylated. The actual steps in its

60

Chapter 4

polyketide

J

2-heptanone

I 0

0

rnalonyl COA

&S-ACP

&S-ACPt3-ketooctanoyl ACP

I

malonvl COA

1, reduce 2, dehydrate

0 hexanoyl ACP

hydrolyse

ds decarboxy1a:e

+ co2

2-heptanone

Figure 4.6 A scheme to show the formation of 2-heptanone through a hypothetical polyketide, and the probable scheme by which it is actually biosynthesized

formation are probably as indicated in Figure 4.6, although it must be emphasized that this has not yet been proven. Condensation of an acetyl group attached to acyl carrier protein (ACP) with a malonyl group gives first acetoacetyl ACP which is reduced to P-hydroxybutyryl ACP, which is dehydrated and the double bond reduced, in the same kind of step as already met for fatty acid synthesis (Figures 3.3 and 3.5). The resulting butyryl ACP is extended again to hexanoyl ACP, and again to p-ketooctanoyl ACP. Decarboxylation of the free p-keto acid gives 2-heptanone (Figure 4.6). Note that, particularly in micro-organisms and insects, methylmalonate (equivalent to a propionic acid unit) can replace malonate (resulting in an acetate unit) in the condensation reaction to give a polyketide. Mycolipenic acid contains three propionate units and nine acetate units, erythromycin is made from seven propionates (Figure 4.7).

CH3 CH3 CH3 CH3(CH2) 17

uCOOH mycolipenic acid

0-sugar

HO

0-sugar erythromycin

Figure 4.7 Acetogenin structures containing propionate units. Mycolipenic acid is produced in the lipids of Mycobacterium tuberculosis and erythromycin is an antibiotic produced by Streptomyces erythreus. The individualpropionyl groups of erythromycin have been indicated by heavy black lines

61

Polyketides and Acetogenins

4.3 VOLATILE PHEROMONES Social insects, living close together in colonies need for communication volatile substances that spread a message quickly and are then dispersed. Many of these substances are volatile acetogenins, made from a mixture of acetate and propionate units. Little work has been done to study their biosynthesis, but it can be easily predicted. A mixture of 2-heptanone (Figure 4.6) and 2-heptanol, or 3-octanone and 3-octanol are often present together as pheromone mixtures. To make 3-octanone requires three acetate units and one propionate unit (Figure 4.8). Alcohol dehydrogenase enzymes are usually present in the glands reducing the initial ketone to a mixture of alcohol and ketone.

/--4JL 3-octanone

Figure 4.8 Illustrating theformation of 3-ketones, typical of ant pheromones. The polyketide shown in square brackets does not have any real existence

The polyketide synthases of micro-organisms are rather unspecific for their substrate and supplying them with a slightly different substrate from the normal can induce them to make different products. If the same is true of insect PKS enzymes, it may explain why a mixture of simple volatile alcohols and ketones of different chain length are frequently found together. For example, the mandibular glands of the ant Myrmica scabrinodis contain 3-hexanol, 3-heptanol, 3-octanol, 6-methyl-3-octano1, 3-nonanol, 3-decanol and 3-undecanol and all their corresponding ketones. The simple substance 4-methyl-3-heptanone (Figure 4.9) is found in secretory glands of a wide variety of insects, including the mandibular glands of several species of leaf-cutting ant. It can serve to illustrate how there are alternative biosynthetic routes possible. The molecule can be seen as composed of three propionate units, but these could be joined in two ways, as shown. The first is more likely, since the second breaks the rule that the keto-group is retained next to the carboxyl that is lost as carbon dioxide. Note that each branch methyl group usually introduces a new chiral centre, unless there is a double bond attached as in Figure 4.10. The final structure shown in Figure 4.9 is (S)-(+)-4-methyl-3heptanone, first identified as the trail pheromone of two leaf-cutting ants, Atta texana and A. cephalotes. This natural enantiomer is 200 times more active in inducing trail-following than the unnatural

62

Chapter 4 3-octanone

ko?koH+ko"-

I

&OH

-

Hod

+Ho$Fo$

0

0

Figure 4.9

O

0

4

,

t

O

H

l

/-

0

(S)-(+)-6methyl-Sheptanone

[Ho-.&&,p]0

0

O

-

0

0

H

O 0

*

h 0

The biosynthesis of 4-methyl-3-heptanone can be supposed to proceed through either of two routes. The imaginary polyketide intermediates are shown, although the former route is more likely because it allows decarboxylation of a P-keto-acid at the$nal stage. Thefinal structure shown in thisfigure is (S)(+) -4-methyl-3-heptanone, an ant trail pheromone

(R)-(-)-enantiomer. Subsequently this same substance has been identified in other ants (frequently in mandibular glands), wasps, caddisflies (Trichoptera), and opilionids. The opilionids (daddy-long-legsor harvestmen), which belong, with the spiders, to the class Arachnida, have defensive glands on the tops of their bodies. Eight species produce a mixture of short-chain branched alcohols and ketones (Figure 4. lo), of the type often used as ant pheromones. The biosynthesis has not been studied, but they evidently are made largely from propionate with some acetate. Other opilionids secrete quinones.

0

R

R

R

R = O or H,OH

Figure 4.10 Alcohols and ketones from opilionid defensive secretions, apparently made from acetate and propionate units

The biosynthesis of the major component of the male-produced aggregation pheromone of Carpophilusfreemuni beetles has been studied in detail by deuterium labelling. It was clear that the molecule was made up of one acetate unit, one propionate and two butyrates. The labelling pattern indicated that decarboxylation was not the final step as would be expected, which left two possibilities, shown in Figure 4.1 1. Some further examples of compounds from the aggregation pheromones of Carpophilus beetles are given in Figure 4.12. These are all

Polyke tides and Ace togenins

Figure 4.1 1

63

Two possibilities for the formation of a Carpophilus beetle aggregation compound suggested by the investigators knowing that the last step is not decarboxylation. The broad lines indicate the individual acid units for clarity. Reduction of carbonyl groups and dehydration of the resulting alcohols are requiredfrom the intermediates to give the$nal products

Figure 4.12 Structures of some other aggregation compounds from Carpophilus beetles. All compounds except c start with acetate, c begins with propionate. They are then extended with propionate (to give methyl branches) or butyrate (for ethyl branches). Compounds a, b and d terminate with propionate and all the others with butyrate

biosynthesized from the same units of acetate, propionate and butyrate. The beetles are pest species and the pheromones have been field-tested. Occasionally one might be led astray when looking at oxygenated alkyl compounds with branched chains. The two compounds shown in Figure 4.13 were found in the mandibular gland of the ant Dinoponeru australis. They are both simply the two possible aldol condensation products of isobutyraldehyde. The clue to their origin was given by other compounds accompanying them which also contained isobutyraldehyde groups. While myrmicine ants generally accumulate their trail pheromones in a part of the poison apparatus (the Dufour gland or the venom reservoir),

Figure 4.13 Two branched compounds from an ant which are not polyketides but aldol condensation products of is0bu tyraldehyde

64

Chapter 4

formicine (non-stinging, formic acid-spraying) ants use the rectal bulb of the hindgut as the source. Two groups of lactone trail pheromones have been identified in formicine ants, one aromatic, of the isocoumarin type, including mellein (see Figure 4.3), and the other aliphatic, of the Slactone type (Figure 4.14). Since mellein is found in so many places, and it could be taken up from food, experiments were first made feeding deuterated mellein to three species, Camponotus rufipes, C. silvicola and Lasius niger. Deuterated mellein was isolated from the rectum, but no labelling was found in the other compounds. Similar experiments were made with deuterated methionine, to see if methylation of mellein was occurring, but there was no labelling observed. When deuterated acetic acid was fed as the sodium salt, the lactones were all labelled, but there was insufficient material to determine just where the deuterium was. With [3-2H,]propionicacid it was clear from the mass spectra of the products that CD, groups were incorporated as indicated in Figure 4.14. These pheromones evidently are of polyketide origins. The compound known as invictolide (Figure 4.14 f) was first identified as part of the queen recognition pheromone of Solenopsis invicta ants and later shown also to be part of the trail pheromone of some Camponotus ants. It is not known if these compounds are made via type I or I1 polyketides. Related in structure is 8-hydroxyisocoumarin, also called centipedin (Figure 4.15). It is an antibiotic substance isolated from the centipede Scolopendra subspinipes multilans (Plate 5). Although centipede bites can

CD3

a

f

OH

OH 0

CD3

b

OH 0 c

d

CD~-

e

no Cp-kJp iCD\,.

t

\/\\.*'

/i . H o

CD3

Figure 4.14

f

OH 0 g

OH 0 h

OH 0 i

Some lactone acetogenin trail pheromones made from acetate and propionate units. The CD, groups indicate where the compounds were labelled when the ants were fed with sodium [3-2H,]propionate. Mellein, compound a, is the trailpheromone of Lasius fuliginosus and Formic rufa. Compound b is the pheromone of Camponotus rufipes and c is the pheromone of C. silvicola, C. inequalis and Lasius niger. Three species of Camponotus use compound d, while C. herculeanus uses a mixture of d andJ and C. ligniperdus has all three of d, e and$ Compounds g,h and i are all found in some of these species, but appear not to act as pheromone components

Po lyke t ides and Ace togenins

65

R

O OH 0

Figure 4.15 Centipedin or 8-hydroxyisocoumarinfrom the centipede Scolopendra subspinipes multilans, acetate-derived but lacking an expected methyl group if a polyketide product

be toxic and painful, there is no direct evidence that this compound is in the venom. It was shown to be formed from [14C]acetate,but not from ['4C]alanineor [14C]tyrosine,but, for an acetogenin, it lacks one methyl group that is found in mellein.

4.3.1 Cyclic Ketals

A number of pheromone compounds have the structures of cyclic ketals. For example, the sex attractant of the female olive fly Dacus oleae is 1,7dioxospiro[5.5]-undecane (Figure 4.16). Because diethyl 5-0x0-1,9nonanedioate has been identified in males of the species, the inference is that the pheromone is made from it, but the ester could be formed by cleavage of oleic acid at the double bond to give the C9 fragment or it could come from acetate units via a polyketide. Various isomers of 2,8dimethyl-1,7-dioxospiro[5,5]undecane(Figure 4.16) have been identified in wasps, solitary bees, fruit flies and bugs. It is, presumably, a polyketide derivative as shown but there is no evidence about its biosynthesis yet. It is not always easy to see a polyketide origin for a substance that at first glance seems to belong to the acetogenins. Chalcogran (Figure 4.17) is part of the aggregation pheromone of the saw-toothed spruce bark beetle Pityogenes chakographus. It is used commercially in pheromone EtO

a

O

0

E

i

0

diethyl5-0~0-1,g-nonanedioate

a hexaketide

OH OH

1,7-dioxospiro[5.5]undecane

Dacus oleae

2,8-dimethyl-l ,-/-dioxospiro[5,5]undecane

Figure 4.16 Two examples of cyclic ketal pheromones and the possible routes to their formation. Note that for the last compound, a dimethylspiroundecane, a number of isomers are possible

66

Chapter 4

traps for this forest pest. It has been pointed out by Francke that many C, compounds like chalcogran could be derived by splitting unsaturated C18 acids. There are many C, compounds used as pheromones. In this case a derivation from linolenic acid can be suggested (Figure 4.17). Note that the (2S,SR)-form is active while the (2S,5S)-form is not, but the presence of the (2S,SS)-form does not affect the activity of the active enantiomer.

Y

v-./-/kA+A.n/ COOH

.-

O=CC -OOH

+

linolenic acid

-c=o

Y

chalcogran

Figure 4.17 Suggested origin of the pheromone chalcogran from linolenic acid

4.4

DEFENSIVE SECRETIONS

A small number of beetles of the genus Paederus when disturbed or crushed exude some haemolymph containing pederine (Figure 4.18) which produces severe blisters. The beetles are hence known as blister beetles. Although the biosynthetic origin may not be very obvious, some early studies showed the incorporation of acetate and propionate, suggesting a polyketide origin. The biosynthetic scheme requires several subsequent methylations on carbon and oxygen (Figure 4.18). Recent work

OH pederine

Figure 4.18

The probable biosynthetic precursors in the formation of the defensive compound pederine. Thefigure summarizes the suggestions of the investigators based on the radio-labelling of the compound and some degradation products

Polyket ides and A cetogenins

67

has shown that pederine is actually produced by Pseudomonas species bacteria which are symbionts (symbiotic micro-organisms)inside the beetles, and it is only produced by females, who pass it to their eggs. Pederine and related compounds have also been found in marine organisms. The coccinellines from ladybirds (Chapter 3), were first thought to be of polyketide origin when it was shown that the beetles incorporated [ l-'4C]acetate and [2-14C]acetate,but subsequently a fatty acid origin has been demonstrated (Figure 3.26).

BACKGROUND AND FURTHER READING

E. Haslam, Metabolites and Metabolism, Oxford University Press, Oxford, 1985, pp. 161 (Chapter 2). J. Mann, Chemical Aspects of Biosyn thesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 3). W. Francke and S. Schulz, Pheromones, in K. Mori, editor, Comprehensive Natural Products Chemistry, Vol. 8, Pergamon Press, Oxford, 1999, pp. 197-261. J. A. Tilman, S. J. Seybold, R. A. Jurenka and G. J. Blomquist, Insect pheromones - an overview of biosynthesis and endocrine regulation, Insect Biochemistry and Molecular Biology, 1999,29,481-5 14. K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapter 5). QUESTIONS 1. Show the polyketide, appropriately folded, from which 2-hydroxy-6methyl-acetophenone is formed. 2. If CD,COOH were used in the biosynthesis of mellein (Figure 4.3), what would be the maximum number of deuterium atoms one could expect to find in mellein? 3. Deduce the polyketide intermediates for each of the three structures in Figure 4.5. 4. What are the arrangements of acetate and propionate units in the pheromone compounds 2-nonanone, 6-methyl-3-octanone and 4-methyl-4-hepten-3-one. 5. Show what further reactions are required on the initial polyketide to give 4,6-dimethyl-4-octen-3-one, an alarm pheromone of the ant Manica rubida. 6. (R)-(+)-4-Methyl-l-nonanolis a sex pheromone of the yellow mealworm Tenebrio molitor (Coleoptera). Using [ 1-'4C]propionic acid, at what positions in the molecule would one expect to find 14Clabelling?

68

Chapter 4

7. Predict the biosynthetic origin of a component of the sex pheromone of the tea tortrix moth below, by drawing its biosynthetic units in position. Careful, it can start with something larger than acetate or propionate!

tea tortrix moth pheromone

0

CHAPTER 5

Experimental Methods 5.1 TRACING BIOSYNTHETIC PATHWAYS In studying biosynthetic pathways, we have to identify (a) the ultimate source in primary metabolism from which the compound of interest derives (for example, fatty acid, polyketide, or others in the following chapters), and (b) the intermediates through which a final product is formed. With so much accumulated knowledge, and with only a few pathways used by nature, the first task of finding the ultimate source is usually not at all difficult. The second objective may be very difficult and subject to all sorts of pitfalls and false clues. The usual method of study is to suggest a possible precursor and to feed it to the biosynthesizing system. The precursor has to be labelled in some way to trace it through the sequence of reactions, and that is usually by some isotopic element. It may be a radio-active isotope, such as 3H, 14C, 32Por 35S,that can be followed by its radiation; or it can be a stable heavy isotope, such as 2H, 13C, I5N, or '*O, that can be traced by mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy (Table 5.1). Another possible way is to use mutant strains of an organism that lack the enzymes to complete a particular synthesis, or to add a specific enzyme inhibitor, so that intermediates accumulate and can be identified. A mutant strain of yeast was important in discovering mevalonic acid and its place in terpene biosynthesis (Chapter 6) and a number of mutants of the bacterium Escherichia coli helped to understand the shikimic acid pathway (Chapter 8). In the simplest kind of experiment, a labelled potential precursor may be fed to, or injected into, an animal or added to an in vitro system and labelling sought in the product of interest. Because many compounds can be broken down to acetate and then re-built into other compounds, this kind of experiment may not be conclusive. This was the case with the labelling study of dendrolasin (Figure 6.17).

69

70

Chapter 5

Table 5.1 Some isotopes used in biosynthesis studies and how they are detected Isotope

Natural abundance

Detection method

Nuclear spin

'H

99.98

112

*H

0.015

'H NMR, I3CNMR coupling NMR, MS, I3CNMR shift Radioactivity 'H NMR I3CNMR, MS Radioactivity

I4N 5N l6O 170

'*O 31P 32P

32s 34s

35s

(half-life 12.26 y) 98.9 1.1 Age and source dependant (half-life 5730 y) 99.63 0.37 99.8 0.037 0.20 100 (half-life 14.28 d) 95.02 4.21 (half-life 87.2 d)

NMR, MS I5NNMR, MS MS 1 7 0 NMR, MS MS, I3CNMR shift 31PNMR, MS Radioactivity MS MS Radioactivity

1 -

0 112 -

1 -112 0 -512 0 112 -

0 0 0

5.1.1 Specific Incorporation If intact plants or animals are used, incorporation of a labelled intermediate can be extremely low (0.01 to 1%) because many processes and organs are competing for the labelled compound. The specific incurporation of the isotope is defined as the specific activity of the desired product divided by the specific activity of the starting material expressed as a percentage. It is an important measure in biosynthesis studies. The specific activity of a labelled compound is in turn defined as the amount of radioactivity in a given amount of material (expressed as counts per minute per mole (cpm mol-I) or cpm g-' or disintegrations per minute, dpm). Radioactivity is generally expressed in becquerels (1 Bq = 1 disintegration sec-') or in curies (1 Ci = 3.7 x 10" Bq). A scale of label incorporation attributed to Sir Derek Barton says that 1% is excellent, 0.1% is good, 0.01% is positive and 0.001% or less is dubious, probably negative. Especially using I3C (which is followed by NMR spectroscopy) it is necessary to obtain at least 1% incorporation for successful deduction of the biosynthesis. Better incorporation of the intermediate can be achieved with micro-organisms, tissue culture of insect cells, or excised glands (see coccinellines, Chapter 3), or by having a cell-free system of partially purified enzymes. Alternatively,

Experimental Methods

71

now genes for making certain enzymes can be inserted into microorganisms (bacteria or yeasts) to carry out the biosynthesis more conveniently. This will become increasingly useful as more genes are sequenced.

5.1.2 Locating the Site of Synthesis

(2)10-Heptadecen-2-one is the major component of the aggregation pheromone of the fruit fly Drosophila buzzatii and 2-tridecanone from the same insect has an inhibiting effect on the aggregation. To find where and how the pheromone was synthesized, insects were separated into heads, thoraces and abdomens (Skiba and Jackson, Insect Biochemistry and Molecular Biology, 1993, 23, 375). Each of these body parts was incubated with sodium [1-14C]acetate. No radioactive pheromone was recovered from the heads or thoraces, but 1.1% of the recovered label was incorporated into pheromone by the abdomens. In addition, 0.2% of label was incorporated into 2-tridecanone (Figure 5.1). Since the site had been traced to the abdomen, in a second series of experiments they separated various parts of the abdomen and found only the ejaculatory bulb (part of the genital organs) of mature male flies synthesized the compounds. This time 3.3% of the recovered label (0.4% of the applied label) was recovered in the heptadecenone, and 1.0% of the recovered label (0.1% of the applied label) in tridecanone.

Figure 5.1 The compounds studied by radio-labelling, which are synthesized in Drosophila buzzatii

As an example of one experiment, 0.2 p1 of sodium [l-14C]acetate containing 440,000 cpm (counts per minute) was incubated with the ejaculatory bulbs at pH 6.8 in phosphate buffer. The products were extracted with hexane and subjected to radio-gas chromatography. The total label recovered was 54,000 cpm, but most of this was in fatty acids, 1162 cpm were recovered in the heptadecenone peak (2.15% of recovered label, 0.26% of applied label) and 625 cpm in tridecanone (1.15% of recovered label, 0.14% of applied label). The experiments effectively proved the site of biosynthesis and showed the compounds were being synthesized from acetate units.

72

Chapter 5

5.2 RADIO-ISOTOPE LABELLING Radioactive isotopes were much used in biosynthetic studies, but stable isotopes are more used now because they can avoid the difficult degradation steps required with radio-labels, as will be illustrated below. Although some common compounds, e.g. 14C-labelled glucose can be purchased, frequently isotopically enriched compounds have to be synthesized from low molecular mass compounds such as 14C02,3H20, K14CN,or CH,35SHand the synthesis of the desired labelled compounds alone may be a major research task. Compounds can be either uniformly labelled or specifically labelled at known atoms. By growing a plant or green alga in the presence of 14C02,uniformly labelled glucose can be obtained, in which all the 12C carbon atoms have an equal probability of being replaced by 14C.More usual is to use a specifically labelled compound, such as [2-14C]-acetic acid, in which labelled atoms are found only in the methyl group. Specific labelling is used in experiments in which a biosynthetic route is suggested, a labelled compound is fed to the system and a plan of degradation of the product compound is made that will show that the radio-labelled atoms are in the expected places. For this, all the common degradative reactions of organic chemistry can be used, e.g. ozonolysis, decarboxylation, double bond cleavage, etc. and the Barbier-Wieland degradation of fatty acids, by which one carbon atom at a time is removed from the chain. The amount of radio-labelled compound obtained at the end of the experiment may be so little that more ‘cold’ or unlabelled final compound may have to be added to have enough to manipulate. In the studies of the biosynthesis of cholesterol (Chapter 7) unlabelled cholesterol for dilution is available in abundance. That is rarely so with insect substances, and a sample of the ‘cold’ target compound may have to be synthesized too. Sometimes it is useful to watch for the appearance of radioactivity in a particular gland or in a compound to obtain information about the sequence of events in biosynthesis. A hormone or pheromone may only be formed at a certain stage of development, or a gland may be activated. A technique not much used but potentially valuable is to take sections through an insect after it has imbibed a radioactive compound and expose the sections on a photographic plate to see in which organs the activity accumulates (whole body autoradiography).

73

Experimental Methods

5.2.1

Examples

The deduction of the biosynthesis of griseofulvin, though an early example, is typical. First was the hypothesis of the biosynthesis (Figure 5.2). This predicted which carbon atoms should be labelled if the compound was made using [l-14C]acetate.Figure 5.3 shows the degradation that was carried out by A. J. Birch and his group. Note the proportion of radioactivity in the various parts is important, and that the degradation of acetic acid formed by strong oxidation, via acetone and then iodoform gives the labelling of the carboxylate carbon atom. Such degradation studies are very skilled and time-consuming.

0 H3C0

Figure 5.2 The hypothesis for the biosynthesis of the antibiotic griseofulvin using sodium [l -14C]acetate, The black dots in the supposed intermediate polyketide and the product indicate which carbon atoms should be labelled

4oH CH3

Bk03

C-

H3C0 +

A = 1.03

OH

CI

OH

NO2

OH

3Ba503 A=0.97 OH

HO

NO2

HO

OH

+

3CBr3N02 A=O

CH~COOH

CH360CH3

+

I

Li2kO3 A = 1.05

I

oxidize

Baa3 A=O

JBa(OBr), 3Bak03 A=1.03

+ 3CBr3N02 A=O

Figure 5.3 The actual degradation made of radio-labelled griseofulvin. A indicates the relative radioactivity at each stage, to show that the labelling is as predicted by the hypothesis

74

Chapter 5

Today acetic acid enriched in 13C would probably be used, which would not require this lengthy degradation. One would look for stronger signals in the 13C NMR spectrum of the product compound for the marked carbon atoms and normal intensity for the unmarked carbon atoms, and also 13C-13Ccoupling (see later). The biosynthesis of the compound 2-hydroxy-6-methylacetophenone by the ant Rhytidoponera aciculata has been studied by Tecle, Brophy and Toia, (Insect Biochemistry, 1986, 16, 333) by injecting sodium [214C]acetateinto mealworm larvae and feeding these to the ants. The radioactive extract of ants was diluted with unlabelled 2-hydroxy-6methylacetophenone, which was then purified and isolated as its 2,4dinitrophenylhydrazone, to give a solid with which the radioactivity could be counted. The compound was oxidized with hydrogen peroxide to remove the acetyl group, which removed one unit of radioactivity. The 3-methylcatechol resulting was oxidized by the powerful Kuhn-Roth method which gives acetic acid from methyl groups while the rest of the molecule is oxidized to CO, (Figure 5.4). Sometimes double or even triple labelling can be useful. Some ponerine ants have dimethyl disulphide in their mandibular glands. Crewe and Ross (Insect Biochemistry, 1975,5, 839) used doubly labelled methionine to show that the methyl and sulphur were incorporated together into the dimethyl disulphide by Paltothyreus tarsatus ants (Figure 5 S).

radioactivity = t

602 1/5 of label

+ GO2

'CH3COOH 1/5 of label

'02

do,

3/5of label

Figure 5.4

The radio-labelling of hydroxymethylacetophenone by R. aciculata ants. Note that the method did not show exactly which carbon atoms were labelled, but did show that the label was consistent with a route through a polyketide

-- -

* ti,?

S-CHC ; H2-cc,ooH methionine

Figure 5.5

NH2

* H3d'S,*,CH3S dimethyl disulphide

Methionine labelled as shown with 14Cand 3sSindicated that the methylthiogroup was incorporated intact into dimethyl disulphide in ants

Experimental Methods

75

5.3 HEAVY ISOTOPE LABELLING Developments in mass spectrometry and NMR spectroscopy have made it more convenient in many cases to study biosynthetic processes with heavy isotopes. The most information can be obtained from a combined use of these methods. The position of labelling can then be deduced from intact molecules, without the degradation process illustrated with griseofulvinin Figure 5.3. Labelling with heavy isotopes however requires rather higher incorporation of the label for secure identification (Table 5.1). The use of l 8 0 in the initial stages of the study of photosynthesis provides a simple example how labelling can be informative. When CI8O2 was supplied to a plant in sunlight, none of the l 8 0 was found in the O2 evolved, but was recovered as H,180. When H2I80was used in the experiment, "0, was produced. The oxygen in the atmosphere is therefore derived from splitting water, not C 0 2(Figure 5.6). '1602

Figure 5.6

+

2H2'80

hv chlorophyll

+ (CH2160) + 1802+ H2l6O

The oxygen released in photosynthesis isfrom the splitting of water, demonstrated by using water containing the heavy isotope of oxygen. The formula (CH,O) represents carbohydrates

Deuterium labelling is the most useful for mass spectrometry. A single 2Hatom incorporated increases the intensity of the M+l ion. There must be sufficient ,H present to make the ion intensity greater than that due to the natural abundance of 13C(1.1Y0) and any other isotopes. Natural ,H (0.015%), 15N (0.36%) and 1 7 0 (0.04%) add little to the strength of the M+l ion, and I8O(0.2%) adds little to the M+2 ion. Incorporation of an intact C2H, group will produce a new ion at M+3 that can be readily seen and its intensity measured to give the degree of incorporation. A thorough knowledge of the capabilities of mass spectrometry and NMR spectroscopy is necessary to make good use of them in biosynthetic studies with heavy isotopes. It is very useful that 13C,"N and l 8 0 all have nuclear spins of n/2 (2H has a spin of 1, see Table 5. l), which means they can all be studied by NMR methods. Some examples only can be given here. A basic knowledge of mass spectrometry and 13C NMR spectroscopy is assumed; for background in this subject D. H. Williams and I. Fleming Spectroscopic methods in organic chemistry 5th Edition, 1995 is recommended.

5.3.1 Examples In Chapter 3 an example was given of the demonstration that isobutyric acid (and methacrylic acid) in an insect was derived from valine by

76

Chapter 5

deuterium labelling (Figure 3.13). In that case the isobutyric acid produced was converted to a less volatile ester of pentafluorobenzyl alcohol in order to show by mass spectrometry that the carbon chain was derived intact from valine. Analysis by gas chromatography and mass spectrometry gave a molecular ion seven mass units greater than 268, so seven deuterium atoms were still present in the isobutyric acid (Figure 5.7)

pentafluorobenzyl isobutyrate mot. mass 268

Figure 5.7

pentafluorobenzyl heptadeuterylisobutyrate mol. mass 275

The ester of isobutyric acid made from fully deuterated valine had a mass of 275, seven units greater than the unlabelled ester, therefore seven deuterium atoms were present in the isobutyric acid

We can set up an imaginary experiment to show that the methyl ester group of methyl 6-methylsalicylate, the trail pheromone of the ant Tetramorium impurum is derived from S-adenosylmethionine (Figure 2.16). First [2H3]-S-adenosylmethionine(indirectly from Sadenosylhomocysteine and ['H,]-rnethyl iodide) is prepared, added to a tissue culture of the excised poison glands of the ant and the two incubated together. There is always a judgement to be made between adding enough labelled compound to get good incorporation of the label, and adding too much so that the metabolism of the tissue is altered. It is often easier to show that a compound can serve as a precursor to the target compound than to show that it is the normal, natural intermediate in the biosynthetic path to the target. The products from the incubation is extracted with hexane and examined by gas chromatography-mass spectrometry. Part of the mass spectrum of the normal pheromone (Figure 5.8) shows the molecular ion at m/z 166 and the daughter ion at m/z 134 formed by loss of methanol. This is known to occur from the methyl ester and the phenol groups together. The product after incubation with deuterated S-adenosylmethionine shows a new peak at m/z 169 (M + 3), and the peak at m/z 166 is correspondingly reduced in abundance. The ion at m/z 134 is unaffected, showing that all the deuterium is lost with the methyl ester group. Note that the molecules with three deuterium atoms will elute slightly earlier from the gas chromatograph. This is because the C-D bond is slightly shorter and stronger than the C-H bond, causing the deuterium-containing molecules to be slightly more volatile (an isotope effect).

Experimental Methods

77 134

c

160

140

rnh

.

I

.

140

.

