ADVANCES IN DENDRlTlC MACROMOLECULES
ADVANCES IN DENDRlTlC MACROMOLECULES
Editor: GEORGE R. NEWKOME Volumes 1-4 were published by:
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ADVANCES IN DENDRlTlC MACROMOLECULES
Editor: GEORGE R. NEWKOME Departments of Polymer Sciences and Chemistry The University of Akron Akron, Ohio, USA
VOLUME5
2002
2002
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CONTENTS LIST OF CONTRIBUTORS
vi i
PREFACE
xi
George R. Newkome AZOBENZENE-CONTAINING DENDRIMERS Ovette Villavicencioand Dominic V McCrath LINEAR-DENDRlTlC BLOCK COPOLYMERS. SYNTHESIS AND CHARACTERIZATION
1
45
/van Citsov DENDRIMERS CONTAINING FERROCENYL OR OTHER TRANSITION-METAL SANDWICH GROUPS Beatriz Alonso, Ester Alonso, Didier Astruc, lean-Claude Blais,
89
Laurent Djakovitch, lean-Luc Fillaut, Sylvain Nlate, Franqoise Moulines, Stgphane Rigaut, )aime Ruiz, and Christine Vakrio MAGNETIC RESONANCE IMAGING CONTRAST AGENTS: THEORY AND THE ROLE OF DENDRIMERS Erik Wiener and Venkatraj V Narayanan
129
INDEX
249
V
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LIST OF CONTRIBUTORS Beatriz Alonso
Laboratoire de Chimie et Organometallique UMRCNRSN°5802 Universite Bordeaux I 33405 Talence Cedex, France
Ester Alonso
Laboratoire de Chimie et Organometallique UMRCNRSN°5802 Universite Bordeaux I 33405 Talence Cedex, France
Didier Astruc
Laboratoire de Chimie et Organometallique UMRCNRSN°5802 Universite Bordeaux I 33405 Talence Cedex, France
Jean-Claude Blais
Laboratoire de Chimie Organique Structurale et Biologique EPCNRSN°103 Universite Paris VI 75252 Paris Cedex, France
Laurent Djakovitch
Laboratoire de Chimie et Organometallique UMRCNRSN°5802 Universite Bordeaux I 33405 Talence Cedex, France
Jean-Luc Fillaut
Laboratoire de Chimie et Organometallique UMRCNRSN°5802 Universite Bordeaux I 33405 Talence Cedex, France VII
LIST OF CONTRIBUTORS
Ivan Gitsov
Faculty of Chemistry State University of New York College of Environmental Science and Forestry Syracuse, NY 13210, USA
Dominic V. McCrath
Department of Chemistry University of Arizona Tucson, AZ 85721, USA
Frangoise Moulines
Laboratoire de Chimie et Organometallique UMR CNRS N° 5802 Universite Bordeaux I 33405 Talence Cedex, France
Venkatraj V. Narayanan
Department of Medical Information Sciences University of Illinois at Urbana-Champaign Urbana,IL 61801, USA
Sylvain Niate
Laboratoire de Chimie et Organometallique UMR CNRS N° 5802 Universite Bordeaux I 33405 Talence Cedex, France
Stephane Rigaut
Laboratoire de Chimie et Organometallique UMR CNRS N° 5802 Universite Bordeaux I 33405 Talence Cedex, France
Jaime Ruiz
Laboratoire de Chimie et Organometallique UMR CNRS N° 5802 Universite Bordeaux I 33405 Talence Cedex, France
Christine Valeria
Laboratoire de Chimie et Organometallique UMR CNRS N° 5802 Universite Bordeaux I 33405 Talence Cedex, France
Ovette Villavicencio
Department of Chemistry University of Arizona Tucson, AZ 85721, USA
LIST OF CONTRIBUTORS
Erik Wiener
ix
Department of Medical Information Sciences University of Illinois at Urbana-Champaign UrbanaJL 61801, USA
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PREFACE
In 1994, I wrote in the original preface to this series that stated, in part, . . . "For nearly two centuries, organic chemists have devised synthetic pathways to millions of new unnatural, as well as naturally occurring, molecules with specific composition. The vast majority of these single organic molecules possess a molecular mass of less than 2000 Dalton. Only a few synthetically prepared biomolecules breached the 5000 Dalton threshold. With the initial cascade synthesis of Vogtle in 1978, and the facile preparation of dendrimers by Tomalia and arborols by Newkome in 1985, this artificial molecular barrier has been permanently removed. Today, combinations of diverse building blocks easily give rise to macromolecules possessing thousands of surface moieties and molecular masses approaching 1,000,000 Dalton. One can predict that there are more molecular combinations above this threshold yet to be devised and constructed than have been created in the past 200 years." As the nanomolecular structural ceiling is elevated, the interaction of fractal construction in the interrelated fields of organic/inorganic, polymer science, materials construction, and into the biological arena, this field will eventually surpass its molecular foundation. This volume further demonstrates the novel and varied growth in this topic and certainly pushes the supramolecular concepts of Lehn into the budding "supramacromolecular" frontier. In Chapter 1, Villavicenio and McGrath present their pivotal work in the creation of azobenzene-containing dendrimers; their Chapter describes the fundamental underpinnings to this interesting family. As they state in their summation, "A continuing combination of fundamental studies on the photomodulation of dendrimer properties in azobenzene-containing dendrimers and the new developments in the application of these materials to new and existing technologies xi
xii
PREFACE
is anticipated." The field of linear-dendritic block copolymers is summarized in Chapter 2 from the eyes of Ivan Gitsov, who along with Professor Frechet were the initiators of this variety of macromolecules. In Chapter 3, Astruc and colleagues present the recent advances in metallodendrimers, which incorporate ferrocenyl and/or other transition metal sandwich components; their Chapter capitalizes on the importance of supramolecular chemistry in these dendritic constructs. Lastly, in Chapter 4, Wiener and Narayanan describe the practical applications of dendrimers to the area of magnetic resonance imaging contrast agents. Each of these unique chapters covers a different perspective of this versatile group of macromolecules. As a final note, I would personally like to thank these authors as well as the other contributors to this series who have each made the field stronger and more diverse by adding their specific scientific disciplines and talents to the mix. It is at each of these multifaceted intersections that new futuristic growths will occur toward the formation of novel molecular forests. George R. Newkome Editor
AZOBENZENE-CONTAINING DENDRIMERS Ovette Villavicencio and Dominic V McGrath
I. II.
III.
IV.
V.
VI.
Abstract Introduction Synthesis and Characterization A. Mono(azobenzene) Dendrimers B. Tris(azobenzene) Dendrimers C. Hexakis(azobenzene) Dendrimers Photoresponsive Behavior A. Mono(azobenzene) Dendrimers B. Tris(azobenzene) Dendrimers C. Hexakis(azobenzene) Dendrimers Photomodulation of Properties A. Polarity B. Molecular Size Alteration Literature Review A. Monolayers/Self-Assembly/Amphiphilic Dendritic Structures . . . . B. Host-Guest Chemistry C. Antenna Effects D. Holography Summary Notes Acknowledgements References
Advances in Dendritic Macromolecules Volume 5, pages 1-44 © 2002 Elsevier Science Ltd. All rights reserved ISBN: 0-7623-0839-7
2 2 4 4 9 11 12 14 18 21 24 24 28 31 31 35 39 41 41 41 42 42
2
OVETTE VILLAVICENCIO and DOMINIC V. McGRATH
ABSTRACT The photochromic azobenzene moiety has a proven versatility as a switching element in many contexts in small molecule and macromolecular systems. This article summarizes our studies on the synthesis, characterization, and investigation of the photoresponsive behavior of azobenzene-containing dendrimers. Benzyl aryl ether-based dendrimers were prepared possessing single and multiple azobenzene units at the core and periphery. In addition, variation in the type of dendrimer subunit (benzyl aryl ether vs. phenylacetylene) was examined. Photochromic behavior of the azobenzene subunits was studied by UV-Vis spectrophotometry and kinetic measurements on the thermal Z^f- E isomerization. In general, no effect of dendrimer incorporation on the photoresponsive behavior of the azobenzenes was observed. Multiple photochromic states, manifested most dramatically in chromatographic behavior, were observed in the multi-azobenzene-containing dendrimers. The effect of azobenzene isomerism on the hydrodynamic volume of azobenzenecontaining dendrimers was studied by size-exclusion chromatography. The magnitude of size alteration is affected by dendrimer structure, number of photochromic units per dendrimer, and the relative placement of photochromic units in the dendrimer. The literature on the application of azobenzene-containing dendrimers in a variety of contexts is reviewed.
I. INTRODUCTION Azobenzenes have enjoyed a major role in the development of photochromic molecular systems during the last several decades as a result of the well-known changes that these chromophores undergo when irradiated with ultraviolet light (Scheme 1).^""^ Three associated changes take place when azobenzenes ZjE photoisomerize: change in absorption profile, change in molecular dimension (ca. 0.5 nm), and change in dipole moment (ca. 3D).^ Wavelength-dependent irradiation induces configurational isomerization in both the £ ^- Z and Z -^ E directions, and since the E isomer is generally more stable than the Z isomer by about 50 kJ/mol, Z ^^ E thermal isomerization is observed. The nature of azobenzene photoisomerization is critically dependent on the aromatic substituents, especially with respect to the lifetime of the Z state which can vary from milliseconds to days at room temperature. In addition, azobenzenes exhibit high photochemical stability. Despite an enormous amount of data, the exact mechanism of azobenzene E/Z photoisomerization is still a matter of debate, yet 5.5 A
9.0 A hv dark or hv' ■180
Scheme 7. Azobenzene photochromism.
N=N
Azobenzene-Containing Dendrimers
3
it is generally accepted that the thermal Z -> E isomerization proceeds through an inversion, rather than rotation, mechanism.^ Incorporation of the photochromic azobenzene moiety in macromolecular systems has enabled the study of a wide variety of phenomena associated with the photochromic nature of the azobenzene chromophore. In general, these studies fall into two main categories: systems that take advantage of the property changes — size, polarity or both — in the azobenzene chromophore as a result of isomerization to the Z state, and systems that take advantage of the reversibility of the isomerization process. Property changes resulting from the photochemical isomerization of azo units can drive subsequent changes in macromolecular conformation.^ This allows reversible switching of a wide spectrum of polymer and polymer solution properties.^"^ Azobenzene-containing polymers have also been prepared and optimized for applications in liquid crystal displays and devices, reversible optical storage systems, nonlinear optical waveguides, photorefractive switches, and holographic gratings.^"^ These applications generally take advantage of the polarized light-induced alignment of azobenzene chromophores by reversible E/Z switching. The incorporation of photoresponsive moieties in dendrimers can potentially widen the application of these new materials in areas such as self-assembly,^"^^ liquid crystallinity,^^"^^ and encapsulation.^^~^^ In contrast to the various azobenzene-containing non-dendritic macromolecular systems mentioned above, dendritic macromolecules with photochromic azobenzene units have only recently been a developing area. Investigators in this new field have considered both traditional azobenzene-containing polymer properties/behavior such as photoinduced birefringence as well as the effect of azobenzenes on more "dendrimer oriented" behaviors such as host-guest encapsulation. These efforts are reviewed in Section V. Our effort in dendrimer chemistry seeks to construct materials that undergo property changes in response to external, non-invasive stimuli, in particular light. ^^~^^ Wishing to probe the nature of azobenzene-based photomodulation of dendrimer properties, we have synthesized several structural classes of azobenzene-containing dendrimers that undergo reversible configurational changes — and concomitant property changes — in response to light energy. These dendrimers possess a single azobenzene as central linkers, multiple azobenzenes proximal to the core, and azobenzenes on the periphery. In addition, variation in the type of dendrimer subunit (benzyl aryl ether vs. phenylacetylene) has been examined. In this manner we have investigated photoinduced dendrimer property changes based on dendrimer generation, type of dendrimer subunit, number of photochromic units per dendrimer, and relative placement of photochromic subunits within the dendrimer. In addition, the photochromic azobenzene moiety has served as a useful probe of dendrimer behavior in solution. The details of our studies are reviewed in Sections II-IV.
4
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
II. SYNTHESIS AND CHARACTERIZATION To delineate the scope of the photomodulation of dendrimer properties with covalently incorporated azobenzene subunits, we chose to prepare several new classes of dendritic materials with azobenzene-based central linkers and peripheral units. In this section, we outline the synthesis and structural characterization of these materials. A. Mono(azobenzene) Dendrimers
We have prepared dendrimers with single azobenzene moieties as the central linker using hydroxy- and amino-substituted azobenzene derivatives 1 and 2 through the attachment of benzyl aryl ether dendrons of the Frechet type of various sizes (Scheme 2)}^''^^~^^ Photochromic dendrimers of the zeroth, first, second and fourth generations were prepared by reaction of azobenzene-derivative central linkers 1^^ and 2^^ with the appropriate dendritic bromides^^ in the presence of K2CO3 in acetone. Zeroth and first generation materials 3a and 3b are yellow-orange crystalline solids obtained in 75 and 91% yields, while second and fourth generation materials 3c and 3d are yellow-orange glasses when concentrated to dryness from methylene chloride (CH2CI2) solutions and were [G-r7]-Br K2CO3 ^
^^N-
3 and 4
acetone A
1:X : 0 H 2:X :NH2
3a: n = 0 3b: n = 1 3c: n = 2 3d: n = 4
4a: n = 0 4b: n = 1
Scheme 2. Synthesis of dendrimers 3a-3d and 4a and 4b.
Azobenzene-ContainingDend
rimers [G-n]-OH DMAP
CI
^—^
'^y<.
^N
acetone A
6a: n = 2 6b: n = 4 Scheme 3. Synthesis of dendrimers 6a and 6b.
isolated in 80 and 81% yield. Compounds 4a and 4b were obtained in low yields (30-40%) according to Scheme 2 due to complications that included over-alkylation. As a result, use of central linker 2 was not continued in these studies. Electrophilic central linker 5^^ has also been used to prepare mono(azobenzene) core dendrimers.^'* Second and fourth generation dendrimers 6a and 6b were prepared by the reaction of benzyl aryl ether dendritic alcohols with 5 in the presence of MA^-dimethylamino-pyridine (DMAP) and benzene under Dean-Stark conditions. These photochromic dendrimers in which the dendritic fragments are attached to the azobenzene core via ester linkages were obtained in 90 and 75% yields, respectively (Scheme 3). Second generation 6a exists as a pink solid and fourth generation 6b is a pink glass when concentrated to dryness from CH2CI2 solution. For the photochromic characterization of these azobenzene-containing dendrimers, we wished to have control compounds where steric interaction between dendrons on either end of the azobenzene was absent. Hence, we prepared dendrimers 8a-8c from the reaction between monomethylated azobenzene 7^^ and benzyl aryl ether dendritic bromides under the same conditions as used for 3 and 4 (Scheme 4). Obtained in 73-81% yields, these dendrimers were either yellow crystalline solids (8a and 8b) or an orange glass (8c) when isolated in pure form. Mono(azobenzene) photochromic dendrimers have also been prepared with modified peripheral units.^^ Photochromic dendrimers 9 and 10 were prepared
OVETTE VILLAVICENCIO and DOMINIC V McGRATH [G-n]-Br K2CO3
H3CO
/
H3CO-
\\
OH
V=/
NN.