1

I

'

160

rn/z

Figure 5.8 Part of the mass spectrum of the pheromone methyl 6-methylsalicylate, and the spectrum after incubation with deuterated S-adenosylmethionine, showing molecular ions at 166 and 169 in a ratio of 8 5 3

The tri-deuterated S-adenosylmethionine is almost 100% pure. Essentially all the hydrogens in the methyl group have been replaced by deuterium. How much of the deuterated methyl group finds its way into methyl 6-methylsalicylate may be very small. Isotopic enrichment refers to the change in isotope content in the biosynthesized compound above the natural abundance and is usually expressed as atom YOexcess. The isotopic enrichment of the methyl group in the pheromone can be estimated from the relative abundances of the peaks at m/z 166 and 169 (Figure 5.8). The ratio is 1:0.18. The enrichment is then 15%. Alternatively, the experiment can be followed by 13C NMR spectroscopy. We can observe 13C-2Hcoupling in the proton-decoupled spectrum. The appearance of the total spectrum is shown in Figure 5.9 (a).The effect of substitution of one, two or three hydrogens in a methyl group by deuterium is shown at (b), and the methyl ester portion of the spectrum after incubation with deuterated S-adenosylmethionine is given at (c). As 85% of the molecules are unlabelled the original peak is strong with a weaker deuterium-coupled septuplet beside it, showing that three deuterium atoms are incorporated into 15% of the molecules. The NMR experiment requires rather more material because 13Cspectra are naturally weak (natural abundance of 13Cis l.lY~),but if the shifts of all the carbon atoms of the molecule are known, deducing the position of labelling can be quite simple. An interesting example of the power of 13C NMR spectroscopy to solve a biosynthesis problem is found in the study of virginae butanolide A (VBA) (Figure 5.10 (a)), one of a series of similar signalling compounds produced by the bacterium Streptomyces antibioticus (S. Sakuda and Y. Yamada, Comprehensive Natural Products Chemistry, Vol. I, Pergamon Press, Oxford, 1999, p. 139). First the culture conditions for the Streptomyces to produce a good yield of VBA had to be studied, then

78

Chapter 5

Figure 5.9 The use of I3C NMR spectroscopy in demonstrating the labelling of the methyl ester of methyl 6-methylsalicylate. ( a ) The proton-decoupled spectrum of the unlabelled compound. ( b ) Substitution of deuterium for hydrogen on a methyl group causes '3C-2Hcoupling and a small upjield shift (0.9 H z ) for CD,. (c). The appearance of the ester methyl of the compound after 15% incorporation of deuterium as in Figure 5.8. The coupled CD, and singlet CH, almost overlap

the conditions for its isolation, and the best conditions for feeding with sodium acetate. Then a mixture of sodium [1-13C]acetate,[2-I3C]acetate and unlabelled acetate were fed to the bacterium. The 13CNMR spectrum of the isolated product showed labelling in carbon atoms 1,2,6 and 7 only (Figure 5.10 (6)). Therefore, these atoms were derived from intact acetate units. The result suggested an experiment with [ 1-13C]isovaleric acid, which gave an enriched NMR peak for carbon atom 8 (Figure 5.10 (c)). The experimenters then thought that the perhaps the remaining three atoms came from glycerol, so they synthesized glycerol labelled at carbon atoms 1 and 3. As expected the NMR spectrum then showed enhanced signals at carbon atoms 4 and 5 of VBA (Figure 5.10 (4).This time there was some labelling at carbon atoms 2 and 7 because there was

virginae butanolide A (VBA)

Figure 5.10 ( a ) The structure and numbering of virginae butanolide A. ( b ) Labelling introduced by supplying a mixture of '3CH3COOH,CH3I3COOHand CH,COOH. ( c ) Labelling with [I-'3C]isovaleric acid. ( d ) Efect offeeding with [l,3-13C2]glycerol.( e ) Incorporation intact of a ready-made P-ketoester

Experimental Methods

79

some degradation of the glycerol to l3CH3COOH,and incorporation of this (Figure 5.10 (6)). The 13C NMR spectrum does not tell whether atoms 4 and 5 are in the same molecule, an important point for a rigorous proof, but the mass spectrum did show that molecules of VBA were either unlabelled or twice-labelled with 13C. Finally to show that the isovaleric acid and the acetate units were linked into a kind of polyketide before linking to glycerol, a di-labelled thioester (Figure 5.10 (e)) was prepared (to resemble a CoA thioester) with two adjacent 13Catoms. The 13CNMR spectrum of unlabelled VBA is shown in Figure 5.1 1 (a), with the same spectrum (on a much smaller sample) after labelling with [l,3-'3C,]glycerol(Figure 5.1 1 (6)). As well as the enhanced intensity of the C-4 and C-5 atoms (about 6.2%), there was unexpected enrichment of

11 & 1 2 1

"

k

n 70

60

50

ppm

Figure 5.11

( a ) The 13CN M R spectrum of VBA, with the carbon atom numbering. Unnumbered lines are solvent or impurities. ( b ) The spectrum after a labelling experiment with [1,3-13C,]glycerol.Labelling of atoms2 and 7 (less than that of 4 and 5 ) was caused by degradation of glycerol to acetate and incorporation of that. ( c ) Part of the spectrum showing coupling between adjacent 13C atoms that shows that the P-ketoester was incorporated intact

(Adapted from S. Sakuda and Y. Yamada, Comprehensive Natural Products Chemistry, Vol. I, p. 139. Pergamon, Oxford 1999)

80

Chapter 5

atoms 2 and 7 (4.5 and 3.7% respectively). This was attributed to breakdown of glycerol to acetate and incorporation of this into VBA. Two adjacent atoms, as in the P-ketoester, gives, on incorporation intact, 13C13Ccoupling, and in the NMR spectrum of the resulting VBA this coupling could be seen, with the doublets superimposed on the unenriched singlets (Figure 5.1 1 (c)). The researchers even went on to show that the glycerol was incorporated as dihydroxyacetone, and other finer points. 5.3.2

Carpophilus Beetle Pheromone

The biosynthesis of the branched acetogenin (2E,4E,69-5-ethyl-3methyl-2,4,6-nonatriene from Carpophilus freemani beetles (Chapter 4 and Figure 4.1 1) provides another good example of deuterium labelling. Biosynthesis of this compound was studied initially by mass spectrometry, and required deuterated acetic, propionic and butyric acids, as well as synthetic model compounds deuterated in selected methyl groups to understand the mass spectrometric fragmentation of the pheromone (Petroski, Bartelt and Weisleder, Insect Biochemistry and Molecular Biology, 1994,24, 69). They found the beetles were able to tolerate relatively large amounts of the deuterated acids in their diet (up to 5% of the wet weight of the diet) and produce as much as 100-250 ng of pheromone per beetle per day. This was collected by trapping from the air, and gave them sufficient labelled pheromone to be able to use 'H and 13CNMR spectroscopy as well. They were able to show that acetic, propionic and butyric acids were all incorporated into the pheromone compound. By mass spectrometry as much as 26 to 49% of the pheromone was labelled with CD3when propionic acid was used. The picture was more complicated with butyric and acetic acids. Their experiments with feeding [13C]aceticacid were not as successful because much of the label was scrambled by being broken down and re-synthesized into a number of compounds. 5.3.3

13C-13CCoupling

In 13C NMR spectra of normal abundance, or even for moderately enriched compounds, the probability of two I3Catoms being next to each other in a molecule is extremely small, so no coupling between 13Catoms is detectable. The 13C nucleus has a spin of 1/2, so it couples like 'H nuclei. Two adjacent 13Catoms each appear as doublets in the (protondecoupled) 13Cspectrum. Taking advantage of 13C-13Ccoupling can be a neat way of showing that a precursor is incorporated intact in a more elaborate molecule, and that the label has not been scrambled. The

Experimental Methods

81

example of virginae butanolide A (VBA, above) illustrates this. In Figure 5.10 (e) a P-keto-thioester was synthesized with C-2 and C-3 labelled with 13C, used in biosynthetic experiments and the VBA isolated. The NMR spectrum of the resulting VBA (Figure 5.11 (c)) showed by the coupled carbon atoms at 47 ppm and 73 ppm that these two labels were still together in the VBA. If the intermediate thioester had been degraded to acetate and then re-incorporated into VBA, there could have been I3C atoms in it but the probability of the two still being in the same molecule and still next to each other would be very small and no 13C-13Ccoupling would have been visible. Doubly labelled acetic acid, '3CH3'3COOH, also gives 13C-13Ccoupling, so whenever it is used and an acetate unit is incorporated intact, this coupling will be seen. It is a useful technique to detect bond-breaking and re-arrangement of the carbon chain.

5.4 ISOTOPE EFFECTS Two effects of replacing hydrogen by deuterium have already been seen. The first concerns volatility. The C-D bond is shorter than the C-H bond, so that deuterated compounds are slightly more volatile and elute towards the front edge of a gas chromatographic peak. The methyl ester of fully deuterated stearic acid elutes as a separate peak before normal methyl stearate. The second example arises in NMR spectroscopy. Not only do 'H and 2H resonate at different radio-frequencies and have different numbers of spin states, but I3Cattached to 2H has a slightly different chemical shift from 13Cattached to 'H (Figure 5.9(c)). Many other isotope effects could be mentioned. It is curious to discover that D 2 0does not support animal or plant life because of the small differences in bond energies.

5.4.1 Kinetic Isotope Effects From quantum mechanics it can be shown that increasing the mass of an atom bonded in a molecule increases the energy between ground and transition states. In other words, the bond with the heavier atom is less easily broken. The rate of reaction of C-D bonds is often four to eight times slower than the reaction of C-H bonds, but it can vary widely, depending upon the bond type. Use can be made of this difference in studying the mechanism of biochemical reactions. For example, in the formation of a double bond in stearic acid (Figure 3.6), by labelling either C-9 or C-10 with two deuterium atoms each, it is found that the rate of formation of oleic acid decreases sharply with deuterium on C-9, showing that the slow, rate-determining step is the breaking of a C-H (or C-D) bond on carbon atom number 9.

82

Chapter 5

Stereochemistry can similarly be studied by deuterium labelling of the pro-R hydrogens on C-9 and C-10 of stearic acid and showing that these are both lost by a syn-elimination to give oleic acid. This is how the stereochemistry given in Figure 3.8 was determined. Similarly our knowledge of the stereochemistry of alcohol oxidation with NAD+, and carbonyl reduction with NADH comes from deuterium labelling (Figure 2.8).

5.5 ANALYTICAL ASPECTS The technique of gas chromatography linked to mass spectrometry is an important tool in the study of volatile insect substances and also for the study of their biosynthesis. The subject of linked chromatographic-mass spectrometric methods, and the newer techniques that can be achieved with mass spectrometry are beyond the subject of this book. We can expect to see much wider use of high resolution mass spectrometry in the study of enzymes and biosynthetic pathways. The position of double bonds in alkenes can be learned by reacting them with dimethyl disulphide (Figure 5.12), the products have characteristic strong fragment ions in their mass spectra, corresponding to cleavage at the S-C-C-S bond. Another way is through chemical ionization mass spectrometry, using special reagents such as acetonitrile.

Figure 5.12

The use of dimethyl disulphide to determine double bondpositions by gas chromatography-mass spectrometry

The position of methyl branches in mono-methylalkanes can also be deduced from mass spectra. As one progresses to more methyl groups per molecule, interpretation becomes more difficult. For a detailed consideration of methylalkane identification see Carlson, Bernier and Sutton, Journal of Chemical Ecology, 1998,24, 1845.

5.6 CHIRALITY Chirality of molecules is an important aspect of their structure in all fields, no less among insect substances. Many pheromones and hormones are chiral, and the behavioural response they produce may vary greatly with the chiral form. For example, the unnatural enantiomer of a pheromone can inhibit completely the response to the natural enantiomer, the natural pheromone may be a blend of unequal proportions of

Experimental Methods

83

two enantiomers, or the response may be equal for both enantiomers. These and several other possibilities have been observed in practice. For any given pheromone compound, it is impossible to predict which enantiomer will be active or whether one or both or a blend of enantiomers will be the naturally occurring substance. It is therefore important to determine chirality of the natural compound or mixture. There are several ways to do this. One is through recording the NMR spectrum with the aid of a chiral shift reagent. Another is by chromatography on a chiral stationary phase in gas or liquid chromatography. With some luck and skill, the enantiomers will be separated by chromatography on a chiral phase. Another is by chromatography of a derivative of the compound made with a chiral reagent. This gives two separable diastereomeric products. Usually it is also necessary to have at least one enantiomer of known chirality for comparison for either of these chromatographic methods. Another possibility is to treat the compound with an enzyme where the reaction proceeds with known stereochemistry. It is rare that an insect substance is available in sufficient quantity to determine its optical rotation, and in some cases, at least, the rotation is very small. Optical rotatory dispersion (ORD) at short UV wavelength, or, if the molecule has a UV chromophore, circular dichroism (CD) can be more useful. These techniques are little used with insects. Frequently all possible isomers and enantiomers have to be selectively synthesized for comparison with the natural compound by gas chromatography.

BACKGROUND AND FURTHER READING I. M. Campbell, Incorporation and dilution values - their calculation in mass spectrally assayed stable isotape labelling experiments. Bioorganic chemistry, 1974, 3, 386-397. L. M. Harwood and T. D. W. Claridge, Introduction to Organic Spectroscopy, Oxford Science Publications, Oxford, 1997, pp. 93 (more elementary spectroscopy). P. Howse, I. Stevens and 0. Jones, Insect Pheromones and Their Use in Pest Management, Chapman and Hall, London, 1998, pp. 369 (Chapters 6 & 8, isolation and structure of pheromones). K. Mori, Overview, in Comprehensive Natural Products Chemistry, Vol. 8, p. 1, Pergamon Press, Oxford, 1999 (chirality). D. H. Williams and I. Fleming, Spectroscopic Methods in Organic Chemistry, 5th Edition, McGraw-Hill Books, London, 1995, pp. 329 (mass spectrometry and nuclear magnetic resonance spectroscopy).

Chapter 5

84

QUESTIONS 1. A total amount of sodium l-[14C]acetatecorresponding to 680,000 cpm was injected into a number of newly emerged female silk moths. After 24 h the moths were killed and the lipids collected. The total radioactivity in the lipids was 48,900 cpm. After removal of the triglycerides and free fatty aids, chromatographic purification gave bombykol of activity of 78 cpm. What was the specific incorporation of radioactivity into the lipids and bombykol? Was this sufficient incorporation into bombykol to study the position of the labelling? 2. If the biosynthesis of 2-heptanone (Figure 4.6) was studied with [ 1-I4C]aceticacid, show where the label should appear in the product. 3. Perform the same exercise with [l-I4C]aceticacid on the synthesis of 6-methyl-3-octanoneand show where the labelling should be found. 4. In a study of the production of the opilionid defensive compound 4-methyl-3-hexanone, excised glands were incubated with sodium 2,3['3C]propionicacid. When later the 4-methyl-3-hexanonewas isolated, there was enhancement of the abundance of I3Cin the compound but no l3C-I3Ccoupling was observed in the 13CNMR spectrum. What would you conclude about the experiment? 5. Write an equation for the reaction of 8-heptadecene with dimethyl disulphide and calculate the masses of the two fragment ions that will be diagnostic. 6. Stegobinone (below) is the sex pheromone of the female drugstore beetle Stegobium paniceum. Work out a possible biosynthetic origin. Propose how this might be proved, given access to deuterium labelling methods to label the proposed building blocks. 0

stego binone

7. Pieces of the abdominal integument (cuticle and attached tissues) from seven-day-old female cockroaches Blatta gevmanica were incubated with sodium [l-I4C]propionate (0.13 pCi) and after eight hours the hydrocarbons were extracted, and found to give 60 x lo3 cpm due to incorporation of propionic acid into the methyl-branched hydrocarbons. What is the specific incorporation into hydrocarbons?

CHAPTER 6

Many of the compounds from plants, important in food, flavours, perfumes and colour, belong to the group called terpenes, made up of five-carbon fragments. Ruzicka in 1922 proposed that the basic building block of the terpenes is an isoprene unit (Figure 6. I), and later Robinson proposed that these isoprene units are joined head-to-tail. The terpenes are sub-divided into groups by their number of isoprene units. Monoterpenes (Cl0compounds) contain two isoprene units, sesquiterpenes (C,5 compounds) contain three isoprenes, diterpenes (C20)four units, triterpenes (C30)six units and tetraterpenes (C4J eight units. There are a smaller number of five-isoprene-unit compounds called sesterterpenes. Some simple examples of terpenes are shown in Figure 6.1, with their structures dissected into isoprene units. Although mainly plant substances, terpenes are frequently found in insects, as pheromones, defensive secretions and a hormone. Our understanding of terpenoid biosynthesis was greatly helped by the efforts of Cornforth, Popjak, Lynen and others on the biosynthesis of cholesterol in the 1950s and 60s.

6.1 MONOTERPENE BIOSYNTHESIS In 1937 it was shown that labelled acetate gave labelled terpenes, but evidently by a different route from the fatty acids and acetogenins. In 1956 it was accidentally found that a substance called mevalonic acid (Figure 6.2) was an intermediate between acetate and terpenes, and this gave the clue needed to study their biosynthesis. In the first step, acetate (as a coenzyme A thioester) plus malonate gives acetoacetate, just as for the fatty acids. The next step is a different kind of reaction. Another acetate unit (as malonate) is added via an aldol condensation, using a HMG-CoA synthase enzyme, to give P-hydroxy-Pmethylglutaryl coenzyme A (HMG-CoA). This is reduced by NADPH with a HMG-CoA reductase to mevalonic acid (MVA) in the ratedetermining step for the whole sequence of reactions that builds up the 85

Chapter 6

86 9

3

or

H2c&,cm2

isoprene

(3-myrcene from myrrh and bay leaves a monoterpene

frans-p-ocimene widepread in plants epecially basil

geraniol from oil of roses

'--"I

L [ & O H

' OH linalool in cinnamon, orange flowers, and bergamot

citronellol in lemon peel and rose oil

-b

$

--

-(TO

OH

H i

H i

H i

n

A \

(+)-limonene in lemon oil

nerol from orange blossom oiloH and usually with geraniol

A \

menthol, in Peppermint oil

carvone in spearmint oil

A & p , & O H

zingiberene from oil of ginger, a sesquiterpene

from lily-of-the-valley, farnesol, a sesquiterpene

Figure 6.1 Isoprene and some examples of simple terpenes, showing the linkage between their isoprene units. Note that some of these substances have chiral centres

0

Claisen

H3$"s,CoA

condensatioe aldol

' b0 ~condensation ' ~ ~ ~0 H3cf

H+ -0oc&~,Co,4 HQ.. CH30

k,

CH2 S' CoA

A-0

HMG-COA

I

MVA

t H20

NADPH + H+

(rate-determining step)

0

CH

+ C02 +

a Hs

0-

0-

isopentenyl pyrophosphate

IPP

L

H3C

0

t

w VO, 0-

0

t

?OH 0

dimethallyl pyrophosphate DMAPP

Figure 6.2 The early stages in terpene biosynthesis to give the two building blocks, IPP and DMAPP Here the diphosphate or pyrophosphate groups are shown in full, in subsequentjgures they are represented by OPOP

87

Terpenes

terpenes. Mevalonic acid is converted to the pyrophosphate (also called diphosphate) and then decarboxylated with simultaneous loss of hydroxyl, to give isopentenyl pyrophosphate (IPP). Three enzymes are required here. IPP is the basic building block of the terpene series, but one more reaction, on an isomerase enzyme, is required to make dimethally1 pyrophosphate (DMAPP), the starting unit for terpenes. Note that labelling experiments with deuterium and tritium have shown that it is the pro-chiral H, of IPP (from the back of the molecule as drawn) that is removed in this step. The combination of isopentenyl pyrophosphate (IPP) and dimethallyl pyrophosphate (DMAPP) on prenyl transferase gives geranyl pyrophosphate, the parent of all the monoterpenes. The IPP is added from above to the DMAPP as phosphate is eliminated from below, so this centre is inverted. The pro-chiral H, on IPP is eliminated from the resulting carbocation (Figure 6.3).

___)

base DMAPP

geranyl pyrophosphate

Figure 6.3 The condensation of IPP and D M A P P to give geranylpyrophosphate. The prochiral hydrogens are here labelled A , B, C and D because they change their chiral labels during the reaction

Hydrolysis of the pyrophosphate with pyrophosphatase gives geraniol (Figure 6.4) directly. Cleavage of the carbon-oxygen bond gives a carbocation and pyrophosphate ion. The carbocation can undergo various changes to provide a number of monoterpene compounds (Figure 6.4). Mevalonic acid, the key intermediate in solving the terpene biosynthesis pathway is an oily liquid, in solution it is in equilibrium with mevalonolactone (Figure 6.5), a crystalline solid, so the latter is used in biosynthetic studies. Fluoromevalonolactone, and the corresponding acid, are powerful inhibitors of terpene formation. If addition of fluoromevalonate to a biosynthesizing system blocks the formation of a compound, then it can be concluded that that compound has a terpene origin. 6.1.1 The Methylerythritol Phosphate Pathway

It was discovered in 1995 by M. Rohmer that micro-organisms, green algae and plastids (membrane-bound organelles of plants, e.g. chloroplasts)

Chapter 6

88 L

O

P

1

O

rearrangement

P

hydrolysis reduction

\

O -H

U

P

linalyl pyrophosphate

geraniol

hydrolysis

O -H citronellol

1

oxidation

U

C

linalool H

citronella1

O

6

I

b'

A \

&OH

I irnonene

borneol

Figure 6.4 The derivation of some simple monoterpenes from geranyl pyrophosphate HO C , HF ,

b mevalonic acid

mevalonolactone

o

6-f Iuoromevalonolactone

Figure 6.5 The key compounds mevalonic acid and its lactone, withjluoromevalonolactone, an inhibitor of the terpene pathway

use a different pathway to mevalonic acid, as outlined in Figure 6.6. It is not surprising that in plants, where sugars are abundant, the starting materials are sugar derivatives. The process begins with pyruvic acid being decarboxylated using thiamine diphosphate (Chapter 2) and the intermediate being condensed with glyceraldehyde 3-phosphate (GAP) (Figure 2.20) to give I -deoxy-D-xylulose 5-phosphate (DXP). This undergoes rearrangement and reduction to methylerythritol4-phosphate. Details of the steps between methylerythritol4-phosphate and isopentenyl phosphate have not yet been studied in detail. The discovery of a new route to terpenes so long after the original elucidation of their formation from acetate and malonate was a great surprise, and has altered our concept of plant terpene production. Incidentally, DXP is also an intermediate in the synthesis of both thiamine and pyridoxal (Chapter 2).

Terpenes

89 CH

0

4COOH

CH,

-co2

pyruvic acid

'. ct- @--

- 6 5-pJH -

thiamine

-N:l

s

H3C-q OH CH-OH H2C,

o@

W, HZC,

O-@

043

DXP

GAP

IPP

Figure 6.6

0p -phosphate

OH OH rnethylerythritol phosphate MEP

The methylerythritol 4-phosphate route to isopentenyl pyrophosphate and terpenes in plant plastids and bacteria. The intermediate in brackets is immediately reduced on the same enzyme which catalyzes the previous rearrangement

6.2 MONOTERPENE PHEROMONES

A large number of insect pheromone compounds are either simple terpenes or are derived from them. The few examples of monoterpenes given in Figure 6.4 illustrates the variety of structures that can be made from geranyl pyrophosphate or linalyl pyrophosphate without much further reaction. Both the Coleoptera and the Hymenoptera make frequent use of terpenes as secretory substances. Bark beetles have particularly employed monoterpenes in their aggregation pheromones. The males of the cotton boll weevil (Anthonornus grandis, Coleoptera, Plate 6), a serious pest of cotton, produce a sex pheromone to attract females. The pheromone, known as grandlure, consists of a mixture of at least four monoterpenes (Figure 6.7). The males make these compounds from geraniol and nerol (the cis-isomer of geraniol), present in the cotton buds on which they feed.

nerol

Figure 6.7

B

C

D

The aggregation pheromone of male Anthonomis grandis made from geraniol and nerol. Thefour compounds A , B, C and D together make up grandlure

90

Chupter 6

It is often difficult to know whether terpene compounds found in insects are made de novo by the insect or whether the insect has altered some terpenes it finds in its food or environment. The boll weevil is known to alter plant terpenes from cotton. Ips beetles which bore under the bark of pine trees produce an aggregation pheromone consisting of ipsdienol or ipsenol (Figure 6.8) which were thought to be conversion products of myrcene from the pine tree. However studies with radiolabelled acetate and mevalonate showed that at least two species, Ips paraconfusus and Ips pini were able to synthesize their pheromones ‘from scratch’. On the other hand, both males and females of Ipsparaconfusus convert a-pinene from the tree to cis-verbenol, which adds to the attraction of the male-produced pheromone.

myrcene

\( S)-ipsdienol (R)-ipsdienol,

Y

in pine trees

(q-ipsenoi

a-~inene

cis-verbenol

Ips phi

Ips paraconfusus

Figure 6.8 The structure of myrcene and the aggregation pheromones of two species of Ips beetles that are now found not to be made from plant-derived myrcene, but Ips paraconfusus males and females convert a-pinene to cis-verbenol

The southern pine beetle Dendroctonusfrontalis is another bark beetle with an aggregation pheromone for which the origin is difficult to see. Taken stepwise, it can be seen to derive from geraniol (Figure 6.9). The mountain pine beetle Dendroctonus ponderosae and other species use exo-brevicomin (Figure 6.10) with a quite different origin in the acetogenin (q-6-nonen-2-one. By labelling the ketone with I8O, and a very detailed argument, it was shown that the epoxide was not opened first but that the carbonyl oxygen atttacked the epoxide, much as shown in Figure 6.10. Both enantiomers of the epoxide were formed but males favoured making brevicomin from the (6S,7R)-keto-epoxide, while the females preferred the (6R,7S)-keto-epoxide as intermediate. The full

geraniol

frontalin

Figure 6.9 The formation of frontalin from geraniol through loss of two carbon atoms from the hydroxyl end, oxidation of a double bond to an epoxide and then ring opening and ketal formation

Terpen es

91

9 0

frontalin drawn similarly for comparison

a-

3-

+

excFbrevicomin

Figure 6.10 Theformation of exo-brevicomin (not a terpene). The retention of labelled oxygen from the ketone to brevicomin indicated the epoxide was not openedfirst. The stereochemistry is more complex than shown here

details are given by Vanderwel and Oehlschlager, Journal of the American Chemical Society, 1992,114, 508 1. The Nasanov gland in the abdomen of honeybees provides an attractant pheromone for marking food sources and the nest. The gland contains geraniol which is enzymically oxidized on release so that the emitted pheromone contains geraniol, geranial, neral, geranic and nerolic acids. Citronellol (Figure 6.4) is a sex attractant produced by male spider mites. The cheese or house mite Tyrophagusputrescentiae (Figure 6.1 1) normally form clusters, but if one of them is crushed, the formate ester of nerol (Figure 6.7) is released and they disperse. Many other oxygenated compounds, clearly monoterpene derivatives, are known from insects. Some are pheromones, for others their purpose is not always known.

6.3 MONOTERPENE DEFENSIVE COMPOUNDS The soldier caste of many termites contains head glands filled with secretion which they spray at invaders of their nests. Terpenes are important constituents. Among the monoterpenes are myrcene, ocimene and limonene (Figure 6. l), a-pinene (Figure 6.8) and P-pinene. 6.3.1 Iridoids

In 1949 Pavan isolated the substance he called iridomyrmecin from the Argentine ant Iridomyrmex humilis (now Linepithema humile). Iridomyrmecin, dolichodial and other monoterpenes with a methylcyclopentanoid structure are called iridoids (Figure 6.12). They act as defensive compounds in ants, stick insects, rove beetles and leaf beetle larvae. Some of the group (e.g. the isomer of nepetalactone shown in Figure 6.12) are also sex pheromones in aphids. Reaction of iridomyrmecin with

Chapter 6

92

Figure 6.11 A scanning electron micrograph of the cheese mite Tyrophagus putrescentiae (source unknown) H

H

HO

'

HO-

0

H iridomyrmecin

chrysomelidial

dolichodial H H

anisomor~hal

a 0

nepetalactone

actinidine

Figure 6.12 Some examples of iridoids. Iridomyrmecin and dolichodial are from ants, chrysomelidial from a leaf beetle larva, anisomorphal from a stick insect, nepetalactone from aphids and the catnip plant, loganinfrom Strychnosfruits and Hydrangea bark, and actinidine accompanies iridoids in many species

ammonia gives actinidine, an insect alkaloid (see Chapter 9). Actinidine has an effect on the central nervous system of cats. Iridoids (including iridomyrmecin and nepetolactone) and iridoid

Terpenes

93

glycosides have subsequently been found in many plants, where they presumably provide defense against insects. Nepetalactone from the catnip plant Nepeta cataria also excites members of the cat family (domestic cats, lions and jaguars but not tigers). Loganin from Strychnos nuxvomica is an example of a plant iridoid glycoside. Since many insects that have iridoids do not feed on iridoid-containing plants, the question arises whether the insects make the compounds entirely themselves or whether they transform simple monoterpenes to iridoids. First ['4C]me~alonolactone was shown to be incorporated into anisomorphal by the stick insect Anisomorpha buprestoides. Then using pentadeuteriomevalonolactone the biosynthesis was studied in leaf beetle larvae (Figure 6.13). The labelled intermediate was first painted onto a cabbage leaf fed to the larvae of Phaedon amoraciae and the defensive secretion collected and analyzed by GC-MS. The mass spectrum showed that at least one unit of deuterated mevalonolactone was incorporated into chrysomelidial. This experiment did not show where the biosynthesis was taking place, so in another experiment a droplet of water containing the labelled compound was placed directly onto a pair of their defensive glands (there are nine pairs on abdominal segments). Now, in addition to some unmetabolized D,-mevalonolactone, a peak for chrysomelidial with a small shoulder (1-5%) for the deuterated compound containing seven deuterium atoms was seen (Oldham, Veith and Boland, Naturwissenschaften, 1996,83,470). In other experiments with four species of leaf beetle it was shown that the early stages of oxidation take place in the gland cells and follow the same route as in plants, that is, the terminal methyl of geraniol is oxidized to alcohol. The molecule is then conjugated to glucose and transported out of the cell into the gland reservoir. It is difficult to study glucosides because they are so easily hydrolyzed, so model experiments are usually made with thioglucosides, which are not hydrolyzed by glucosidases. There are enzymes in the reservoir. The glucoside there is hydrolyzed back to 8-hydroxygeraniol by P-glucosidase and then both hydroxyls are oxidized by an oxidase and oxygen to 8-oxocitral by removal of the pro-R hydrogens at C-1 and C-8, as in Figure 6.14. The

Figure 6.13 The formation of D,-chrysomelidial from D,-mevalonolactone in Phaedon larvae defensive glands

Chapter 6

94

OH

transfer

glucosidase

___)

to reservoir

CHO

chrysornelidial

plagiodial

Figure 6.14 The sequence of reactions from hydroxygeraniol to iridoids, deduced from studies with labelled compounds, in the defensive glands of beetle larvae

D5-norgeraniol

deuterated diol

Figure 6.15 The incorporation of a deuterated norgeraniol and its corresponding diol into a deuterated noriridoid in leaf beetles

oxocitral in some species is cyclized directly to chrysomelidial, but in Phaedon it is first converted to plagiodial and then isomerization of the double bond gives chrysomelidial. In this work the synthetic norgeraniol (Figure 6.19, rather than labelled geraniol was used to make it easier to separate the labelled product and analyze it.

6.3.2 Degraded Terpenes Sometimes with oxygenated compounds not having 10, 15 or 20 carbon atoms, it is difficult to see if they have a terpenoid or polyketide origin. 6-Methyl-6-hepten-2-oneand its corresponding alcohol (also known as sulcatol) occur widely as insect pheromones. Seen as direct retro-aldol products from geranial (Figure 6.16) their terpene origin is clear. By analogy with the formation of brevicomin (Figure 6. lo), the bicyclic ketal in Figure 6.16 can be seen as formed from 6-methyl-6-hepten-2-one. A whole series of these compounds have been found in the army ant Aenictus rotundatus and in some stingless bees. 6.4 SESQUITERPENES

Addition of another isopentenyl pyrophosphate (IPP) to geranyl pyrophosphate with prenyl transferase enzyme, in the same way that gave

95

Terpenes

Figure 6.16 How 6-methyl-6-hepten-2-one is a terpene derivative and how thefamily of bicyclic ketals may be formed by retro-aldol reaction and epoxidation from terpenes

geranyl PP

farnesyl pyrophosphate

farnesol

a-farnesene

B-farnesene

nerolidol

oH

Figure 6.17 The formation of farnesylpyrophosphate by addition of another molecule of isopentenyl pyrophophate (IPP)to geranyl pyrophosphate, and some simple sesquiterpenes

geranyl pyrophosphate, gives farnesyl pyrophosphate, the parent of the sesquiterpenes (Figure 6.17). Hydrolysis, or rearrangement followed by hydrolysis, or dehydration leads to a number of products as for the monoterpenes. By suitable folding, further cyclic products are obtained. Some monocyclic sesquiterpenes are shown in Figure 6.18. There are many bicyclic (and more complex) sesquiterpenes in plants. Juvabione comes from some species of fir tree, particularly the American Abies balsamea. It was accidentally discovered to be a mimic of insect juvenile hormone (see below) but it only affects the growth of a small number of insects. Dendrolasin, a defensive compound from the mandibular glands of an ant, may be seen as an oxidation product of p-farnesene. Labelled sodium acetate, sodium mevalonate and glucose were all incorporated into dendrolasin by the ant, but the label was scattered over all the carbon atoms, so that firm conclusions about the biosynthetic route could not be made. The aggregation pheromone of the rusty grain beetle Cryptolestes ferrugineus is a mixture of large ring lactones called cucujolides

96

p- ci'l-@ /

I

/

Chapter 6

/

0

OPOP

farnesyl PP

a-bisabolol used in perfumery

juvabione

COOH dendrolasin, alarm substance of Lasius ants

Figure 6.18 Some monocyclic sesquiterpenes

cucujolide I

t

cucujolide ti

Figure 6.19 Cucujolides I and II. Whenfarnesene was labelled with deuterium and l80on C-1, both isotopes were incorporated into cucujolide I. Cucujolide 11 is synthesized from an unsaturated acid, possibly oleic. Labelled hydroxyl oxygen is retained in the Eactone oxygen of the product

(Figure 6.19). Cucujolide I might not at first look like a farnesene derivative, but when farnesene was labelled with deuterium and l8O,both were incorporated into the product, indicating the biosynthetic plan shown, which requires oxidation at the terminal double bond to a carboxylic acid, However cucujolide I1 has no methyl branches and biosynthetic studies show that it has a fatty acid origin (see Figure 3.38). Neither lauric (dodecanoic) nor 11-hydroxydodecanoic acid were effective precursors of cucujolide 11, but 3-(Z)-dodecenoic and 1 1-hydroxy-3-(Z)dodecenoic acids were. It seems therefore that oleic or palmitoleic acids are the natural precursors. These are chain-shortened to a 3-dodecenoic acid (Figure 6.19). 6.4.1 Sesquiterpene Pheromones Simple sesquiterpenes like farnesols, dihydrofarnesols and nerolidols, esters of these and farnesenes are used as pheromones. (E)-P-Farnesene is an alarm pheromone of some aphids (Figure 6.20); nomadone, slightly more modified, comes from the mandibular glands of Nomada bees. Its function is unknown. More complex are the monocyclic periplanones A and B, sex attractants from the females of the American cockroach Periplaneta americana (Figure 6.20). Periplanone A is very unstable and twice the wrong structure was proposed for it, synthesized and found to

Terpenes

97

* \

\

pp

(€)+-famesene

periplanoneA

Periplanone B

nornadone

dpy? -

1

n

y-cadinene

caryophyllineoxide

E-co ancistrodial

gerrnacrene A

ancistrofuran

caparrapioxide

Figure 6.20 Some examples of insect sesquiterpenes used as pheromones or defensive secret ions

be inactive before the correct structure was found. Other species of cockroach use related periplanones. The larvae of swallowtail butterflies have a horn-like organ, called an osmeterium, on their heads, normally hidden, but everted when disturbed. They attempt to smear its secretion onto the disturber. In one species, Papilio memnon, the osmeterial secretion contains caryophylline oxide (Figure 6.20), which is also found in the oil of some tropical plants and there acts as a deterrent to leaf-cutting ants. Papilio protenor produces germacrenes A and B in its osmeterium. When the organ was treated with D,-acetic acid, both compounds incorporated the deuterium. The monoterpenes in the defensive gland of soldier termites have already been mentioned. A great variety of monocyclic and bicyclic sesquiterpenes like y-cadinene and germacrene A are also present in their secretions. While well-known in plants, little is yet known about how they are biosynthesized in insects. Ancistrodial is the principal constituent of the defensive secretion of minor soldiers of the termite Ancistrotermes cavithorax. The compound is repellent to ants, which are the chief invaders of termite mounds. Ancistrofuran is the chief repellent of the major soldiers of that species. Caparrapioxide is found in the defensive secretion of Amitermes. All these can be seen as further oxidation and cyclization products from farnesol, or possibly p-farnesene.

6.4.2 Cantharidin Cantharidin is a defensive secretion of meloid beetles (another type of blister beetle, Plate 10). It forms about 0.25-0.5% of the body weight, and is stored in the haemolymph and male genitalia. It is present in all life

98

Chapter 6

stages. When disturbed, adult insects bleed as a reflex from the leg joints, early larvae regurgitate a milky secretion from the mouth. Cantharidin is highly toxic in humans and an extreme irritant to all tissues, it is known to inhibit protein phosphatases. Cantharidin is synthesized by both sexes as larvae, but only by the male adult beetles. Females acquire it from males through frequent copulation and it passes thence to eggs. Anyone attempting labelling experiments with adult female beetles would be puzzled to find no incorporation of the label. Cantharidin (C,,H,,O,) may look something like a monoterpene, but by use of labelled acetate, mevalonate, and multiply labelled farnesol, it has been shown to be formed through farnesol with loss of carbon atoms 1 and 5 to 7 (Figure 6.21). The whole synthesis apparently takes place on one enzyme without formation of an intermediate that can be isolated.

@ 4 -O 0 cantharidin

Figure 6.21 The biosynthesis of cantharidin. The asterisks and black dots represent two diferent experiments with labelled farnesol that showed these particular carbon atoms are retained in cantharidin. The two teminal methyls (asterisked) of farnesol become scrambled during the synthesis. The excision of atoms 5 to 7, ring closure between C-3 and C-11, insertion of the oxygen bridge, oxidation to acid and anhydride formation all occur without the molecule dissociating from the enzyme. No mechanism has been proposed for this unusual reaction

6.4.3 Lac Insects A group of homopterous insects, chiefly Laccifera Zacca, feeding on various forest trees produce an excretion on the bark of the tree which eventually covers the insects. This has been a commercial product for a long time as lac for making varnish or in a purified form as shellac. As with many insect products, it is not yet certain whether lac is made by the insects themselves or by symbiotic micro-organisms in them. Lac consists of a mixture of polyhydroxylated palmitic acids and sesquiterpenes. The palmitic acids are hydroxylated at C-10 and C-1 1 or C-16 or all three, as in aleuritic acid (Figure 6.22). The principal terpenes are jalaric acid and laccijalaric acid (Figure 6.22), which have a cedrene structure. It is known from plants that

Terpenes

H

99

O

A

o

H

OH aleuritic acid

__t

0 H t a R y H

H

+

R = CHzOH jalaric acid ‘ 6

R = CH3 laccijalaric acid

.)

A

H

-*

Figure 6.22 The principal substances in lac produced by Laccifera insects. The lac consists of a mixture of hydroxylated palmitic acid and tricyclic sesquiterpenes. The formation of cedrene according to the plant route is shown

cedrenes are formed there from farnesyl pyrophosphate via linalyl pyrophosphate (Figure 6.17) as shown in Figure 6.22, but there is no information on the insect route. While a-cedrene and cedrol are found in Juniperus trees, there is no cedrene in the trees that the lac insects attack, and, for insects, a different isomer of cedrene is involved. 6.5

HOMOSESQUITERPENES

Simple sesquiterpenes are common substances in insects, but they also make sesquiterpenes with one, two or three extra carbon atoms, by using one, two or three molecules of homomevalonate in place of mevalonate. Labelling experiments by Schooley, Judy, Bergot, Hall and Siddall (Proceedings of the National Academy of Sciences, USA, 1973,70,2921), have shown that homomevalonate starts with a propionic acid molecule as shown in Figure 6.23. In some insects it is known that the propionate is derived from isoleucine or valine only. The intermediate 3methylenepentyl pyrophosphate in Figure 6.23 can be isomerized to two other structures. From these are obtained by reaction with IPP or DMAPP, compounds with either a terminal ethyl branch, an internal ethyl branch or two vicinal methyl branches (Figure 6.23). All three possibilities are found among insect compounds but the one on the left seems more common. Such sesquiterpene homologues seem plentiful, particularly in ants. The three farnesene homologues from the Dufour glands of Myrmica ants are an example (Figure 6.24). Their purpose there is unknown. In the Dufour gland of the ant Manica rubida nine isomers and homologues of farnesene were found. Their function is again unknown. Faranal (Figure 6.25) is the trail pheromone of Pharaoh’s ant Monomorium pharaonis, a tropical species that has become a pest inside warm buildings in temperate climates. It contains two homomevalonate

100

Chapter 6

----)-

f-0~

0

SCoA

-

L O P O P

3-rnethylenepentyl PP

hornomevalonate

Figure 6.23 The biosynthesis of homomevalonate from acetate (as malonate) and propionate units. The black dot on carbon indicates a labelled carbon of propionate. Isomerization of 3-methylenepentyl pyrophosphate gives rise to three possible kinds of branching. The one shown on the left is most common and the formation from it of a homosesquiterpene is illustrated

hornofarnesene

bishornofarnesene

trishornofarnesene

Figure 6.24 Homologues of farnesene found in Myrmica ants, containing one, two or three homomevalonate units replacing mevalonate

faranal

hornohirnachalene

Figure 6.25 Further examples of homosesquiterpenes from insects. Faranal has two homomevalonate units, homohimachalene has one

units incorporated in different ways. Homohimachalene (Figure 6.25) is the sex pheromone of the sandfly Lutzomyia Zongipalpis one of the species which carries the protozoal disease Leishmaniasis in South America.

101

Terpenes I

I

0

Figure 6.26 Some homomonoterpenes. Compounds a and b, decyl and dodecyl esters of 4methylgeraniol, form the trailpheromone of the ant Gnamptogenys striatula, compound c, a bishornornonoterpene is also present in the gland mixture but does not contribute much to the pheromone blend. These compounds come from the Dufour gland

The first homomonoterpene and bishomomonoterpene have recently been discovered. A mixture of decanoate and dodecanoate of the third form of homogeranyl pyrophosphate shown in Figure 6.22, (4S,2E)3,4,7-trimethyl-2,6-octadienol (Figure 6.26) have been found to be the trail pheromone of the ponerine ant Gnamptogenys striatula. The dodecanoate of the bishomomonoterpene (4S,2E,6@-3,4,7-trimethyl2,6-nonadienol was also present but only marginally active in the pheromone. 6.6 JUVENILE HORMONE An important hormone in insects is the so-called juvenile hormone, produced in the corpora allata, a part of the brain. It acquired that name because it is required at each moult between immature stages (see Figure 1.4). When JH is absent, the insect moults to the adult form. But the hormone is also produced in adult insects and has other functions there. Pheromone release in Lepidoptera and Coleoptera is often under the control of JH. The juvenile hormone first discovered, known as JH I, is a bishomoterpene (Figure 6.27). Later, all the forms from JH I to JH 111, corresponding to the farnesene homologues shown above have been found, but only JH I11 is found in all insect orders. The hormones JH 0 to JH I11 are found only in Lepidoptera. Notice that a different isomer, 3-methyl3-pentenyl pyrophosphate is added in 4-methyl-JH I compared to the others (Figure 6.27). Similar homofarnesene skeletons are also found in ants. JHB,, an epoxide form of JH 111, has been found in all the higher flies (Diptera) so far studied. Biosynthetic studies have firmly shown that JH I11 is produced via mevalonate or homomevalonate, as required, and then through the steps already discussed to farnesyl pyrophosphate or homofarnesyl pyrophosphate. The action of pyrophosphatase releases free farnesol, which is oxidized by a dehydrogenase and NAD' to

102

Chapter 6

H

JH I1

JH 0

OCH3 4-methyl-JH I

Figure 6.27 The known forms ofjuvenile hormone found in insects. The structure of 4-methyl-JH I is explained in Figure 6.22

farnesal and then to farnesoic acid. This is esterified using S-adenosyl methionine and a methyl transferase. Finally methyl farnesoate is epoxidized by a cytochrome P450to juvenile hormone. These stages have been thoroughly investigated, for example in the yellow fever mosquito Aedes aegyp ti. It is interesting that methyl farnesoate is present in some Crustacea, and a mixture of methyl farnesoate and JH I11 in the embryos of primitive insects such as cockroaches. There appears to be a kind of evolution through appearance of successive epoxidases of the cytochrome P,,, family. Some form of juvenile hormone is present in ticks and other arthropods. Juvenile hormone is inactivated by hydrolysis of the methyl ester followed by opening of the epoxide ring to a diol. The epoxide hydrase works 17 times faster on the JH 111acid than on the JH 111itself. BACKGROUND AND FURTHER READING W. Francke and S. Schulz, Pheromones, in K. Mori, editor, Comprehensive Natural Products Chemistry, Vol. 8, Pergamon Press, Oxford, 1999, pp. 197-261. E. Haslam, Metabolites and Metabolism, Oxford University Press, Oxford, 1985, pp. 161 (Chapter 5). J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 4). K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapter 6). J. A. Tilman, S. J. Seybold, R. A. Jurenka and G. J. Blomquist, Insect pheromones - an overview of biosynthesis and endocrine regulation, Insect Biochemistry and Molecular Biology, 1999,29,48 1-5 14.

103

Terpenes

QUESTIONS 1. Show how a-pinene (Figure 6.8) can be derived from geranyl pyrophosphate. What kind of enzyme is required to convert it to cis-verbenol? 2. a-Sinensal, found in orange peel, has the structure below. What is it made from and how has it acquired the functional group indicated with the question mark?

3. If mevalonic acid was labelled in its methyl group, show where this label should appear in iridomyrmecin. 4. Show farnesene folded as it must be as a precursor to the formation of periplanone A (Figure 6.20). Indicate what further changes have been made to give periplanone A. 5. Fold the molecule of farnesene in a way that is probable as an intermediate to caryophyllene oxide (Figure 6.20) and indicate further changes necessary to arrive at the final structure. 6. Suggest a route from (E)-9-farnesene to ancistrodial and ancistrofuran (both Figure 6.20). 7. Gyrinidal and gyrinidone are compounds from the defensive glands of whirligig water beetles (Gyrinus sp.). Suggest schemes for their biosynthesis from farnesol. Note the resemblance to iridoids. 'f

gyrinidal

Y

gyrinidone

CHAPTER 7

Higher Terpenes and Sterols 7.1 DITERPENES

Addition of another unit of isopentenyl pyrophosphate (IPP) to farnesyl pyrophosphate by the same process as described in Chapter 6 gives geranylgeranyl pyrophosphate (Figure 7. l), the parent of the diterpenes. Diterpenes are very common in higher plants; 110 are listed as biologically active in the Phytochemical Dictionary (Taylor and Francis, London, 1999 p. 701). They may be acyclic, macrocyclic, or polycyclic. Diterpenes are less volatile because of their greater molecular mass, and cyclized ones are generally solids, but some diterpenes are found in insect glands where they probably act as pheromones. Uncyclized diterpenes, like isomers of springene (Figure 7.1), simple alcohols such as geranylgeraniol, geranyllinalool, and geranylcitronellol and their esters are particularly found in Hymenoptera, but also in termites. These compounds and uncyclized sesquiterpenes are also found in mammalian scent glands. Geranylgeraniol has been found in the labial glands of male bumblebees, in the Dufour glands of the stingless bee Nannotrigona testaceicornis, the ant Ectatomma ruidum, in the female sex pheromones of click beetles (Agriotes, Sinapus and Melanotus species, Coleoptera,

farnesyl pyrophosphate

IPP

geranylgeranyl pyrophosphate

r-* \

geranylcitronellol \ \

a-springene

geranylgeraniol geranyllinalool

Figure 7.1

OH

Theformation of geranylgeranyl pyrophosphate, the parent of the diterpenes and some simple uncyclized diterpenes found in insects

104

Higher Terpenes and Sterols

105

Elateridae) and even in a mammalian secretion (the collared peccary, Tayassu tajacu). Geranylcitronellol (Figure 7.1) has also been found in male bumblebees, and springene, first discovered in facial glands of the South African springbok has been found in Dufour glands of ants, stingless bees and a parasitic braconid wasp. The corresponding aldehydes geranylgeranial and geranylcitronellal are also bumblebee substances. The use of a homomevalonate is also observed. The first acyclic homoditerpene from the parasitic wasp Habrobracon hebetor has recently been isolated. Examples of cyclized diterpenes in insects are also known. The cembrene structure (Figure 7.2) seems much used by insects. Neocembrene (or cembrene A) is the trail pheromone of some termites, and it, or an isomer of it, is also the sex pheromone of some sandflies, and is the queen pheromone of the ant Monomorium pharaonis. Related structures, crematofuran and isocrematofuran have been found in the defensive or offensive secretions from the Dufour glands of the Brazilian ant Crematogaster brevispinosa (Figure 7.2). These are quite different from the compounds from some European Crematogaster (see Figure 3.34).

cembrene

Figure 7.2

crematofuran

isocrematofuran

Cembrene, a diterpene from termites and ants and two derivatives of it from the oflensive secretion of Crematogaster ants

7.1.1 Termites The star performers on the diterpene stage are the soldier caste of some termites. Termites (Isoptera) have developed many means of defense against predators, both physical and chemical. The Nasutitermitinae are the largest, most advanced, and most widely distributed subfamily of higher termites. They have a sterile soldier caste with large frontal glands, and heads modified into a turret shape for discharging the frontal gland secretion. Many of these termites produce a gluey secretion of terpenes. In addition to the monoterpenes, which act as a ‘solvent’ for the glue, and some sesquiterpenes in the secretion, are a mixture of acyclic and cyclic diterpenes. The mixture oxidizes in the air to produce the sticky secretion which traps ants or other invaders. In Reticulitermes species geranyllinalool is the principal component. The cyclic diterpenes are

Chapter 7

106

hydrocarbons of the cembrene and cubitane type and oxygenated bi-, tri-, and tetracyclic diterpenes (Figure 7.3). The tetrapropionate derivative of a trinervitene with an extra methyl group has been reported, without comment, from HospitaZitermes umbrinus. This is the first example of a cyclized homoditerpene, made with a homomevalonate starter unit. Thirty-nine diterpenes from frontal glands of termites are listed by Wheeler and Duffield in Handbook of Natural Pesticides Vol. IVB, Pheromones (ed. Morgan and Mandava, CRC Press, Boca Raton, 1985, pp. 159), more have been isolated since. It is clear these are insectproduced via the isoprenoid route. When the soldiers of Nasutitermes octopilis were injected with 14C-labelledacetic acid or mevalonolactone, the 14Cwas incorporated into tri- and tetra-cyclic diterpenes (Prestwich, Jones and Collins, Insect Biochemistry, 1981, 11,33 1). In spite of the large number of diterpenes identified from termites, and the interest they have aroused among structural and synthetic chemists, no further biosynthetic studies appear to have been carried out since the work of Prestwich et aE. in 1981.

6 &-&-& /

cubitene

H+ cembrene

seconervitene type

trinervitene type

&C\,..' @

HO" seconervitene

kempane type

-

3a-hydroxy-7,16secotrinervita-7,11,15(17)triene

HO trinervi-2p,3a,Sa-triol 9-0-acetate

a trinervitene derived from hornocernbrene

kempene-2

Figure 7.3 Some cyclic diterpenes from termites. Cembrene is shown with its isopropenyl group turned inwards. Rearrangement as indicated may lead to bicyclic seconervitenes, tricyclic nervitenes and tetracyclic kempenes. Oxygenated examples of each type are given. The tetrapropionate is a homoditerpene

Higher Terpenes and Sterols

107

7.2 SESTERTERPENES Still higher homologues of terpenes are found in insects, presumably made by extending the synthesis scheme already described. The sesterterpenes, C25compounds, contain five isoprene units. Acyclic sesterterpenes and C30compounds, of unknown function, with structures similar to springene have been found in Dufour glands of stingless bees, and geranylfarnesol was identified in the frontal glands of Reticulitermes santonensis, and as esters in the wax of scale insects. A group of homopterous scale insects make insect wax that is an article of commerce (Plate 8). In China the insects are Ericeruspela, and in India Gascardia cerifrea, while in Central and South America various Ceroplastes species provide the same product. These waxes consist principally of esters of long chain acids with long chain alcohols, but hydrolysis of Ceroplastes albolineatus wax gives geranylfarnesol (Figure 7.4) and the cyclized derivatives of it, albolineol, ceroplastols I and 11, and ceroplasteric acid and albolic acid (Figure 7.4). The mechanism shown is how they are probably formed, though no studies have been made.

+ & -& '

/

r'

OPOP

geranylfarnesyl pyrophosphate

/

POP9

GR \"

'H

R = CH20H ceroplastol I1 R = COOH albolic acid

$+&J

\

8.

,'H

R

R = CH20H ceroplastol I R = COOH

ceroplasteric acid

\

,OH

1

HOalbolineol

Figure 7.4

Sesterterpenes from Ceroplastes albolineatus, homopteran scale insects. The mechanism of formation is only conjecture

108

Chapter 7

7.3 TRITERPENES AND STEROIDS The linking of two farnesyl groups head-to-head leads to the C30terpene hydrocarbon squalene, a compound first found in the liver oil of sharks (Squalus species), but is now known to be a widely distributed compound (Figure 7.5). It is found, for example, in the oil of human skin. The coupling reaction to give squalene occurs in plants and vertebrates, but is not available to insects. The head-to-head joining of the two farnesyl groups is a complex reaction with cyclopropyl and cyclobutyl intermediates, and requires one molecule of NADPH. The essential steps of the coupling are shown in Figure 7.4. Squalene is most important as the compound from which all the plant triterpenoids and the steroids are biosynthesized. From mevalonate to squalene there are no less than 14 stereospecific steps, all well described. The triterpenes derived from squalene are generally polycyclic compounds. Plants and animals (excluding insects) make them by folding squalene, oxidizing one double bond to give squalene epoxide which is then cyclized into a four-ring triterpenoid (Figure 7.6). The squalene epoxide is held on the active site of the enzyme in the correctly folded form for the first concerted cyclization, which leaves a carbocation at C-20. By a series of 1,2-shifts of two hydrogen atoms and

L :base

presqualene pyrophosphi

famesyl pyrophosphate

R = c1d17

R

\

squalene

'''I

B

H

famesyl pyrophosphate

famesyl pyrophosphate

squalene

Figure 7.5

Theformation of squalene from two farnesyl pyrophosphate molecules joined head-to-head requires the elimination of the pyrophosphate groups and the use of one molecule of NADPH. The hydrogen transferredfrom NADPH is labelled with a dot for identlJcation

109

Higher Terpenes and Sterols acetate

-

mevalonate

-

farnesyl

---+

pyrophosphate

squatene

squalene-3Pepoxide

HO

HO

lanosterol

Figure 7.6 Outline of the biosynthesis of the triterpenoid lanosterol from acetate and mevalonate via squalene

w

reduced

HO

Y

0x1 ized to

COOH and removed

removed as HCOOH

cholesterol

lanosterol

Figure 7.7 A summary of the steps by which the triterpene lanosterol is converted to the sterol cholesterol

two methyl groups, and loss of one hydrogen atom a stable structure is reached. In animals the triterpenoid is lanosterol (Figure 7.6), first identified in wool wax. Lanosterol does not normally accumulate but is converted by quite a large number of further reactions (summarized in Figure 7.7) to cholesterol, the characteristic animal sterol. While triterpenes are C30 compounds, in sterols, three methyl groups of the triterpenes have been lost. By the action of a cytochrome P450,the methyl group on C-14 is oxidized to a formyl group and then removed by folic acid. The pair of methyls on C-4 are sequentially oxidized to carboxylic acids and lost by decarboxylation (Figure 7.6). The side chain double bond of lanosterol is reduced in cholesterol and the A8 double bond is isomerized to a A5 double bond in cholestrol. The essential features of sterols are three cyclohexane rings and one cyclopentane ring fused together in the way shown. They always have an oxygen function at C-3, the position of the hydroxyl in cholesterol.

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Chapter 7

Animals make and use cholesterol. It is a constituent of all vertebrate cell walls, of blood lipoproteins and is the precursor of the bile acids and a number of mammalian hormones. Plants make sterols through cycloartenol, another triterpenoid, made by folding squalene differently and further alkylate the sterol formed (see Mann, Secondary Metabolism, 2nd edit. Oxford University Press, 1987, p. 138). The typical higher plant sterol is sitosterol, with campesterol and stigmasterol less widely found (Figure 7.8). Micro-organisms make a still greater variety of sterols. The extra side-chain methyl groups in plant sterols come from S-adenosyl methionine. Insects that feed on vertebrates have a ready supply of cholesterol. Those feeding on leaves or phloem of plants tend to convert the plant sterols to cholesterol. Feeders on fungi and bacteria have an even wider variety of sterols to assimilate. The de-alkylation of plant sterols in insects takes place in the gut (Figure 7.8). Cholesterol or some equivalent sterol is therefore an essential nutrient for insects. It has been found recently that rice planthoppers and some anobiid beetles harbour yeastlike symbionts that make sterols which the insect can use, making those insects less dependant upon food sources.

p-sitosterol

carnpesterol

R = H or CH3 ',a*.

A

f?

stigmasterol

/

HO cholesterol

Figure 7.8

The de-ulkylution ofplunt sterols in insect gut to cholesterol

I

Higher Terpenes and Sterols

111

7.3.1 Sterols in Insects It is perhaps surprising that insects use sterol derivatives both for a few pheromones and more widely for defensive compounds when they are dependant upon their diet for a supply of them. Cholestanone is used as a trail pheromone by tent caterpillars (Malacosoma species) (Figure 7.9), cholesteryl oleate and other cholesteryl esters are attractant pheromones for some ticks, and two sterol glucosides, blatellostanosides A and B, are aggregation pheromones of the cockroach Blatta germanica. Note that these not only retain the side-chain ethyl group of sitosterol but have been chlorinated as well. It is noteworthy that a modified sterol androstenone known as ‘boar taint steroid’ acts as a sex pheromone in pigs.

Figure 7.9 Some sterolpheromones. Cholestanone is a trail pheromone of tent caterpillars and the two blatellostanosides are aggregation pheromones of the German cockroach

Some water beetles store in their defensive glands the same sterols that are adrenal hormones in mammals (Figure 7.10). These compounds provide protection against fish, frogs and small mammal predators. Clearly the beetles possess enzymes with which they can degrade cholesterol further and remove most or all of the side chain (Figure 7.10). cholesterol

cybisterone

6,7-dehydrocortexone

6,7-dihydrocybisterone

Cortexone

Figure 7.10 Corticosteroids from water beetles, used as defensive secretions. Other hydroxylated derivatives of these compounds have also been found in water beetles

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[4-14C]Cholesterol,when injected, served as a precursor of all the corticosterones. Insects injected with labelled mevalonolactone, as expected, produced no labelled sterols. The pathway to dienones and enones presumably diverges at an early stage since there was a greater incorporation of [4-'4C]cholestadienone into the two dienones (Figure 7. lo), while [4-14C]progesteronewas better incorporated into the two enones. Specific incorporation of labelled cholesterol varied from 0.5 1% for cortexone to 1.20% for dihydrocybisterone. Labelling experiments with cholesterol showed that the 4P and 7p hydrogen atoms were eliminated in forming the diene. There is about 1 mg of cortexone (also known as deoxycorticosterone or systematically as 4-pregnene-21-01-3,20-dione) in the prothoracic defensive glands of each beetle of Cybister limbatus. In IZybiusfenestratus there is testosterone, oestradiol, three compounds related to cortexone and the quinoline compound in Figure 7.11, which too is toxic to small mammals.

COOCH3 OH methyl 8-hydroxyquinoline-2-carboxylate

HO testosterone

oest radio1

Figure 7.11 Defensive compoundsfrom Ilybius fenestratus

The adults of another group of beetles, the Chrysolinina, have cardiac glycosides in their defensive glands. These are steroids (cardenolides) with sugar attached, first found in plants of the Asteraceae, Ranunculaceae and Apidaceae, in which the steroid side-chain is converted to an a,P-unsaturated lactone. The cardiac glycosides from plants, like

CH3COOH

0&8H

do RY..

xylose

---

0

R2

Cardenolides R, = OH, R2 = H sarrnentogenin xyloside R1 = H, R2 = OH periplogenin xyloside R1 = OH, R2 = OH bipindogenin xyloside

Figure 7.12 Cardiac glycosides containing xylose made by the beetle Chrysolina coerulans from cholesterol. The asterisk indicates a 14C-labelwhich is lost in the synthesis

Higher Terpenes and Sterols

113

digitoxin, originally used in arrow poisons, cause vomiting in mammals and affect their nervous systems. In plants it is known that the sterol sidechain is degraded to two carbon atoms and the lactone re-built with an acetate unit. The beetles that make these compounds do not feed on plants that contain them. It has been shown with [1,2-2H,23''C]cholesterol that the beetles make the cardenolide from cholesterol (Figure 7.12). In the process the deuterium is retained but the 14Cis lost. It is concluded that the beetles make the cardenolides in much the same way as plants do. Other chrysomelid beetles attach 2-deoxyhexoses and others make the steroids without the sugars.

7.3.2 Saponins from Triterpenes Saponins are plant compounds, close relatives of the cardiac glycosides. They consist of a sterol or triterpenoid joined to a sugar. They make a strong foam when shaken with water. They are bitter-tasting and generally toxic to lower forms of life. They have been much used by primitive people for killing fish. The defensive glands of adult beetles of Platyphora, Leptinotarsa and Desmogramma have been found to contain triterpene saponins. Since insects cannot make triterpenes, and the plants they were feeding on do not contain saponins, biosynthetic studies were made to find how the insects acquired them. Feeding Platyphora kollari with [2,2,3-2H,]Pamyrin (a-and P-amyrin are widely distributed plant triterpenoids) produced up to 40% of the labelled saponin shown in Figure 7.13. The enzymes necessary for the oxidation of a methyl group of P-amyrin and coupling of the product with sugars are generally present in all living organisms.

deuterated pamyrin

a triterpenoid saponin

Figure 7.13 By feeding deuterated P-amyrin to adult Platyphora kollari beetles, they have been shown to be able to convert it into a labelled saponin

114

7.4

Chapter 7

INSECT MOULTING HORMONE - ECDYSTEROIDS

An essential stage in the growth and development of insects is moulting or ecdysis. Growth is a discontinuous process, initiated at each stage by the moulting hormone (see Figure lS), a derivative of cholesterol. As there is not just a single compound involved in all insects, the group of hormones are called ecdysteroids. Although insects are unable to make squalene and do not possess the enzymes necessary for cyclization and sterol formation, they require cholesterol or an equivalent to make their moulting hormone. A protein prothoracotropic hormone (PTTH) stimulates the prothoracic gland to begin the synthesis that results in the formation of 20-hydroxyecydsone, the main hormone. The early stages of hydroxyecdysone synthesis are still not accurately known after some years of intensive study. Cholesterol is converted in the prothoracic gland to 7-dehydrocholesterol, probably through 7J3-hydroxycholestero1(Figure 7.14). The next step may be epoxidation to 7-dehydrocholesterol 5,6aepoxide. By three more steps not yet clear, the epoxide is re-arranged to a 6-ketone, an a-hydroxyl group is introduced at C-14, and the 3P-OH is oxidized to a ketone. The first well established intermediate is 3P,14adihydroxycholest-7-en-6-one(A in Figure 7.14). From here it seems that two sequences run in parallel, the relative amounts of each varying with species. In one series, the 3P-hydroxyl is oxidized to a ketone and we have a 3-deoxy-group. The other retains the 3P-hydroxyl, but the two routes seem to be interconnected. Certainly the next step for each sequence is hydroxylation at C-20, followed by C-2. All of this occurs in the prothoracic gland. The end product in the gland is ecdysone, the first compound that was isolated, and thought for a time to be the hormone. Later it was found that hydroxylation of ecdysone at 20(S), which occurs in the peripheral tissues, was necessary to give 20-hydroxyecdysone, the true hormone. The hydroxyl groups are inserted by cytochrome P,,, enzymes. The enzyme for the last step, in the peripheral tissues, ecdysone 20-mono-oxygenase (and NADPH) has been studied in great detail. The story of the moulting hormone is not so neat and clear-cut as that of the juvenile hormone. There are a number of compounds of similar structure, all called ecdysteroids. There are plant-eating insects in at least four orders (Diptera, Coleoptera, Hemiptera and Hymenoptera) including the honeybee Apis mellifera, that are unable to remove the side-chain alkyl groups of plant sterols, and use makisterone A as their moulting hormone (Figure 7.15). It has the structure of 20-hydroxyecdysone, but with an extra 24(R)-methyl group. Ecdysteroids are also found in mature ovaries and pass into eggs, and stored there as conjugates, that is, either phosphate esters or esters of fatty acids, attached through the C-22

Plate 1 A unit of the castor oil stearoyl-ACP desaturuse, with a molecule of stearic acid (white stick model, grey space-Jilling outline) modelled into the active site, in a gauche conformation. The protein chain with its a-helix coils is shown green. This conformation orients both C-9 and C-I0 pro-R hydrogens towards the activated oxygen (not shown), bound to the di-iron active site (red spheres) allowing removal of hydrogen and introduction o f a cis double bond (Illustration: J. Shanklin and W. McGrath, Brookhaven Natl. Lab.)