//
%
»>
acetone A
-^
2"-1
8a: n = 0 8b: n = 1 8c: n = 2
Scheme 4. Synthesis of dendrimers 8a-8c.
/
p-CH3C02[G-4]-Br K2CO3
VN
9 and 10
THF A
1:X = Y = 0 H 7: X = OH; Y = OCH3
Me02C RO-/
\—N
,
, OR'
9(R = R' = 11) 10(R = 11;R' = Me) 11 Scheme 5. Synthesis of dendrimers 9 and 10.
in a fashion similar to the preparation of dendrimers 3 and 8 (Scheme 5). However, a fourth generation dendritic bromide with carbomethoxy groups on the periphery, previously reported by Frechet, was used in the synthesis. In addition, due to the insolubility of this dendritic bromide in acetone, the alkylation reactions were performed in anhydrous tetrahydrofuran (THF).
Azobenzene-Containing Dendrimers T M S - ^
X-nf V-N=N—(\ /y~x 12a:X = OTf 12b: X = I
Pd(dba)2 PPha
TMS- - ^ — ^
'^N=N-<(
Cul EtgN
))—^E—TMS
13 fBu4N"'F~| THF-H2O
14
[G-"]-'
/
/Pd(dba)2 PPh3 Cul EtgN
Scheme 6. Synthesis of dendrimers 15a-15e.
Carbomethoxy-terminated dendrimers 9 and 10 were isolated as yellow-orange glasses when concentrated to dryness from CH2CI2 solutions in 96 and 66% yields, respectively. In order to explore the effect of dendrimer subunit structure on the behavior of photochromic dendrimers, we prepared mono(azobenzene) dendrimers with a rigid framework using phenylacetylene subunits as analogs of the dendrimers above (Scheme 6).^^ The azobenzene-containing core is the key component of these dendrimers because coupling of the azobenzene configurational switch to the remainder of the dendrimer requires rigid linkages between the dendrons and the azobenzene. Hence, we designed an azobenzene core (14) that contains two acetylenic groups, which was prepared by coupling trimethylsilylacetylene and 4,4'-iodoazobenzene (12b)^^ under Sonagashira conditions to
OVETTE VILLAVICENCIO and DOMINIC V McGRATH Table 1. Dendrimer 3a 3b 3c
3d 4a 4b 6a 6b 8a 8b 8c 9 10 15a 15b 15c 15d 15e
Physical characterization of mono(azobenzene) photochromic dendrimers Molecular formula
FW
Mass spec. ^
>^max (nm) ^
C26H22N2O2
394.47 818.91 1667.85 6761.92 392.50 817.00 1723.87 6817.54 318.34 530.58 955.05 8618.67 4430.46 606.89 1231.80 2481.62 4981.26 9980.53
392^ 820^ 1670.3 (M+H); 1691.5 (M+Na) 6765.79 (M+H) 392.2013^ 817.3729 (M+H) ^ 1724.9 (M+H); 1747.4 (M+Na) 6859.1 (M+K) 319^ 531^ 956^ 8644.4 (M+Na) 4463.1 (M+K) 606.46 (M+H) 1231.8 (M+H) 2481.6 (M+H) 4983.6 (M+H) 10037 (M+Cu)
359 359 359 359
C54H46N2O6 C110H94N2O14 C446H382N2O62 C26H24N4 C54H48N4O4 C112H94N2O16 C448H382N2O64 C20H18N2O2 C34H30N2O4 C62H54N2O8 C510H446N2O126 C262H230N2O64 C44H50N2 C92H98N2 C188H194N2 C380H386N2 C764H770N2
331 331 359 359 359 358 358 384 385 383 382 383
^ MALDI-TOF data. ^ Absorbances of CHCI3 solutions (40 |JLM). ^ FAB data.
provide 13 (92% yield), followed by removal of the trimethylsilyl groups (89% yield) by treatment of 13 with tetrabutylammonium fluoride in a mixed solvent (THF: H2O = 2:1). Both potassium carbonate in methanol-methylene chloride and tetrabutylanmionium fluoride in THF were unsuccessful at effecting this deprotection. First through fourth generation dendrimers 15b-15e were prepared by Pd(0)-catalyzed coupling of diacetylene azobenzene 14 with the iodo-functionalized phenylacetylene dendrons reported by Moore^^ in 49-63% yields. Zeroth generation dendrimer 15a was prepared through the direct coupling of 3,5-di-f^rr-butylphenylacetylene^^ and 4,4'-dihydroxy-azobenzene^'* ditriflate (12a). Mono(azobenzene) dendrimers were characterized by a combination of ^H and ^^C NMR spectroscopy, combustion analysis, and mass spectrometry. Selected physical characterization data are shown in Table 1. The different linking functionality (e.g., ether, amine, ester) between the azobenzene and dendritic sector of the materials serves to alter the position of the TI-TT* band of the azobenzene chromophore (Fig. 1). For dendrimers with azobenzene cores based on 1 or 2, this band is at ca. 360 nm, while for cores based on 5, this band is blue-shifted to ca. 330 nm. Acetylenic central linker 14, however, results in a red-shifted n-n* band at ca. 380 nm. In addition, generational growth of benzyl aryl ether based dendrimers was confirmed by the increase in absorbance of the 280 nm band that corresponds to the dioxygenated aromatic chromophores of these dendrons relative to the azobenzene chromophore at ca. 360 nm (Fig. 1).
Azobenzene-ContainingDendhmers
400
X/nm
300
400
^/nm
Figure 1. Absorbance spectra of solutions (CHCI3) of (left) second generation dendrimers 3c and 6a and zeroth generation dendrimer 15a, (middle) first, second, and third generation dendrimers 3b, 3c, and 3d (normalized at 360 nm), and (right) zeroth through fourth generation dendrimers 15a-15b (normalized at 382 nm).
A similar increase in absorbance of the phenylacetylene dendron chromophore at 295 nm was observed for dendrimers 15a-e. B. Tris(azobenzene) Dendrimers Two classes of dendrimers with three azobenzene subunits, tris(azobenzene) dendrimers, have also been prepared (Scheme 7).20,2i,35,36 j ^ ^^ic first class, second and fourth generation dendrimers 17a and 17b contain three azobenzene units at their core. In the second class, second and fourth generation dendrimers 18a and 18b contain three azobenzene units at their periphery. Note that compound 16 serves as the smallest member of both dendrimer types 17 and 18. To obtain tris(azobenzene) dendrimers 16, 17a, and 17b compounds 20a20c with azobenzene focal units were first prepared in 67-87% yield by alkylation of azobenzene derivative 4-(p-hydroxyphenylazo)benzyl alcohol (19) with iodomethane or second and fourth generation benzyl aryl ether dendrons in the presence of K2CO3 in acetone (Scheme 8).^^'^^ Compounds 20a20c (3 equiv.) were then allowed to react with Cs-symmetric central linker 1,3,5-benzenetricarbonyl trichloride in refluxing benzene under Dean-Stark conditions in the presence of DMAP. Compounds 16, 17a, and 17b were isolated as yellow-orange solids in 80-90% yield.^^ Dendrimers 18a and 18b are macromolecular isomers^^ of dendrimers 17a and 17b, respectively. To access dendrimers 18a and 18b, it was necessary to prepare dendrons with a single functional group on the periphery and then attach them to the central core. Since the standard convergent method for
10
OVETTE VILLAVICENCIO and DOMINIC V McCRATH Me
Key:
X = 1 =
oy
o
A:oJa' Note: arrow indicates direction of central linker (dendrimer core)
Scheme 7. Tris(azobenzene) dendrimers.
dendrimer synthesis only provides dendrons with identical peripheral subunits,^^ a protection/half wedge generation/deprotection/whole generation synthetic strategy was applied.^^'^^ Similar extended strategies have been used in the
Azobenzene-Containing
Dendrimers
11 CO2H
.a'
H^ > ^^
<^^^^!r^OH ^
R O — '
RBr K2CO3 acetone
^
19:R = H
HO2C -^
CO2H ^
^ ~ - ^
16:R = CH3 17a: R = [G-2] 17b:R = [G-4]
benzene, A
20a: R = CH3 13 20b: R = [G-2] 20c: R = [G-4]
Scheme 8. Synthesis of tris(azobenzene) dendrimers 16 and 17a-17b.
p^g|. 38,39 jj^g ^jjyj gj.Qup ^as chosen to protect the phenolic residue on the periphery of the dendritic wedges not only because several methods for its removal are available,^^ but also because it is readily monitored by its distinctive ^H-NMR signals. Second and fourth generation monofunctionalized dendrons 23a and 23b were prepared using standard coupling strategies.^^'^^ For the preparation of 18a and 18b from dendrons 23a and 23b, deprotection, alkylation with 4-(4^-methoxyphenylazo)benzyl bromide, and coupling to the central linker was performed (Scheme 9). Note that deprotection removes not only the allyl protecting group but the benzyl subunit to which it was attached as well. This lability of /7-hydroxybenzyl ethers has been previously observed."^^ Although second generation dendrimers 17a (C195H162N6O27) and 18a (C177H150N6O27) and fourth generation dendrimers 17b (C699H594N6O99) and 18b (C681H582N6O99) are not exact structural isomers of each other, the relative placement of the azobenzene subunits in the center and on the periphery makes them effectively macromolecular isomers.^^ Dendrimers 16, 17a-b, and 18a-b were characterized by ^H/^^C NMR, MALDI MS, and combustion analysis for confirmation of primary structure (Table 2). Generational growth in both dendrons 20a-20c and dendrimers 16, 17a, and 17b was confirmed by their respective absorbance spectra (Fig. 2, left and middle). The TT-TT* band at ca. 350 nm is clearly present in the spectra of all compounds and is of essentially the same molar absorptivity within each series. Relative to this band, the band at 280 nm grows with increasing molecular weight. This band is given rise to by the benzyl aryl ether fragments that are absent in 16 and 20a, and present to a greater extent in fourth generation compounds (20c and 17b) relative to second generation compounds (20b and 17a). C. Hexakis(azobenzene) Dendrimers First and third generation hexakis(azobenzene) dendrimers 24a and 24b were prepared (Scheme 10).^"^ Compounds 20a and 20b were converted into
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
12
AllylO
4-(4'-methoxyphenylazo)benzyl bromide
^^=/
u
KgCCb 18-crown-6 acetone
Scheme 9. Synthesis of dendrimers 18a and 18b.
their corresponding bromides and coupled to 3,5-dihydroxybenzyl alcohol to yield dendrons 26a and 26b (Scheme 11). The reactions of 26a and 26b with 1,3,5-benzenetricarbonyl trichloride in the presence of DMAP in benzene under Dean-Stark conditions provided dendrimers 24a and 24b, respectively. Structural characterization of 24a and 24b was performed by ^H and ^^C NMR, MALDI mass spectrometry, and combustion analysis, with selected data shown in Table 2. A similar rise in the 280 nm absorbance band in hexakis(azobenzene) dendrimers 24a and 24b, indicative of generational growth, is also observed (Fig. 2c).
III. PHOTORESPONSIVE BEHAVIOR Prior to our initial work in the synthesis of azobenzene-containing dendrimers,^^ only one example of an azobenzene-containing dendrimer was reported in the literature.^^ Hence, it was of paramount importance to characterize the
Azobenzene-Containing Dendrimers Table 2.
13
Physical characterization of tris- and hexakis(azobenzene) dendrimers
Dendrimer
Molecular formula
FW
Mass spec.
16 17a 17b 18a
C51H42N6O9 C195H162N6O27 C699H594N6O99
882.85 3021.25 10661.74 2793.16
884 (M+H)^ 3048.10 (M+Na) 10663.40 (M+H) 2794.09 (M+H); 2816.02 (M+Na); 2832.06 (M+K); 2855.95 (M+Cu) 10434.94 (M+H) 1923.8 (M+H) 6200.31 (M+H)
18b 24a 24b
C177H150N6O27
C681H582N6O99 Cl14H96Ni20i8 C402H336N12O54
10434.09 1922.08 6199.14
:(nm)^ 348 351 352 349
349 349 350
^ MALDI-TOF data. ^ Absorbances of CHCI3 solutions (40 [itA). ^ FAB data.
400 ^/nm
500
300
400 A,/nm
300
500
400 ^/nm
Figure 2. Absorbance spectra (CHCI3) of (left) azobenzene-dendrons 20a-20c, (middle) tris(azobenzene) dendrimers 16, 17a and 17b, and (right) hexakis(azobenzene) dendrimers 24a and 24b.
Me
Me
24b Scheme 10, Hexakis(azobenzene) dendrimers.