Plate 2 Female silkworm moth Bombyx mori with her pheromone glands extended (arrowed), em it ting bombykol (Photo: Jacques Six, with permission of Societk Nouvelle des Editions Boubee)

Plate 3

The seven-spotted ladybird Coccinella septempunctata, which produces precoccinelline and coccinelline by reflex bleeding. The larva, /eft (Photo: Jim Kalish) Adults mating, right (Photo: Steve McWilliam)

Plate 4 Pupa of the Mexican bean beetle Epilachna varivestis, showing its many hairs with droplets ofj7uid containing epilachnine and related macrocyclic compounds; and a close-up view of some of the hairs and droplets (Photo: Maria Eisner)

Plate 5

The centipede Scolopendra subspinipes multilans which produces the antibiotic centipedin. It also has an extremely painful bite of unknown composition (Photo: Tom Larsen)

Plate 6

The boll weevil Anthonomis grandis, an importantpest of cotton andproducer ofa terpene aggregation pheromone. Insects are a favoured subject for philately

Plate 7 Larva of the ladybird Hippodamia convergens, which produces hippodamine and convergine, eating aphids of Aphis nerii, which in turn live on oleander bushes, and are pigmented with the naphthol glucoside B (Figure 8.17). This aphid collects and stores three cardiac glycosides from oleander (Photo: Mike Quinn)

Plate 8

W a x mounds on an Acacia branch produced by Ceroplastes scale insects. Anoplolepis ants are searching the branch (Photo: Hamish Robertson)

Plate 9 Thefire bug Pyrrhocorus apterus coloured by red erythropterinpigment and other pterins (Photo: Anthony Papadopoulos)

Plate 10 The blister beetle Lytta magister which produces cantharidin (Photo: Warren E. Savary)

Plate 11 Larva of the moth Arctia caja, with its long urticating hairs, which contain the alkaloid serotonin (Photo: Jens Christian Schou)

Plate 12 Larva of the Monarch butterfly Danaus plexippus, with aposematic (warning) colours. The larva acquires the cardiac glycoside calotropin from its food plant (Photo: Mike Quinn)

Plate 13 The millipede Harpaphe haydeniana, aproducer of hydrogen cyanide. It has been used to study the biosynthesis of mandelonitrile (Photo: Markku Savela)

Plate 14 Adult female lone star tick Amblyomma americanum, which produces 2,6-dichlorophenol as a sexual attractant (Photo: School of Veterinary Medicine, State U. Oklahoma)

Plate 15 Larva of the tobacco hornworn Manduca sexta (Photo: Paul Choate)

Plate 16 Two adult wasps ofPolistes dominulus on a newly constructed nest. Their yellow colour is due to xanthopterin. The upper wasp is marking the pedicel of the nest with abdominal secretion (Photo: Stefan0 Turillazzi)

Plate 17 Adults andnymphs of the milkweed bug Oncopeltus fasciatus on a milkweedpod. Their cuticle is partly black (melanin) and partly transparent, showing the pterin pigments underneath (Photo: Mike Quinn)

Plate 18 A worker of the garden ant Myrmica rubra following an artiJicia1 trail of synthetic 3-ethyl-2,5-dimethylpyrazineon paper. Note the position of the antennae. The sting lance is protruded, probably re-enforcing the trail with more pheromone. The paper is ruled in 0.I inch (2.54m m ) squares (Photo: Terry Bolam)

Higher Terpenes and Sterols

115

HO

HO

cholesterol

OH HO HO

25-deoxy-20-hydroxyecdysone ponasterone A

H I OH

0

20

OH

HO

HO OH

HO 0

ecdysone

HO

20-h ydroxyecdysone

Figure 7.14

A summary of the formation of the moulting hormone 20-hydroxyecdysone from cholesterol. The early stages are not yetfirmly known. All the stages except the last occur in the prothoracic gland. Ponasterone A is a crustacean moulting hormone

hydroxyl. Ecdysteroids are inactivated by conversion to C-22 sulphates or oxidized to ecdysonoic acid (Figure 7.15) or to a lesser extent converted to glycosides. In some arthropods (for this hormone is shared with other classes besides insects), the series are not hydroxylated at C-25. Many crustaceans use 20-hydroxyecdysone as moulting hormone, others use ponasterone A (Figure 7.14). Ecdysteroids have also been found in plants. These phytoecdysteroids are widely distributed in the plant

Chapter 7

116

HO

0

0

ecdysone 22-palmitate

makisterone A

HO

HOT ajugasterone c

20-hydroxyecdysone

0

0 ecdysonoic acid

Figure 7.15

Some ecdysteroid examples. Makisterone A is used by some insects, including Apis mellifera and Drosophila melanogaster, and by crabs and is a phytoecdysteroid in Podocarpus trees and the wildflower Lychnis flos-cuculi (ragged robin). Ecdysone 22-palmitate is an example o f a storage product found in insect ovaries and eggs. Ajugasterone C is an example of a phytoecdysteroid from the Labiatae, Compositae and Verbenaceae,but also identiJied in an Anthozoa (corals and sea anemones)

kingdom, and many variations of the ecdysone structure have been identified. The number of phytoecdysteroids is now in the hundreds. They tend to be concentrated in the growing tips of plants and are presumed to act as anti-hormones, inducing premature moulting in the insects feeding on the plant (see The Ecdysone Handbook, edited by Harmatha, MarionPoll and Wilson, http://ecdybase.org). By a strange twist in the battle between plants, insects, and other predators. 22-acetyl-20-hydroxyecdysone has been found in high concentration in the defensive glands of the beetle Chrysolina carnifex. Further sterols will be encountered among plant substances sequestered by insects (Chapter 10). 7.5 TETRATERPENES Tetraterpenes are constructed in a manner similar to squalene, by headto-head condensation of two molecules of geranylgeranyl pyrophosphate, also through a cyclopropane intermediate. The initial product is phytoene (Figure 7.16). It is desaturated to carotenes, which have long conjugated systems of double bonds, usually all trans, and are strongly coloured. Tetraterpenes appear now to be synthesized in the plastids of plants via the methylerythritol phosphate process (Figure 6.6). They were

117

Higher Terpenes and Sterols 2 x geranylgeranyI

lycopene

&carotene

oxidation

p-carotene monoepoxide

zeaxanthin

HO

HO

0

astaxanthin

violaxanthin

abscissic acid

HO

Figure 7.16 Theformation of tetraterpenes by condensation of two molecules of geranylgeranyl pyrophosphate, and conversion to carotenes, with some examples of insect carotenes, retinal or vitamin A and absicic acid, a degradation product of carotenes. Theformation of phytoene is very similar to that of squalene (Figure 7.4)

thought to be made only by plants, but that is now uncertain. Bovine corpus luteum has been shown to synthesize carotene. Carotenes are highly unsaturated and are very unstable in air, nevertheless they are found in many places, from butter to flamingos. Lycopene is the red pigment of tomatoes, paprika and other fruit. pCarotene is an essential part of the photosynthetic process in plants. Cleavage of p-carotene by oxidation gives two molecules of vitamin A aldehyde or retinal, an important part of the visual pigment of insects as well as higher animals. Other carotene derivatives give colour to many fruit and vegetables, e.g. zeaxanthin is the yellow pigment of maize (Zea mais). Astaxanthin is a further oxidation product of carotene and gives the pink colour of shrimps, boiled lobsters, salmon and the pink bollworm. Violaxanthin is a common plant pigment, which on oxidation gives abscissic acid, a plant hormone that controls loss of leaves (Figure 7.16). It is also widely found in Lepidoptera. Lycopene, p-carotene,

Chapter 7

118

p,p-carotene

p,y-carotene [major product]

green aphid

y,y-carotene

pink aphid

Figure 7.17

i

p,P-carotene

torulene [major product]

3,4didehydro-~,cp -carotene

The carotene pigments in the two colour forms of Macrosiphum liriodendri aphids. The systematic names are used here. p,P-Carotene is more commonly known as j?-carotene

zeaxanthin, violaxanthin, astaxanthin, xanthophyll and p-carotene monoepoxide and at least eight other carotenes have been found in insects. Xanthophyll, also known as lutein is found in almost all Lepidoptera examined (the name xanthophyll is also used for all oxygenated carotenes). As retinal combines with the protein opsin to form the purple visual pigment rhodopsin, so carotenes combine with proteins to give green, blue-green, blue and red pigments in insect integument and haemolymph. The carotenes of one aphid (Macrosiphum Ziriodendri), that occurs in two colour variants have been studied in detail. This aphid, living on tulip trees (Liriodendron tulipifera), exists in green and pink forms which appear to differ in their level of cyclizing enzymes. The green form have all cyclized carotenes, while the pink form have two partly cyclized and two uncyclized carotenes (Figure 7.17). It must be presumed the aphids have the necessary enzymes to cyclize lycopene. Many other insects, particularly aphids, contain carotenes along with other pigments.

Higher Terpenes and Sterols

119

BACKGROUND AND FURTHER READING

G. Britton, The Biochemistry of Natural Pigments, Cambridge University Press, 1983, pp. 366 (Chapter 2, carotenes). W. Francke and S. Schulz, Pheromones, in K. Mori, editor, Comprehensive Natural Products Chemistry, Vol. 8, Pergamon Press, Oxford, 1999, pp. 197-261. L. I. Gilbert, R. Rybczynski and J. T. Warren, Control and biochemical nature of the ecdysteroidogenic pathway, Annual Review of Entomology, 2002,47,883-9 16. J. B. Harborne, Introduction to Ecological Biochemistry, 4th Edition, Academic Press, London, 1993, pp. 319 (Chapter 4, hormones between plants and insects). E. Haslam, Metabolites and Metabolism, Oxford University Press, Oxford, 1985, pp. 161 (Chapter 5). J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 4). A. E. Needham, Insect biochromes: their chemistry and role, in M. Rockstein, editor, Biochemistry of Insects, Academic Press, New York and London, 1978, pp. 233-305. H. H. Rees, Insect Biochemistry, Chapman and Hall, London, 1977, pp. 64. H. Schildknecht, The defensive chemistry of land and water beetles, Angewandte Chemie, International Edition in English, 1970,9, 1-9. J. A. Tilman, S. J. Seybold, R. A. Jurenka and G. J. Blomquist, Insect pheromones - an overview of biosynthesis and endocrine regulation, Insect Biochemistry and Molecular Biology, 1999, 29,48 1-5 14. K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapter 6).

QUESTIONS 1. Consider the double bonds in the ring of cembrene as drawn in Figure 7.2. How many are cis and how many trans? How many possible ring isomers are there for cembrene, presuming there are no stereochemical restrictions? 2. What is the isoprene unit structure of trinervitriol diacetate in Figure 7.3? There is a unique solution. 3. The first cyclized triterpene in plants is cycloartenol. Suggest a way in which the cyclized carbocation formed in the cyclization of squalene

Chapter 7

120

cycloartenol

epoxide (Figure 7.6) is converted to cycloartenol. Hint: a different hydrogen atom is eliminated. 4. Suggest a way in which lycopene is cyclized to p-carotene (a part structure is sufficient). 5. [la-3H]Cholesterol(total activity 160 x lo6cpm) was injected into the larvae of the blowfly CaZZiphotra stygia. Each larva received 2 pl of emulsion. Later the pupae which formed were extracted with ethanol, and after purification 20-hydroxyecdysone (activity 30 x lo3cpm) was recovered. What was the specific incorporation of cholesterol into 20-hydroxyecdysone? 6. In another experiment 3p, 14a-dihydro~y-5~-[3a-~H]cholest-7-en-6one (see Figure 7.14 structure A) (28 x lo6 cpm) was similarly used with Calliphora stygia larvae. When the pupae were collected 12 h later the activity isolated in 20-hydroxyecdysone was 140,000 cpm. What was the specific incorporation, and does it suggest compound A is an intermediate between cholesterol and 20-hydroxyecdysone?

d

'\

CHAPTER 8

Aromatic Compounds 8.1 AROMATIC COMPOUNDS IN NATURE

Plants and micro-organisms have an exclusive route to benzene-ring compounds. The great majority of aromatic compounds in nature therefore are produced by plants and micro-organisms, and animals are dependant upon plants for many aromatic compounds, either directly or indirectly. A small number of aromatic compounds are made from polyketides, some of these have already been encountered. The latter usually have a number of oxygen functional groups, pointing to their polyketide origin. Simple substances containing benzene rings, such as the amino-acids phenylalanine and tryptophan must be obtained by animals, including insects, in their food. Plants and micro-organisms make aromatic amino-acids by the shikimic acid pathway. When a mutant strain of E. coli bacterium was produced by ionizing radiation, that was unable to make the three aromatic amino-acids, as well as paminobenzoic acid and p-hydroxybenzoic acid, it was discovered that shikimic acid could restore to the bacterium the ability to make the amino-acids. The process was therefore called the shikimic acid pathway. While the shikimic acid route is unavailable to insects, their metabolism is vitally dependent upon it.

8.2 THE SHIKIMIC ACID PATHWAY The shikimic acid pathway requires the C, sugar erythrose-4-phosphate, and phosphoenol pyruvic acid (a derivative of pyruvic acid, locked in its enol form, see Figures 1.1 and 2.19) as starting materials. The route to aromatic compounds has more steps than those met earlier, and, not surprisingly, for a plant process, uses sugar derivatives as starting materials. A total of ten carbon atoms are required, four from erythrose, and six from two molecules of pyruvate; one of these is later lost as CO,. The final product therefore is a C , compound, so that such products are 121

Chapter 8

122

phosphoenol pyruvate erythrose-&phosphate

/

OH dehydroquinic acid

6H DAHP

qOOH

OH dehydroshikimic acid

(second molecule of pyruvate)

c--

chorismic acid

OH

phenylpyruvic acid

Figure 8.1 The shikimic acid pathway from erythrose-4-phosphate and phosphoenolpyruvic acid to phenylpyruvic acid

referred to as phenyl-C, compounds. Many plant substances have this phenyl-C, structure. The details of the shikimic acid pathway are shown in Figure 8.1. In the first step, a molecule of phosphoenol pyruvic acid adds on to a carboxyl group on the enzyme. The resulting ester loses phosphate to give an enolpyruvate ester, which is in a reactive form to add to erythrose-4-phosphate. The product is 3-deoxy-D-arabinoheptulosonic acid 7-phosphate, a name mercifully shortened to DAHP. The enzyme catalyzing this condensation is DAHP synthase. Again loss of phosphate and cyclization, through the nucleophilic addition of the enol group to the a-keto-group derived from pyruvic acid, gives the six-membered ring

Aromatic Compounds

123

that will become a phenyl group. This first cyclized product is dehydroquinic acid, and the enzyme reponsible for it is dehydroquinate synthase. By dehydration of a P-hydroxy-ketone in the usual way, followed by reduction of the ketone (actually through a Schiff s base formed with an NH, group on the enzyme), shikimic acid is reached, the key compound in the series. This is phosphorylated (with adenosine triphosphate and shikimate kinase) before addition of the second molecule of phosphoeno1 pyruvic acid. Loss of another phosphate gives an enol ether combining shikimic acid phosphate and pyruvic acid. Loss of phosphate for the fourth time gives a second double bond, and another key intermediate called chorismic acid. This compound undergoes a Claisen rearrangement to prephenic acid, followed by a concerted loss of C 0 2and H,O to give the third double bond and the first aromatic phenyl-C, compound, phenylpyruvic acid (Figure 8.1).

8.3 PHENYL-C, COMPOUNDS One of the first important derivatives of phenylpyruvic acid is the ‘essential’ amino-acid phenylalanine, produced with the aid of pyridoxamine (Figure 2.17, with that reaction running in reverse). It is an essential amino-acid because all animals must have it in their diet. Tyrosine in plants is made directly from prephenic acid by oxidation concurrently with decarboxylation. Mammals can make tyrosine from phenylalanine, but insects must obtain it in food. Some other simple phenyl-C, compounds and derivatives are shown in Figure 8.2. The mechanism of formation of tyrosine in animals is shown in Figure 8.3. An epoxide of phenylalanine is formed which is opened in two ways. Labelling experiments show that it goes about 90% with a 1,2shift of hydrogen frompara to meta position. This pathway is known as the NIH shift, from the National Institutes of Health (USA) where it was discovered. The remaining 10% gives direct loss of hydrogen from the para position. There is evidence from a number of experiments that insects cannot convert phenylalanine to tyrosine, but can hydroxylate tyrosine further. 8.3.1 Aromatic Pheromones

A number of compounds easily recognized as derivatives of the phenylC, series have been identified as pheromones (Figure 8.2). For example, ethyl cinnamate has been found as a component of the male sex pheromone of the oriental fruit fly Grapholitha molesta. 2-Phenylethanol has been found in the mandibular glands of the ant Camponotus clarithorax,

Chapter 8

124

phenylpyruvicacid

1

/

cJyH

phenylalanine

O

C

H

tyrosine

O

-

benzaldehyde ~

&OH 2-phenylethanol

Cinnamic acid

phenylacetic acid

ethyl cinnarnate

(R)-1-phenylethanol

-OH caffeic acid

coniferyl alcohol

Figure 8.2 Some simple phenyl- C,derivativesfrom phenylpyruvic acid, including some insect pheromones. Theformation of I-phenylethanol has not been investigated. Benzaldehyde can be formed in more than one way. Coniferyl alcohol, an attractant for a fruit fly is the precursor of the important plant product lignin

-90%

tyrosine

9-

HO + H".

H

CHzT
H

Figure 8.3 The oxidation of phenylalanine with the NIH sh$t and without it. The dot on hydrogen represents a label

and provides the male sex pheromone of the lepidopteran bertha armyworm Mamestra configurata. In the latter case labelling showed that the 2-phenylethanol was derived from phenylalanine, and the route probably

125

Aromatic Compounds

went via cinnamic acid, since the pheromone was also produced from [3''C]cinnamic acid. It was suggested that phenyllactic acid was also an intermediate and the final precursor was the glucoside of phenylethanol. (R) - 1-Phenylethanol is the trail pheromone of the ant Aphaenogaster cockerelli. Benzaldehyde has been found widely in Coleoptera and in some Hymenoptera, and is often associated with HCN in millipedes and some insects as a defensive secretion (Chapter 10). Benzoic acid (in Coleoptera) and phenylacetic acid (in ants) are also widely found, as well as a number of simple esters of phenylacetic acid. Coniferyl alcohol is an attractant for females of the oriental fruit fly, but its immediate precursors are obtained from flowers (Chapter 10). Adult males of a number of Lepidoptera use simple phenyl-C,, and - C, compounds like benzyl alcohol, benzaldehyde, p-hydroxybenzaldehyde, phenylacetaldehyde and 2-phenylethyl acetate as copulating pheromones. Adult males of leaf-footed bugs (Leptoglossus species) use a variety of simple aromatic compounds in abdominal glands, apparently for species recognition. Among these are guaiacol, vanillin, cinnamyl alcohol and syringaldehyde (Figure 8.4). Their origin has not been studied. The curious example of 2-nitroethenylbenzene (2-nitrostyrene) (Figure 8.4) has been found in the defensive secretion of the millipede Eucondylodesmus elegans, and shown to be repellant to ants. When [2H,]phenylalaninewas fed to the millipedes, [2H7]-2-nitroethenylbenzenewas detected by mass spectrometry.

8.3.2 Compounds from Chorismic Acid Various other aromatic compounds are obtained from the intermediate chorismic acid in Figure 8.1, including the other 'essential' amino-acid tryptophan, which is made only by plants and micro-organisms. These routes are shown in Figure 8.5. Attack of ammonia on chorismic acid gives (Figure 8.5, mechanism a) after further steps, anthranilic acid, while addition of ribose phosphate to anthranilic acid gives after several

vanilIin

guaiacol

OCH3 syringaldehyde

cinnamyl alcohol

hNO2 \ /

\

2-nitro-1(€)-ethenylbenzene

Figure 8.4 Some simple aromatic compounds found in abdominal glands of adult male leaf-footed beetles, probably recognition pheromones. 2-Nitroethenylbenzene (nitrostyrene) is a defensive compound from a millipede

Chapter 8

126 COOH

COOH

anthranilic acid chorismic acid

NH

OP

glomerine

0Y

I-

1

- H20 -co2 y

1

y

3

CH3COCOOH

H2ND C O O H paminobenzoicacid

dirnethylquinazoline

amcv H

indole

\

H tryptophan

H skatole

Figure 8.5 Aromatic compounds from chorismic acid. Mechanism a in the top left structure leads to anthranilic acid and tryptophan, while mechanism b leads to p-aminobenzoic acid. The route from chorismic acid to tryptophan is found in plants and micro-organisms only Glomerine is a millipede defensive compound, dimethylquinazoline is found in Triatoma bugs. Indole and skatole are used in various insects as pheromones

more stages, tryptophan. Many insect compounds and related substances are derived from this pathway. Displacement of hydroxyl on the other side of the ring of chorismic acid by ammonia as in Figure 8.5 (mechanism b), and the same steps gives p-aminobenzoic acid. Dimethylquinazoline is from Triatoma bugs which carry trypanosomiasis parasites, and indole and skatole are evil-smelling compounds found as trail pheromones in ants and defensive secretion of caddis flies (Trichoptera). Methyl anthranilate has been found in the mandibular gland of males of the ant Camponotus nearticus, and is one component of the trail pheromone (with methyl nicotinate) of the ant Aenictus rotundatus. Glomerine (Figure 8.5) and pseudoglomerine, from anthranilic acid, form the defensive secretion of the myriapod Glomeris marginata that is toxic to mice and causes paralysis in spiders. It was shown that

Aromatic Compounds

127

A

, CH3 CH3 glomerine

CH3

0

I

I

CH3

pseudoglornerine

Figure 8.6 Anthranilic acid labelled with I4C in the carboxyl group gives labelled glomerine andpseudoglomerine. The label is still found in the carboxyl goup of N-methylunthranilic acid produced by degradation of glomerine

anthranilic acid labelled with 14Cin the carboxyl group was incorporated into both compounds. When the labelled glomerine was hydrolyzed, N-methylanthranilic acid was recovered with approximately the same level of activity as the glomerine, showing that all the radioactivity was still located in the carboxyl group (Figure 8.6).

8.4 AROMATIC AMINES The sequence of important reactions forming amines from the essential amino-acids phenylalanine and tryptophan is worthy of attention because of the physiological role of these amines in many animals. Little is known of their effects in insects but they have been shown to be present in insects as neurotransmitters. 8.4.1 Adrenaline Group Tyrosine is synthesized from phenylalanine in mammals in quantity (Figure 8.3), while a tiny amount of tyrosine is converted to DOPA (dihydroxyphenylalanine) (Figure 8.7). The second hydroxyl is inserted without a shift of hydrogen, unlike the formation of tyrosine (Figure 8.3). DOPA is decarboxylated to dopamine, which in turn gives noradrenaline (norepinephrine), an important neurotransmitter in the autonomic nervous system, and noradrenaline is methylated to adrenaline, a hormone from the adrenal glands of mammals (Figure 8.8). Both dopamine and noradrenaline are found in the venom of wasps and the honeybee, adrenaline is a minor component of wasp venom. There is about 1 mg of dopamine per g of bee venom. These substances appear to be present in the nervous systems of insects and their relatives too. Dopamine, or something like it, stimulates production of pheromone in female ticks after they have taken a blood meal.

Chapter 8

128

dihydroxyphenylalanine

Figure 8.7 The oxidation of tyrosine in animals to DOPA, without hydrogen shift

phenylalanine

adrenalin or epinephrine

tyrosine

dihydroxyphenylalanine L-DOPA

doparnine

noradrenalin

or norepinephrine

Figure 8.8 The sequence of compounds from phenylalanine to adrenaline

tryptophan

tryptamine

5-hydroxytryptamine or serotonin

Figure 8.9 The serotonin group of important brain chemicals that also occur in some insect venoms

8.4.2 Serotonin Group

A relationship similar to that between tyrosine and dopamine exists between the amino-acid tryptophan and the brain substance serotonin or 5-hydroxytryptamine (Figure 8.9). Tryptamine is known in the venom of scorpions, while serotonin is found widely in the venom of honeybees, centipedes, and at least two spiders. Serotonin is not found in the venom of ants or solitary wasps but social wasps have it in quantity, as much as 1 pg per insect. It is present in the barbs of the larvae of the Tiger moth Arctia caja (Plate 11) and a saturnid butterfly larva. Their origins in insect venoms are presumably from phenylalanine but this has not been proven. The same substance is in the hairs of the common stinging nettle Urtica dioica. It is interesting to compare the structure of some alkaloids (Chapter 9) with these brain chemicals, dopamine, noradrenaline and serotonin. Many of the alkaloids are made from phenylalanine and tryptophan. For

Aromatic Compounds

129

example, the opiates alter the release and breakdown of at least four substances, noradrenaline, serotonin, dopamine and acetylcholine, all of which are believed to act as transmitters in different parts of the brain. A complex system of neurotransmitters also exists in insects, which use some of the same compounds. 8.5

PHENOLS

Phenols and quinones are both found widely among insects, but so far have been found mainly in Coleoptera, Orthoptera (crickets and locusts), Isoptera (termites) and Dictyoptera (cockroaches). Phenols provide protection upwards against predators and downwards against microorganisms. They can be formed through a variety of biosynthetic routes. Some phenols have already been encountered among acetogenins (Chapter 4). They can also be formed from phenylpyruvic acid as in the formation of tyrosine from phenylalanine (Figure 8.3). Phenol itself is widely scattered, but not frequently encountered, from millipedes (diplopods) and opilionids (daddy-longlegs, or harvestmen) to grasshoppers and beetles. Beetles are the most frequent users of phenols, often mixed with other compounds. o-Cresol and m-cresol are common in beetle defensive glands and p-ethylphenol is found in the glands of the cockroach Periplaneta americana and is probably responsible for that insect’s characteristic odour. Both o-cresol and p-cresol are known in the defensive secretion of a grasshopper Romalea microptera. Phenols like salicylaldehyde that are obtained from plants on which insects feed are considered in Chapter 10. The large metapleural glands of the ant Crematogaster deformis contain a mixture of m-substituted phenols and resorcinols shown in Figure 8.10. The presence of mellein (see Figure 4.3) with the phenols suggests they are all of polyketide origin. Simple phenols are found occasionally in the defensive secretion of millipedes and more commonly in opilionids. Three species of millipede have been shown to produce phenol and 2-methoxyphenol from tyrosine, but phenylalanine is not incorporated, which is an indication they are unable to hydroxylate phenylalanine. Other alkylphenols (e.g. 2,3-dimethylphenol, guaiacol or 0-methylcatechol, and 5-ethyl-2methylphenol, of unknown origin) are present in some opilionids. The centipede Scolopendra subspinipes multilans (Plate 5) secretes 8hydroxyisocoumarin or centipedin (Figure 8. lo), which has an antibiotic effect. In biosynthetic studies [14C]aceticacid was efficiently incorporated, indicating it is probably produced through a polyketide like mellein. Two species of ticks (Amblyomma ainericanum (Plate 14) and A . maculatum) were found to use the unusual 2,6-dichlorophenol (Figure 8.10) as

Chapter 8

130

Ho& mpropylphenol

mpentylphenol

5-prop ylresorcinol

HO 5-pentylresorcinol

OH

mellein

8-hydroxyisocoumarin

2,3-dimethylphenol

2-methyl-5-ethylphenol

&'\CH3 guaiacol, 2-methoxyphenol or catechol monomethyl ether

2,6-dichlorophenol

Figure 8.10 Phenols and resorcinolsfrom metapleural glands of an ant and from defensive glands of millipedes and opilionids. Their function is undetermined but is probably antibiotic and defensive. 2,6-Dichlorophenol is the sexual pheromone of a number of species of ticks

a sexual pheromone. They incorporated 36Clfrom Na36Clinto the pheromone, presumably by chlorination of tyrosine or another precursor. In all, 14 species from five genera of ticks use this one compound as a femaleproduced sexual attractant. The tick Amblyomma variegatum on the other hand uses o-nitrophenol,methyl salicylate and nonanoic acid (ratio 2: 1:8 pg per female). The biosynthesis in ventral glands has been demonstrated to occur after feeding, but the source of the compounds has not been studied. It is known that primitive insects like Collembola (springtails) do produce pheromones, but so far only the alarm pheromone of Neanura muscorum has been identified as 1,3-dimethoxybenzene. Its origin is unknown. Other simple aromatic compounds like 2,4-dimethoxyaniline, phenol and 2-aminophenol were also found in whole body extracts. 8.6 QUINONES Quinones are distributed widely from opilionids and millipedes to grasshoppers, cockroaches and caddis flies, but are most frequently found in beetles (Coleoptera). Phenols have been shown to be oxidized to quinones in both millipedes and beetles. It was shown some time ago in the beetle EZoides Zongicollis, that quinones can arise by two independent pathways. Labelled tyrosine, acetic, propionic and malonic acids were all used. Benzoquinone itself was preferentially made from labelled tyrosine. Simple alkylquinones were produced by the acetate pathway (Meinwald, Happ, Labows and Eisner, Science, 1966, 151, 79). Propionic acid was

Aromatic Compounds

131

rnethylhydroquinone

homogentisic acid

phydroxyphenylpyruvicacid

Figure 8.11 An unconfirmed way in which quinones can be produced from phenyl-C, compounds

only incorporated well into ethylbenzoquinone. Similar results were obtained with another tenebroid beetle Zophobas rugipes. It has been said that opilionids and millipedes make quinones from pre-existing aromatic substances while insects can make them from acetates. The work of Meinwald et al. indicates that this is not completely so. The benzoquinone and methylbenzoquinone in the secretion of the millipede Narceus gardanus were shown to be made from 6-methylsalicylic acid (Figure 4.2), indicating a polyketide pathway. Another research group found that three other millipedes required tyrosine, while acetic and malonic acid were not incorporated into quinones. Tyrosine can also be degraded via homogentisic acid (Figure 8.1l), which requires a 1,2-shift of the side chain and oxidation. If this is followed by another decarboxylation, methylhydroquinone is produced. The explosive mixture produced by bombardier (brachynid) beetles has been studied in detail by Schildknecht. A pygidial gland produces a mixture of hydroquinones in an aqueous solution of hydrogen peroxide, the concentration of the latter can be up to 28%. This mixture is stored in an inner sac. When the beetle is disturbed the mixture is discharged into an outer chamber, which is supplied with many small glands secreting a mixture of catalase and peroxidase. The resulting reactions are given in Figure 8.12. Quinones, gas and heat are evolved in a very rapid reaction, with an audible 'plop', taking the temperature of the exploded mixture to 100 "C It is noteworthy that the temperature optimum of the catalase is between 70 and 80 "C.The beetle ca.n turn its abdominal tip through 360" to direct the explosion at any predator. Quinone, hydroquinone, methyl-, methoxy- dimethyl-, and ethylquinones have all been identified in beetles, and some of them in cockroaches and grasshoppers. The tenebrionid beetle Argoporus alutacea 2

catalase H202

+

0 2

0

+ H202

OH

eR+

2 H20

9" peroxidase

+

R = HorCH3

2H20

?J

Figure 8.12 The defensive reaction of' bombardier beetles

+ heat

132

Chapter 8

a

b

C

R

d

e

R = Me, Et, Pr and Bu 0 6-alkylnaphthoquinones

Figure 8.13 Compounds a, b and c are examples of millipede quinones, d and e are from opilionids. The naphthoquinones are from the beetle Argoporus alutacea

produces a viscous, orange defensive secretion consisting of four naphthoquinones (Figure 8.13) as well as benzoquinone and its methyl and ethyl derivatives. The aphin pigments (see later) are also accompanied by simpler naphthoquinones.