14
OVETTE VILLAVICENCIO and DOMINIC V McGRATH R0^\
I
RO
^g^
N - """^ -"-XJ
11
'
!>. iJ
F
PP^3 L
/h-N^
O^
K2CO3 18-crown-6 acetone
20a:X = OH;R = Me 20b: X = OH; R = [G-2]
A
i>-o y ^
,—,
bH
N-
RO-T^^V-M' MU-\^^^^^^^N
25a: X = Br; R = Me 25b:X = Br;R = [G-2]
26a: R = Me 26b: R = [G-2] COCl
cioc" ^^-^^^coci 18-crown.6 DMAP benzene, A
^
24a: R = Me 24b: R = [G-2]
Scheme 11. Synthesis of Hexakis(azobenzene) dendrimers 24a and 24b.
materials in the previous section based on their photoresponsive behavior. Photoisomerization of the azobenzene subunits in the photochromic dendrimers we have prepared in the previous section will result in different diastereomeric forms based on the configurational state of the azobenzene(s) (Fig. 3). Two questions needed to be addressed. First, what is the effect of dendrimer incorporation on the photochromic character of the azobenzene subunits? And second, what is the effect of the azobenzene photochromism on dendrimer properties? In this section, we will primarily address the former question, while in the following section we will address the latter. In general, our results demonstrate that the photochromic behavior of the azobenzene is not perturbed by incorporation into the dendrimer structure. A. Mono(azobenzene) Dendrimers As anticipated, these dendrimers undergo configurational E -^ Z isomerization in response to visible light (Fig. 3A). The photoinduced conversion of E- to Z-dendrimer was easily observable by ^H NMR. As a representative illustration, consider the isomerization of dendrimer 8c followed by ^H NMR (Fig. 4). Upon initial dissolution, the methoxy group in £-8c appears as a singlet at 3.87 and the azobenzene protons give rise to two AA'BB' in the aromatic region (ca. 7.0 and 7.8 ppm). Upon irradiation at 350 nm, the methoxy peak at 3.87 for E-Sc yields to a new methoxy peak for Z-8c at 3.74 ppm. Simultaneously, the AA'BB' resonances of the azobenzene moiety also shift upfield to ca. 6.8 ppm. Interestingly, the effect of the azobenzene isomerization is felt more strongly by
Azobenzene-Containing
Dendrimers
15
o<
EEZ
EZZ
Figure 3. Schematic representations of the photoisomerization of the photochromic azobenzene subunits in mono- and tris(azobenzene) dendrimers.
the aromatic protons on the benzyl subunit directly attached to the photochromic moiety. These protons shift slightly upheld relative to the aromatic protons of the more remote benzyl subunits. Extensive irradiation resulted in a change in the ratio of £'-8c/Z-8c from 87 :13 to 8 :92 based on integration of the methoxy singlets. In all dendrimers derived from azobenzene 1, the photostationary states of dark incubated and irradiated (350 nm) samples were similar. Azobenzene photochromism is traditionally studied by absorption spectroscopy. For example, dark incubation of a chloroform solution (40 |JLM) of second generation dendrimer 3c served to maximize the absorption at 360 nm corresponding to the E azobenzene chromophore. Irradiation of a dark-incubated solution of 3c with 350 nm light resulted in clean photoisomerization to the predominantly Z-dendrimer as evidenced by a decrease in the absorbance at 360 nm and an increase in absorbances at 314 and 450 nm (Fig. 5). A photostationary state was reached within approximately 60 s, although this time period varies depending on the concentration of the sample, the temperature, and the intensity of the irradiating light. When left in the dark, the Z-dendrimer reverted to the original E form, as indicated by an increase in the original absorbance at ca. 360 nm, over the course of several hours at ambient temperature. We studied the kinetics of this process by absorption spectroscopy to obtain a more quantitative measure
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
16
I—'—I—'—I—'—I—'—I—'—I—'—I—•—I—■— 7.8 7.6 7.4 7.2 70 6.8 6.6 ppm
3.8
ppm
3.8
ppm
cis^c
Auik^
rwM»i^»nM^i%a>ii«i>in<(»ii'to)«i>i**i«
7.8
7.6
7.4
7.2
6.8
7.0
6.6
S^iTAK^t
ppm
Figure 4. ^ H NMR spectra (CDCI3) of dendrimer 8c after dark incubation (top) and after subsequent irradiation (350 nm, 10 min).
400 500 wavelength (nm)
0
60
120 180 tinne(min)
240
0
60
120 180 time (min)
Figure 5, Plot of (left) absorption spectra of dendrimer 3c (40 |xM in CH2CI2) under irradiation conditions (350 nm; 0, 5 , 1 0 , 15, 20, 25, 30, 60 s), (middle) absorbance at 359 nm of a sample of 3c kept in the dark at 292, 313, and 333 K after irradiation (10 min at 350 nm), and (right) first-order rate constant of ln[Aoo - A] vs. time (s) for absorbance data at the middle.
Azobenzene-Containing Dendrimers Table 3.
17
Rates and activation energies for thermal isomerization of mono(azobenzene) benzyl aryl ether dendrimers
Compound
/C27 f x
3a 3b 3c 8a 8b 8c 9 10
9.79 11.3 10.0 8.66 8.92 10.0 9.48 9.74
7 0 S ^;
k4o (xlO^s 9.82 9.51 9.45 10.8 10.7 10.2 8.47 8.04
0
/ceofx 70^ s-'')
Eact (kcal/mol)
6.30 6.75 7.23 6.52 7.39 7.18 7.59 6.93
20.7 20.3 21.3 21.4 21.8 21.2 21.8 21.2
of the effect of dendrimer incorporation on azobenzene photochromism. The thermal Z ^^ E isomerization was monitored at three different temperatures for the benzyl aryl ether based dendrimers in Table 1, and the results are shown in Table 3 (representative data are shown in Fig. 5). The results of these measurements lead to the following two conclusions. First, the relative invariance of the first-order rate constants and activation energies (^act) of these dendrimers indicates no strong steric influence on the Z -> £" thermal isomerization. The activation energy (^act) for this process is invariant with respect to increasing dendrimer size (Table 3)?^ Second, the values of the same data indicate no alteration of the photochromic behavior of azobenzene by incorporation into dendritic architecture. The first-order rate constant for the thermal process in all dendrimers studied is ca. 10"^ at ambient temperature, and increases approximately an order of magnitude for each jump in 20 K.^^ While initially surprising, we believe that these results reflect the highly flexible nature of benzyl aryl ether dendrons in solution. Indeed, in studies of linear azobenzene-containing polymers, perturbation of azobenzene photochromism was not observed at high dilution but only in a rigid glass matrix.^'^ To probe the effect of dendritic incorporation on the photochromism of azobenzenes more fully, we prepared the shape persistent dendrimer series 15a15e, which range in molecular weight from approx. 600 to 10,000. With rigid phenylacetylene dendrimer subunits as well as an acetylenic linkage between the azobenzene and the TT-framework of the dendron, the geometrical aspects of the azobenzene photochromism must be translated to the entire dendrimer structure. In other words, when the azobenzene bends, so will the dendrimer. Absorption spectroscopy indicates that these dendrimers also undergo photochromic switching of the central azobenzene moiety upon photoirradiation at the appropriate wavelength (380 nm), and Z ^^ E thermal isomerization occurs in the dark. The kinetics of this process were monitored and the results are shown in Table 4. Based on initial inspection, it appears that there may be an effect of dendritic incorporation on azobenzene photochromism in this series
18
OVETTE VILLAVICENCIO and DOMINIC V McGRATH Table 4,
Rates and activation energies for thermal isomerization of mono(azobenzene) phenylacetylene dendrimers
Compound
/C27 rx 70^ s-'^)
k4o (x 70^ s-0
keo (X 1(P s-^)
Eact (kcal/mol)
15a 15b 15c 15d 15e
6.82 6.28 6.73 11.7 17.8
4.17 4.56 4.93 8.23 12.9
2.35 3.03 3.25 4.71 8.03
■\1.1 19.4 19.3 18.5 19.0
of dendrimers. The rate constants forZ^^E thermal isomerization appear to increase on going from the second to fourth generation structure (Note a, see Section VII); however, this rate increase is consistent at all temperatures studied and likely results from a deviation in the pre-exponential factor. Therefore, the activation energy is essentially constant over the generation range studied here, and is again consistent with typical azobenzenes. We conclude, based on these data, that the azobenzene moiety is, again, not perturbed by incorporation into the dendritic interior. These studies have established the photochromic behavior of mono(azobenzene)-containing dendrimers to be essentially identical to that of small-molecule azobenzenes. This information is of importance for the use of these materials in reversible optical data storage applications and other contexts that require reversible E/Z isomerization of the azobenzene moiety. B. Tris(azobenzene) Dendrimers With each azobenzene capable of E/Z isomerization, tris(azobenzene) dendrimers can potentially exist in four discrete states, ZZZ, ZZE, ZEE, and EEE (Fig. 3B,C). These individual diastereomeric states of 16, 17a, and 17b are easily detected by ^H NMR in solution (Fig. 6). The resonances assignable to the central linker aromatic protons (Fig. 6c) for Ca-symmetric EEE and ZZZ isomers are singlets, whereas the formally C2v-symmetric EEZ and EZZ isomers give rise to two mutually coupled multiplets. Irradiation of individual dark-incubated NMR samples (CDCI3) of 16,17a, and 17b resulted in a dramatic change in the ratio of these resonances reflective of the increasing Z content in the mixture of isomers (Fig. 6a,b) (Note b). Other protons within these structures also reflect the individual diastereomeric content of samples of 16, 17a, and 17b. For example, the benzyl protons directly adjacent to the central linker are also sensitive to the diastereomeric state of the dendrimers (Fig. 6e). As the four possible isomers ZZZ, ZZE, ZEE, and EEE provide six possible environments for these benzyl protons, we can observe this in the appropriate region of the ^H NMR spectrum (Fig. 6d). Most importantly, the relative appearance and dis-
I
'
8.95
-
4
1
8.90
8.85
.
8.W
J
"
, ' . . . I " '
ppm
8.95
8.96
I
8.85
'
I . .
8.80
','"',~* 8.75
8.70
ppm
I'
5.5
.".'
I
5.4
.' .'
,
5.3
5.2
ppm
Figure 6. ' H NMR spectra of dendrirners (a) 17a and (b) 17b in the region corresponding to the protons of (c) the trimesate central linker. (d) 'H NMR spectra of dendrirner 16 in the region of the (e) benzyl protons adjacent to the central linker. Stack plots are of a dark-incubated sample and after irradiation (350 nm) for 5, 10, and 20 rnin (bottom to top).
20
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
Table 5. Rates and activation energies for thermal isomerization of tris(azobenzene) and hexakis(azobenzene) benzyl aryl ether dendrimers and building blocks Compound 16 17a 17b 20a 20b 20c 24a 24b
k2i (x W^ s''') 4.48 4.09 4.35 3.65 3.86 3.36 10.5^ 13.4^
/c^o (x 10^ s''') keo U 10"^ s"^; fact (kcal/mol) 5.16 3.03 20.9 3.29 2.65 20.8 4.89 2.90 20.9 4.85 3.03 21.9 4.06 2.91 21.5 4.61 2.92 22.2 3.08 2.46 21.1 3.89 2.50 19.5
^ Measured at 30°C (i.e., /C30 (x 10^ s"^)). appearance of all four isomers during the course of thermal E/Z isomerization, also followed by ^H NMR, indicate independent,"^^ rather than simultaneous"^ isomerization of the azobenzene units — the behavior of each dendrimer arm is unaffected by the presence of the other two. Therefore, there is no cooperativity to the azobenzene photoresponsive behavior by incorporation into this dendritic architecture. Since it is possible to treat these compounds as ensembles of individual decoupled azobenzene groups, we can use their kinetic behavior to determine the effect of the molecular environment on their photoresponsiveness. Compounds 16, 17a, and 17b all exhibited photoresponsive behavior characteristic of azobenzene-containing materials.^""^ Dark incubation of chloroform solutions of 16, 17a, and 17b served to maximize the 71 -> it* transition at 352 nm, and irradiation with 350 nm light resulted in photoisomerization as evidenced by a decrease in the absorbance at 352 nm and an increase in the n -> Tt* transition at 442 nm. The presence of observed sharp isosbestic points in the absorption spectra during isomerization was expected based on the independence of the chromophores. Thermal back-isomerization occurred, the rates of which were measured at several temperatures. The results of these measurements (Table 5) lead to similar conclusions as seen above for the mono(azobenzene) benzyl aryl ether dendrimers: (1) the relative invariance of the first-order rate constants and activation energies (£'act) of 16, 17a, and 17b indicates no strong steric influence on the Z-E thermal isomerization; and (2) there is no alteration of the photoresponsive behavior of azobenzene by incorporation into dendritic architecture. In addition, the strict adherence to first-order kinetics further supports the independent behavior of the azobenzenes. Dendrimers 18a and 19a exhibit photochromic behavior essentially identical to dendrimers 16, 17a, and 17b that is characteristic of azobenzene-containing materials.-^^
Azobenzene-Containing Dendrimers
21
C. Hexakis(azobenzene) Dendrimers The different possible configuration isomers of dendrimers 24a and 24b with six radially configured azobenzene moieties are represented in Fig. 7. Although at first it may seem that there should be 7 configurational isomers possible of 24a and 24b, the constitution of these molecules dictate that there are actually 10 different isomers. While there is only one possible constitutional arrangement for isomers with EEEEEE, EEEEEZ, EZZZZZ, and ZZZZZZ azobenzene distributions, isomers with EEEEZZ, EEEZZZ, and EEZZZZ azobenzene distributions have two possible constitutions. For example, the EEEEZZ isomer can have both Z azobenzenes on the same dendron or two different dendrons. Therefore, dendrimers 24a and 24b can exist in 10 different diastereomeric states rather than merely 7. Multiple photoisomeric states of hexakis(azobenzene) dendrimer 24a in solution were detected using ^H NMR spectroscopy (Fig. 8). The aromatic protons of the azobenzene subunits reflect a decrease in E azobenzenes and a concomitant increase in Z azobenzenes (Fig. 8, middle); however, little information is available in this region as to the distribution among the 10 possible diastereomeric states. The aromatic protons of the trimesate core also did not display dispersion significant enough for identification of individual diastereomers (Fig. 8, left). However, the methoxy protons proved to be quite sensitive to the isomeric state of the azobenzene to which they were attached as well as those surrounding them. This allowed greater distinction among diastereoisomers (Fig. 8, right). Considering first generation dendrimer 24a, the 10 possible diastereomeric states strictly give rise to 24 possible chemical environments for the methoxy groups, 12 corresponding to E-methoxy groups and 12 corresponding to the Z-methoxy groups (Fig. 8, inset). For example, when dendrimer 24a is in the EEEEEE form, all methoxy groups are chemical shift equivalent (in a single environment), while the EEEEEZ form contains three different methoxy environments (two E environments and one Z environment). Hence, one might expect to see up to 24 resonances for the methoxy groups in the ^H NMR spectra, 12 in the E region and 12 in the Z region. However, examination of the methoxy region of the spectra reveals that only 10 resonances were observed for dendrimer 24a, 5 in the E region and 5 in the Z region (Fig. 8, right). Since we conclude that it is highly unlikely for any of the 10 isomers to be absent from an equilibrium mixture of these dendrimers, it appears that there is significant overlap in this region of the NMR, although we cannot conclusively account for all 10 isomers in solution (Note c). The rates and activation energies for thermal Z -^ E isomerization of hexakis(azobenzene) dendrimers 24a and 24b (Table 5) reveal typical azobenzene behavior similar to that seen for all of the dendrimers presented above.
N N
EZZZZZ
EEEEZZ
EEEZZZ
EEZZZZ
Figure 7. Ten possible diastereomeric states for hexakis(az0benzene) dendrimers 24a and 24b.
zzzzzz
Z
$ 17
0
5
z n S
6 5
EE EE EE EE EE EZ
0 1
EE EE ZZ EE EZ EZ
EE EZ zz EZ EZ EZ
1
8.90
ppm
EE zz zz EZ EZ ZZ
I 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 ppm
1
EZ
zz zz
0 ZZ ZZ ZZ
5 6
I
3.85
3.80 3.75
ppm
'
Figure 8. H NMR spectra (CDC13)of dendrimer 24a in the region corresponding to the protons of (left) the 1,3,5-triacylbenzene central linker, (middle) the azobenzene protons, and (right) the methoxy groups. Stack plots correspond to irradiation of the sample with 350 nm light (0, 5, 10,15, and 25 min). Inset: E and 2 azobenzene content of the ten possible isomers of 24a and 24b
N W
24
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
IV. PHOTOMODULATION OF PROPERTIES With the above dendrimers in hand, and their photoresponsive behavior investigated, we next sought to probe the effect of azobenzene photochromism on dendrimer physical properties. The photomodulation of dendrimer properties is affected by dendrimer generation, type of dendrimer subunit, number of photochromic units per dendrimer, and relative placement of photochromic subunits within the dendrimer. In particular, a comparison of the properties of these dendrimers reveals that the relative placement of responsive groups within the structure can dramatically alter their effect on the photomodulation of dendrimer properties. However, the magnitude of the alteration is markedly dependent on the property observed. In the following sections, we summarize our studies on the photomodulation of dendrimer polarity and molecular size in solution. A. Polarity Since Z-azobenzene is more polar than E,^ it is not unexpected that an analytical technique sensitive to molecular polarity would be able to distinguish between the configuration isomers of the multi-azobenzene-containing dendrimers prepared above. For example, as detailed below, retention times on standard chromatographic supports (Si02) increase with increasing Z content of the diastereomeric forms of the multi-azobenzene-containing dendrimers in a predictable fashion. With tris(azobenzene) dendrimers 16, 17a, and 17b, in which the azobenzenes are at the core, we were able to probe the insulation of the dendrimer interior with increasing dendrimer generation.^^ Somewhat surprising to us, this insulation is not, at least of the fourth generation, all that effective. Hence, a comparison of this behavior with that of the dendrimers representing limiting isomeric forms, 18a and 18b, was carried out.^^ This comparison provides information about the effect of relative placement of photoresponsive subunits on the photomodulation of dendrimer properties. In addition, it demonstrates that the azobenzene subunits serve as effective probes of the accessibility of different dendrimer regions to the outside environment. Isomerization of the interior azobenzene moieties in 16, 17a, and 17b markedly affects their macroscopic polarities in a predictable fashion. For example, monitoring the distribution of the isomers of 16 as a function of irradiation time was possible by thin layer chromatography (TLC) since each discrete state was easily separable based upon azobenzene configurational content (Fig. 9, left). The higher the Z-azobenzene content in the interior of a dendrimer, the higher the chromatographic retention time of that isomer. This difference in chromatographic retention times among the four isomers was also observed for second generation dendrimer 17a (Fig. 9, middle). Remarkably, even in compound 17b, with a molecular weight of over 10.6 kD
Azobenzene-Containing
1
2
Dendrimers
3
25
1
2 3
1
2
-1
^
3
f I 1t 1
f
1 1
u
Figure 9. Thin layer chromatography (TLC) of 16,17a, and 17b on Si02-coated plates. Left: TLC of 16 (24 :1 CH2Cl2-Et20). Middle: TLC of 17a (48 :1 CH2Cl2-Et20). Right: TLC of 17b (99 :1 CH2Cl2-MeOH). Lanes 1 - 3 in each set are after dark-incubation (lane 1), 1 min irradiation (lane 2), and 2 min irradiation (lane 3) at 350 nm.
and the azobenzenes buried in the core, differences in retention time between the isomers are still readily observable, albeit with a much different solvent system (Fig. 9, right). The difference in solvent systems suggests that increasing generation on going from 16 to 17a to 17b results in more effective insulation of the core. Nevertheless, the apparent facility with which we could separate the isomers of fourth generation 17b was quite intriguing. It is likely the case that these observed property differences among the isomers of 17a and 17b were given rise to by the changes in polarity of the individual azobenzene moieties and not, for example, by a molecular size effect. However, modeling suggested that the azobenzenes in 17b are insulated within the interior of the dendrimer (Fig. 10). This behavior, then, may be a manifestation of the high degree of flexibility of dendrimers of this type."^^"^^ To further probe the nature of these dendrimers in solution with respect to the insulation of the core, we decided to investigate the effect of the relative placement of azobenzene subunits within the architecture of tris(azobenzene) dendrimers using dendrimers 18a and 18b. Similar to the behavior detailed above, monitoring the distribution of the isomers of 18a and 18b as a function of irradiation time was possible (Fig. 11). Hence, we directly compared the chromatographic behavior of second generation dendrimers 17a and 18a, dendrimers that differ only in the placement of azobenzenes within the dendritic architecture. After irradiation with 350 nm light, four spots, which represent the four isomers (EEE, EEZ, EZZ and ZZZ), were observed for both compounds (Fig. 11a). Despite a small difference in the absolute /?F values of the
26
OVETTE VlLLAVlCENClO and DOMINIC V. McCRATH
L
t ._ C
U
Azobenzene-Containing
27
Dendrimers (b)
(a)
♦♦
Figure 11. Thin layer chromatography (TLC) of second generation dendrimers 17a and 18a and fourth generation dendrimers 17b and 18b on Si02-coated plates: (a) 17a (left) and 18a (right) with 1 :24 Et20-CH2Cl2 as eluent; (b) 17b (left) and 18b (right) with 3 : 9 0 : 7 Et20CH2Cl2-hexane as eluent. Samples were irradiated with 350 nm light in acetone solution for approximately 1 min prior to experiment.
individual isomers, the range of R^ values for the isomers of both compounds 17a and 18a are identical. This indicates that the effect of the azobenzene on the polarity properties of both dendrimers is similar (by TLC), even though their structures are significantly different. Even the chromatographic behavior of fourth generation dendrimers 17b and 18b exhibit a remarkable similarity in the observed R^ values of the corresponding configurational isomers (Fig. lib). Indeed, the range of/?F values for the isomers of 17b, with interior azobenzenes, is somewhat smaller than the corresponding range for the isomers of dendrimer 18b, with exterior azobenzenes. Yet overall, we find that photomodulation of dendrimer polarity is insensitive to the relative placement of the responsive groups within the structure. Presumably the flexibility of the benzyl aryl ether dendrons"^^"^^ as well as a lack of secondary interactions between the end groups"^^ precludes these dendrimers from adopting a fully insulating core-shell morphology,'^^'^^ at least to the fourth generation. Hence, the apparent increase in insulation of the core of dendrimers 16,17a, and 17b was not really the observed effect at all. Rather, the different solvent systems necessary to separate the isomers of these three dendrimers were probably more a reflection of the azobenzene subunit/benzyl aryl ether subunit ratio. Hexakis(azobenzene) dendrimers 24a and 24b also exhibit polarity properties which can be photomodulated. TLC is effective at resolving these materials into seven polarity levels (Fig. 12). Clearly, the added constitutional complexity discussed above (Section IILC) for these dendrimers is not a factor in the
28
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
I w
w
I
I
Figure 12. Thin layer chromatography (TLC) of 24a and 24b on Si02-coated plates. Left: TLC of 24a (19:1 CH2Cl2-Et20). Right: TLC of 24b (39 :1 :1 CH2Cl2-Et20-hexane). Lane 1 in each set is after dark-incubation. Subsequent lanes are after increasing irradiation at 350 nm, except lane 4, left, which is after exposure to bright sunlight.
discrimination of the E/Z content of the core by TLC. A difference in the chromatographic behavior of first generation 24a and second generation 24b is evident in the different solvent systems necessary to resolve their respective configurational isomers. B. Molecular Size Alteration
With 1, 3, or 6 azobenzenes capable of E/Z isomerization in the photochromic dendrimers presented above, several different diastereomeric forms exist in solution as confirmed by NMR and TLC analysis. Since the end-to-end distance in Z azobenzene is approximately 0.45 nm longer than E azobenzene, then the hydrodynamic volume of these dendrimers in solution should decrease on irradiation and this photomodulation of size should be observable by size-exclusion chromatography (SEC or GPC). This is indeed the case, as detailed below. A comparison of different photochromic dendrimers reveals the structural factors that determine the effect of the azobenzene subunits on the photomodulation of hydrodynamic volume in solution. These structural factors include the number of azobenzene moieties, the relative placement of azobenzene moieties, and the type of dendrimer subunits used to construct the dendrimer. For the purposes of our studies on photomodulation of hydrodynamic volume, we considered certain compounds prepared in Section II as belonging to three structural classes of azobenzene-containing dendrimers: those with a single azobenzene at the core (Fig. 3A); those with multiple azobenzenes at
Azobenzene-Containing
Dendrimers 17a
(a)
29 1
ft:17b
1 li
1
c•
■ 1
p 1
(6
5 1'
1
'1
1 1
/ 1
IJII ■ ■ . ■ ■ ■
.1
•1 ' 1 < •1 1
inJ 1
1
25
30 Ret. Vol. (mL)
25
30 Ret. Vol. (mL)
Figure 13. GPC traces for dendrimers (a) 17a and 17b and (b) 18a and 18b before (solid line) and after (broken line) irradiation with 350 nm light. Conditions: CH2CI2; 1 ml/min; 500 A, 1000 A, 10,000 A DVB Gordl) columns (250 x 10 mm); ambient temperature.
the core (Fig. 3B); and those with azobenzenes at the periphery (Fig. 3C) of the dendrimer (Note d). Consideration of these three dendrimer classes has allowed us to assess the effect of the number of azobenzene subunits as well as their relative placement in the dendritic structure on the photomodulation of hydrodynamic volume. In addition, we have also been able to assess the effect of dendrimer subunit type (flexible benzyl aryl ether vs. rigid phenylacetylene) by comparing benzyl aryl ether dendrimers (3a-3d) and shape-persistent phenyl acetylene dendrimers (15a-15e). For all dendrimers studied, we observed GPC peaks at greater elution volumes after irradiation indicating a decrease in hydrodynamic volume. Fig. 13 gives representative GPC traces of samples of type B dendrimers 16a, 16b, 17a, and 17b before and after irradiation. Although all four isomers of these tris(azobenzene) dendrimers are present together in solution (see Section III.B), we did not observe four individually resolved peaks in the GPC of equilibrium mixtures after different irradiation times. However, a reproducible limiting elution volume was always observed for both dark incubated and extensively irradiated samples of both mono- and tris(azobenzene) dendrimers (Note e). From the elution volumes we calculated hydrodynamic radius (Note f) and plotted this versus generation for all dendrimers studied (Fig. 14, left). In addition, we calculated the percent difference in hydrodynamic volumes between irradiated and non-irradiated forms versus generation (Fig. 14, right). Several conclusions can be drawn from these data. First, within the same family of dendrimers, the percent change in hydrodynamic volume caused
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
30
2.5
-3c-3d -15a-15e -17a-17b -18a-18b
-3c-3d -15a-15e -17a-17b -18a-18b
E 1.5h 1 h 0.5
Generation
Generation
Figure 14, Plots versus generation of (left) hydrodynamic radius before (solid line) and after (broken line) irradiation (top) and (right) % difference in hydrodynamic volume before and after irradiation for mono- and tris(azobenzene) dendrimers.
by azobenzene isomerism was smaller at higher generation than at lower generation. This indicates that the effect of the azobenzene unit on the molecular size of a dendrimer in solution decreases with increasing dendrimer size. Second, when the same dendrimer subunits were used (e.g., benzyl aryl ether) to construct the dendrimers, multiple azobenzenes were more efficient than a single photochromic unit at changing dendrimer volume in solution (3c-3d vs. 17a-17b). Third, when multi-azobenzenes were contained in a dendrimer, the change in hydrodynamic volume caused by azobenzene isomerism was significantly smaller when the azobenzenes are on the exterior rather than the interior of the dendrimer (17a-17b vs. 18a-18b). Fourth, a comparison of hydrodynamic volumes between 3a-3d and 15a-15e reveals a significant difference in change in hydrodynamic volume for the same generation of dendrimer. For example, for second generation dendrimers 3c and 15c, the percent differences in hydrodynamic volume between the E and Z forms were 18.2 and 28.8%, respectively. This indicates that dendrimers consisting of rigid phenylacetylene dendrimer subunits reflect the configuration of a central azobenzene more efficiently than those with flexible benzyl aryl ether subunits. These studies reveal that the hydrodynamic volume of azobenzene-containing dendrimers can be significantly changed when azobenzene units are subjected to irradiation with Z-azobenzene-containing dendrimers smaller than their ^'-isomers. However, the magnitude of this size alteration is affected by dendrimer structure, number of photochromic units per dendrimer, and the relative placement of photochromic units in the dendrimer.
Azobenzene-ContainingDendrimers
31
C15H31
N //
C15H31
HO
V-N=N \-=/
OH
C15H31
25
,-iv/
C15H31
N=N
\=z/
HO
OMEM
f
26
28a: n = 1 28b: n = 2 28c: n = 3 28d: n = 4
Scheme 12. Synthesis of donor/acceptor azobenzene-containing dendrons by Yokoyama et ai;i-53
V. LITERATURE REVIEW A. Monolayers/Self-Assembly/Amphiphilic Dendritic Structures Yokoyama et al. investigated dipolar, amphiphilic dendrons with electron donor/acceptor azobenzene branches for use in possible optoelectronic and electronic applications.^^"^^ Azobenzene-containing dendrons 27 and 28 were prepared up to the fourth generation through repeated carbodiimide-catalyzed ester couplings (Scheme 12).^^'^^ In these structures, donor/acceptor azobenzene subunits are present at each branch point, the focal point is a carboxyl residue, and the periphery consists of hydrophobic hexadecanoyl esters. The amphiphilic character generated by the carboxyl focal point and the hydrophobic periphery allows these dendrons to be incorporated into self-assembled molecular films using Langmuir-Blodgett techniques. In this fashion, the donor/acceptor azobenzenes were maintained in a non-centrosymmetric environment appropriate for second harmonic generation (SHG) activity. Films of both pure dendrons and dendrons in a mixture with arachidic acid were fabricated. Transmission SHG measurements revealed that the SHG was fully coherent. This suggests that the thin film was of uniform polar order or molecular orientation. In addition, SHG activity was greater in the films diluted with arachidic acid (64 times quartz for 27d) compared to pure films (2 times quartz for 27d). The scaled SHG results versus the fraction of number density of dendron in the diluted films indicate a plateau region between films of 1/10 and 1/80 ratios of dendron-arachidic acid. Presumably, arachidic acid improves
32
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
the molecular orientation and packing arrangement of the film. A greater than expected enhancement in the SHG was observed for fourth generation dendron 27d in the diluted films. The authors claim that the polar order of these dendrons increases with larger generations. A study of related dendrons in solution was recently reported by the same workers.^^ The molecular hyperpolarizability of the NLO azobenzenes was measured by the hyper-Rayleigh scattering (HRS) method. The pi^^^/^l^^ value of 4.5 was estimated from depolarized HRS signal intensities of a chloroform solution. Close to the theoretical value of 5.0 for a linearly polarized molecule with one dominant ^ component, this value correlated to the thin rod model suggesting that the azobenzene units are arranged non-centrosymmetrically in solution. This is in apparent contradiction with evidence that dendritic macromolecules tend to be spherical or globular with spreading branches. The nonresonant hyperpolarizabilities of the dendrons not only increased with the number of chromophoric branching units, but also were enhanced by up to 33% greater than just simply adding the monomer ^o values. This increase is attributed to the intermolecular non-centrosymmetric orientation of the azobenzene units within the dendron resulting in each chromophoric unit coherently contributing to the SHG. This effect was also found to be solvent dependent, highly supporting that the effect is conformational in nature. Meijer and his group have prepared amphiphilic dendrimers of five generations based on a relatively hydrophilic poly(propylene imine) core and hydrophobic hydrocarbon periphery units and studied their self-assembly at the air-water interface and as aggregates in solution (Scheme \?>).^^ Their results illustrate the extreme conformational flexibility available to dendrimers under certain structural and environmental circumstances. Three different kinds of periphery units were studied: palmitoyl groups (29); alkyl chains containing azobenzene chromophores (30); and adamantane substituents (31). While the pressure-area isotherms indicate that dendrimers 31 are forming multilayers at the air-water interface, dendrimers 29 and 30 form stable monolayers with the molecular area per molecule exhibiting a linear increase with the number of peripheral alkyl chains. The assumed orientation of the dendritic amphiphiles is where the hydrophilic dendritic poly(propylene imine) core is pointing at the aqueous phase and all the hydrophobic tails attached to the core are oriented perpendicularly to the water surface in parallel fashion (Fig. 15a). The azobenzene moieties in dendrimers 30 serve as effective probes of conformation in this system. A significant blue shift of the azobenzene TT-TT* transition to 316 nm indicates H-type aggregates in the alkyl tail region of the structures. With the adamantane dendrimers, the persistent globular structure prevents the flat conformation of the core and thus explains the non-linear increase of the observed molecular area with the number of adamantane units attached to the different generation of dendrimers.