8.7 INSECT PIGMENTS The colours of insects are as varied as those of a fashion designer’s dress show; from the satiny black of the elytra of some beetles and cuticle of black wood ants, through the gaudy colours of some butterflies, to the pinstripes of the Colorado potato beetle (Leptinotarsa)and the polka dots of the ladybirds. Some insect colours are due to the physical effect of interference, such as found on the wings of some butterflies and the surface of scarab beetles; these colours are called schemochromes. The beautiful irridescent blue of some butterflies like Morpho rhetenor and M. didius is due to schemochromes. Chemical substances in the cuticle, on scales or in the haemolymph that cause colours are called chemochromes. The colours may carry a message for the species (recognition, mating, camoflage) or for predators (warning colours of aposematic species). Strictly it must be remembered that the spectra of colours seen by vertebrates and insects are different, that of insects being shifted about 100-150 nm towards the ultraviolet. Carotenes in insects have already been considered in Chapter 7. They are the most widely distributed of all natural pigments.

8.7.1 Melanin Melanin is an insoluble, unreactive and irregular polymer derived from tyrosine. It is therefore perhaps unreasonable to ask if the melanin of insects is the same product as the melanin of human skin, of animal hair, and the sepia of squid. Biosynthesis of insect melanin, like the others, begins with the oxidation of tyrosine to DOPA (Figure 8.7), which is further oxidized by polyphenol oxidase to dopaquinone (Figure 8.14)

Aromatic Compounds

133

dihydroxyphenylalanine L-DOPA

O=$H

o

--2H

/

H

dopaquinone

i-1 '"/ indole-5.6-q:none indole-5,6-quinone

leucodopachrome

-

c c02 --

"n';CH HO

/

N

ii

dihydroxyindole

dopachrke

0- 0

Figure 8.14

Theformation of melanin from DOPA, showing a fragment of the melanin structure with two of the resonanceforms that contribute to the conjugated system of double bonds that gives it its strong light absorption

and then cyclized to leucodopachrome. This in turn is oxidized to another o-quinone, dopachrome, which is re-aromatized by loss of CO,, and oxidized yet again to indole-5,6-quinone.Oxidative dehydrogenation of this quinone links the aromatic units together to give the polymer, a section of which is represented in Figure 8.14. Very little is known about this final stage or the degree of cross-linking. The extended conjugated system of double bonds absorbs all over the visible spectrum so that the polymer appears black. The black pigment is also called eumelanin, while the yellow to red cuticle pigment containing sulphur is called phaeomelanin. It is not clear whether the yellow or red cuticle of some ants is coloured with phaeomelanin. The oxidative dehydrogenation of phenols and quinones to give dimers and polymers (as here in melanin and in the aphins, see later) is a common reaction in both animals and plants. Compare the production of lignin and tannins in plants. The reaction is usually seen as a radical reaction. The in vitro coupling of p-cresol to give the compound known as Pummerer's ketone, using ferricyanide, peroxidase or phenol oxidase provides something of a model (Figure 8.15).

8.7.2 Quinones There are two groups of the order Homoptera that have shown great originality in producing pigments. These are the superfamily Coccoidea

Chapter 8

u>Gu

H3C

A

H3C

CH3

CH3 Purnrnerer'sketone

Figure 8.15 An in vitro reaction which is a possible model for the oxidative coupling of phenols to give melanin and other polymers

(scale insects and mealy bugs) and Aphidoidea (the aphids or plant lice). Both groups feed on phloem sap of plants. The Coccoidea make anthroquinones, and the Aphidoidea make complex naphthoquinones. All pigments of the scale insects are polyketide anthraquinones. The artist's colour, Venetian red, is produced by Kermococcus ilicius feeding on an oak, Quercus coccifera. The pigment is kermesic acid (Figure 8.16). It is probably the oldest used insect pigment. Cochineal, the food colouring, is obtained from dried females of Dactylopius coccus (formerly COCCUS cacti), a bug feeding on Opuntia cactus (prickly pear). The pigment, carminic acid, has the same structure as kermesic acid but with a Cglucoside attached. It gives a deep red colour in water. These pigments are present as the potassium salts in vivo. Lac insects (Chapter 6) also produce pigments, consisting of more polar laccaic acids and less polar anthroquinones. The most commonly encountered examples are given in Figure 8.16. Shellac is the resin after the pigments have been removed. The additional benzene ring in laccaic acids is from a tyrosine molecule linked to the naphthoquinone by oxidative dehydrogenation. The anthroquinones can comprise up to 50% of the female body weight, but most of what is produced is secreted externally in the lac. Emodin is an example of a very widely distributed anthraquinone, from the Australian scale insect Eriococcus species, to the roots of rhubarb. It comes from an octaketide folded differently from the others illustrated here. Incidentally, the cochineal bug D. coccus, also produces a long-chain ester wax, which is a biosynthetic curiosity, 15-oxotetratriacontanyl 13-oxodotriacontanoate (Figure 8.16). 8.7.3 Aphins Aphids make their own pigments, called aphins, not found in any other insects. Their complex chemistry was studied by Lord Todd in the 1950s and 60s. Aphins are dimeric naphthoquinones. The two most important are protoaphin-fb (isolated first from the common bean aphid (Aphis

Aromatic Compounds 0

0

135

CH3 +--+

OH

OH 0 kermesic acid

0 laccaic acid D

PI

COOH

HO

OH

OH R

O

R = OH, laccaic acid

R = H, xantholaccaic acid 0

HO 0

R

carrninic acid OH R=OH, erythrolaccin R = H, desoxyerythrolaccin

emodin

Figure 8.16 Some anthroquinone pigments of scale insects, with a presumed parent polyketide. Kermesic acid is the artist’s pigment Venetian red and carminic acid is the food colouring cochineal

fabae) and protoaphin-sl (first isolated from the brown willow aphid Tuberolachnus salignus and only differing from the first at one chiral centre) (Figure 8.17). There is restricted rotation about the bond joining the two parts of the molecule. The protoaphins are found in the haemolymph. The isolated material is brownish-yellow, but they are substantially ionized at physiological pH, and then give a deep purple colour. Different species of aphid vary in colour through shades of green, brown and red to almost black. Aphins are characteristic of the darker species. The green pigment is aphinin. Some species, e.g. greenfly, Macrosiphium rosae, contain only aphinin. The extraordinary situation exists that the structure of this pigment has been left incompletely solved for 35 years. It is probably as given in Figure 8.17. It was long presumed that these pigments were made by symbiont micro-organisms, but surprisingly, it has been shown that aphids treated with antibiotics and having no bacteria in them still produced aphins. The aphid Aphis neri feeding on the toxic shrub Nerium oleander is bright orange, possibly as a warning colour (Plate 7). It contains glucoside B (Figure 8.17) and a number of naphthalene derivatives related to it. Some are shown in Figure 8.18. The structure with the quinone methide a in Figure 8.18 is probably formed by condensation of neriaphin (before the glucoside is attached) with biacetyl and elimination of water, a rare example of biacetyl taking part in the formation of a natural product. The condensation can be repeated in vitro in weakly alkaline conditions.

Chapter 8

136

HO

COOH presumed polyketide

differ here

HO glucoside B OH

quinone A

OH- -0

a perspective view aphinin-fb (?) OH

Figure 8.17 The formation of the aphins, pigments of aphids. The naphthalene derivative first formed is converted into quinone A and glucoside B, which linked together give the uphins. Note that aphinin-fb and aphinin-sl difer by only one chiral centre. There is restricted rotation about the central bond. Structure a shows the twist in the molecule with the right side of the upper portion turned toward the reader and the right side of the lower portion turned away. The structural study of aphinin-fb has never been completed

"CH3

HO

"CH3 0

neriaphin

HO

6-hydroxyrnusizin

H3C

OHCH3 a

8-OP-D-glucoside

Figure 8.18 Some of the pigments from Aphis neri. Compound a cun be made by reaction between biacetyl and a ketone derivative of the first intermediate in Figure 8.17

8.7.4 Pterins The pigments of butterflies were first identified by Heinrich Wieland in the 1930s. They belonged to a whole new class of nitrogen heterocycles, which he called pteridines (Greek pteros = wing). The structure of the white pigment of Pieris brassicae and l? rapae, leucopterin, and the yellow pigment xanthopterin (from Gonepteryx rhamni) were the first elucidated (Figure 8.19). Later it was found that compounds with the

Aromatic Compounds

137

popopo-w Hd bH guanosine triphosphate

xanthopterin

chrysopterin

Figure 8.19 Theformation of the parent of thepterins from GTE with the structures of some insect pterin pigments. Leucopterin is colourless, xanthopterin and chrysopterin are yellow, erythropterin is red. Biopterin isfound generally in all cells. Note the similarity between the third and fourth steps in this sequence and the third step in the formation of tryptophane in Figure 8.5

pteridine structure are widely found in nature, e.g. tetrahydrofolic acid (Figure 2.15) and flavin (Figure 2.10). The compound biopterin (Figure 8.19) occurs in every animal cell or tissue as a co-factor of some enzyme reactions, e.g. the hydroxylation of phenylalanine to tyrosine and tyrosine to DOPA, but mammals are unable to make pterins. Biopterin is thought to be a growth factor for some insects. As well as forming some butterfly wing pigments, pterins are also found as body pigments of Lepidoptera and Hymenoptera. Xanthopterin, as well as being present in the wings of Gonepteryx rhamni, is particularly widely distributed in insects and other animals. It provides the yellow colour of common wasps (Vespa vulgaris, Polistes dorninulus (Plate 16), and I? crabro, the hornet). Little is known about pterin biosynthesis in insects, but it is probably as in mammals. In mammals it begins from the nucleotide guanosine triphosphate (GTP). The imidazole ring of guanine is opened up and loses one atom of carbon as

138

Chapter 8

formic acid (Figure 8.19), leaving a ribose triphosphate derivative of a diaminopyrimidine. The ribose undergoes ring opening to give an open-chain keto-sugar, which cyclizes with the free amine group to give dihydroneopterin triphosphate. Surprisingly all these steps are catalyzed by the one enzyme, GTP cyclohydrolase. The black and orange warning colour of the milkweed bug OncopeEtus fasciatus (Plate 17) is due to part opaque black melanin and part transparent cuticle that allows the underlying pterins to show through. Five different pterins were identified in the red heteropteran fire bug Pyrrhocoris apterus (Plate 9), with erythropterin the most abundant.

8.7.5 Tetrapyrroles The bilins are linear tetrapyrroles produced by ring cleavage of protoporphyrins. They have been identified in most major groups of animals. In mammals they are known as bile pigments. They are found in Phasmida, Mantida, Orthoptera and Lepidoptera. The green colour of many grasshoppers and lepidopteran larvae is due to bilins of the biliverdin type (Figure 8.20). Biosynthesis of the tetrapyrroles begins with a Claisen condensation between glycine attached to pyridoxal phosphate (Chapter 2) and SUCcinyl CoA. After cleavage from the pyridoxal, 5-aminolaevulinic acid is obtained. Condensation of two molecules of the latter gives the first pyrrole, porphobilinogen (Figure 8.20). Porphobilinogen molecules are coupled by deamination to give a dimer, trimer and tetramer, which can be cyclized to give a variety of tetrapyrroles, the important one here being protoporphyrin IX. Oxidative ring opening at the a-meso position of protoporphyrin IX (between rings A and D) gives the blue-green pigment biliverdin. Reduction of the central -CH= gives the orange coloured bilirubin (Figure 8.20). Chironomus larvae store both biliverdin and bilirubin in their fat body. Cleavage of protoporphyrin IX between rings C and D gives pterobilin, first isolated from the larvae and pupae of Pieris brassicae (cabbage white butterfly), but has been found in the majority of 100 species of Lepidoptera examined. Irradiation of pterobilin causes rotation about the -CH= joining rings A and B and then reaction of the vinyl group on ring B with ring A. Further reaction between the vinyl group of ring A and ring D also occurs to give sarpedobilin. More details of the biosynthesis of this group can be found in Torssell or Britton (see list of further reading).

Aromatic Compounds

O ''H

H

2 x 5-aminolaevulinic acid

- Z H 2 O b g C o o -+ H

a

139

+

9'

NH2 H porphobilinogen

HN /

'

5

protoporphyrinIX

haem

'

NH

HN

'

00

COOH

COOH

pterobilin or biliverdin IXy

bilirubin

COOH

HOOC

hv

phorcabilin

Figure 8.20 A brief summary of the biosynthesis of some insect bilins, starting from 5-aminolaevulinic acid. Pterobilin is found only in the wings of pierid butterflies, while phorcabilin is found in the wings of papilionid, arctid and microlepidop tera together with isophorcabilin and sarpedobilin (not illustrated)

8.7.6 Ommochromes and Ommins This discussion of pigments does not exhaust the list of coloured substances from insects. Some mention should be made of the ommochrome pigments from insect eyes. They function as screening pigments to cut out stray light. They can also be found in insect integument. They are divided into the ommatins of lower molecular mass, labile to alkali, and ommins, of higher molecular mass and stable to alkali. They are extracted from the ommatidia of the compound eyes. The ommatins are formed from the amino-acid tryptophan via kynurenine and hydroxykynurenine, which undergoes oxidative dimerization to give the ommatins. Yellow xanthommatin (the most frequently encountered,

Chapter 8

140 C-C-COOH

d F

t -COOH NH;!

N-CHO

H

kynurenine 1

&Z0H-

H2NY 0

xanthornmatin

rhodommatin

ommin A

Figure 8.21 Theformation of ommochromes and ommins from tryptophan via kynurenine

Figure 8.21) and red dihydroxanthommatin are examples of ommochromes, derived from tyrosine. Use of labelled xanthommatin has shown it is converted to rhodommatin. Much has been learned about ommatins from studies of the fruit fly Drosophila melanogaster and its many eye-colour mutants. There are about 10 to 15 of these pigments in insects, from brown-yellow through deep red to purple. Loss of the amino-acid chain from hydroxykynurenine and dimerization gives cinnabarinic acid, a compound found only in the commercial silk moth Bombyx rnori. Ommins are polymeric, insoluble and some contain sulphur. They are less well investigated. The structure of the trimer ommin A (Figure 8.21) suggests that they may all be based on linear polymers of hydroxykynurenine. Using 35S-labelling,it has been shown that the sulphur in ommins can be derived from cysteine or methionine, but not from sulphate, thiocyanate or sulphide.

BACKGROUND AND FURTHER READING M. S. Blum, Biosynthesis of arthropod exocrine compounds, Annual Review of Entomology, 1987,32,381-413. G. Britton, The Biochemistry of Natural Pigments, Cambridge University Press, 1983, pp. 366 (Chapters 1, 5 & 7, insect pigments). J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 5).

Aromatic Compounds

141

A. E. Needham, Insect biochromes: their chemistry and role, in M. Rockstein, editor, Biochemistry of Insects, Academic Press, New York and London, 1978, pp. 233-305. H. Schildknecht, The defensive chemistry of land and water beetles, Angewandte Chemie, International Edition in English, 1970,9, 1-9. K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapter 4). J. Weatherstone and J. E. Percy, Venoms of Coleoptera, in S. Bettini, editor, Arthropod Venoms, Springer, Berlin, 1978, pp. 97 (for a lengthy list of Tenebrionid beetle quinones up to that time).

QUESTIONS 1. Show two possible polyketides that could give rise to emodin (Figure 8.16). 2. Show a common biosynthetic route that accounts for m-propylphenol and mellein (Figure 8.10). 3. Suggest biosynthetic precursors for the two aromatic compounds in the queen substance of honeybees, methyl p-hydroxybenzoate and 4-hydroxy-3-methoxyphenylethanol (Figure 3.32). 4. What kind of experiment would you suggest to decide whether the plant compound eugenin was derived by the polyketide route or the shikimic acid route?

H3c'0w OH

6

eugenin

5 . Marginalin is a yellow pigment in the pygidial glands of the water beetle Dytiscus marginalis. Also present are p-hydroxybenzaldehyde and homogentisic acid (Figure 8.11). Suggest biosynthetic origins for all three.

marginalin

142

Chapter 8

6. The pterins are shown in their amide forms, because these are the more stable tautomers. Below is the violet-blue pterorhodin in its amide form, draw it in its fully aromatic imidol form.

pterorhodin

7. The enzyme tyrosine phenol lyase can convert tyrosine to phenol and phenol back to tyrosine. Working on tyrosine, it also produces pyruvic acid and ammonia. The tyrosine is attached to pyridoxal phosphate during the reaction, and the phenol goes through a quinonoid form. Write the reaction going either from tyrosine or from phenol. This route may be a way by which insects can de-toxify phenol they themselves have produced.

CHAPTER 9

Alkaloids and Compounds of Mixed Biosynthetic Origin 9.1 ALKALOIDS Alkaloids were formerly defined as basic, nitrogen-containing plant substances, but examples have also been found in fungi, marine organisms, amphibians, insects and even mammals. There are over 10,000 plant alkaloids known, very varied in structure, from simple ones like coniine to complex structures like strychnine and reserpine (Figure 9.1). The biosynthesis of alkaloids in plants has received a lot of study. Less is known about the biosynthesis of the much smaller number of insect alkaloids, except for a few examples, like the coccinellines, adaline and epilachnine (Chapter 3). All the available evidence suggests that plants make alkaloids to deter predators. Some, like the tobacco alkaloids are strongly toxic to insects. Nicotine, anabasine and other related alkaloids are produced in the roots of the tobacco plant and translocated to the leaves. Nicotine is certainly toxic to most insects. Formerly, a crude preparation of nicotine was used commercially as an insecticide, but the tobacco hornworm (Manduca sexta) (Plate 15) has adapted itself so that its larvae feed only on tobacco

strychnine

Figure 9.1 Some examples of plant alkaloids. Coniine is from the leaves and seeds of hemlock and very toxic to motor nerves. Strychnine, from Strychnos nux-vomica, is a central nervous system and respiratory stimulant, also very toxic. Reserpine, from Rauwolfia serpentina roots, is used clinically against hypertension and formerly as a tranquillizer

143

144

Chapter 9

leaves, and others, like the cigarette beetle Lasioderma serricorne live only on dried tobacco leaves. Nearly half of all plant families have at least one species that contains alkaloids, but of about 10,000 plant genera, less than 10% are known to make alkaloids, so the distribution is very patchy. Alkaloids are particularly common in the Leguminosae (peas and beans) and the Solanaceae (tomato, potato and tobacco). 9.1.1 Alkaloid Precursors

The amino-acids phenylalanine, tryptophan (and the physiological amines derived from them), plus the amino-acids ornithine and lysine, and nicotinic acid, are the precursors of most alkaloids (Figure 9.2). There is not, however a uniform biosynthetic route to alkaloids as for those compound types in earlier chapters.

tN%COOH #I NH2 histidine

-C

A

ocH2 H ,C-COOH

N H p+

cop

'NHp

phenylalanine

histarnine

tryptophan

H2NYCooH - cadaverine lysine NH2 H2N-NH2

+

cop

qcooH

nicotinic acid

Figure 9.2 Three important amines, histamine, putresine and cadaverine, derived from amino-acids, together with the four amino-acids and nicotinic acid from which the great majority of alkaloids are made

Physiological amines from phenylalanine and tryptophan have been discussed in Chapter 8. A non-aromatic example to add is histamine, derived from the cyclic amino-acid histidine (Figure 9.2). Histamine is released in large amounts in allergic reactions in humans and other animals. Histamine is a characteristic component of wasp and bee venom. It is also in the venom of some Australian ants and in the spines on larvae of some Lepidoptera. Putrescine and cadaverine are two diamines with repulsive odours, first identified in decaying meat. Their odours are warnings against eating such material that contains bacterial toxins. Cadaverine is also a skin irritant and poison.

Alkaloids and Compounds of Mixed Biosynthetic Origin

145

9.1.2 Plant Alkaloid Biosynthesis Coniine from the hemlock plant Conium maculatum is a simple example of an alkaloid, although its biosynthesis is unusual. It was thought that coniine would be derived from the amino-acid lysine, but labelled lysine was not converted to coniine. It is unusual in being derived from a type I polyketide (Chapter 4). Both [6-14C]-5-oxo-octanoicacid and [6-'4C]-50x0-octanal are well-incorporated into labelled coniine (Figure 9.3). A more typical example of the biosynthesis of an alkaloid is given by the tobacco alkaloid nicotine, which has been shown to be derived from nicotinic acid and ornithine. Its biosynthesis has been studied in detail. A dihydropyridine is an intermediate because if nicotinic acid is labelled at C-6, that label is lost during condensation. The methyl group in the pyrrole ring comes from S-adenosyl methionine (Figure 9.4). In the final stage, the labelled hydrogen is lost as H- to NADP". Anabasine (another tobacco alkaloid) is produced similarly from nicotinic acid and lysine.

5-0x0-octanoic acid NH3

1

transaminase

coniine

Figure 9.3 The route to coniine (in plants) from a tetraketide via 5-0x0-octanoic acid, which was labelled in the 6-position

9.1.3 Insect Alkaloids

Anabasine, anabaseine and bipyridyl have been found in the venom of several species of Aphaenogaster and Messor (seed-gathering ants) but we do not know anything about their biosynthesis there. In Aphaenogaster rudis anabasine, anabaseine and bipyridyl are all part of the trail pheromone. Alone, each compound shows no activity, but with N-isopentyl-2-phenylethylamine(Figure 9.4) they form the active pheromone. The anabasine in some Messor ants was shown to have the same 2'-(S) configuration as it has in plants, but in some Aphaenogaster it is of mixed enantiomers. Cotinine, a metabolite of nicotine, is produced by the American and German cockroaches (Periplanata americana and Blatta germanica) and the housefly Musca domestica.

Chapter 9

146

ornithine

t

c,

Q' H '

anabasine

Figure 9.4

anabaseine

bipyridyl

cotinine

N-isopentyl2-phenylethyiamine

The biosynthesis of nicotine from ornithine and nicotinic acid. Anabasine, anabaseine and bipyridyl, all ant alkaloids, are made by plants using lysine in place of ornithine. Cotinine is a metabolite of nicotine, N-isopentyl-2phenylethylamine is an important constituent of the trail pheromone of the ant Aphaenogaster rudis

The alkylpiperidines and tetrahydropyridines form a powerful venom in Solenopsis ants (Figure 9.5). The sting of these ants is so painful, they are known as fire ants. Both cis and trans arrangements of the methyl and alkyl groups are found; the cis-linked are always (R,S)and the trans are (R,R). Because S. geminata makes essentially only cis- and transsolenopsin A, it was chosen for the biosynthesis experiments. Other species make more complicated mixtures. Feeding experiments with 3,000 to 4,000 Solenopsis geminata, using sodium [ 1-14C]-acetate and

o kp * g 2-methyl-6-alkylpiperidines

A

n ' ' ' ' ' @ 2 m C H 2 g

n=3,5or7 2-methyl-6-alkenylpiperidines

CH2

fl "'"'TH

3'

2 10

2-methyl-6-alkyltetrahydropyridines

n = 6 to 14

cissolenopsin A

franssolenopsin A

NP-dimethyl6-alkylpiperidines n =8 or 10

Figure 9.5 2-Methyl-balkylpiperidines and tetrahydropyridines and some N-methyl derivativesfound in the venom of Solenopsisfire ants. The two solenopsins A form almost the total venom of S. geminata

Alkaloids and Compounds of Mixed Biosynthetic Origin

147

[2-14C]-acetate, followed by dilution with ‘cold’ synthetic 2-methyl-6undecylpiperidine indicated that the compounds are biosynthesized as shown in Figure 9.6. As a first estimate, the pathway seems similar to that of coniine in plants. The synthesis begins with the linking together of 9, 10 or 11 acetate units. It is not clear whether this is through a fatty acid or a polyketide. At some stage a keto-acid must be formed, which is decarboxylated; addition of an amino-group, cyclization and (for most of the compounds) reduction gives the solenopsins. Another group of Solenopsis (thief ants) and some species of Monomorium ants have alkyl-pyrrolidines and -pyrrolines (Figure 9.7). The sting of these ants does not seem to be so painful, but whether this is due to the alkaloids or the protein part of the sting is not known. More recently some of the same and similar pyrrolidines have been found in Megalomyrmex ants. Their biosynthesis is not yet known.

Figure 9.6 Labelling experiments indicate that the synthesis of solenopsins in S. geminata goes by one of the routes shown

Pf4 H p*tI. & *: n =4,6or a

m = l or3,n=4or6

e4pY4 CH3

Me

rn = 1,3,4 or 5, n = 4, 5.6,or 8

Figure 9.7 Alkyl- and alkenyl-pyrrolines, -pyrrolidines and N-methylpyrrolidines of unknown origin from Solenopsis thief ants and some Monomorium ants

An unrelated group of alkylpyrrolidines have been found in the venom glands of the slave-making ant Harpegoxenus sublaevis and its slaves Leptothorax acervorum and L. muscorum (Figure 9.8). The amounts are

Chapter 9

148 OH

6 most

d

i b

1'

least

Figure 9.8 Substititedpyrrolidines from the venom of a slave-making ant and its slaves. The compounds are arrangedfrom the one in greatest quantity on the left to the least on the right

very small, 10 ng down to 5 pg, but there is more in the slave-maker than in the slaves. The alkyl groups certainly suggest amino-acid origins (leucine, methionine, phenylalanine and isoleucine). The coccinellid beetles (ladybirds) evidently have enzymic resources to make a variety of alkaloidal defenses. The coccinellines, adaline and epilachnine have already been encountered, since their biosynthetic origin is known. Another ladybird, Hyperaspis campestris produces hyperaspine (Figure 9.9), which has a partly constructed precoccinelline (see Figure 3.22) with pyrrole carboxylic acid esterified to it. Exochomus quadripustulatus produces a more complex compound, exochomine (Figure 9.9), which has an azaacenaphthalene linked to hippodamine (see Figure 3.22). Yet another coccinellid Chilocorus cacti produces the complex alkaloid chilocorine (Figure 9.9), which has hippodamine linked by two bonds to the same azaacenaphthalene as that in exochomine. An amino-acid amide of quinaldinic acid occurs in Subcoccinella vigintiquatuorpunctata. For a series of eight coccinellid alkaloids, some met previously, see Attygalle, Xu, McCormick, Meinwald, Blankspoor and Eisner (Tetrahedron, 1993,49,9333). Finally the fatty acid derivative (2)1,17-diamin0-9-octadecene is found in several species. Its structure indicates an origin in oleic acid but it has no obvious relationship to the wealth of polycyclic coccinellid amines. These compounds have been relegated to this section because their biosynthesis is incompletely known. Some ants make products reminiscent of the coccinellid alkaloids. Three indolizidines are known from the venom gland of Monomorium pharaonis (Figure 9.10), and a pyrrolizidine from a Solenopsis ant has been known for some time. They would appear to be formed from polyketides or fatty acids as in the case of the coccinellines. More pyrrolizidines and indolizidines with two alkyl and alkenyl groups have been found in Monomorium and Solenopsis species. Tetraponerine ants smear the venom of their poison glands onto prey. The venom consists of tricyclic alkaloids called tetraponerines (Figure 9.10). They are divided

Alkaloids and Compounds of Mixed Biosynthetic Origin

niiocorine

149

N-a-quinaldinyl-L-arginine

Figure 9.9 Some alkaloids with bridge-head nitrogen and a diamine from ladybirds. The biosynthetic scheme for hyperaspine is the author’s present suggestion only. No scheme for the formation of the azanaphthacene part is ofered

tetraponerines

Figure 9.10 More complex alkaloids from ants. The indolizidines where R is butyl, hexyl and 3-hexenyl have been known from the venom of Monomorium pharaonis, pharaoh’s ant, for some time. The 2-heptyl-8-methyl-pyrrolizidine is from a Solenopsis ant. The keto-indolizidine is from an African Myrmicaria ant. There are eight of the tetraponerines from Tetraponera species from New Guinea

into two groups, designated 6,6,5 and 5,6,5 by the size of their three rings (Figure 9.10). The biosynthesis of the tetraponerines has been studied using sodium [ 1-14C]-acetate and [2-14C]-acetate, [ 1,4-’4C]putrescine dihydrochloride and L-glutamic acid, y-aminobutyric acid and Lornithine, all uniformly labelled with 14C. Both groups were shown to have a mixed biosynthetic origin, from an amino-acid and a chain of acetate units, as shown in Figure 9.11. Note that y-aminobutyric acid, produced by decarboxylation of glutamic acid is not a protein aminoacid, but is another of the physiologically active amines. It is a potent neurotransmitter. Topical application of a tetraponerine mixture to Myrmica ants indicated a toxicity ten times greater than that of nicotine.