Azobenzene-ContainingDendrimers
33 29a: n = 4 29b: n = 8 29c:n = 16 29d: n = 32 29e: n = 64
32: n = 32; m = 32
Scheme 13. End group modifications of poly(propylene imine) dendrimers.^"^ Black circles represent PPI dendrimers of varying generation.
When these amphiphilic dendrimers were dispersed in buffered water (pH 1), aggregation into vesicles with bilayer walls (Fig. 15b) was supported by TEM, dynamic light scattering, critical aggregation concentration, fluorescence depolarization, XRD, and osmotic behavior measurements. In addition, UV-Vis measurements indicate H-packing in dispersions of dendrimers 30. The authors suggest that protonation of the poly(propylene imine) core leads to an extended dendritic structure as a result of Coulombic repulsion. This in turn facilitates changes in dendrimer shape allowing the formation of parallel-packed bilayers (Fig. 15b). Vesicles of this type were extensively investigated by the group of De Schryver.^^ Fifth generation poly(propylene imine) dendrimers functionalized with 64 palmitoyl (29e), 32 palmitoyl and 32 azobenzene (32), or 64 azobenzene groups (30e) were shown to form giant spherical vesicles up to 22 |xm in size in aqueous solution at pH 1 or pH 5.5. The size distribution and morphology of the vesicles were affected by pH and substituents on the dendrimers. At higher pH (less than 8), smaller vesicles are formed, and with increasing azobenzene substituents, larger vesicles are formed. The azobenzene-containing vesicles were fluorescent (A,ex = 420, Aem = 600) and showed a blue-shifted absorption band at 310 nm with multiple shoulders to the red. Confocal laser scanning microscopy z-scans indicated that azobenzene fluorescence was fairly
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
Air Water
M
Air Water
Air Water (b)
(c)
48.8 A
Figure 15. (a) Schematic representation of the organization of amphiphilic dendrimers in a monolayer at an air-water interface, (b) Schematic representation of bilayer formation of amphiphilic dendrimers in acidic water.^"^ (c) Schematic representation of individual bilayers of azobenzene-containing vesicles in aqueous solution.
constant through the entire vesicle structure. Based on these and other data, the authors propose a multilaminar onion-like structure with individual bilayers consisting of interdigitated dendrimer end groups (Fig. 15c). The fluorescence of the vesicles, as well as the blue-shifted absorption, is strong evidence for the indicated aggregation of the azobenzene moieties within the bilayers.^^ Irradiation with a focused laser beam of either 420 or 1064 nm light
Azobenzene-Containing Dendrimers
35
induces an increase in fluorescence intensity of the vesicles. The enhancement, including a shift in emission maximum to 530 nm, is consistent with cis-trans isomerization cycles of the azobenzenes leading to a reorganization to a different state of aggregation. The nature of this new state of aggregation may involve an increase in head-to-tail stacked azobenzenes as well as a decrease in the pH in the vicinity of the azobenzenes. Recently, Weener and Meijer demonstrated that Langmuir monolayers of dendrimers with a mixture of palmitoyl and azobenzene-containing periphery groups (32) exhibit distinctly different photochromic behavior from the corresponding monolayers fabricated of all azobenzene-containing periphery groups (30a-e).^^ Whereas the azobenzenes in the latter monolayers showed evidence of aggregation and did not undergo E-Z isomerization when irradiated with 365 nm light, the azobenzenes in the former mixed monolayers show no evidence of aggregation and undergo reversible E-Z isomerization. The E-Z isomerization is accompanied by a change in surface area at a constant pressure (20 mN/m). A LB film of 32 was prepared and also shown to exhibit E-Z isomerization behavior upon irradiation. The advantage of preparing mixed monolayers of azobenzene-containing and non-azobenzene-containing amphiphiles attached to a dendritic scaffold, as opposed to monomeric units, is that phase separation within the monolayer is suppressed. A different approach to photochromic Langmuir monolayers was reported by the group of Tsukruk.^^'^^ Dendrons 33 have a single photochromic moiety proximal to the focal point of these amphiphilic benzyl aryl structures (Scheme 14).^^'^^ The architecture with the two dissimilar bulky terminal fragments (i.e., the dendritic fragment and the crown ether) was designed to provide a loose packing of the central azobenzene and suppression of crystallization. Film formation at the air-water interface is observed with the molecular crosssection equivalent to the number of alkyl chains on the periphery of the dendron up to the first generation (33b). Above this, the molecular cross-section is greater (ca. 30%) than the sum of the peripheral alkyl chains suggesting a loose packing of the dendrons because of steric constraints in the architecture. This picture is also supported by a dramatic reduction of the in-plane elastic modulus with increasing generation. The films are photochromic, and the change in surface area per molecule decreases as well with increasing generation. While the molecular surface area of reference compound 34 increased 25% in a Langmuir monolayer in the liquid state, 33a and 33b increase by only 7-10% and 33c and 33d by 3%. B. Host-Guest Chemistry Vogtle and his group have prepared dendrimers with azobenzenes on the periphery,^^'^^ including the first example of an azobenzene-containing dendrimer 35,"^^ and most recently provided evidence in support of these types
36
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
33a: n = 0 33b: n = 1 33c: n = 2 33d: n = 3
34 Scheme 14. Amphiphilic azobenzene-containing dendrons reported by Tsukruk, McCrath and co-workers.^^~^^
of structures acting as photoswitchable hosts.^^ The dendrimers prepared most recently by Vogtle were poly(propylene imine) dendrimers with azobenzenes attached to the periphery by carboxamide linkages (Scheme 15). The position of the amide linkage with respect to the azo function was either para (36a-d) or meta (37a-d). Absorption spectra of the para and meta carboxamide substituted azobenzene show that the Amax of the TT-TI* and n-n* absorption bands do not change within each family of dendrimers (generations 1-4) indicating a lack of strong interchromophoric interactions. Photoisomerization experiments on these dendrimers demonstrate conclusively that there are no effective steric constraints imposed on the azobenzene by incorporation into dendrimers of all generations studied. All the azobenzene type compounds undergo EtoZ and Zio E photoisomerization and Zio E thermal isomerization. The m^m-substituted dendrimer had higher quantum yields for both the E to Z and Zio E photoreactions than the para-substituted azobenzene. This is due to the electronic factors caused by the different substitution. In addition, the overall quantum yields decreased with increasing chromophoric groups but the quantum yield due to a single chromophoric unit remained almost constant implying that there is no steric constraint towards photoisomerization on increasing generations of dendrimers. In collaboration with the group of Balzani, fourth generation dendrimers 36d and 37d were studied as potential hosts for eosin Y (38),^^ which was chosen for its strong fluorescence and its ability to sensitize the photoisomerization of the peripheral azobenzenes in the dendrimer. Fluorescence experiments indicated that when dendrimers were present in an eosin Y DMF solution,
Azobenzene-Containing
O
• ( IH
37
Dendrimers
^
//
N=N
36a: n = 4 36b: n = 8 36c: n = 16 36d:n = 32 37a: n = 4 37b: n = 8 37c:n = 16 37d: n = 32
para linked
meta linked
Scheme 15, Azobenzene-containing dendrimers reported by Vogtie and co-workers.^^'^^
the fluorescence intensity was decreased. When eosin was present with a non-dendritic azobenzene, no quenching was observed. Both the E and Z forms of 36d and 37d were active at quenching eosin Yfluorescence,although the Z form was more effective. Quenching of thefluorescenceof eosin Y is smaller for first generation 36a as compared to that of 36d. It was determined that the quenching most likely occurred by a static mechanism of electron transfer from the dendrimer amine units to the lowest singletfluorescentexcited state of eosin
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
38
Boc-HNBocHN,
TFA'HgNP
Boc»HN
2"-1
TFA-HgN40a: n = 3 40b: n = 4
1.H0~ 2. tBuOCOCI, EtgN 3. 1,3-PrO[4]CH2NH2
41a: n = 3 41b: n = 4
Scheme 16. Synthesis of azobenzene-containing dendrimers with 1,3-alternate calix[4]arene
Y. These results lead the authors to conclude that both forms of the dendrimers are indeed hosts for eosin Y and that the Z form is a more effective host. Using a 1,3-altemate calix[4]arene as a core, Nagasaki et al.^"^ synthesized third and fourth generation dendrimers with azobenzene-based subunits (Scheme 16). Amide bond formation between the branching subunits was achieved using isobutyl chloroformate mediated mixed anhydride couplings. Both dendrimers 41a and 41b showed typical switching of the azobenzene moieties with the E'-azobenzene absorption band at 347 nm decreasing upon 365 nm irradiation light while the absorption at 441 nm increased due to the Z form of the azobenzene. Dendrimer 41a reached a 20/80 E/Z mixture upon irradiation while dendrimer 41b reached an equilibrium of 35/65 in THE The authors claim that this difference in photostationary states between the two dendrimers is due to increased steric repulsions in the branches of the larger dendrimer. Upon irradiation at 436 nm, £'-azobenzenes were recovered in both dendrimers.
Azobenzene-Containing Dendrimers
39
The authors surmised that these materials might exhibit photocontrolled size differences. Size differences were confirmed in the second generation version of dendrimers 41 modified with lysine residues on the periphery by gel filtration chromatography in aqueous medium.^^ This was corroborated by dynamic light scattering measurements in ethanol on Boc-protected versions of this lysine modified dendrimer. UV irradiation purportedly decreased the size of this dendrimer from 7.3 nm to 5.6 nm. The lysine modified dendrimer in this latter paper of Nagasaki et al.^^ was prepared with the intention of using the photochromic nature of the azobenzene to alter the size to charge ratio, or surface cation density of the structure. Indeed, the zeta potential was repeatedly switched upon 365 nm UV irradiation between 16.7 ± 1 . 7 mV and 13.3 ± 0.9 mV. The authors claim that this indicates a decrease in cationic density that is ascribable to the deprotonation of surface primary amines due to an increase in electronic repulsion. The interaction of this poly cationic dendrimer and plasmid DNA (pCHllO) was confirmed by light scattering and electrophoresis. Apparently the irradiated form of the dendrimer has a higher affinity for DNA. The transfer of pCHllO containing LacZ encoding ^-galactosidase into COS-1 cells was demonstrated. C. Antenna Effects Jiang and Aida^^~^^ have reported that mono-azobenzene-containing dendrimers 42a-d exhibited remarkable generation-dependent energy harvesting capabilities. This series of dendrimers containing an azobenzene core with four attached benzyl aryl ether dendrons was prepared and structurally characterized (Scheme 17). Proton spin relaxation data (Ti) show a transition between third generation (42b) and fourth generation dendrimer 42c. The Ti values for the exterior methoxy protons decrease sharply at this transition while the Ti values for the interior aromatic protons remain fairly constant over the entire range of structures. This suggests that 42c and 42d possess relatively rigid exterior shells while the interior of the dendrimer remains non-constrained. As expected, compounds 42a-42d undergo ultraviolet photoinduced isomerization from the E form to the Z form of the core azobenzene and thermally isomerize in the reverse direction with typical half-lives. However, when individual solutions of the Z forms of 42c and 42d were irradiated with infrared irradiation (75 W glowing nichrome source), isomerization to the E form was accelerated over 250 times that of the thermal isomerization and, remarkably, over 20 times that of the rate found on irradiation with visible light (440 nm). Yet, the rates of Z -> £ isomerization of 42a and 42b were unaffected by the infrared irradiation. Two control experiments suggested that spatial isolation of the azobenzene is crucial for this effect. First, the infrared irradiated isomerization of 42a was carried out in the presence of 4 equiv. of a large generation
40
OVETTE VILLAVICENCIO and DOMINIC V McCRATH
42a: n = 1 42b: n = 3 42c: n = 4 42d: n = 5
2"-1
2"-1 43: n = 5 Scheme 17. Dendrimer system reported by Jiang and Aida to exhibit low-energy photon harvesting.^^"^^
dendritic alcohol (unanchored to the azobenzene unit). Second, the infrared irradiated isomerization of 43, comparable in molecular weight to 42d but with a monosubstituted azobenzene, was carried out (Note g). In neither case was the isomerization accelerated by the infrared irradiation. The Z -^ E isomerization for 42d was further investigated. Infrared irradiation of Z-42d was carried out using three specific wavelengths: (a) a stretching vibration for aromatic rings (1597 cm~^); (b) a stretching vibration for CH2-O (1155 cm~^); and (c) a transparent region (2500 cm~^). Only the 1597 cm~^ radiation accelerates the Z -> £" isomerization reaction. In addition, irradiation of Z forms of the entire series of dendrimers (42a-42d) with 280 nm light (^max of the benzyl aryl ether dendrimer framework) accelerated the Z -> £" isomerization, but only for 42c and 42d and not 42a or 42b (Note h). These two results taken in concert strongly suggest that a matrix to core intramolecular energy transfer is partly responsible for the acceleration effect. Hence, the authors postulate that the dendrimer frameworks in 42c and 42d insulate the interior units from collisional energy scattering as well as serve as light harvesting antenna. Photon harvesting is necessary to account for how 1597 cm~^ light (0.2 eV) could accelerate a process that has an activation free energy (AG*) of 19.4 kcal mol~^ (0.84 eV) at 2 r C . Indeed, photon flux experiments indicated
Azobenzene-Containing Dendrimers
41
that 4.9 photons at 1597 cm~^ (0.98 eV total) are involved in this photochemical process. D. Holography
The first investigation of the use of azobenzene-containing dendrimers for holographic applications was carried out by Archut et al.^^ Thin films of the m^ta-substituted dendrimers 37b-37d (second to fourth generation) and the first generation para-substituted dendrimer 36a were prepared by casting chloroforms solutions on glass substrates. Diffraction gratings were written into the films using two orthogonally circularly polarized beams at 488 nm (argon ion laser at 1400 mW cm~^). Diffraction efficiencies (x) of ca. 20% are achieved after 60 s of irradiation. Interestingly, the 36a grating was erased by heat (75°C), while the grating of 37b remained stable. AFM investigation revealed that the film of 37b had large surface relief (up to 1500 nm), while the film of 36a had no observed surface relief. The difference in the packing of the chromophores in the meta and para constitutions are possibly responsible for the different surface relief and stability of the diffraction gratings. It would be of interest to compare the results reported with those for linear azobenzene-containing polymers of similar structure.
Vl. SUMMARY The preparation of a wide variety of azobenzene-containing dendrimers has followed the initial report of the incorporation of this photochromic chromophore as a dendrimer subunit by Mekelburger et al.^^ Studies of these materials have both shed light on the nature of dendritic macromolecules as well as expanded the utility of dendrimers in a variety of contexts. A continuing combination of fundamental studies on the photomodulation of dendrimer properties in azobenzene-containing dendrimers and new developments in the application of these materials to new and existing technologies is anticipated.
NOTES ^ The rate constants for thermal isomerization of 15a-15e are approximately three orders of magnitude greater than those of dendrimers based on central linker 1. Since this is the case for all generations of these dendrimers, it is must be a consequence of the electronic nature of this particular azobenzene structure.^ ^ The inherently globular nature of dendrimers is evident in the increased dispersion of the resonances arising from the central linker with increasing molecular size. For example, the chemical shift difference between the singlets for the EEE and ZZZ isomers increases from 0.07 (16) to 0.11 (17a) to 0.17 (17b) ppm.
42
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
^ Attempts to investigate this system using ^H NMR at 600 MHz and ^^C NMR at 150 MHz yielded no additional information. ^ Hexakis(azobenzene) dendrimers 24a and 24b do not fall into these classifications but will be discussed as well in terms of the photomodulation of their hydrodynamic size in solution. ^ Based on a photostationary state ratio of 1:9 E/Z for these experimental conditions, the isomer ratio for 16a, 16b, 17a, and 17b after extended irradiation was approximately <0.1:3 :24:72 EEE/EEZ/EZZ/ZZZ and the reverse for a dark incubated sample. ^The GPC elution volumes observed for all dendrimer samples were converted to corresponding hydrodynamic volume values using the relationship Vh = OAKM^'^^, where K and a are the Mark-Houwink constants for poly-styrene in 1,2-dichloroethane and M is the co-eluting polystyrene molecular weight. Hydrodynamic radius was calculated from the volume assuming a spherical shape and using the formula for the volume of a sphere (V = (4/3)7rr^. ^ The structure of the azobenzene unit in 43 is significantly altered relative to 42a-42d, so the appropriateness of this control experiment is in question. ^ This result in itself is remarkable in that the azobenzene itself has a finite absorbance at 280 nm and irradiation at this wavelength markedly accelerates Z ^^ E isomerization.
ACKNOWLEDGEMENTS We thank the National Science Foundation, Research Corporation, Air Force Office of Scientific Research, and the Camille and Henry Dreyfus Foundation for support of this research. DVM is grateful to all of the co-workers and collaborators, whose names appear in the references, who contributed to the research detailed in sections II, III, IV, and V.A of this monograph.
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44
OVETTE VILLAVICENCIO and DOMINIC V McGRATH
54.
Schenning, A. P. H. J.; Elissen-Roman, C ; Weener, J.-W.; Baars, M. W. P. L.; van der Gaast, S. J.; Meijer, E. W. /. Am. Chem. Soc. 1998,120, 8199-8208. Tsuda, K.; Dol, G. C ; Gensch, T.; Hofkens, J.; Latterini, L.; Weener, J. W.; Meijer, E. W.; De Schryver, F. C. / Am. Chem. Soc. 2000, 122, 3445-3452. Shimomura, M.; Kunitake, T. J. Am. Chem. Soc. 1987, 109, 5175-5183. Weener, J.-W; Meijer, E. W. Adv. Mater. 2000, 12, 741-746. Sidorenko, A.; Houphouet-Boigny, C ; C. Greco, A.; Villavicencio, O.; Hashemzadeh, M.; McGrath, D. V.; Tsukruk, V. V. Am. Chem. Soc, Div. Polym. Chem., Preprints 2000, 41(2), 1487-1488; Sidorenko, A.; Houphouet-Boigny, C ; Villavicencio, O.; Hashemzadeh, M.; McGrath, D. V.; Tsukruk, V. V. Langmuir, 2000,16, 10569-10572. Tsukruk, V. V.; Luzinov, I.; Larson, K.; Li, S.; McGrath, D. V. / Mater Sci. Lett. 2001, 20, 873-876. Hashemzadeh, M.; McGrath, D. V. Am. Chem. Soc, Div. Polym. Chem., Preprints 1998, 39(2), 338-339. Li, S.; Sikder, S.; McGrath, D. V. Am. Chem. Soc, Div Polym. Chem., Preprints 1999, 40(2), 267-268. Archut, A.; Vogtle, P.; De Cola, L.; Azzellini, G. C ; Balzani, V.; Ramanujam, P. S.; Berg, R. H. Chem. Eur J. 1998, 4, 699-706. Archut, A.; Azzellini, G. C ; Balzani, V.; De Cola, L.; Vogtle, E J. Am. Chem. Soc. 1998, 120, 12187-12191. Nagasaki, T; Tamagaki, S.; Ogino, K. Chem. Lett. 1997, 717-718. Nagasaki, T.; Shinmori, H.; Shinkai, S. Tetrahedron Lett. 1994, 35, 2201-2204. Jiang, D.-L.; Aida, T. Nature 1997, 388, 454-456. Aida, T.; Jiang, D.-L.; Yashima, E.; Okamoto, Y. Thin Solid Films 1998, 331, 254-258. Wakabayashi, Y; Tokeshi, M.; Jiang, D.-L.; Aida, T.; Kitamori, T. / Lumin. 1999, 83/84, 313-315.
55. 56. 57. 58.
59. 60. 61. 62. 63. 64. 65. 66. 67. 68.
LINEAR-DENDRITIC BLOCK COPOLYMERS SYNTHESIS AND CHARACTERIZATION
Ivan Gitsov
I. II.
III.
Abstract Introduction Synthetic Pathways A. Coupling of Reactive Preformed Blocks B. Polymerization of Dendritic Macromonomers C. Divergent Growth of Dendrimers on a Preformed Polymer Chain D. Growth of Polymer Chains on a Preformed Dendrimer Characterization Techniques A. Size Exclusion Chromatography B. MALDI-TOF Mass Spectrometry C. Atomic Force Microscopy D. Nuclear Magnetic Resonance Notes Acknowledgements References
. .
45 46 50 50 58 65 72 79 80 82 83 83 84 84 85
ABSTRACT This chapter provides thefirstcomprehensive review on a new class of materials that is build up by dendritic fragments and linear blocks. These linear-dendritic block copolymers were introduced Advances in Dendritic Macromolecules Volume 5, pages 45-87 © 2002 Elsevier Science Ltd. All rights reserved ISBN: 0-7623-0839-7
45
46
IVAN GITSOV
a decade ago and are attracting considerable attention because of their intriguing properties and broad application field. The major architectural types are reviewed together with the synthetic strategies for their construction and the main analytical techniques for their structure elucidation.
I. INTRODUCTION The linear-dendritic block copolymers are a relatively new class of polymeric materials that are composed of two or more fragments with fundamentally different molecular architectures and physical properties. The combination of predominantly flexible linear chains and semi-rigid perfectly branched globules yields macromolecules with a broad spectrum of interesting properties that do not necessarily behave as a simple combination of their initial constituents. These block copolymers can be tailor-made to serve as simplified theoretical models for various biological macromolecules, but could also find practical usage in surface modification, sustained drug delivery, phase-transfer catalysis, blend compatibilization and many other areas. Since the first publication in 1991 this area of research has expanded steadily and lately enjoys increased interest because of its appeal to a broad scientific audience. The aim of this chapter is to review the current status of the field with an emphasis on the synthetic strategies used in the formation of the linear-dendritic block copolymers and the methods for their analysis and characterization. By definition a "linear-dendritic block copolymer" (LDBC) should contain linear and dendritic segments of sufficient length and size (generation), respectively, to be considered as "blocks". Therefore, the review will be restricted to systems composed of linear chains with nominal molecular weight above 1000 Da and perfectly branched (dendritic) moieties of generation two and higher. For an excellent review of copolymers containing linear and broadly defined branched units see Roovers and Comanita.^ Despite the fact that only two types of blocks are used to construct the LDBCs, multiple combinations are possible. They are presented schematically in Figs. 1-4 in increasing structural order and complexity. The simplest members of the series are DL (Fig. 1, A), DLD (Fig. 1, B) and LDL (Fig. 1, C)
0% B Figure 1,
Linear-Dendritic Block Copolymers
47
D
E
F Figure 2.
copolymers where the Hnear chain (L) is linked to a monodendron (A, B) or a dendrimer (C), denoted as D (see Note a, Section IV). They should possess a high degree of structuralflexibilityand could possibly form different phases in bulk and solution. A characteristic feature of the second group of macromolecules is the presence of many highly branched blocks (dendrimers and monodendrons) in the copolymer (Fig. 2). They can be directly incorporated in the main chain to afford an alternating (LD)n copolymer (Fig. 2, D) or grafted onto it producing
48
IVAN GITSOV
%
^
«iii^
H Figure 3.
either L-graft-D (Fig. 2, E) or C-graft-LD (Fig. 2, F) copolymers. Depending on the length of the linear segments in the main and side chains these copolymers can change their shape from a relatively flexible coil/globule (long main or side chains and small dendrimers, respectively) to a more stiff, tubular frame (short side chains and large dendrimers, respectively). The third variety of linear-dendritic block copolymers consists of rather centro-symmetrical star-branched macromolecules like D(L)„ — with a dendrimer serving as the core of the star (Fig. 3, G) or (LD)„ where monodendrons are attached at the extremities of the arms (Fig. 3, H). The structures could possibly collapse or extend depending on their chemical composition and the nature of the surrounding medium.
Linear-Dendritic
49
Block Copolymers
y&
J Figure 4.
Polymer networks constructed by dendrimers as the large shape-persistent cross-linking agents (Fig. 4,1) and "super dendrimers" with a dendritic core and monodendrons serving as branching units (Fig. 4, J) make the final group of
50
IVAN CITSOV
linear-dendritic copolymers. The formation, the precise placement of reactive units with distinctive functions at specific positions in these macromolecules, and their characterization require a multitude of synthetic strategies and analytical procedures at the intersection of three disciplines: organic chemistry, polymer science and chemical biology.
11. SYNTHETIC PATHWAYS A vast array of organic reactions can be utilized to produce linear-dendritic block copolymers. Interestingly, the ten major architectures shown in Figs. 1-4 can be build with only four principal synthetic strategies. They are based on: (a) coupling of preformed linear and dendritic blocks; (b) polymerization of macromonomers containing dendritic fragments; (c) growth of dendrimers on linear chains with suitable reactive groups placed along the chain or at the chain ends; and (d) linear chain growth initiated by dendrimers, as macroinitiators. A. Coupling of Reactive Preformed Blocks
This method could possibly produce all macromolecular structures from A to I (Figs. 1-4) using suitable chemistries. Even J (Fig. 4) could be formed by this technique with additional protection-deprotection sequences. The synthesis of F (Fig. 2) is shown in Scheme 1 to illustrate the principle. This strategy was the first one to be used in the synthesis of a LDBC. Poly(styrene) was initially formed by "living" polymerization with potassium naphthylide and subsequently terminated by two poly(benzyl ether) monodendrons with a benzyl bromide reactive group at their "focal" point to yield a DLD (Fig. 1, B) copolymer.^ The good molecular weight control provided by the anionic polymerization enabled the formation of a variety of copolymers with different molecular weight characteristics and the side reactions of metal-halogen exchange were eliminated by reducing the reactivity of the polystyrylpotassium with 1,1-diphenylethylene.^ "Living" poly(styrene) generated with butyllithium, as the initiator, is capable of forming an LD copolymer (Fig. 1, A) by the same type of chemistry."^ The change of the reactive group at the "focal" point of the Frechet-type dendrimer from benzylic bromide to methyl ester directs the reaction towards LDL LDBCs (Fig. 1, C) via a nucleophilic attack on the ester carbonyl, elimination of MeO~, and second addition of polystyryllithium.^ The structure of the resulting copolymer 1 is presented in Fig. 5. The reaction is also possible with aldehyde functions placed at the "focal" point of the monodendron. The same interaction can be used again for the formation of (LD)„ alternating copolymers (Fig. 2, D) by a simple switch from mono- to bifunctional "living" polystyrene."^ At high dilutions, this process produces cyclodendritic structures 1^ by intramolecular ring formation
Linear-Dendritic Block Copolymers
51
Scheme 7.
(Fig. 5). Despite the lack of published data other reactions of reactive monodendrons with different "living" polymers could be easily envisioned. The use of dendrimers in this type of coupling will be discussed later. The "living" polystyrene is undoubtedly a versatile reagent for the synthesis of LDBCs, but it requires work at low temperatures, vacuum or inert atmosphere and elaborate laboratory techniques. The final coupling of the two blocks is the most difficult stage of the process because the reactive monodendrons must be quantitatively titrated with the polystyryl anions at —70°C in vacuum.^ Therefore alternative strategies were tested in order to facilitate the production of LDBS in sufficient quantities. They consist of two steps: anionic synthesis of monodisperse polystyrene with specific functional groups at the chain ends and controlled molecular weight, and subsequent coupling with reactive poly(benzyl ether) monodendrons equipped with suitable moieties at their focal points. The attempted Williamson reaction of polystyrene with a single oxyethylene end group and poly(styrene)-^/oc^-poly(oxyethylene) and Frechet-type monodendrons with benzylic bromide at the focal point failed to produce the desired DL copolymers.^ The transesterification of the same linear
52
IVAN GITSOV
4r
Figure 5.
blocks by reactive monodendrons with an ester group at the "focal" point was also unsuccessful. Medium to high yields of DL copolymer were achieved by reductive amination of amino-terminated poly(styrene) with monodendrons containing aldehyde groups at the "focal" point.^ The reaction was catalyzed by sodium cyanoborohydride in a mixed solvent (DMF/water is 40).