150

Chapter 9

glutamic acid

6 X CH3COOH

ornithine

4 x CH&OOH

\

tetraponerine 8

y-aminobutyric acid

tetraponerine 6

Figure 9.11

The formation of two examples of the tetraponerines from ants. Glutamic acid, ornithine and y-aminobutyric acid uniformly labelled with 14C,sodium acetate labelled in C-1 and C-2 with 14C,and [1,4-l4C]putrescine were all used to show the synthesis is as shown

Recently a whole series of indolizidine and pyrrolo-indolizidine alkaloids have been found in Myrmicaria ants (Figure 9.12), which evidently have a similar origin to the indolizidines in Figure 9.10. Note that they are all constructed from straight carbon chains. The structures of the complex dimer and trimer were deduced from NMR and mass spectral studies. H

a pyrroloindolizidine

Figure 9.12 Some complex alkaloids from the venom of Myrmicaria ants from Africa. Their biosynthesishas not been studied, but they can been viewedasproducedfrom the diketopiperidine shown in the smaller structures in the lower part of thefigure

Alkaloids and Compounds of Mixed Biosynthetic Origin

151

9.1.4 Other Examples Some rather strange one-off examples of alkaloids have been found. The water skater Stenus comma produces stenusin, or 3-(2'methylbuty1)piperidine (Figure 9.13) in its pygidial gland, probably as an antibiotic substance. A phasmid insect (Oreophoetes peruanu) stores quinoline, as the single component in its thoracic glands. It is probably made from anthranilic acid. The compound is repellent and topically irritant to ants, spiders and cockroaches. The pale-brown chafer beetle Phyllopertha diversa uses an alkaloid as its female sex pheromone. The 1,3-dimethy1-2,4-quinazolinedione(Figure 9.13) evidently comes from anthranilic acid. Equally curious is the discovery that only the males of this species possess a cytochrome P,,, specifically for breaking down this compound (by demethylation of N-1 and hydroxylation of the aromatic ring). The sex pheromone of the longhorn beetle Migdolus fryans, which

QT H

stenusin

N

quinoline

QJ5r3 CH3 dimethylquinazolinedione

+N?

H

i

N-2-methylbutyl 2-methylbutanamide

& pohonamine

Figure 9.13 More unusual insect and arthropod alkaloids

does so much damage to woodwork, N-2-methylbutyl 2-methylbutanamide is apparently derived from L-isoleucine. A millipede Polyzonium rosalbum produces polyzonamine (Figure 9.13) which has a very noticeable camphor-like odour. Polyzonamine evidently has a isoprenoid origin but its biosynthesis is not known. The philanthotoxins (Figure 9.14) are a group of compounds from the venom of the bee-wolf Philanthus triangulutus, which preys on worker bees, stinging and paralyzing them. The compounds of the group differ in the number of carbon atoms between the nitrogens. The components of these compounds are easily recognized, a butyric acid amide of tyrosine in turn linked to putresine. Note the similarity of this part of the structure to spermidine and spermine, trace substances widely found in cells but of uncertain function. The bee-wolf lays its egg on the live but paralyzed bee, which provides fresh food for the larva when it hatches. Polyamines like spermine and spermidine are found in the venom of the funnel-web spider Atrux robustus, and have been identified in the venom of tarantula spiders some time ago. Compounds similar to the philanthotoxins have been identified in many spider venoms. The compound designated CNS 2103, based on tryptophan, has been found in the

Chapter 9

152

H-iA N 4i&NH2 H2 philanthotoxin-433

H2

putresine

Figure 9.14 Philanthotoxins are alkaloidsfrom wasp venom, spermine and spermidine occur in venoms and as part of these larger compounds. Compounds CNS 2I03 and Agel 489a are from spider venoms

venom of a fishing spider (Figure 9.14). It consists of hydroxylated indoleacetic acid amide linked to cadaverine that has been extended with three C3-NH2units. About 70 such compounds are known in spider venoms, some have N O H or N-CH, groups. Agel 489a from AgeneZopsis uperta is another example. Such compounds are described as polyazaalkanes coupled to an aromatic head group. 9.1.5

Alkylpyrazines

Alkylpyrazines are not usually considered as alkaloids, although they are basic nitrogen compounds; they are better known as flavour components, present in very small quantities, and apparently detectable by humans and insects in parts per billion. They are very common among ants, as trail pheromones in their venoms and of unknown function in the mandibular glands of many ponerine ants. Their biosynthesis can be reasonably predicted from the known chemistry of their formation and the knowledge that the amino-acid threonine decomposes by way of oxidation to a P-keto-acid and decarboxylation to amino-acetone (Figure 9.15). The intermediate dihydropyrazine can react with an aldehyde in a way that has been demonstrated in the laboratory, to give the final alkylpyrazine. Trisubstituted pyrazines with C3to Cs alkyl groups and tetrasubstituted pyrazines with an additional isopentyl group (hydroxylated or unsaturated) are found in the mandibular glands of a number of ponerine ants. The trisubstituted pyrazines can be converted to the tetrasubstituted ones in vitro by free radical addition of isopentanal followed by reduction

Alkaloids and Compounds of Mixed Biosynthetic Origin 0

153

H2N

HOOC

1

threonine

RCHO

Figure 9.15 The probable biosynthetic route to alkylpyrazines. The trisubstituted pyrazines have been converted to tetrasubstituted hydroxylated or unsaturated examples in the laboratory by addition of isopentanal, followed by NaBH, reduction and sometimes dehydration. All such compounds are found in nature. 2,3-Dimethyl-5- (2’-methylpropy1)pyrazine seems to have a diferent biosyn thet ic origin

CHO H

leucine

COOCH3

methyl 4-methylpyrrole2-carboy late

Figure 9.16 A suggested route to the pyrrole trail pheromone of some leaf-cutting ants

of the initially formed keto-group to an alcohol and possible dehydration to the unsaturated examples. This suggests the possible biosynthetic route. The presence of the aldol dimer of isopentanal in the same secretion offers support for this suggestion. Some alkylpyrazines are of the 2,3-dimethyl type, with a less obvious biosynthetic origin. An example is 2,3-dimethyl-5-(2’-methylpropyl)pyrazine (Figure 9.15), the trail pheromone of the ant Eutetramorium mocquerysi from Madagascar. Methyl 4-methylpyrrole-2-carboxylate(Figure 9.16) is non-basic. It is the trail pheromone of some leaf-cutting ants. It is probably made from leucine by oxidation and cyclization. The subject of alkaloids is one with no clear boundaries. Other compounds already discussed, indole and skatole (Figure 8.5, non-basic), actinidine, a defensive compound of several species of rove beetles

154

Chapter 9

(Figure 6.1 l), pterins and ommachromes, could be included under this heading, while some neutral plant amides like colchicine are always considered among alkaloids. 9.2 COMPOUNDS OF MIXED BIOSYNTHETIC ORIGIN Some compounds of mixed biosynthetic origin have already been met in earlier chapters. Epilachnine (Figure 3.29) is made from a fatty acid and an amino-acid, loganin is composed of a sugar bonded to a monoterpene (Figure 6.1 l), the blatellostanosides have a sugar bonded to sterols (Figure 7.9) and ecdysone palmitate is a sterol with a fatty acid attached (Figure 7.15). Philanthotoxin-433 in Figure 9.14 has a phenyl-C, portion, a simple aliphatic acid and a polyamine part. It is useful to look at some further examples of compounds of mixed origin so that they present less difficulty in the practical elucidation of structure of insect substances. Plant substances, as might be expected, give the most frequent examples of mixed biosynthetic type. Humulone, one of the bitter substances of the hop vine (HumuZus ZupuZus) that gives beer its characteristic taste has a polyketide core, with isoprene units attached to it (Figure 9.17). These isoprene units are now known to be made by the methylerythritol phosphate pathway (Chapter 6). The important groups of plant substances, the anthocyanins (flower pigments), the flavonoids and isoflavonoids have a phenyl-C, unit extended with a polyketide (Figure 9.17). The polyketide added here is called a type I11 polyketide. Both flavones and anthocyanins are found in insects. The Common Blue butterfly, Polyammatus icarus, contains three flavones and an isoflavone, 3-methoxykaempferol and quercetin-3’,4’-diglucoside (Figure 9.17). All are found in the larval plant food and are therefore not really insect products. The Marbled White butterfly, Melanargia galanthea, has twelve flavones. In all cases they are probably obtained from their food plants. A very remarkable example of a pheromone is the oviposition deterrent of the cherry fruit fly RhagoZetis cerasi. This substance is placed on the cherry fruit by the female fly after she has laid her egg in it (Ernst and Wagner, Helvetica Chimica Acta, 1989, 72, 165). Its purpose is to stop other females of that species from laying eggs in the same fruit. The pheromone must be stable and non-volatile and remain intact in sun and rain. It consists of sugar, fatty acid and amino-acid portions, which together ensure a non-volatile, UV transparent and insoluble substance (Figure 9.18). Adult chrysomeline beetles, when disturbed, release from their elytra (the hardened fore-wings of beetles) and from their thorax curious

Alkaloids and Compounds of Mixed Biosynthetic Origin

H

o

\

I

~

o

(

isoflavones H

I

155

\W'')

anthocyanins 0-glucose

1

OCH3 OH 0 3-methoxykaempferol

1

quercetin 3,4'-diglucoside

Oh v

Figure 9.17 Humulone, an example of mixed biosynthetic origin, is made of a triketide with three isoprene units grafted onto it (shown with thick black lines). The phenyl-C, unit of p-coumaric acid is extended with a triketide to give at least four series of important plant compounds. Flavones, isoflavones and anthocyanins have been found in insects. 3-Methoxykaempferol and quercetin 3t,4'-diglucoside are from the Common Blue butterfly. OH

H

N

-S03H

0

Figure 9.18 The oviposition deterrentpheromone of the cherryfruitfly. It consists of a dihydroxystearic acid with a glucose molecule attached as an ether and an amide of the amino-acid sarcosine

isoxazole glycosides esterified to nitropropionic acid (Figure 9.19). By feeding adults of Chrysomela tremulae with ~-[U-'~C]aspartic acid (i.e. uniformly labelled aspartic acid) it was shown that the beetles convert this into both the isoxazole and nitropropionic acid parts. An equally unusual structure has been found in the venom of a funnelweb spider Hololena curta. The funnel-web spiders are noted for their particularly deadly venom. The compound, designated HF-7 is a disulphate of guanosine, linked to an acetylated fucose sugar (Figure 9.20). It acts as many neurotoxins do by blocking calcium channels in cell membranes. This compound and the oxalic acid amide of agmatine, or decarboxylated arginine (Figure 9.20) are further examples of spiders

156

Chapter 9 co2 COOH

HOOC-

A

HOOC-NH2

e

y

NH2 aspartic acid

H

r

HOOCor

HOOCTCooHA02 HN-OH

.N-OH

-

HN>o

3-isoxazolin-5-one UDP-glUCOSe

H O O C Y

COOH

c02

HOOCbNo2

t

NO2

Figure 9.19 Theformation of the defensive secretion of Chrysomela tremulaefrom labelled aspartic acid, Both the isoxazolinone part and the nitropropionic acid part are derivedfrom aspartic acid. The 6'-nitropropionic ester as well as the 2',6'-bis-(nitropropionic) ester shown, are present in the secretion

0

HF-7

Figure 9.20

The guanosine derivative designated HF-7 from venom of Hololena curta, a funnel-web spider, and the amide of oxalic acid with agmatine (decarboxylated arginine) from a hunting spider Plectreurys tristis

adapting derivatives of primary metabolites (compare section on spiders in Chapter 3, p. 52). a-Tocopherol acetate (Figure 9.21) is present in the secretion of the pupae of the squash beetle Epilachna borealis. It is made from chorismic acid via p-hydroxybenzoic acid with isoprene units added. Tocopherol contains a completely reduced diterpene chain fused onto a trimethylhydroquinone. The extra methyl groups on the quinone part come from S-adenosylmethionine. Tocopherol is an antioxidant and is vitamin E in humans, so this application of the compound is curious. It is related to the ubiquinones, also known as coenzyme Q, they are primary metabolites, found in all cells, with a function in electron transport.

Alkaloids and Compounds of Mixed Biosynthetic Origin

157

YOOH + P O P O y y - )

Q OH

n

+

P O P O + q q H 4

Figure 9.21 Outline of the formation of a-tocopherol acetate from p-hydroxybenzoic acid, a reduced terpene chain and additional methyl groups, and, for comparison, the formation of ubiquinones

9.2.1 Luciferin Bioluminescence, the ability of living organisms to emit light, is widely distributed throughout more primitive orders. It occurs in insects (fireflies and glow-worms) among the Collembola, Hemiptera, Diptera, Lepidoptera and Coleoptera. It is found particularly in three Coleoptera families: Lampydidae, Elateridae and Phengodidae. All the insects examined share the same system of luciferin and luciferase to produce light. Luciferin is synthesized by the fusion of quinone and cysteine to give first 6-hydroxybenzothiazine-2-carboxylicacid. This condenses with a second molecule of cysteine before oxidation and re-arrangment in an unexplained way to give luciferin (Figure 9.22). Luciferin is oxidized enzymically to oxyluciferin, which is formed in two excited states (Figure 9.23). To reach ground state, one form emits red light, the other green, the resultant effect is close to white light. One third of the molecules that undergo oxidation emit light (White, Miano and Umbreit, Journal of the American Chemical Society, 1975, 97, 198). Insects emitting light make an easy target for prey. It is not surprising that lampyrid beetles also employ chemicals, lucibufagins (Chapter lo), to make themselves unpalatable.

HS)

Figure 9.22

The biosynthesis of luciferin. The carboxyl group of cysteine was labelled with 14C

158

Chapter 9

aN\HNrcyH luciferase + ATP

HO

s

Mg2+_

[ luciferin - luciferase - ATP] + pyrophosphate

s

\red

hv

green hv

Figure 9.23 The light-emitting reaction of luciferin. When "0,gas was used, labelling was found equally in CO, and oxyluciferin. The asterisks indicate the excited electronic states

9.2.2 Volicitin Some lepidopteran larvae and grasshoppers have been shown to induce plants on which they are feeding to release volatiles (mostly terpenes) that attract predators and parasitoids of the plant-eating insects, by the saliva or regurgitated juices from the mouths of the larvae. The cause in at least one insect, Spodopteru exiguu, feeding on corn seedlings, is called volicitin, an elicitor of plant volatiles. Volicitin has been identified as N-( 17S-hydroxylinolenyl)-~-glutamine (Figure 9.24). The linolenic acid, taken from the plant, is hydroxylated by the insect and conjugated with glutamine. The saliva also contains 17-hydroxylinolenic acid, 17-hydroxylinoleic acid, linolenyl-glutamine and linoleyl-glutamine, but none of these show activity comparable to volicitin. There does not seem to be any obvious benefit to the Spodopteru, nor is it clear how different insect species affect the bouquet of plant volatiles differently, so that parasitic wasps, specific to that insect, are attracted. Probably other compounds, with action similar to volicitin, will be isolated. The subject is new and more will be learned as research progresses.

*YNH2 volicitin

Figure 9.24

Volicitin, a derivative of linolenic acid from a plant and glutamine from the insect, which stimulates the plant to release volatile compounds that attract predators of the insect

Alkaloids and Compounds of Mixed Biosynthetic Origin

159

BACKGROUND AND FURTHER READING M. S. Blum, Biosynthesis of arthropod exocrine compounds, Annual Review of Entomology, 1987,32,381-413. M. S. Blum, Biochemical defenses of insects, in M. Rockstein, editor, Biochemistry of Insects, Academic Press, New York and London, 1978, pp. 465-513. D. Daloze, J. C. Braekman and J. M. Pasteels, Ladybird defensive alkaloids: structural, chemotaxonomic, and biosynthetic aspects (Col.: Coccinellidae), Chemoecology, 1995,516, 173-1 83. E. Haslam, Metabolites and Metabolism, Oxford University Press, Oxford, 1985, pp. 161 (Chapter 3, alkaloids). J. B. Harborne, Introduction to Ecological Biochemistry, 4th edition, Academic Press, London, 1993, pp. 3 18 (Chapter 7). J. Mann, Chemical Aspects of Biosynthesis, Oxford University Press, Oxford, 1994, pp. 92 (Chapter 6, alkaloids). K. B. G. Torssell, Natural Product Chemistry, Swedish Pharmaceutical Society, Stockholm, 1997, pp. 480 (Chapter 8).

QUESTIONS 1. Note that reserpine (Figure 9.1) has a mixed biosynthetic origin. The centre part is more difficult, but what are the precursors of the leftand right-hand parts? 2. Note that both portions of the molecule of exochomine (Figure 9.9) have 13 carbon atoms. Suggest a biosynthetic scheme for the formation of the dimethylaza-acenaphthalenoneportion. 3. The biosynthesis of solenopsin (cis and trans) was studied by feeding sodium [ 1-14C]acetate(activity 60 mCi mmol-') to several thousand ants of 5'. geminata over 60 h. The solenopsins were then isolated and found to contain 6.1 x mCi mmol-'. Calculate the YOincorporation of acetate into solenopsins. 4. (a) In the experiment above with sodium [1-l4C]acetate,the methyl group on the pyrimidine ring of the solenopsins was selectively removed (for details of the method, see Leclercq, Braekman, Daloze, Pasteels and Vander Meer, Naturwissenschaften, 1996,83,222). What fraction of the total radioactivity would be expected in this carbon atom? (b) In a repeat of the experiment with [2-14C]acetate,used in exactly the same way, how much of the radioactivity would be expected in this same carbon atom? 5. The pupae of the ladybird Subcoccinella vigintiquatuorpunctata are

160

Chapter 9

protected by hairs which contain oily droplets. HPLC-mass spectrometry showed the secretion consisted mainly of three compounds that have molecular masses of 530, 532, and 534. They are derived from the compounds below (compare Figure 3.29). Suggest molecular structures for the three compounds. The three compounds all contain free NH groups but are not acidic. 0

T -

OH

HN\/-OH

mol. mass 283

0

OH HN-OH

mol. mass 285

6. In a study of luciferin biosynthesis in the beetle Pyrearinus termitilZuminans uniformly labelled cysteine (''C(U)cysteine, 30 nCi per insect) was injected into five insects and after two days the luciferin was separated by thin layer chromatography and counted. In the total extract of five insects, 480 dps were found. What is the specific incorporation of cysteine into luciferin?

CHAPTER 10

Plant Substances Stored, Changed or Unchanged, by Insects 10.1 TOXIC PLANT SUBSTANCES IN INSECTS In their constant evolutionary struggle against insects that attack them, plants have evolved many kinds of toxins to defend themselves. While most plant-eating insects have to avoid these toxins, some insects, in turn, have evolved the ability to overcome them, and some even to store them in their bodies as their own defence against predators (other insects, arthropods or higher animals). Butterflies and moths, followed by beetles, stand out prominently as insect sequesterers of plant chemicals. Day-flying, brightly coloured butterflies and moths would otherwise be particularly attractive prey. The plant substances may be stored unchanged, or changed slightly or metabolized so much that their connection with the plant may not be immediately apparent. Insects use a variety of enzymes to degrade or modify the plant products, including oxidases, reductases, hydrolases, esterases and transferases. The insect may be able to absorb selectively certain compounds and reject others or control the level of toxin they store. The ingestion of toxic compounds for protection is sometimes called pharmacophagy, and the insects are called pharmacophagous species. The earlier chapters of this book have been organized by fundamental biosynthetic route. No system can be totally logical. It might be argued that the section on sterols and the carotenes in Chapter 7 and the whole of Chapter 8 on aromatic compounds should be included here, but all insects modify dietary sterols to make moulting hormones and all animals modify the aromatic amino-acids to make important compounds. Here we are considering small groups of insects and essentially compounds normally toxic to insects. It is, moreover, a compilation, and cannot be expected to cover all known examples.

161

162

Chapter 10

10.1.1 Cardiac Glycosides The first example of the collection of toxic plant compounds to protect insects was demonstrated by Miriam Rothschild. She showed that the larvae of the Monarch butterfly Danaus plexippus (Plate 12) feed on milkweed, Asclepias curassavica which contains cardiac glycosides like calotropin (used as an arrow poison) (Figure 10.1). The compounds are stored unchanged in the larva and through metamorphosis, making the adult butterfly unpalatable to predators like birds. Calotropin is highly toxic to vertebrates but evidently has no ill effects on the insect. In addition to calotropin, the butterfly also stores three volatile alkylmethoxypyrazines (Figure 10.1) from the plant. The warning odour of these volatile compounds, associated with calotropin, is enough to deter a bird on close approach from trying to eat a butterfly charged with calotropin. It does not follow that because an insect feeds on a toxic plant that it will sequester the toxins. Another danaid butterfly Danaus chrysippus does not seem to sequester calotropin from milkweed.

0

calotropin

Figure 10.1 The structures of calotropin and the three volatile pyrazines sequestered by the Monarch butterfly

The popular flowering tropical shrub oleander (Nerium oleander) contains toxic cardenolides. The principal one is oleandrin (Figure 10.2), but the bug Aspidiotus nerii feeding on it sequesters only a minor component, adynerin. The ladybird Coccinella undecempunctata preying on Aspidiotus nerii sequesters the adynerin from its prey while another ladybird, C. septempunctata feeding on the same bugs does not. The aphid Aphis nerii (see Chapter 8, and Plate 7) on the same plant collects and stores three of its cardiac glycosides. The formation of cardiac glycosides from non-toxic plant sterols (Figure 7.12) and of related saponins from common triterpenes (Figure 7.13) have already been described.

Plant Substances Stored, Changed or Unchanged, by Insects

EQG0 H3CO

163

H

oleandrin

Figure 10.2 Two cardiac glycosides from oleander used by insects. Oleandrin is not sequestered by the aphid C. undecempunctata, but only the minor product adynerin. The sugar in oleandrin is oleandrose, in adynerin it is digitalose

10.1.2 Veratrum Alkaloids The veratrum alkaloids are a group of particularly toxic steroidal alkaloids. A study of the specialist sawfly Rhadinoceraea nodicornis has demonstrated how they store in their haemolymph ceveratrum alkaloids from the host plant, false, or white hellebore Veratrum album. The alkaloids in sawflies can either be directly sequestered, partly metabolized and sequestered, excreted intact or destroyed. The principal alkaloid stored in the haemolymph is 3-acetylzygadenine (Figure 10.3). 3Angeloylzygadenine of the plant is probably hydrolyzed in the gut to zygadenine and then acetylated. At the same time protoveratrines A and B (Figure 10.3) are degraded. Another sawfly, Aglaostigma sp., fed on false hellebore leaves neither had alkaloids in their haemolymph nor excreted any (Schaffner, Boeve, Gfeller and Schlunegger, Journal of Chemical Ecology, 1994,20, 3233-3250).

A

R=

do.. HO '

protoveratrine A

R= or

R=

OH

HO

zygadenine esters

9.. HO

COCH~

*

protoveratrine 6

Figure 10.3 Alkaloids from Veratrum plants. The angeloyl ester of zygadenine is present in the plant, the acetyl ester in the insect. Protoveratrines are, at the same time, metabolized completely by the sawfly R. nodicornis

164

Chapter 10

10.1.3 Pyrrolizidine Alkaloids The pyrrolizidine alkaloids such as seneciphylline from ragwort, Senecio jacobaea, and monocrotaline from Crotolaria species (Figure 10.4) are well-studied examples of metabolized plant substances converted to insect use. The subject of pyrrolizidine alkaloids and their presence in insects is too detailed to consider fully here. Taxonomically unrelated insect groups feed on unrelated plants, chiefly in the Asteraceae and Boraginaceae which contain these alkaloids and store the compounds in their bodies. The alkaloids consist of two parts, the basic pyrrolizidine (the necine part), and various hydroxy and branched acids esterified to it (the necic acid part). A feature of pyrrolizidine alkaloids is that they exist in two interchangeable forms, the non-toxic N-oxides, and the pro-toxic free bases. They only become toxic when the free bases are metabolized to highly reactive pyrroles by cytochrome P450oxidases. In most plant taxa the alkaloids are stored as the N-oxides. On ingestion by many insects they seem to be reduced in the gut to free bases and then re-oxidized for storage. The origin in the plant of the nitrogen-containing necine part is putrescine, which by exchange with spermidine gives first homospermidine (Figure 10.4), while the carboxylic acid portion of seneciphylline comes from amino acids, with many changes. In monocrotaline, the dicarboxylic acid comes from two molecules of 2-methylbutanoic acid.

spermidine homospermidine

OH HO

CHzOH hydrolysis of ester

retronecine

- 2H 0 monocrotaline N-oxide

hydroxydanaidal

by insect

+

danaidone male sex attractant

0 seneciphylline N-oxide

Figure 10.4 Some examples of pyrrolizidine alkaloids and the sex attractants made from them by danaid butterflies and ornate moths. The biosynthesis in plants of the retronecine part of the pyrrolizidine alkaloids begins with reaction between putrescine (from ornithine, Chapter 9 ) and spermidine

Plant Substances Stored, Changed or Unchanged, by Insects

165

Some adapted insects can metabolize the alkaloids and use the metabolites as pheromones. For example, male danaid butterflies collect pyrrolizidine alkaloids from Senecio plants, part of the alkaloid they store, and part degrade to danaidone (Figure 10.4) to use as a sex attractant. The danaidone encourages the females to copulate with them. It shows the females how rich in alkaloids the males are. The danaidone is presented on organs called hairpencils. Female butterflies are able to detect which males have more toxin by the amount of danaidone they secrete, and choose males with most alkaloid. The males pass on the alkaloids with their sperm, and the females invest their eggs with the alkaloids in turn. The ornate moth Utethesia ornatrix produces (R)-(-)-hydroxydanaidal (Figure 10.4) from pyrrolizidine alkaloids like monocrotaline N-oxide, and uses it as a sex attractant for females as the danaid butterflies do. The aphid Aphis jacobaeae also feeds on Senecio plants and stores these alkaloids. The ladybird Coccinella septempunctata (Plate 3) feeding on the aphids accumulates the pyrrolizidine alkaloids in turn, but does not accumulate cardiac glycosides (see earlier). Two groups of chrysomelid beetles have evolved different ways of dealing with these alkaloids. The genus Oreina feed on Senecio and Adenostyles plants. They make their own cardiac glycosides (see Figure 7.12), and accumulate pyrrolizidines as N-oxides, in their haemolymph and glands. One species has only the pyrrolizidines for its defence. Generally the insects do not alter the alkaloids, but two examples, one of hydrolysis and one of epoxidation are known. A typical Oreina alkaloid and these two metabolized products are shown in Figure 10.5. The mechanism for handling alkaloids in the genus Phtyphora is different. Only open-chain pyrrolizidines are accumulated, along with the saponins these insects make themselves (Figure 7.13). The alkaloids are stored as the tertiary amines, not the N-oxides, and only in the haemolymph of larvae and defensive glands of adults. Some alteration of their structures is possible by the insects as shown in Figure 10.6. These species

t

0

t

0

t

0

Figure 10.5 Small changes made in the pyrrolizidine alkaloid acetylseneciphylline N-oxide by Oreina species. The epoxide example is not found in plants

166

Chapter 10

rinderine

lycopsamine

interrnedine

0

OH

retronecine

+i$ OH

L%

OH

OH

N

Figure 10.6 Changes made in open-chainpyrrolizidine alkaloid by Platyphora boucardi by epimerization, and new alkaloids made by this species from retronecine

were also shown to be able to take ingested retronecine and esterify it with propionic, lactic and a-hydroxyisovaleric acids to make new alkaloids. It has been suggested that the toxic effect of pyrrolizidine alkaloids does not really protect insects, because the toxicity only comes into effect when the compound is metabolized, which is well separated from the eating or tasting of the insect, rather it is only the bitterness of the taste of the compounds that offers protection.

10.1.4 Cyanogenic Glucosides Many plants store cyanogenic glucosides, which on removal of the glucose can decompose to release hydrogen cyanide. As HCN is a powerful toxicant to all haem groups containing complexed iron (present in both plants and insects), it is remarkable how such compounds can be safely sequestered by plants or insects. More than 40 cyanogenic glucosides are known in plants, mandelonitrile glucoside, prunasin (Figure 10.7), and its epimer sambunigrin, are typical examples. A glucosidase cleaves the prunasin when the plant is damaged to give glucose and mandelonitrile. The released mandelonitrile is either cleaved spontaneously, at acid pH, or by oxynitrilase in alkaline medium, giving benzaldehyde and HCN. Among animals, this type of defence is practised by millipedes, centipedes and insects only. The larvae of the Australian beetles of Puropsis and Chrysophtarta, feeding on Eucalyptus leaves produce mandelonitrile and prunasin. When freeze-dried insects were treated with pglucosidase and nitrilase, HCN was released (Figure 10.7). The larvae

Plant Substances Stored, Changed or Unchanged, by Insects

w:sRH HHo

Q+I

+

HCN

or oxynitrilase

H

mandelonitrile P-glucoside or prunasin

Figure 10.7

uncatalysed

167

mandelonitrile

benzaldehyde

Conversion of plant-producedprunasin to HCN in Australian beetles

have special glands that secrete the HCN, but eggs, pupae and adults also release some HCN but without specific glands. In beetles and millipedes, which do not live on plants that produce cyanogenic glycosides, the compound is almost certainly made by the insect or arthropod itself. In millipedes mandelonitrile is stored in an inner chamber of their defensive glands, which is separated from the outer chamber which holds enzymes that catalyze the breakdown of mandelonitrile to benzaldehyde and HCN. A muscle controls the valve between the two chambers. The millipede Oxidus gracilis converted racemic [2-14C]phenylalanine to HCN, but did not make HCN from [2-'4C]tyrosine.The steps from phenylalanine to mandelonitrile have been studied in the millipede Harpaphe haydeniana (Plate 13) by feeding a wide range of potential precursors, from which the biosynthetic scheme shown in Figure 10.8 was constructed. Phenylalanine was much less well incorporated into mandelonitrile than N-hydroxyphenylalanine, but that may be because there are many competing uses for phenylalanine. Phenylpyruvic acid oxime was also incorporated, but not as well as phenylacetaldehyde oxime, which leaves the two possible routes open in the middle of the sequence. Cyanogenic species are much more common among Lepidoptera, and many of them are able to synthesize the cyanogens. The bright red-andblack burnet moth Zygaena trifolii obtains the cyanogenic glycosides linamirin and lotaustralin from Lotus corniculatus. This species is also

rnandelonitrile

Figure 10.8 The investigated route to mandelonitrile in the millipede Harpaphe haydeniana. The actual route was notJirmly established, but with small possible variation, it is the same as the route in plants

168

Chapter 10 H3C, CH-CH I ,COOH H3C'

.hH

Fe2+ * 2 0 2 NADPH

H3C\~

-co2

II

CH-CH H3C'

____c

.L

H3C,, H3C' CH-? .\I

02

NH-OH

valine

H

N-OH

I

linamarin

- -

H2 H3C-C, ,COOH CH-CH H3C' L NH2 isoleucine

H2 H3C-C, ,O-glucose

4

/c,

H3C

CSN

lotaustralin

Figure 10.9 The biosynthesis of the cyanogenic glycosides linamarin and lotaustralinfrom amino-acids in the burnet moth. The dot on nitrogen and the prime and double prime on carbon represent 15N and I3C respectively. Labelling showed that these atoms were retained in place during the synthesis

able to synthesize more linamirin and lotaustralin from valine and isoleucine (Figure 10.9). This may be a case of co-evolution according to the suggestions of J. M. Pasteels. Other cyanogenic species do not feed on cyanogenic plants at all. The ability to make and store these compounds seems general in the heliconiine tribe of butterflies. About one third is stored in the haemolymph and two thirds in the integument. Biosynthetic studies in both Zygaena and Heliconius species showed that larvae fed with uniformly labelled 13C-valineor isoleucine were able to synthesize linamarin and lotaustralin respectively. Studies showed that the carbon skeleton was retained intact except for the carboxyl group. For release of HCN, the glucose is enzymically cleaved by linamarinase and the cyanohydrin spontaneously decomposes (Figure 10.10). The cyanide once released can be detoxified as in plants, by conversion to cyanoalanine and asparagine (Figure lO.lO), or by the enzyme rhodanese, which converts cyanide into the relatively innocuous thiocyanate. The formation and decomposition of HCN in millipedes and insects is therefore a remarkable example of parallel evolution, where insects and plants both produce HCN and benzaldehyde in similar ways and avoid the toxic effect of HCN by converting it to the same cyanoalanine. It is not easy to perform tests for the presence of rhodanese and so it is uncertain how widely it occurs in insects. It is the view of at least one author, L. Kassarov, that the cyanogenic effect has no influence on rejection of butterflies and moths by birds and animals. The release of HCN is too slow to protect the insect. Not even the bitterness of the compounds is responsible, since the compounds are

Plant Substances Stored, Changed or Unchanged, by Insects

COOH CN-

1

-

/!F H

-

NH2 cysteine

N-c--\

,COOH CH

H20

A

NH2

*

169

H2N7fYcooH 0 NH2 asparagine

cyanoalanine

pyridoxal phosphate

R-CEN

rhodanese

*

R-S-EN

Figure 10.10 The release of cyanide from linamarin and the detoxiJication of cyanide either by conversion of cysteine to asparagine or by oxidation to thiocyanate

internal and water soluble, not on the cuticular surface, which is hydrophobic. He suggests the whole concept needs to be re-thought. Some of the primitive cycad plants produce the highly toxic glycoside cycasin (Figure 10.11). Yet some butterflies and moths can feed on the plants and sequester the cycasin. The hairstreak butterfly Eumaeus atala florida absorbs the toxic glucoside, so that larvae contain 0.02% and adults 1.O to 1.8%cycasin by weight. When the moth Seirarctia echo was fed on the aglycone, methylazoxymethanol, they stored cycasin. They probably hydrolyze cycasin in their gut and then re-glycosylate it again to store it. Cycasin and methylazoxymethanol are carcinogenic, hepatotoxic, mutagenic and radiomimetic, all of which can be summarized by saying that they are active alkylating agents. OH

cycasin

methylazoxymethanol

Figure 10.11 Cycasin and its aglycone methylazoxymethanol

10.1.5 Glucosinolates

Glucosinolates, or mustard oil glycosides, are like the cyanogenic glycosides, non-toxic substances which give toxins after the enzymic cleavage of the glycoside. About 100 of them are known, and some have been found in all species of Cruciferae examined. Their synthesis in the plant begins with N-hydroxylation of an amino-acid or modified amino-acid and decarboxylation. Further steps are not known with certainty, but probably proceed as shown in Figure 10.12.