Linear-Dendritic Block Copolymers
53
Figure 6.
The Williamson ether synthesis was successfully applied in the coupling of Frechet-type dendritic benzyl bromides and poly(ethylene glycol)s, PEGsJ The reaction is initiated by NaH in dry THF and proceeds at room temperature with a slight excess of the monodendron. Both DL, DLD copolymers and (LD)„ star-blocks (Fig. 6, 3) were produced in nearly quantitative yields depending on the functionality and the architecture of the initial linear polyether block.^ Monodendrons modified with fluoroalkyl chains at the periphery were also reported to form DLD and (LD)„ copolymers after reaction with linear- or star-branched PEGs.^ No information on the reaction conditions and yields of the copolymers was given. The placement of cyano groups at the periphery of the monodendron reduced the yields of the DLD copolymers to 60%,^^ ester groups at the same position practically prevented the copolymer formation. ^^ The ester groups both at the periphery and at the "focal" point were successfully used in LDBC synthesis, but under different reaction conditions — melt transesterification with mono- and bifunctional poly(ethylene glycol)s under the action of tin or cobalt compounds. DL copolymers with a single polyether chain attached at either the "focal" point or the periphery of the monodendron were formed with yields between 60 and 80%.^^'^^
54
IVAN GITSOV
The transesterification of a poly (benzyl ether) didendron containing 16 methyl ester groups at the periphery with methoxy poly(ethylene glycol) was attempted with a tin catalyst at 95°C in toluene. ^^ After 5 days, an D(L)„ star-block copolymer (Fig. 3, G) with a dendritic core and 16 arms was formed in 64% yield. The long reaction time and relatively low yield are caused most probably by the steric hindrance experienced at the dendrimer surface by the approaching PEG chains. This assumption is supported by other published data. For example, the reaction of PEGs containing A^-succinimidyl groups with PAMAM dendrimers yields star-branched copolymers where only a portion of the surface amino groups was substituted.^"^ Kopecek and coworkers also attempted the synthesis of multi-arm stars from dendrimers of the same type and poly[A^-(2-hydroxypropyl)methacrylamide] activated with succinimide and reported similar results (substitution between 19 and 50% depending on the generation).^^ The coupling of "living" polybutadienyllithium with molecular weight between 6400 and 72,000 to third- and fourth-generation carbosilane dendrimers containing 32 and 64 dichlorosilane groups required 6 days to 8 weeks to produce the D(L)„ LDBCs with moderate yields (Fig. 7, 4).^^ Other more efficient synthetic pathways for the formation of D(L)„ star-block copolymers with a dendrimer in the core and poly(ethylene glycol) in the periphery were explored in some detail by Frechet and coworkers using activated PEG derivatives — bromide, mesylate and tosylate and a first-generation tridendron modified with hydroxymethyl groups at the surface. ^^ Because of the block size limitations set for this review, this star-branched macromolecule could not be regarded as a true block copolymer and will not be further discussed. Dendrimers seem to be ideally suited as multi-functional cross-linking agents. In combination with reactive linear blocks they can form gels (Fig. 4, I) with composition and cross-linking density that can be conveniently controlled by the generation of the dendrimer fragment. In distinction to the previous studies^^ three ester groups placed at the periphery of a Frechet-type tridendron were quantitatively transesterified within 24 h by a PEG with molecular weight of 3000.^^ The product formed is an amphiphilic network that could be also prepared under the action of DCC/4-(dimethylamino)pyridinium /7-toluenesulfonate from PEG and the same tridendrons, but with COOH groups at the periphery (Fig. 8, 5).^^ Other chemistries were also tested in the crosslinking procedure. It was shown that dendrimers with amino groups at the periphery (PAMAM dendrimers and Frechet-type tridendrons) can form gels with different dendritic content and cross-linking density through surface coupling with telechelic PEGs containing isocyanate-,^^'^^ acrylate-^^ and oxirane-^^ end groups. L-graft-D copolymers (Fig. 2, E) could also be formed by reactions of preformed blocks using grafting of reactive monodendrons onto linear chains
Linear-Dendritic
55
Block Copolymers
4 Figure 7.
with suitable functional groups placed along the polymer backbone. Schluter and coworkers conducted numerous studies to find the optimal chemistry and reaction conditions for the grafting process.^^ It was found that for mediumsized monodendrons (generations 2 and 3) the reactivity of the functional groups participating in the reaction was the success-determining factor and not steric hindrance.^^ Thus, Frechet-type monodendrons with isocyanate "focal" points react quantitatively with hydroxyethyl groups in one of the co-monomer units of an alternating poly(/7-phenylene) copolymer 6 (Fig. 9).^^ On the other side, only a 50-60% monodendron attachment was observed in a classical Williamson ether reaction between the same linear copolymer and
56
IVAN GITSOV
s
.6°
^^-^o'^x
fO5
-°^.^ oi^
X = -CH2NHCONH-; -COO-
5 Figure 8.
second-generation monodendrons with benzylic bromide groups at their "focal" points. Interestingly, a 100% grafting efficiency under nearly identical reaction conditions was achieved by a simple reversal in the position of the leaving group — benzyl alcohol in the monodendron and methyl iodide in the linear polymer. Using this approach, it was possible to attach even a third-generation monodendron in a higher yield (70%).^^ Similar conclusions for the importance of the functional group reactivity and the relative size of grafted monodendrons can be made from a recent study by Hawker et al.^^ They investigated the reaction of monodendrons with benzyl amine "focal" points and polystyrene with randomly distributed 7V-oxysuccinimide functional groups. It was found that monodendrons of generations 2-5 could react with almost all active ester groups when their content in the linear chains was between 10 and 21 wt%. The increase in the density of reactive sites in the polymer backbone leads to a
Linear-Dendritic
57
Block Copolymers
Figure 9,
dramatic decrease in the grafting efficiency especially at high generations (only 20% grafting of generation 5 with 40% content of active ester groups). The last group of investigations shows vividly the limitations of this method in the synthesis of E-type (Fig. 2) grafted LDBCs. The steric hindrance problem could be possibly circumvented in the synthesis of C(LD)„ comb-grafted copolymers (Fig. 2, F and Scheme 1) by a favorable balance between the length of the side arms in the linear block and size of reactive monodendrons. So far, no experiments have been performed in this direction. The strategy, based on preformed blocks coupling, is one of the most versatile tools that is widely used for the construction of LDBC with different macromolecular architectures. In most cases, the molecular size of the reagents does not interfere with the normal flow of the coupling reaction, which is influenced only by the chemical reactivity of the participating groups.^'^^'^^ The steric hindrance becomes a yield determining factor only in those cases where the synthesis aims at the formation of LDBCs with voluminous constituents (linear or highly branched) and high dendritic content.^^~^^'^^'^^ The major advantage of the method is that the desired materials are formed in a single step. This greatly simplifies their isolation and purification due to the great
58
IVAN GITSOV
disparity in molecular weights and solubility between the initial blocks and the final product. The analysis of the purified linear-dendritic macromolecules is also quite straightforward because of the significant differences in the chemical nature of the two constituents. B. Polymerization of Dendritic Macromonomers
This method provides an alternative access to L-graft-D (Fig. 2, E) or C-graft-LD (Fig. 2, F) copolymers. It is also a fine tool for the creation of materials with specific macromolecular geometry. Thus, depending on the degree of polymerization (DP) and generation of dendritic fragments, the configuration of monodendrons along the polymer backbone would ultimately transform the shape of the polymer macromolecules from a random coil to a sphere or a nanocylinder (Scheme 2). Indeed, several elegant studies by Percec and coworkers demonstrated the ability of second-generation dendritic macromonomers (Fig. 10, 7) to form macromolecules with spherical (DP < 15-20) or cylindrical shape (DP > 1520) under the action of radical initiators.^^ The concept was further validated when the same monodendrons were attached to a 7-oxanorbomene moiety and the resulting macromonomer was polymerized via "living" ring-opening metathesis polymerization using a ruthenium carbene initiator.^^ It should be noted that the long alkyl substituents at the periphery of monodendrons 7 (Fig. 10) impart a high degree of structural flexibility to the macromonomers and facilitate their transformation to polymers with relatively high DPs. The incorporation of monodendrons with more rigid structures in close proximity to the polymerizable group significantly decreases the ability of resulting compounds to homopolymerize due to the severe steric congestion at the propagating centers. For example, the first series of dendritic macromonomers reported in 1992 by Hawker and Frechet (Fig. 11, 8) was able to form polymers only by copolymerization with styrene.^"^ At low initial
Scheme 2,
Linear-Dendritic Block Copolymers
59 X
X: }-0
^-'
or
/ - o ' ^
Figure 10.
concentrations of the dendritic co-monomers (3 wt%), they were quantitatively incorporated in the copolymers, but the copolymer yields decreased with the increase of the monodendron's generation: 76-72% for [G-3]; 69% for [G-4]; and 68% for [G-5]. The yields were also significantly lower at high initial concentrations of dendritic comonomer: 76% at 1 wt% and only 52% at 40 wt% of [G-3].^^ The sequence distribution in the copolymers was not investigated, and no copolymerization rate constants were calculated. Later, it was reported that styrenic macromonomers containing monodendrons of the same type (generations 1-3) could be radically homopolymerized in concentrated toluene solutions or in the bulk.^^ Polymers with very broad molecular weight distributions {M^/M^ > 10) were obtained with the yields exceeding 50%. The change of the reactive group at the focal point of monodendron 8 (Fig. 11) from styrene to methacrylate did not improve the polymerization ability of the resulting macromonomer.^^ Only oligomers with DP of 3-6 were formed under the action of anionic and radical initiators at various reaction conditions. The copolymerization of the same macromonomer with "living" bifunctional polystyrene was also investigated in an attempt to form a rodcoil block copolymer.^^ Regrettably, only a DLD-type copolymer (Fig. 1, B) was formed — the (co)polymerization stopped after the initial addition of one macromonomer unit at both ends of the polystyrene block. Regardless of the existing literature data, other attempts to polymerize the same type of methacrylate monodendron with the same radical initiator were published.^^
60
IVAN GITSOV
8 Figure 11.
Despite the prolonged polymerization time, again only oligomers with DP of 7 were produced. The use of atom-transfer radical polymerization for the synthesis of amphiphilic LDBCs with similar dendritic methacrylate derivatives was also reported.^^ The polymerizability of the methacrylic macromonomers was investigated in detail by Schliiter and coworkers in an attempt to trace the influence of the steric crowding on the polymerization reactivity of these compounds.^^*^ It was found that the [G-2] macromonomer could form homopolymers with relatively high molecular weights and satisfactory yields only after incorporation of a spacer (p-benzyloxy group) between the "focal" point of the monodendron and the polymerizing methacryl moiety. The same research group pioneered the synthesis of alternating LDBCs by polycondensation of properly designed comonomers.^^"^^ In this way, copolymers like 6 (Fig. 9) could be formed in good yields by Suzuki cross-coupling of dibromobenzenes containing Frechet-type monodendrons of generations 1-3 and dialkyl-substituted phenylene diboronic acid under the action of FdiFip-Tol)^]^}^ This reaction was successfully utilized for the construction of unique amphiphilic LDBCs where the hydrophilic and hydrophobic blocks are placed on the two opposite sides of the polymer backbone (Scheme 3)?^ Obviously the cross-coupling with esters of the phenylene diboronic acid is a promising technique that might eventually produce alternating LDBCs even with a fourth-generation monodendritic macromonomer.^^ The essential features
Linear-Dendritic
Block Copolymers
61
vv°^
\
I
Scheme 3.
of the reaction that facilitate the formation of the desired products are (1) high reactivity of the participating groups, (2) very small size and favorable shape of the co-monomer, and (3) stiffness of the resulting copolymer chain. The synthetic strategy, based on these common principles, was successfully applied in the construction of aromatic polyamides coated with Percec-type monodendrons using the polycondensation of symmetrical biphenyl diamines with two secondgeneration monodendrons and terephthaloyl chloride (Fig. 12).^^ The polymers 10 were isolated in nearly quantitative yields (90%), but no information on their molecular weight characteristics was provided. The fulfillment of all the three requirements is an essential prerequisite for completion of the polymerization process. For example, in spite of the massive synthetic effort, the oxidative coupling of rran^'-enediynes substituted with two
62
IVAN GITSOV
^
%f
^
"^o V ° / r ° ^
10 Figure 12.
Frechet-type monodendrons of generations 1-3 failed to yield products with DP higher than 5 (for [G-1]), 3 (for [G-2], Fig. 13, 11) and 2 (for [G-3]) because of the severe steric hindrance at the reactive sites.^^ Another acetylene macromonomer containing rigid Moore-type monodendrons of second generation produced a polymer in low yield (36%) after a single polymerization experiment with a rhodium catalyst;^^ however, the molecular weights of the polymer 12 (Fig. 14) were surprisingly high. In a similar fashion, the radical copolymerization of [1.1.1] propelane derivatives and dendronized acrylates affords copolymers with modest yields and DPs between 8 and 24 depending on the proximity of the monodendron to the acrylate moiety. ^"^ So far the polymerization of dendritic macromonomers was successfully accomplished along two main avenues: (a) incorporation of flexible monodendrons by chain-growth polymerization,^^'^^ and (b) construction of stiff main chain through step-growth polymerization of suitably designed reactive monodendrons.^^'^^"^^ The potential of the method, however, is not fully de-
Linear-Dendritic Block Copolymers
63
11 Figure 13.
pleted. An interesting and very promising procedure was developed recently by Schliiter's research group in a clear attempt to circumvent the negative influence of the steric overcrowding and the flexibility in the LDBC backbone (requirements 2 and 3, see above) on the outcome of the polymerization of dendritic macromonomers.^^ In a double-stage approach, styrenes containing second-generation monodendrons with protected groups at the periphery were initially converted to polymers under typical radical polymerization conditions, the products were then deprotected and quantitatively functionalized with reactive monodendrons of first generation. In this way, the method provides an alternative and relatively easy access to LDBCs with complete dendritic coverage that might be difficult to obtain by the previous synthetic strategies. It should be noted that the increased flexibility of the monodendrons used in the study in comparison to the Frechet-type monodendrons (their aryl/alkyl branching units
64
IVAN CITSOV
Q, P /
Si
Si
\
12 Figure 14,
have an additional methylene group between the branching point) favorably supplements the advantage of the double-stage approach. The polymerization of DL-type LDBCs containing a polymerizable group at the end of the linear (L) block (Scheme 2) provides the second alternative route for the construction of C-graft-LD copolymers (Fig. 2, F and Scheme 1). The flexible linear chain would lift the steric restrictions not only at the reactive site, but along the resulting graft copolymer backbone as well. One example is shown in Scheme 4. The linear-dendritic macromonomer 13 was obtained by a two-stage Williamson ether coupling of Frechet-type dendritic bromide andp-chloromethyl styrene to PEGs with molecular weights between 1000 and 5000.^^ The polymerization afforded LDBCs with apparent molecular weights in the range of 50,000-2,000,000 and yields between 20 and 85% depending on (a) the molecular weight of the macromonomer, (b) the type of initiator, and (c) the nature of the polymerization medium.^^ It was suggested that dendrimers attached through a long spacer to the main backbone of the LDBC would have a negligible effect on the overall conformation of the copolymer (see reference 1, p. 208). It is interesting to note, however, that due to its amphiphilic character polymer 14 (Scheme 4) was able to reorganize in a broad variety of distinct supramolecular structures depending on the hydrophilic/hydrophobic balance in the macromolecule and the relative sizes of the two main constituents.^^