170

Chapter 10

HN-OH

-cS,-

0-D-glucose

II

N

4

-

‘o-so3

glucose POSO3

Na+

SH

R-c:,

N-OH

a t hiohyd roximic acid

a glucosinolate

Figure 10.12 The partly speculative route by which glucosinolates are formed in plants. POS03- represents a phosphoric-sulphuric anhydride which donates sulphate

On decomposition they release isothiocyanates or mustard oils, but can also give thiocyanates, and at low pH can give cyanides (Figure 10.13). They generally act as feeding deterrents but some insects, including the Cabbage white butterflies (Pieris sp.) have adapted to feed only on Brassica plants and use the glucosinolates to attract them to the plants for egg-laying and larval feeding. As well, they sequester one of these glucosinolates, sinigrin (Figure 10.13) and its corresponding allyl isothiocyanate. The latter is both toxic and repellent to most insects. There is in the foregut of the Diamondback moth PZuteZZa xyZusteZZa a sulphatase which converts the glucosinolate to a non-toxic oxime thioglucoside (Figure 10.13) which is excreted in the faeces. Glucosinolates are toxic to the thyroid and liver of domestic animals and humans, but many humans are attracted by their pungent effect in cabbage, horseradish and cress.

HzC=HC-CH

-?,S-P-D-glucose

sinigrin N. o-sO3-

H&=HC-CH2-s-C-N allyl thiocyanate

N

+

11

H2C=HC-CH2-C~~ allyl cyanide

,+c/S-~-D-g~ucose “

mvrosinase* glucose

~af

-

%-so3

Naf

sulphatase H20

@-H

‘NLO-SOJ

N i

+

NaHS04

allyl isothiocyanate

*

R-

c, S-P-D-g lucose II

N . ~ - ~

non-toxic

Figure 10.13 The spontaneous release of allyl isothiocyanate and lesser amounts of other compounds from sinigrin in damaged Brassica leaves after the cleavage of the thioglycoside by myrosinase. Sinigrin can also be detoxified by some insects by selective cleavage of the sulphate group

Plant Substances Stored, Changed or Unchanged, by Insects

171

10.1.6 Coniferyl Alcohol A small example, similar to the conversion of pyrrolizidine alkaloids to male copulating pheromones by some Lepidoptera, is the conversion of flower compounds to attractants by males of the oriental fruit fly Bactrocera dorsalis. These adult males feed on the fragrant flowers of Fagraea berteriana which contain (E)-3,4-dimethoxycinnamylalcohol and smaller amounts of (E)-3,4-dimethoxycinnamylacetate (Figure 10.14). The male flies convert these to (E)-coniferyl alcohol (Figure 10.15) and store it in rectal glands. The coniferyl alcohol is very attractive to females, although they do not appear to derive any benefits from the compound. A neat use of the simple thin layer chromatography plate to locate the position of the attractive compounds on the plate with the fruit flies is illustrated in Figure 10.16. 10.1.7 Other Types Harborne (Ecological Biochemistry, pp. 98-1 00, see Further Reading) lists 12 types of plant toxin sequestered by insects, including the cardiac glycosides, veratrine and pyrrolizidine alkaloids, cyanogenic glycosides, and glucosinolates already considered. The others are aristolocic acids, quinolizidine alkaloids, iridoids (Chapter 7), and phenols. Nishida (Annual Review of Entomology, see Further Reading) lists 17 types for Lepidoptera alone. Even so, insects have only harnessed a small fraction of the hundreds of types of plant toxins that they can encounter. Another example of partial modification in the insect is found with the beetle Diabrotica speciosa. It feeds normally on plants of the Cucurbitaceae (cucumbers) which contain bitter triterpenoids (Chapter 7) called cucurbitacins. D. speciosa when fed on ['4C]-cucurbitacinB converts it by removing the C-25 acetate and reduces the side-chain double bond to give dihydrocucurbitacin D (Figure 10.17). Other species raised on diets free of cucurbitacins, when fed cucurbitacin D, were able to glycosylate it (at the 2-OH), hydrogenate, desaturate and acetylate it. The water beetles of the Dytiscidae family convert dietary sterols (e.g. cholesterol, see Chapter 7) to mammalian hormones like oestrone and testosterone, but most interesting is the cortisone-type cortexone (Figure 7.10). A Dytiscus beetle can contain as much as 1 to 3 mg of cortexone, which it can discharge from glands if attacked by a predator. A cantharid beetle, Cauliognathus lecontei, feeding on Compositae that contain acetylenic esters, has been found to contain dihydromatricaria acid (Figure 10.18), the first acetylenic compound found in insects, and which the beetles use as a defensive secretion.

Chapter 10

172

Figure 10.14 Males of the Orientalfruitfly Bactrocera dorsalisfeeding on aflower of Fagraea berteriana to obtain dimethoxycinnamic alcohol and acetate (Photo R. Nishida. Reproduced from Fig. 1 in J Chem Ecol., 1997, 23, p. 2277, ‘Acquisition of female-attracting fragrance by males of oriental fruit fly from a Hawaiian Lei flower Fagrea berteriana’ by Nishida, Shelley and Kaneshiro. By kind permission of Kluwer Academic Publishers)

CH30

H

O

P

*

H

(a-coniferyl alcohol

Figure 10.15

The production of the sex attractant coniferyl alcohol by fruitfliesfrom dimethoxycinnamic alcohol and acetate fromflowers

Plant Substances Stored, Changed or Unchanged, by Insects

173

The lampyrid beetles (fireflies, Chapter 9) produce lucibufagins (Figure 10.18) to make themselves unpalatable to predators. These substances come from dietary sterols, and therefore represent a group of more highly metabolized compounds. They are related in structure to the cardiac glycosides and toad poisons.

Figure 10.16 A thin layer chromatogram, origin arrowed right, of a solvent extract of the Fagraea berterianaflowers was developed with benzene-ethyl acetate (2:l). The solvent front, arrowed, is on the left. Male B. dorsalis flies were then allowed to gather on the plate to show where the active substances were located (Photo R. Nishida. Reproduced from the same source as Figure 10.14 with the kind permission of Kluwer Academic Publishers)

HO H3C

\

0

',,

'

cucurbitacin 6

dihydrocucurbitacin D

Figure 10.17 An example of a triterpenoid slightly mod$ed and sequestered, dihydrocucurbitacin D in chrysomelid beetles

dihydromatricaria acid R = H3CCH-C0 H3C. CH,CH,CO

H RO

RO romallenone

OH

R ' = CH3C0

CH3CO

CHSCO

CH3CO

CH&O

H

lucibufagins

Figure 10.18 Dihydromatricaria acid is found in cantharid beetles feeding on Compositae which contain polyacetylenic acids. Lucibufagins are similar to cardenolides, sequestered by fireflies. Romallenone is present in the defensivefoam of a grasshopper

174

Chapter 10

The grasshopper Romalea microptera discharges a foam from glands when disturbed. The principal constituent of the foam is romallenone (Figure 10.18) an allenic sesquiterpene. This compound is thought to be OH P-glucosidase

salicin

L.d

salicyl alcohol

salicylaldehyde

Figure 10.19 Two chrysomelid beetles (Phratora vitellinae and Chrysomela tremulae) produce sulicyluldehyde us a defensive secretion.

a degradation product of carotenoids (see Chapter 7) from its food plants. Two chrysomelid beetles (Phratora vitellinae and Chrysomela tremulae) produce salicylaldehyde as a defensive secretion (Figure 10.19). They both feed on willow and poplars, which produce salicin, which they have been shown to convert to salicylaldehyde.

10.1.8 A Parting Thought

A number of highly toxic plant substances manipulated by insects have been considered here. How do they do it? The tobacco alkaloids, represented by nicotine, were considered in Chapter 9. Here are a group of simple substances, highly toxic to higher animals and insects, long used as a commercial insecticide. Nicotine affects acetylcholine receptors in the central nervous system. Yet Lepidoptera of the subfamilies Macroglossinae and Sphinginae are able to tolerate large quantities of nicotine. The tobacco hornworm, Manduca sexta and the cigarette beetle Lassioderma serricorne have adapted to live only on fresh tobacco leaves or cured tobaco respectively. Manduca larvae thrive better on plants with lower nicotine levels than on those on artificially high nicotine, so the alkaloid is no help to them. Yet they prefer to feed on young leaves, and nutritionally thrive much better on them although they have twice a much nicotine in them as older leaves. How do they cope with the toxins? It has been demonstrated for M. sexta that their acetylcholine receptors are not different from those of other insects. They do not tolerate the nerve poisons, rather they appear to have a rich variety of detoxifying enzymes to break down the nicotine, particularly in their central nervous system. Certainly only 10 to 20% of nicotine injected into tolerant larvae can be recovered as nicotine, nicotine N-oxide or cotinine (Figure 9.2). What happens to the rest? We do not know.

Plant Substances Stored, Changed or Unchanged, by Insects

175

We have, as yet, few clues to how insects overcome plant defences. With insects, nothing can be taken for granted and their chemistry still holds many surprises.

BACKGROUND AND FURTHER READING D. Daloze, J. C. Braekman and J. M. Pasteels, Ladybird defensive alkaloids: structural, chemotaxonomic, and biosynthetic aspects (Col.: Coccinellidae), Chemoecology, 1995, 5/6, 173-1 83. J. B. Harborne, Introduction to Ecological Biochemistry, 4th edition, Academic Press, London, 1993, pp. 318 (Chapters 5 & 7, plant defenses). T. Hartmann and D. Ober, Biosynthesis and metabolism of pyrrolizidine alkaloids in plants and specialized insect herbivores, Topics in Current Chemistry, 2000,209,207-243. R. Nishida, Sequestration of defensive substances from plants, Annual Review of Entomology, 2002,47,57-92.

QUESTIONS 1. Leaves of Nerium oleander were injected with sodium [2-2H,]acetate. Aphis nerii were allowed to feed on the leaves and then the aphids were fed to the ladybirds Coccinella undecimpunctata. The cardiac glycoside oleandrin was isolated from the ladybirds and subjected to mass spectrometry. There was a small, but distinct mass spectral peak at M+3. What do you conclude? 2. What experiment do you suggest to discover whether pyrrolizidine alkaloids are reduced from the N-oxide form of the plants to the free base in the gut of an insect and later re-oxidized? 3. By NMR spectrometry, how would you check that the labelling of valine in Figure 10.9 had remained intact in linamarin? 4. In a study of the conversion of phenylalanine to mandelonitrile, benzaldehyde and HCN, DL-phenylalanine (0.17 pmoles) labelled with 14Cin the phenyl ring (specific activity 82 pCi mmol-') was injected into each of 10 individual millipedes. The specific activity of the benzaldehyde isolated from all ten at the end of the experiment was 7.39 x pCi mmol-'. Allowance must be made for the dilution with endogenous precursors, because 2.3 pmoles of benzaldehyde was isolated while only 0.17 pmoles of phenylalanine was injected. Presuming that only the radioactivity in L-phenylalanine is used for incorporation, what was the %age conversion of radio-labelled phenylalanine to benzaldehyde? No radioactivity was found in the HCN.

176

Chapter 10

5. The secretion of the grasshopper Romalea microptera contains, in addition to romallenone (Figure 10.18) verbenone, isophorone and 2,6,6-trimethylcyclohex-2-en1,4-dione, below. What is the likely source of these compounds?

verbenone

isophorone

trimethylcyclohexendione

THE BONUS QUESTION Silkworm silk is composed essentially of four amino-acids, glycine, alanine, serine, and tyrosine. The silk, fibroin, is produced from two lateral glands, and the two fibres are glued together with another protein called sericin. The fibres are approximately triangular of dimensions 0.01 mm on each side. A silkworm larva spins its cocoon of silk at the rate of 1 cm sec-’. The unit cell of fibroin has a length of 70 nm and contains two amino-acids, that means the average length of an amino-acid is 35 nm. The amino-acids can be considered as cylinders with a radius of 47 nm. How many amino-acids does the silkworm incorporate into each fibre per second?

Answers to Questions Chapter 1

Ql. From COz to glucose, acetyl CoA, malonyl CoA, fatty acids to hydrocarbons. 4 2 . E=hv of sunlight. Q3. Primary: alanine, deoxyribose, glucose. Secondary: vitamin A, camphor, penicillin. Q4. A synomone, assuming the bee gets nectar from the flower.

Chapter 2

Q1. Haem. More water soluble (and hence more easily excreted). Q2. CH,COCH, + NADH + H’Cl- + CH,CHOHCH, + NAD’ C143. CH,CH,CH,COOH + FAD -+ CH,CH=CHCOOH + FADH2 Q4,

-N

H-enzyme a-keto-isovaleric acid

part of lipoic acid

isobutyryl CoA

For more intermediate steps see Figures 2.11 and 2.13. Q5. Methyl hexanoate. Q6.

177

1

178

Answers to Questions

Note that 2-deuterioacetic acid is not pro-chiral, according to the priority rule, because replacing another hydrogen by deuterium means there is one 'H and two 2Hson the methyl group. Chapter 3

Acetyl-S-ACP to malonyl-S-ACP to stearic acid, desaturated to oleic acid, chain extended by three acetate units to a C,, unsaturated acid, and decarboxylation gives (Z)-9-tricosene. As for question 1, as far as stearic acid, then addition of one propionate (= methylmalonate) unit and two more acetate units gives 6-methyltetradecanoic acid. Decarboxylation gives 5-methyltricosane. Palmitic acid is converted to 11-hexadecenoic acid, then chain-shortened to 9-hexadecenoic acid, and again desaturated by a All-desaturase to (9E, 11E)-tetradecadienoic acid. The remaining steps are obvious. Three deuterium atoms in 9-hydroxydecenoic acid and two in 10hydroxydecenoic acid. The 2-fluoro-atom blocks the first stage of chain-shortening of stearic acid so the whole process is halted. Nine of the ten deuterium atoms would remain.

(a-

p

D3C,C,CD.

3

C D ,

0

.kOH

NH2 Dlo-isoleucine D2

-

p 3 0 D3C.C,CDroH D2

-

0

--c

-..

D

3

7D3 C

.

C

,

C

D

~

o

D2 OH Dg-14-methylhexadecanoic acid

Q7. Bruchin A appears to be made from oleic acid by chain extension with two more acetate units and reduction to the alcohol to give docosanol, which is a-oxidized to an alcohol at the other end of the chain (not necessarily in that order). The 3-hydroxypropionic acid (hydracrylic acid) is an unusual metabolite, with no obvious precursors. It is possibly made here by cooxidation of propionic acid. Both C2* and C24diols are found in the mixture from the insect. Chapter 4 CH3 0 I

enolize

Q2. Seven deuterium atoms maximum.

II

2-hydroxy-6-methylacetoDhenone

'soom

Answers to Questions

e

179

0

H

0

0

0

0

0

0

requires oxidation here polyketide for endocrocin

0 0

0

0 OH

0

O

0 polyketide for griseofulvin

polyketide for alternariol

Q4. 2-Nonanone: Ac-Ac-Ac-Ac-Ac 6-Methyl-3-octanone: Ac-Pro-Ac-Pro 4-Methyl-4-hepten-3-one: Pro-Pro-Pro Q5. Ac-Pro-Pro-Pro

-

*-*-J O

O

+ I

reduction to CH2

O

O

OH 0

H

t

0

Jehy ciration

decarboxylation

pheromone

reduction to CHOH

Q6. A

O

H

Q7. Because of the (@-configuration of the methyl branch, it looks like the chain is begun with 2-methylbutyric acid (see Figure 3.12) but that is without proof. It might be made from acetate and propionate.

CH3COOH

k

O

Y 0

t OH

Chapter 5

Specific incorporation into the lipid fraction was 7.19% and into bombykol 0.1 15%. The amount in bombykol is just about enough to study further by addition of more 'cold' bombykol. If the experiment had been conducted when the females were a few days older, better incorporation might have been achieved.

The propionic acid had not been incorporated intact, or there would have been 13C-13Ccoupling. We must conclude that the propionic acid had been broken down and the label scrambled, so the experiment was unsuccessful. Propionic acid is easily degraded to acetic acid. When using propionic acid

180

Answers to Questions

it is much safer to use it labelled in the carboxyl group. See Question 7 below for an example. Q5. H3C-s

gCH3

> --

m/z 173

m/z 159

The molecule can only be constructed from acetate and propionate by starting on the right-hand side. It contains two acetates and three propionates. Any of a number of ways of labelling could be used. Radio-labelling would be less useful because the molecule would have to be degraded to find where the label was located. CD, labelling of propionic acid would show up in the mass spectrum, but best would be labelling with 2,3['3C]propionic acid and -the 13C-13Ccoupling at the places indicated by asterisks should be seen. 0

HOOC I*

I*

Incorporation is 20.79%. It is much easier to get high incorporations like this when using excised tissues or glands. Chapter 6 Q1. An oxidase will convert a-pinene to cis-verbenol.

42. Made from a-farnesene. An a-oxidation is required (-CH, -+-CH,OH -+ CHO) to produce the aldehyde at the 'wrong' end of the chain. 4 3 . To check, see Figure 6.12, although there can be some mixing of the label in the terminal dimethyl group of geranyl pyrophosphate. *

+

Answers to Questions Q4-

181

rzO

epox'd

p & + , H -

-

h '

-

oxidize

- 2H

periplanone A

Q5.

0

1

caryophylline oxide

H-wor reduce

-

-oxidize

*

enoiize

\ oxid. ancistrodial

\

ancistrofuran

h-&

Q7. The ring closure from gyrinidal to gyrinidone is as for iridoids.

a 2 0 b

farnesol

oxid.

oxid.

OH OH

gyrinidal

Chapter 7

Q1. All the double bonds in the cembrene isomer in Figure 7.2 are trans. There are eight possible isomers, one with all trans double bonds, one all cis, three with one trans and two cis, three with two trans and one cis. 42.

A $J OH

geranylgeraniol

182

Answers to Questions

Q3.

Iycopene

p-carotene

Q5. Specific incorporation was 0.0 19% Q6. Specific incorporation was 0.50%, which is good indication that this compound is further along the route to 20-hydroxyecdysone. Chapter 8 Q1. COOH

O

or

m W C 0 0

mpropylphenol

O 0

O

H

w 0

0

0

rnellein

Q3. Since insects do not have chorismic acid available, both compounds must be derived from tyrosine. Q4. It looks very like a polyketide, so one should try incorporating sodium 2[13C]acetatein the predicted positions without scrambling the label. Both homogentisic acid (Figure 8.1 1) and p-hydroxybenzaldehyde can be Q5derived from tyrosine. Marginalin is a condensation product of homogentisic acid and p-hydroxybenzaldehyde.

Answers to Questions

183

Q7. The sphere represents the remainder of the pyridoxal phosphate co-enzyme.

Chapter 9

The left-hand part is derived from tryptophan and the right-hand part is a phenyl-C, compound.

Rate of incorporation is 0.010% (a) Expected O%, actually found was 0.25%. (b) Expected 11% or 1/9th. Actually found was 10%. See Schroeder, Smedley, Gibbons, Farmer, Attygalle, Eisner and Meinwald, Proc. Natural Academy of Sciences, USA. 1998,95, 13387.

mol. mass 532

mol. mass 530

mol. mass 534

Remember there are two cysteine molecules incorporated into one luciferin molecule. The yield is 4.32%. This maximum is reached after one day of incubation.

Answers to Questions

184 Chapter 10

The oleandrin, which contains an acetate group, was labelled with the trideuterated acetate and this has passed unchanged from plant to aphid to ladybird. The alkaloids are extracted from the plant, reduced to free bases, reoxidized using l 8 0 and fed to the insect. Later the alkaloids are extracted from the insect and examined by mass spectrometry. If the l 8 0 is still intact, there will be a distinct M+2 ion in the spectrum. There should be enhancement of the peaks for the two labelled carbon atoms in the 13CNMR spectrum and there should be 13C-13C coupling. There was 0.002% conversion. Since romallenone is probably a carotene degradation product from the carotenes in the leaves on which the grasshopper feeds, the isophorone and the trimethylcyclohexendione are from the same source. It is difficult to see how verbenone can be formed in the same way, so it has either been made by the grasshopper or from a monoterpene in its food. The Bonus Question

The average volume of an amino-acid in silk is 2.4 x m, and the volume of 1 cm of a silk fibre is 8.66 x m. Therefore there are approximately 360,000,000 molecules per cm length and this number added per second per fibre. The fibres are actually hollow, so a minimum dimension for the fibre has been used. Fibre dimensions vary from 0.023 to 0.009 mm during the spinning process

Appendix Common Abbreviations Ac AcCoA ADP ATP Ci COA-SH D DAHP DMAPP DOPA DXP FAD FADHz GC GC-MS GTP HMG-COA HPLC IPP JH LC-MS MEP MH MVA NAD+,N A D F NADH, NADPH NMR PLP Pr PTTH T

acetyl acetyl coenzyme A adenosine diphosphate adenosine triphophate Curie unit of radioactivity coenzyme A (free form) deuterium (2H) 3-deoxy-D-arabinoheptulosonic acid 7-phosphate dimethallyl pyrophosphate 3,4-dihydroxyphenylalanine 1-deoxy-D-xylulose 5-phosphate flavin adenine dinucleotide reduced flavin adenine dinucleotide gas chromatography linked gas chromatography and mass spectrometry guanosine triphosphate P-hydroxy-P-methylglutarylcoenzyme A high performance liquid chromatography isopentenyl pyrophosphate juvenile hormone linked liquid chromatography (HPLC) and mass spectrometry methylerythritol phosphate moulting hormone mevalonic acid nicotinamide adenine dinucleotide (phosphate) nicotinamide adenine dinucleotide (phosphate), reduced form nuclear magnetic resonance pyridoxal phosphate propyl or propionyl prothoracotropic hormone tritium (3H)

185

Subject Index Normal type entries are page numbers, bold numbers, e g , 1.1 are figure numbers A Abbreviations, 185 Acanthoscelides obtectus, 45,3.25 ACP, see acyl carrier protein Acetic acid, 18,42, 57-58, 64, 85, 106, 130, 2.13,4.1 Acetogenins, 57-68, 129, 130, 131, 1.1,4.5,4.6,4.7 from butyric acid, 62-63,4.11,4.12 from propionic acid, 60-63,4.7, 4.8,4.9,4.10,4.11 Acetylcoenzyme A, see Coenzyme A thioacetat e Acetylenase, 34 Acetylenic acids, 171, 10.18 Actinidine, 92,6.12 Active site, 10, 11, 12, 14, 108 Acyl carrier protein, 29, 31, 60,3.5 Adalia beetles, 47 Adalia punctata, 47 Adaline, 47,3.28 Adalinine, 47, 3.28 S-Adenosyl homocysteine, 19, 76, 2.16 S-Adenosyl methionine, 19, 76, 102, 110, 145, 156,2.16 Adrenalin, 127,8.8 Adynerin, 162, 10.2 Aedes aegyptii, 102 Aenictus rotundatus, 94, 126 Agelenopsis aperta, 152 Aglaostigma sandflies, 163 Agmatine, 156,9.20

Agroporus alutacea, 131,8.13 Albolic acid, 107,7.4 Albolineol, 107,7.4 Alcohol dehydrogenase, 25,61 Alcohol oxidation, 15-1 6, 2.9 Aldolase, 22 Aldol condensation, 63, 85,4.13,6.2 Aleuritic acid, 98,6.22 Alkaloids, 47, 128-129, 143-154,l.l biosynthesis, 145,164,9.3, 9.4, 10.4 bitterness, 166 of insects, 145-1 54 of plants, 143-145, 9.1 metabolized and sequestered, 163-166 precursors, 144,9.2 pyrrolizidines, 164-1 66, 10.4-10.6 toxicity, 143, 149, 166, 174-175 veratrum, 163,10.3 Alkane oxidation, 12 Alkenes double bond position, 40, 82,3.19, 5.12 oxidation, 13,41, 50 Alkylpiperidines, 146, 9.5,9.6 Alkylpyrazines, 152-1 53, 162,9.15, 10.1 Alkylpyrrolidines, 147, 9.7, 9.8 Allelochemicals def., 4 Allenes, 45, 172,3.25, 10.18 Allomone def., 2 , 4 Alydus eurinus, 37,3.14 187

188 Amblyomma americanum, 129, Plate 14 Amblyomma rnaculatum, 129 Amblyomma variegatum, 130 p-Aminobenzoic acid, 126,8.5 5-Aminolaevulinic acid, 138,8.20 Amitermes, 53, 97, 3.37 Ammonia, 20,3.26 Amyrin, 113,7.13 Anabasine, 145,9.4 Ancistrodial, 97,6.20 Ancistrofuran, 97,6.20 A n cistrotermes ca vitho rax, 97 Ancistrotermes pakistanicus, 3.31 Anisomorpha buprestoides, 93 Anisomorphal, 6.12 Anomala cuprea, 45,3.25 Anteiso-acids, 36, 37, 39,3.12,3.17 Anthocyanins, 154,9.17 Anthonomus grandis, 89, Plate 6,6.7 Anthranilic acid, 125, 126, 150,8.5 Ants, 21, 51-52, 57,63-64, 91,98, 105, 126, 144, 146-148,2.19,9.8 Aphaenogaster cockerelli, 125 Aphaenogaster rudis, 145,9.4 Aphidoidea (aphids), 46,96, 134 Aphinin, 135,8.17 Aphins, 134-1 35,8.17 Aphis fabae, 135 Aphis jacobaeae, 165 Aphis neri, 135, 162, Plate 7,8.18 Apis mellifera, 50-5 1, 59, 114 Nasanov attractant, 9 1 queen substance, 50,3.32 sex attractant, 50 venom, 127, 128 Apterygota, 8 Arachidonic acid, 35-36,3.10,3.11 Arachnida, 62 Arctia caja, 128, Plate 11 Argyrotaenia velutinana, 42,3.21 Aromatic compounds, 6, 121-132 amines, 127-1 29,8.9 pheromones, 123,8.2,8.4,8.5 Arthropods, 7, 8, 102, 114

Subject Index

Asparagine, 24, 168,2.21, 10.10 Aspartic acid, 155,9.19 Aspidiotus nerii, 162 Asymmetric induction, 24-26, 2.23 Atrax robustus, 151 Atta cephalotes, 61 Atta texana, 61 Autoradiography, whole-body, 72

B Bactrocera dorsalis, 171, 10.14, 10.16 Becquerel unit, 70 Bees bumblebees, 104, 105 colletine, 54-55 halictine, 54 honeybees, see Apis mellifera Nomada, 96 solitary, 65 stingless, 94, 104, 105, 107 Biacetyl, 135 Bilins, 138, 8.20 Bilirubin, 138,8.20 Biliverdin, 138,8.20 Biopterin, 137,8.19 Biosynthesis, def., 1 analytical aspects, 82 compounds of mixed pathway, 154-158 de novo, 90,93 dynamic equilibrium, 5 in plants, 6,40, 87-88, 110, 115, 121-123, 143-145,154, 158, 161, 164, 166, 169,6.6,9.17, 10.12 reaction types, 5, 6, 1.3 routes, 1.1 sequence of events, 72 site of, 69, 71 Blatta germanica, 48, 84, 111, 145, 7.9 Blattodea (cockroaches), 48, 102, 130, 131 Blister beetles, see Coleoptera Bombykol, 9,44, 83-84,3.23

Subject Index Bombyx mori, 9,44, 140, Plate 2 Brevicomin, 90,6.10 Brood cells, 55 Bruchus pisorum, 56 Bugs, see Hemiptera C 13C-2H coupling, 77,5.9 13C-13Ccoupling, 74, 80-81,5.11 13Clabelling, 70, 74, 93, 168, 10.9 and NMR spectroscopy, 77,78, 5.9,5.11 14Clabelling, 40-41,46,65,7 1,73-74, 93, 106, 112, 113, 125, 127, 129, 145, 146, 149, 155, 157, 167, 168, 171,3.20,5.2, 5.3,5.4, 7.12,8.6,9.3,9.22, 10.9 36Cllabelling, 130 Caddis flies, see Trichoptera Cadaverine, 144, 152,9.2 y-Cadinine, 97,6.20 Cahn-Ingold-Prelogrules, 24-25,2.22 Calliphora stygia, 120 Calotropin, 162,lO.l Campesterol, 110,7.8 Camponotus clarithorax, 23, 123 Camponotus herculeanus, 4.14 Camponotus inequalis, 4.14 Camponotus ligniperdus, 4.14 Camponotus nearticus, 126 Camponotus rujpes, 64,4.14 Camponotus silvicola, 64,4.14 Camponotus ants, 64 Cantharidin, 97-98,6.21 Caparrapioxide, 97, 6.20 Carabid beetles, 57 Carboxypeptidase, 12-1 3,2.3 N-Carboxybiotin, 29,3.4 Cardiac glycosides, 112-1 13, 162, 165, 172, 7.12, 10.1, 10.2 Carminic acid, 134,8.16 Carotenes, 6, 116-118, 132,1.1,7.16, 7.17 Carpophilusfreernani, 62,80,4.11, 4.12

189 Carvone, 24,2.21 Caryophylline oxide, 97,6.20 Cauliognathus lecontei, 171 Cedrene, 98-99,6.22 Cembrene, 105, 106,7.2, 7.3 Centipedes (Chilopoda), 64, 128, 129, 166 Centipedin, 64, 129,4.15,8.10 Cerambycid beetles, 57 Ceroplasteric acid, 107, 7.4 Ceroplastes albolineatus, 107,7.4 Ceroplastols, 107,7.4 Chalcogran, 65,4.17 Chelicerata, 7 Chemical parsimony, 50 Chemochromes, 132 Chilocorine, 148,9.9 Chilocorus cacti, 148 Chirality, 5,23-26, 36,45, 61, 82, 2.22,3.3 chiral shift reagent, 83 diastereomers, 83 optical rotation, 83 optical rotatory dispersion, 83 circular dichroism, 83 Chironomus flies, 138 Cholesterol, 24, 85, 109-113, 7.7,7.8 Chorismic acid, 123, 156,8.1 compounds derived from, 125-127, 156,8.5 Choristoneurafumiferana, 3.23 Chrysolina carnifex, 116 Chrysolina coerulans, 112-1 13,7.12 Chrysomela tremulae, 15&155, 174, 9.19, 10.19 Chrysomelidial, 93,6.12,6.13 Chrysophtarta beetles, 166 Chrysopterin, 138,8.19 Cinnabarinic acid, 140,8.21 Citronellol, 91,6.1,6.4 Claisen condensation, 28, 138,6.2 Claisen rearrangement, 123 Coccinella septempunctata, 47, 162, 165, Plate 3 Coccinella undecempunctata, 162,175

190 Coccinellids (ladybirds), 4 6 4 8 , 148 Coccinellines, 4647, 67,3.26,3.27 Coccoidea (scale insects and mealy bugs), 107, 133 Cochineal, 134,8.16 Co-enzyme A, 14,31,2.6 thioacetate, 14, 29, 85, 1.1, 2.6, 3.3,6.2 Co-enzymes, 10, 13-2 1 Cold-hardy insects, 23 Coleoptera (beetles), 4, 8,45 bark beetles, 65, 89-90 benzaldehyde in, 125 benzoic acid in, 125 blister beetles, 66-67, 97-98 bombardier beetles, 131,8.12 cantharid beetles, 173 carabid beetles, 21 Chrysolinina, 112-1 13,7.12 chrysomelids and cucurbitacins, 171, 10.17 click beetles, 104 defensive secretions, 4 5 4 8 , 91 lampyrid (fireflies), 173, 10.18 leaf beetles, 93-94 luciferin in, 157 mandelonitrile in, 167 meloid beetles, 97, Plate 10 phenols in, 129 pheromones, 45, 54,3.25,3.38 quinones in, 130 sequestering plant toxins, 161 water beetles, 111, 171 Collembola (springtails), 130 Communication chemicals, 4 Coniferyl alcohol, 125, 171, 8.2, 10.15 Coniine, 143, 145, 9.1, 9.3 Convergine, 47, 3.26 Corpus allatum, 101 Cotinine, 145, 174,9.4 Crematofuran, 105,7.2 Crematogaster ants, 51-52, 105 Crematogaster brevispinosus, 105,7.2 Crematogaster deformis, 129,S.lO

Subject Index Crematogaster scutellaris, 3.34 Crustacea, 7, 102, 115,7.14 Cryptolaemus montrouzieri, 47 Cryptolestes beetles, 3.38 Cryptolestes ferrugineus, 95,3.38, 6.19 Cucujolides, 95-96,6.19 Cucurbitacins, 171, 10.17 Cupiennius salei, 52 Curie unit, 70 Cuticular hydrocarbons, 3 7 4 0 , 8 4 Cyanoalanine, 168,lO.lO Cyanogenic glucosides, 166-1 69, 10.7-10.10 Cybister limbatus, 112 Cycasin, 169, 10.11 Cycloartenol, 110 Cysteine, 157,9.22 Cytochromes, 33,41, 5 1,2.5 cytochrome P450,13, 38, 51, 102, 109, 114, 151, 164

D Dactylopius coccus, 134 Dacus oleae, 65, 4.16 Danaidone, 165, 10.4 Danaus chrysippus, 162 Danaus plexippus, 162, Plate 12 Decarboxy lation of amino-acids, 20,2.18 of fatty acids, 38,3.15 of pyruvic acid, 16-18, 2.13 of sterols, 109,7.7 Defensive substances, 2, 5, 7, 2 1, 36, 37,40,4648, 51-52, 53, 57,62, 66-67,91,97, 105, 111-1 12, 116, 125, 126, 129, 131, 161-170, 171-174,6.17,7.3, 7.10 Dendroctonus frontalis, 90,6.9 Dendroctonus ponderosae, 90,6.9 Dendrolasin, 69,95,6.18 Deoxyarabinoheptulosonic acid phosphate, 122,S.l Deoxyxylulose 5-phosphate, 88,6.6

Subject Index

Desaturase, 32-35,43,3.6-3.8,3.22, Plate 1 A9 type, 32-34 A1' type, 34,43,3.22 Desmogramma beetles, 113 Deuterium labelling, 17,26,37,40,47, 48,62, 64, 75-76, 80, 81, 87,93, 96, 97, 113, 125,2.24,2.25,3.13, 3.19,3.28,3.29,4.14,6.13,6.15 and gas chromatography, 76 and stereochemistry, 8 1-82 and volatility, 81 Deuterium oxide (heavy water), 6, 81 Diabrotica speciosa, 171 Dichlorophenol, 129,8.10 Dimethallyl pyrophosphate, 87, 99, 6.2 Dimethyl disulphide, 74, 82, 5.5 Dimethylquinazoline, 126,8.5 Dinoponera australis, 63 Diplopoda (millipedes), 53, 125, 126, 129, 130, 131,151,166, 167,8.13 Diptera (flies), 40,65, 101 Disparlure, 3.23 Diterpenes, 85, 104-106,7.1-7.3 Dolichodial, 9 1, 6.12 DOPA, 127, 132,8.7,8.8,8.14 Dopamine, 127,8.8 Double bonds, 82,5.12 reduction, 16,2.10 oxidation, 41,3.20 Drosophila buzzatii, 71,5.1 Drosophila melanogaster, 140, 7.15 Dytiscus marginalis, 141 E Ectatoma ruidum, 104 Ecdysone, 114,7.14 Ecdysteroids, 5, 114-1 16,7.14,7.15 conjugates, 114,7.15 inactivation, 114,7.15 phytoecdysteroids, 115-1 16,7.15 Eicosanoids, 35, 1.1,3.11 Eloides longicollis, 130 Emodin, 134, 8.16

191 Emulsin, 5 Enantiomers, 23, 50, 61, 66, 2.22 Endopterygota, 8 Enzymes, 5, 10-13 Epiblema scudderiana, 23 Epilachna borealis, 156 Epilachna varivetis, 47, Plate 4 Epilachnine, 47,3.28 Epiphytas postvittana, 55 Epoxidase, 34 Ericerus pela, 107 Eriococcus scale insects, 134 Erythropterin, 138, Plate 9,8.19 Erythrose 4-phosphate, 121-1 22, 1.1,8.1 Estigmene acrea, 44,3.24 Ethanol, 18, 22, 25-26, 2.24 Eucondylodesmus elegans, 125, 8.4 Eumaeus atalajlorida, 169 Eumelanin, 133 Eupoecilia ambiguella, 42 Eutetramorium mocquerysi, 153 Evolution chemical, 17, 102 parallel, 7, 113, 168 Exochomine, 148,9.9 Exochomus quadripustulatus, 148 Exopterygota, 8 F Faranal, 99,6.25 Farnesyl pyrophosphate, 95, 99, 101, 104,6.17, 7.1 Farnesoic acid, 102 Farnesol, 98,6.1,6.17,6.21 Farnesenes, 96,99,6.17,6.20,6.24 Fatty acids, 28-35, 1.1 allenic, 3.25 biosynthesis, 29-30,3.3,3.5 branched, 36-38,3.12 chain lengthening, 38,3.15 degradation, 29-30,3.3 odd-numbered, 31, 33, 36, 37 synthetase, 29

192 unsaturated, 31-35,40, 53,3.8, 3.19 Feeding deterrence, 143, 170 Flavin adenine dinucleotide, 16, 32, 33, 137, 2.10,3.3 Flavones, 154,9.17 Fluoromevalonolactone, 87,6.4 Formic acid, 8, 19,40 biosynthesis, 21,2.19 Formica rufa, 4.14 Formyl group, 19, 109 Frontalin, 6.9

G Gascardia cerifera, 107 Gas chromatography, 76,81, 82,93 chiral stationary phase, 83 -mass spectrometry, 76, 82,93 radio-, 71 Geraniol, 87,91,6.1,6.4,6.7,6.9, 6.13 Geranylcitronellol, 104,7.1 Geranylfarnesol, 107 Geranylgeraniol, 104,7.1 Geranylgeranyl pyrophosphate, 104, 116, 7.1,7.6 Geranyllinalool, 104,7.1 Geranyl pyrophosphate, 87, 94, 6.3, 6.17 Germacrenes, 97, 6.20 Glands (secretory) of insects, 61 abdominal, 42, 125 corpus allatum, 101 defensive, 62,93, 113, 116, 165, 167 Dufour, 51, 54, 63, 99, 104, 105, 107 ejaculatory bulb, 7 1 elytra, 154, frontal, 105, 106, 107 hairs, 48, 128, 144, Plate 4 hairpencils, 165 labial, 104 mandibular, 61, 62,95, 123, 126, 152,4.3

Subject Index

metapleural, 129,4.3 metathoracic, 37 Nasanov, 91 osmeterium, 97 prothoracic, 112, 114, pygidial, 37, 58, 131, 151 rectal, 64, 171 sternal, 49 thoracic, 150 venom or poison, 51,63, 148, 152 ventral, 130 Glomerine, 126,8.5 Glomeris marginata, 126 Glucose, 17, 22, 1.1 Glucosidase, 93, 166 Glucosinolates, 169-170,10.12,10.13 detoxified, 170, 10.13 Glutamine, 46, 158 Glyceraldehyde 3-phosphate, 22, 88, 1.1,2.20,6.6 Glycerol, 23,24, 79,2.23,5.10 Glycine, 19,21, 138 Gonypteryx rhamni, 136, 137 Gnamptogenys striatula, 101, 6.26 Grandlure, 89,6.7 Grapholitha molesta, 123 Green leaf volatiles, 53,3.36 Griseofulvin, 73-74, 5.2,5.3 Guanosine triphosphate, 137,8.19 Gyrinidal, 103 Gyrinidone, 103

H Habrobracon hebetor, 105 Haem, 12,42,46, 166,2.4, 2.5,8.20 Haemolymph, 38,42,46,66,97, 135, 163, 165, 168 Harpaphe haydeniana, 167, Plate 13, 10.8 Harpogoxenus sublaevis, 147,9.8 Heavy water, see deuterium oxide Heavy-atom labelling, 42,69, 75-8 1 Heliconius butterflies, 168 Helicoverpa zea, 3.23 Hemimetabola, 8,1.5

Subject Index Hemiptera (bugs), 65 aggregation, 53 defensive secretion, 53 Hexapoda, 7 Hippodamia convergens, 47, Plate 7 Hippodamine, 47,3.26,3.27 Histamine, 144,9.2 Hololena curta, 155,9.20 Holometabola, 8, 1.5 Homofarnesenes, 99, 6.24 Homohimachalene, 100,6.25 Homomevalonic acid, 99, 101, 105, 6.23 Homomonoterpenes, 101,6.26 Homoptera (plant-sucking bugs, scale insects), 107, 133-134, Plate 8 Homoterpenes, 99-101, 106,7.3 Honeybees, see Apis mellifera Hormones, 2,4, 35, 1.1 def., 2 Hospitalitermes umbrinus, 106 Humulone, 154,9.17 Hydrocarbons, 1.1 chiral, 38, 39,45,3.24 of cuticle, 37-39 melting temperature, 39 methyl-branched, 38-40,48,3.16, 3.17 pheromones, 40-41,44 Hydrogen cyanide, 166-1 69 detoxified, 168, 10.10 possibly not deterrent, 168-169 Hydrogen peroxide, 131,8.12 Hydroxydanaidal, 165, 10.4 20-Hydroxyecdysone, 114,7.14 Hydroxylase, 34,3.9 2-Hydroxy-6-methylacetophenone, 74, 5.4 p-Hydroxy -p-methylglutaryl coenzyme A, 85,6.2 Hymenoptera, see ants, bees and wasps, 89, 104 pigments, 137

193 Hyperaspine, 148,9.9 Hyperaspis campestris, 148

I Ilybius fenestratus, 112, 7.11 Immune response, 35 Indole, 126,8.5 Indolizidines, 148, 150,9.10, 9.12 Ingested substances, 13 Insecta, 7, 8 Invertin, 5 Invictolide, 64,4.14 Ips paraconfusus, 90,6.8 Ipspini, 90,6.8 Iridoids, 9 1-94 Iridomyrmecin, 9 1,6.12 Iridomyrmex, see Linepithema Iso-acids, 36, 39,3.12,3.17 Isobutyric acid, 36, 37,75,3.12,3.13, 3.23 Isocrematofuran, 105,7.2 Isoflavones, 154, 9.17 Isoleucine, 36, 99, 148, 151, 168, 3.12, 10.9 Isopentenyl pyrophosphate, 87, 88, 94,99, 104,6.2,6.17,7.1 Isoprene, 85,6.1 Isoprenoids, 1.1 Isoptera (termites), 49-50, 9 1, 104, 105-1 06 Isothiocyanates, 170, 10.13 Isotopes, 69-70, Table 5.1 isotope effect, 76,81-82 isotopic enrichment, 77 kinetic isotope effect, 8 1-82 Isovaleric acid, 52, 78,5.10 Isoxazole glycosides, 154-1 55,9.19 J Jalaric acid, 98, 6.22 Juvabione, 95,6.18 Juvenile hormone, 24, 101-102, 1.5, 6.27 inactivation, 102 mimics, 5,7,95

194 K Kairomone, def., 4 Kermesic acid, 8, 134 Kermococcus ilicius, 134 Ketals, cyclic, 65-66, 94,4.16,4.17, 6.16 P-Ketosynthase, 30,3.5 Kuhn-Roth degradation, 74 Kynurenine, 139-140,8.21

L Laccaic acids, 134, 8.16 Laceifera lacca, 98 Laccijalaric acid, 98,6.22 Lac insects, 98-99, 134 Lactic acid, 16-18,22, 2.12 Lactones, 4.14 macrocyclic, 53-55, 96, 3.37,3.38, 3.39,6.19 Ladybirds, see coccinellids Lanosterol, 109,7.6,7.7 Lasioderma sericorne, 144, 174 Lasius fuliginosus, 4.14 Lasius niger, 64,4.14 Lepidoptera, 4, 8,40, 117, 136, 144, 158, 161, 171, 3.18 cardiac glycosides in, 162, 10.1 copulating pheromones, 125 cyanogenesis, 167, 168,10.9 and cycasin, 169 juvenile hormone, 101 pigments, 137, 138,8.20 pyrrolizidine alkaloids in, 164-165, 10.4 sequestering plant toxins, 121 sex pheromones, 4 1 4 5 Leptinotarsa beetles, 113 Leptoglossus bugs, 125 Leptothorax acervorum, 147,9.8 Leptothorax muscorum, 147,9.8 Leucine, 52, 148, 153 Leucopterin, 136,8.19 Limonene, 6.1,6.4 Linalool, 6.1,6.4 Linalyl pyrophosphate, 89,6.4

Subject Index Linepithema humile, 54, 91,3.39 Linoleic acid, 35,49,3.10,3.31 Linolenic acid, 35,44, 66, 158,3.10, 3.26,4.17 Limonene, 24 Linamirin, 167, 10.9, 10.10 Linamirinase, 168 Linyphia triangularis, 52 Lipoic acid, 18,2.14 Lipophorin, 38,49 Loganin, 93,6.12 Lotaustralin, 167, 10.9 Lucibufagins, 157, 173, 10.18 Luciferin, 157,9.22, 9.23 Luciferase, 157 Lutzomyia longipalpis, 100 Lymantria dispar, 3.23 Lysine, 144,9.2 Lysozyme, 10-12,2.1,2.2

M Macrosiphon Eiriodendri, 118,7.17 Macrosiphon rosae, 135 Macrotermitinae, 49 Makisterone A, 114,7.14 Malacosoma moths (tent caterpillars), 111,7.9 Malonyl CoA, 29, 85,3.4,3.5, 6.2 Mamestra configurata, 124 Mandelonitrile, 166-1 67, 10.7, 10.8 Manduca sexta, 143, 174-1 75, Plate 15 Manica rubida, 67,99 Mannich reaction, 47, 3.28 Marginalin, 141 Mass spectrometry for heavy isotope labelling, 64, 75, 76, 80, 82, 93, 125, 150, 5.7,5.8 Mating disruption, 4,43 Megalomyrmex ants, 147 Melanargia galanthea, 154 Melanin, 132-1 33, 138,8.14 Mellein, 64, 129,4.3,4.14 Messor ants, 145 Methacrylic acid, 37, 75,3.13

Subject Index Methionine, 148 deuterated, 64 doubly labelled, 74,5.5 2-Methylbutyricacid, 36,54,164,3.12 3-Methylenepentyl pyrophosphate, 99,6.23 Methylerythritol phosphate pathway, 6, 87-88, 116, 154,6.6 Methyl group transfer, 19, 2.16 6-Methyl-6-hepten-2-one, 94,6.16 Methylmalonyl CoA, 38, 60 Methyl 4-methylpyrrole-2carboxylate, 153,9.16 Methy1 6-methylsalicylate, 57, 76-77, 1.2, 5.8,5.9 Methyl oxidase, 34 6-Methylpelleterine, 47,3.28 Mevalonic acid, 69, 85, 87, 1.1,6.2 Mevalonolactone, 87,93, 106, 112, 6.5,6.13 Migdolus fryans, 151 Millipedes, see Diplopoda Monomorium ants, 147, 148 Monomorium pharaonis, 99, 105, 148,9.10 Monoterpenes, 85-94,6.1,6.2,6.3, 6.4,6.6 biosynthesis, 85-89,6.2-6.4 defensive compounds, 9 1-94 pheromones, 89-91 Morpho didius, 132 Morpho rhetenor, 132 Moulting hormone, see also ecdysteroids, 114-1 16, 1.5 Musca dornestica, 40, 145 Myrcene, 6.1,6.8 Myriapoda, 7, 126 Myristic acid, 28,3.1 Myrmica ants, 99, 6.24 Myrmica rubra, Plate 8 Myrmica scabrinodis, 61 Myrmicaria ants, 150,9.10, 9.12 N 15Nlabelling, 168, 10.9

195

NIH shift, 123, 8.3 NMR spectroscopy, 75-77, 78, 80-81, 83, 150,5.9,5.11 Nannotrigona testaceicornis, 104 Naphthoquinones, 131,8.13 Narceus gardanus, 131 Nasutitermes octopilis, 106 Nasutitermitinae, 105 Natural products, 2 Neanura muscorum, 130 Nepetalactone, 9 1,6.12 Neriaphin, 135,8.18 Nerol, 6.1,6.7 Nerolidol, 6.17 Neryl formate, 91 Neurotransmitters, 127, 149 Nicotinamide adenine dinucleotide (and its forms), 15-16, 17, 22, 30, 33, 82,85, 101, 108, 145,2.8, 2.9,3.3,7.5 Nicotine, 143, 145, 174-175,9.4 Nicotinic acid, 144,9.2 Nomadone, 96,6.20 Norgeraniol, 94,6.15 0 '*Olabelling, 75, 90, 96, 5.6, 6.10, 6.19, 9.23 Ocimene, 6.1 2-Octynoic acid, 46 Odour receptors, 23,2.21 Oleandrin, 162, 10.2 Oleic acid, 28, 38,45,48,49, 65, 81-82, 148,3.1,3.10,3.29,3.31 Ommins, 139, 8.21 Ommochromes, 139,8.21 Oncopeltusfasciatus, 138, Plate 17 One-carbon fragment, 19,21,2.15 Opilionids, 62, 129, 130,4.10 Oreina beetles, 165, 10.5 Oreophoetesperuana, 151 Orgyia pseudotsuga, 3.23 Ornithine, 144, 9.2 Orthoptera (grasshoppers and locusts), 129, 130,131, 138

196 Oxidative dehydrogenation, 133, 134, 8.15 Oxidus gracilis, 167 Oxygenases, 12 Oxynitrilase, 166, 10.7 P Paederus beetles, 66-67 Palmitic acid, 28, 32,42,44, 98, 3.1 Palmitoleic acid, 32,45,3.25 Paltothyreus tarsatus, 74 Papilio memnon, 97 Papilio protenor, 97 Paropsis beetles, 166 Pect inophora gossyp iella (pink bollworm), 117,3.23 Pederine, 66,4.18 Periplanata americana, 96, 129, 145 Periplanones, 96-97,6.20 Pest control, 2,4, 63, 66, 143 Phaedon amoraciae, 93,6.13 Phaeomelanin, 133 Pharmacophagy def., 161 Phenols, 129-1 30,S.lO Phenylacetic acid, 125,8.2 Phenylalanine, 121, 123, 125, 127, 137, 144, 148, 167,8.2, 10.8 Phenyl-C, compounds, 122, 123-125, 154, 1.1, 8.2 Phenylpyruvic acid, 123, 129,8.1,8.2 Pheromones, 2,4,94, 1.1 aggregation, 54, 62,65-66, 7 1, 89, 90, 95,4.12,4.17, 6.7,6.8 alarm, 9697, 130,6.20 attractant, 37, 91, 111 blends, 43 and chirality, 82-83,90 contact, 48 copulating, 125, 165 def., 2 dispersing, 9 1 hydrocarbon, 40 oviposition deterrent, 154,9.18 queen, 64, 105 response inhibition, 42-43

Subject Index

sex attractant, 8, 37,4045, 50, 52, 65, 89,91, 100, 104, 105, 123, 124, 130, 151,3.12,3.21-3.25, 3.31 swarm, 3.33 trail, 49-50, 57,61,64,76,99, 101, 105, 111, 125, 126, 145, 150, 152, 153, 3.31, 4.14, 6.25, 6.26 volatile, 61-65 Philanthus triangulatus, 151 Philanthotoxins, 151,9.14 Phorcabilin, 8.20 Phosphoenol pyruvate, 22,121, 123, 1.1,2.20,8.1 Photosynthesis, 16, 75, 1.1, 1.4,5.6 Phragmatobia fuliginosa, 44,3.24 Phratora vitellinae, 174, 10.19 Phyllopertha diversa, 15 1 Phytoecdysteroids, see ecdysteroids Pieris brassicae, 136, 138, 170 Pieris rapae, 136 Pigments of insects, 117-1 18, 132-140,7.16,7.17,8.14, 8.16-8.21 a-Pinene, 90,6.8 Pityogenes chalcographus, 65,4.17 Plagiodial, 94,6.14 Plant substances metabolized by insects, 35, 44, 53, 89-90, 110-115, 116, 121, 125, 127, 129, 130-131, 132, 154, 158, 161-167,169, 170, 171-174 Platyphora beetles, 113, 165 Platyphora boucardi, 10.6 Platyphora kollari, 113,7.13 Plectreurys tristis, 9.20 Plutella xylostella, 170 Polistes dominulus, 137, Plate 16 Polyamines, 151-152,9.12,9.14 Polyketides, 57, 78, 121, 131, 145, 154,1.1,4.1-4.4,4.6,4.8,4.9,9.3 Polyketide synthase, 61 type I, 59, 145 type 11, 59 type 111, 154,9.17

Subject Index Polyomrnatus icarus, 154 Polyphenol oxidase, 132 Polyunsaturated acids, 6, 35,44 Polyzonamine, 151,9.13 Polyzonium rosalbum, 151 Ponasterone A, 115,7.14 Porphobilinogen, 138,8.20 Precoccinelline, 47,3.26,3.27 Prephenic acid, 123,g.l Prenyl transferase, 87,94 Primary metabolites, 1, 28 Pro-chiral atoms, 15,25,82, 87, 2.9,2.23-2.25,3.9,6.3, 8.1 def., 25 Propionic acid, 37,38,48, 60,62, 64, 99, 130,3.16,3.17,3.30, 4.7,4.9,4.10-4.12 Prostaglandins, 35,3.11 Prothoracotropic hormone, 114 Protoaphins, 134-1 35,8.17 Protoporphyrin IX, 138,2.4, 8.20 Prunasin, 166, 10.7 Pterins, 136-138, Plate 17,8.19 Pterobilin, 138,8.20 Pterygota, 8 Putresine, 144, 151, 164,9.2 Pyrearinus termitilluminans, 160 Pyridoxal phosphate, 19-21,88, 138, 2.17,2.18, 10.10 Pyridoxamine, 20, 123, 2.17 Pyrophosphatase, 87, 101 Pyrrhocoris apterus, 138, Plate 9 Pyrroles, 153 Pyrrolizidine alkaloids, see alkaloids Pyrrolizidines, 148,9.10 Pyruvic acid, 17-18,22,88, 1.1,2.12, 2.13,2.20,6.6

Q

Queen substance, 50,3.32 Quinoline, 150 Quinones, 130-132, 157 pigments, 133-1 36

197

R Radio-labelling, see also I4Cand tritium tritium, 21,42, 57, 69, 72 dilution, 72,73, 147 double labelling, 74 specific, 72 uniform, 72, 149 Reflex bleeding, 46, 66,98 Reticulitermes santonensis, 107 Reticulitermes termites, 105 Retronecine, 166, 10.4, 10.6 Rhadinoceraea nodicornis, 163, 10.3 Rhugoletis cerasi, 154, 9.18 Rhinotermitidae, 50 Rhodommatin, 140,8.21 Rhodanese, 168 Rhytidoponera aciculata, 58, 74, 78, 5.5 Rhytidoponera chalybaea, 4.3 Romalea micropteru, 129, 174, 176 Romallenone, 174, 10.18 Royal jelly acid, 5 1,3.33

S 35Slabelling, 69,70,73, 140,5.5 Salicylaldehyde, 174, 10.19 Sandflies (Diptera), 105 Saponins, 113, 165,7.13 Sarpedobilin, 138 Scale insects, see Homoptera Scarites subterraneus, 37 Schemochromes, 132 Schizura concinna, 21 Scolopendra subspinipes multilans, 64-65, 129, Plate 5,4.15,8.10 Secondary metabolites def, 1 Seirurctia echo, 169 Semiochemistry,4,5 1 Sequestered substances from plants, 161-1 74 Serine, 19,21,48 Serotonin, 128-129,8.9 Sesquiterpenes,85,94-99,6.17,10.19 pheromones, 96-97,6.20

198 Sesterterpenes, 85, 107,7.4 Shellac, 98, 134 Shikimic acid, 6, 69, 1.1 pathway, 121-123,S.l Silk, 176 Sinigrin, 170, 10.13 Sitosterol, 110,7.S Skatole, 126,S.5 Social insects, 37, 61 Solenopsins, 146-1 47,9.5,9.6 Solenopsis ants, 148 fire ants, 146, 9.5 thief ants, 147,9.7 Solenopsis geminata, 146,9.5,9.6 Solenopsis invicta, 64 Species recognition, 125 Specific activity def., 70 Specific incorporation, 70, 112 Spermidine, 151, 164, 9.14, 10.4 Spermine, 151,9.14 Spider mites, 90 Spiders, 52 sex attractant, 52 venom, 128, 151, 152, 155,9.14 Spodoptera eridania, 39,3.18 Spodoptera exigua, 158 Springene, 104, 105, 7.1 Squalene, 108, 114,7.5,7.6 Stearic acid, 28,32,38,48, 51,81, 82, 3.1,3.15,3.33 Stegobinone, 84 Stegobium paniceum, 84 Stenus comma, 151 Stenusin, 151,9.13 Steroids, 6, 12, 109-1 16,1.1,7.7-7.11 as insect nutrient, 110,7.S as defensive secretions, 111-1 13, 116 as pheromones, 111 Stigmasterol, 110,7.S Subcoccinella vigintiquatuorpunctata, 148, 159 Substrate, 10, 33 Succinyl CoA, 138 Sulcatol, 94, 6.16

Subject Index Sulphatase, 170 Symbionts, 67,98, 135 Synomone def., 4

T Taste receptors, 23, 2.21 Tenebrio molitor, 67 Termites, see Isoptera Terpenes, 85, 158, 1.1 degraded, 94,6.16 Tetradecenyl acetate, 44,3.23 Tetrahydrofolic acid, 19, 21, 109, 137, 2.15,2.16 Tetramor ium impurum, 76 Tetraponerine ants, 148 Tetraponerines, 148,9.10,9.11 Tetrapyrroles, 138,8.20 Tetraterpenes, 85, 116-1 18,7.16 Thiamine diphosphate, 17, 88, 2.11, 2.13 Thin layer chromatography, 171, 10.16 Thioesters, 14, 29, 79, 2.6,2.7,3.2, 5.10 Thioglucosides, 93 Threonine, 152 Ticks (Ixodoidea), 102, 111, 127, 129-130, 8.10 Tocopherol acetate, 156,9.21 Transaminase, 19-20,2.17 Triatoma bugs, 126,8.5 Tribolium confusum, 40 Trichoplusia ni, 43 Trichoptera (caddis flies), 62, 126, 130 9-Tricosene, 40,41,3.20 Triglycerides, 28,42,3.1 Triterpenes, 85, 108-1 10, 171, 10.17 Tritium labelling, 26,41, 76, 87,2.25, 3.20 Trogoderma beetles, 37,3.12 Trogoderma granarium, 37 Tryptophan, 121, 125-128, 139, 144, 151,S.5 Tuberolachnus salignus, 135 Tyrophagus putrescentiae, 9 1,6.11

Subject Index Tyrosine, 123, 127, 130-132, 134, 137, 140, 151, 8.2,8.3 U Ubiquinones, 157, 9.21 Unsaturated acids, 32-35,3.8, 3.10 Uric acid, 20 Utethesia ornatrix, 165 V Valine, 36, 37, 75-76, 99, 168,3.12, 10.9 Venetian red, 8, 134,8.16 Venom components, 5,21, 127, 128, 144-148, 151,152, 155, 156 Veratrum alkaloids, see alkaloids cis-Verbenol, 90,6.8 Vespa crabro, I37 Vespa vulgaris, 137 Violaxanthin, 117 Virginae butanolide A, 77-79, 8 1, 5.10

199 Visual pigment, 117, 139-140, 7.16,8.21 Visual spectrum, 132 Vitamins, 21,29, 117, 156 Volicitin, 158,9.24

W Wasps, 62,65, 127 social, 128 parasitic, 53, 105, 158 pigments, 137

X Xanthommatin, 139,8.21 Xanthophyll, 118,7.16 Xanthopterin, 136, Plate 16, 8.19 Z Zophobas rugipes, 130 Zygaena trifolii, 167, 168, 10.9