Linear-Dendritic Block Copolymers
65
u
0-U
o
k->o-
.2 L
U
14
13 Scheme 4.
C. Divergent Growth of Dendrimers on a Preformed Polymer Chain In analogy with the classic scheme of divergent dendrimer synthesis pioneered by Vogtle, Newkome and Tomalia^^ dendrimers could be grown not only from a multifunctional core, but at the ends of a linear chain or on specific reactive sites along a polymer backbone as well. The method could be applied for the construction of macromolecular structures like A and B (Fig. 1), E (Fig. 2), H (Fig. 3) or their combinations. The synthesis of B is shown in Scheme 5 as an example. The use of linear polymers with reactive side groups as elongated cores for dendrimer growth was initially patented in 1987^^ and subsequently mentioned in a seminal paper by Tomalia and coworkers.^^ A more detailed study was published recently."^^ A poly (ethylene imine) core with DP of 10-500 was formed by "living" cationic polymerization of 2-ethyl-2-oxazoline and subsequent hydrolysis of the resulting polymer. The imine groups in the polymer backbone were used as the growing sites of PAMAM monodendrons, their number being equal to the DP. Enormous excess of ethylenediamine (lOOOx to 10,000x!) and prolonged reaction time (5-8 days) were used to ensure the complete transformation of all ester groups at the amidation step. Despite the substantial synthetic challenges the process was successfully carried out to the third (15,
66
IVAN GITSOV
Scheme 5.
Fig. 15) and even the fourth generation. The efficiency of the process could be hardly estimated because the molecular weight characteristics of the starting core and the cyHndrical dendrimers derived therefrom were not reported. However, the size-exclusion chromatograms provided in the supplementary materials of the paper indicate that the polydispersity of the LDBCs increases with the generation of the coating monodendrons.^^ The same type of chemistry was used by Hammond and coworkers'*^ for the synthesis of copolymers with different composition. After the first unsuccessful attempt to form LDBCs of the rod-coil type via copolymerization of dendritic macromonomers,^^ block copolymers with this architecture were synthesized by growing monodendrons on one of the two blocks in the linear chain. Poly(ethylene oxide) tosylate was used as a macroinitiator for the polymerization of 2-ethyl-2-oxazoline and the resulting block copolymer was coated with PAMAM monodendrons on the poly(oxazoline) block using the same chemical procedures as previously described. Again no data for the molecular weights and the polydispersities of the starting materials and the final products are currently available. The polymeric multi-functional core strategy was also used recently for the construction of the same type of LDBC, but having entirely different chemical composition. Carbosilane monodendrons were grown on a poly(methylsiloxane) by repetitive hydrosilation, alkenylation and alkynylation reactions with dichlorovinylsilane, dichloromethylsilane, allylmagnesium bromide and phenylacetylene/ lithium phenylacethylide, respectively."*^ No data for the yields and the molec-
Linear-Dendritic Block Copolymers
67
w-^^ HN-^O
r" r 0=< NH
„ H2N
>=0 HN
r^ ° S Oo=( NTT NH ) H2N
)=0^X ' "O-^xTTT '^NH
tnvT HN
( NH2
\ H2N
Is HN. ) NH.1-2
15 f/gwre 75.
ular weight characteristics of the copolymers with the second-generation monodendrons were given, but the authors did mention the broadening of the molecular weight distribution in analogy with the previously reported synthesis"^^ of the same type of LDBC (E, Fig. 2). The largest group of studies in this category concentrates the efforts on the divergent monodendron growth from a single reactive center typically placed at the chain end of the linear precursor. Chapman and coworkers were the first who utilized this concept to produce a LDBC of the LD type."^^ In the original paper^^^ they described a synthetic procedure based on the modification of a methoxy-PEG (M^ = 4700) with r-Boc-glycine and subsequent coupling/deprotection cycles with pentafluorophenyl A^,A^-di-r-Boc-/-lysinate/trifluoroacetic acid. The molecular weight distributions of the copolymers remain narrow even at generation 4 (16, Fig. 16). Later the synthesis was extended to generation 7"^^^ and the surface of the dendritic block was modified with perfluorooctanoyl groups.^^'^'^ Essentially the same approach and the same chemistry were reported recently by Park and coworkers for the synthesis of surprisingly similar (if not identical)
68
IVAN GITSOV
16 Figure 16,
LDBCs of LD and DLD types.^ Several other studies also used PEGs and their derivatives as suitable "templates" for dendrimer growth due to their solubility in aqueous and organic media, their activity towards a broad variety of reagents and their biomedical importance. For example, poly(ether monodendrons) based on a pentaerythritol branching moiety (for the synthesis of pentaerythritol-based dendrimers see Padias et al."^^) were grown at the extremities of 3-, 4- and 8-arm star-branched PEGs using the Williamson ether synthesis."^^ The materials formed (Fig. 17, 17) could be regarded as the first true "architectural" copolymers (see Note b) because both blocks in the macromolecule consist of repeating units with essentially the same chemical composition — a poly(alkyl ether), and the only difference between them is their geometry (architecture) — linear vs. dendritic. Frechet-type monodendrons could be grown divergently using PEGs with molecular weights between 3000 and 5000 as bifunctional cores. Scheme 1^'^ The overall yields however, were lower than those achieved by the coupling of preformed blocks^ (60%"^^^ vs. 95%^ for generation 2). Similar copolymers were obtained in moderate overall yields from bismesylate PEG with molecular weight 4600 and dimethyl S-hydroxyisophthalate."^^ The procedure requires an additional step to afford a structure similar, but not identical to 18 (Scheme 6). It is interesting to note that both strategies of "preformed blocks — coupling" and "linear chain — monodendron growth" afford LD (19, Fig. 18) and DLD block copolymers in similar yields from mono- or bifunctional PEGs with molecular weights between 1000 and 5000 and Hult-type monodendrons."^^^'"^^ In a further study, methoxy-PEGs with molecular weights 2000 and 5000
Linear-Dendritic Block Copolymers
69
12 Figure 17,
\4?* < Y = -OH, -NHj
Z = -CHzBr, <:00R, -CHO
^J^jf^
X = -CHjO-, -C00-, -CHjNH-
^""O^
R = -Si(Ph)jC(CH3)3
18 Scheme 6.
were exploited to produce LD-type LDBCs with PAMAM monodendrons. The yields were generation- and conditions-dependent and decreased from 95 (generation 1) to about 80% (generation 3).^^ It should be mentioned that PAMAM monodendrons up to generation 5.5 could also be formed on a poly(2-methyl-oxazoline) with ethylenediamine end group according to a study published independently from Tomalia's"^^ and Hammond's^^ reports.^^ Another fine example of PEG-mediated LDBC synthesis was reported recently by Kim et al.^^ Carbosilane monodendrons up to the third generation (20, Fig. 19) were formed at the chain end of a low-molecular-weight methoxy-PEG by repetitive hydrosilylation and alkylation reactions with yields between 45 and 63%.
70
IVAN GITSOV
12 Figure 18,
20 Figure 19.
Poly(styrene) formed by "living" anionic polymerization and containing reactive end groups could also serve as a suitable linear platform for the growth of monodendrons of different chemical composition. In a series of elegant procedures, Meijer and coworkers were able to construct a new class of amphiphilic LDBCs containing this linear segment and poly(propylene imine) monodendrons.^^ Starting with amino-terminated poly(styrene) of molecular weight 3000 a fifth-generation monodendron was formed (21, Fig. 20) after five consecutive cycles of acrylonitrile coupling via Michael addition and catalytic hydrogenation. The careful selection of the reaction conditions was a primary factor to ensure high yields at each synthetic step. Thus, the cyanoethylation proceeded smoothly only in a two-phase system (toluene/water) with acetic acid as the catalyst, while the quantitative reduction of all nitrile groups was
Linear-Dendritic Block Copolymers
71
21 Figure 20.
22 Figure 21.
achieved at high hydrogen pressures (80 bar) in a unique combination of NHs/methanol/toluene is 1/1/3 (v/v).^^ The monodendron could be selectively modified not only at the periphery (-CN to -CO2H groups)^^^ but also throughout the entire structure (quatemization of the primary and tertiary amines with methyl iodide).^^ In a similar attempt, bifunctional poly(styrene) obtained by a "living" anionic polymerization under the action of potassium naphthylide and subsequent termination with a reactive protected pentaerythritol branching fragment [4-(bromomethyl)-l-methyl-2,6,7-trioxabicyclo[2.2.2]-octane^^ was used for the synthesis of DLD-type LDBCs that contain polyaromatic chain as the linear (L) block and dendritic poly(alkyl ethers) as D fragments (Fig. 21, 22).^^ The initial
72
IVAN GITSOV
quenching of the "Uving" polymer proceeded quantitatively even at —70°C and the second-generation copolymer 22 was isolated in 65% yield after purification. The product formed could be regarded as the reverse analog of the previously described DLD copolymers composed of a poly(alkyl ether) as the linear L segment and polyaromatic dendritic D blocks.^ The published data suggest that the method based on the monodendron growth from a preformed linear chain is a versatile tool for the construction of several classes of LDBCs. In distinction to the first two strategies discussed in this chapter, it offers a better control over the functionality on the surface of the monodendrons."^^""^^'^^'^^'^^'^^ Through suitable chemical procedures practically all major groups of divergent dendrimers could be attached to a single polymer chain.'^^"'*^'^^'^^ In many occasions, the linear precursor facilitates the synthesis and purification of resulting copolymers by changing their solubility in selective solvents.^^'^^ The deficiencies of this approach arise from the increased steric hindrance around the growing sites exercised by the polymer chain that necessitates use of a substantial excess of dendritic building blocks and prolonged reaction times."^^'^^''^^^'^^*''^^ Because of this "polymer effect" the useful molecular weight range of the linear chains is rather narrow — practically all published studies used polymers with molecular weights between 500 and 7000. Some preliminary results indicate that these limits could be pushed higher (to 10,000-15,000) for systems with favorable chemistry."^^'^^'^^ It should be pointed out, however, that long linear blocks make the spectral identification, control of purity, and the presence of defects in the dendritic portion of the LDBC particularly difficult because of the overwhelming intensity of their characteristic signals. D. Growth of Polymer Chains on a Preformed Dendrimer
This synthetic strategy enables the single step formation of LDBCs with different architecture: DL and LDL types (Fig. 1, A and C, respectively), as well as a D(L)„ type (Fig. 3, G). Linear blocks with well-defined molecular weight characteristics could be formed on a dendritic fragment by various chain-growth polymerization processes. Several other structures are achievable in a few additional steps (Scheme 7). The use of monodendrons as macroinitiators was pioneered in 1994 when the anionic ring-opening polymerization of 8-caprolactone was initiated by Frechettype monodendrons with an alcoholate active center created at the "focal" point.^^ It was found that large monodendrons were able not only to initiate the polymerization, but also to exercise a kinetic control over the propagation process by temporal suppression of the back-biting side reactions. It was assumed that the active chain ends could be partially immobilized through a complex formation inside the monodendrons and by the steric screening from
Linear-Dendritic
Block Copolymers
73
M Scheme 7.
the bulky dendritic "wedges".^^'^^ Thus, the favorable microenvironment surrounding the active center in the growing linear block enabled rapid formation of a LDBC with a relatively high molecular weight and narrow molecular weight distribution (M^ = 307,000; Mw/Mn = 1.07).^^ The amount of cycHc oligomers increased and the molecular weight characteristics of the copolymers deteriorated with the decrease in the generation of the dendritic initiator and the extension of the polymerization time — a clear sign that intramolecular transesterification could be temporarily suppressed, but not fully eliminated. The anionic ring-opening polymerization initiated by Frechet-type monodendrons was also tested on other monomers with mixed success. It was found, for example, that hexamethylcyclotrisiloxane is able to polymerize under the action of these macroinitiators.^^ Initially, the polymerization mixtures contained only propagating LDBC species and unreacted monomer as evidenced by size-exclusion chromatography with double detection. However, when the process was extended to the full consumption of the monomer only a poly(dimethylsiloxane) homopolymer and the dendritic initiator could be isolated regardless of the
74
IVAN GITSOV
23 Figure 22.
generation of monodendron used.^^ In the total absence of a competing initiator in the system (the monodendritic alcohol was quantitatively titrated in vacuum with potassium naphthylide), the homopolymer is formed most probably through a cleavage of the labile C-O-Si link at the focal point by the silanolate chain end residing in a close proximity (Fig. 22, 23). On the other side, polymerization of ethylene oxide under the action of the same initiators proceeded smoothly and LDBCs with narrow polydispersities were produced in quantitative yields.^^ The fact that DL-type copolymers still contain "living" alcoholate chain ends could be further exploited for construction of other analogues. Besides the "trivial" examples like B (Fig. 1) and H (Fig. 3) unique structures could be formed by coupling with dissimilar monodendrons (Scheme 7a, L) or dendrimers with reactive groups at the surface (Scheme 7b, M).^^ The ring-opening polymerization of cyclic esters (e-caprolactone, lactides, and derivatives) could proceed by a coordinative mechanism as well. The process is typically free of side reactions and was widely used in the production of various aliphatic polyesters.^^ Thus, Frechet-type monodendrons of generations 2-4 with diethyl aluminum alkoxide at the "focal" points were able to initiate the polymerization of these monomers to form LDBCs with molecular weights between 1800 and 13,000 with fairly narrow molecular weight distributions (Mw/Mn is 1.1-1.25).^^ The "living" character of the polymerization process allowed not only a good control over molecular weight characteristics of the
Linear-Dendritic Block Copolymers
75
24 Figure 23.
copolymers, but also the formation of a DL-type macromonomer and unusual comb-graft LDBCs. This was accomplished by a suitable modification of the hydroxyl groups placed at the chain ends or along the backbone of the linear segment of the copolymer. Polymerization of other heterocyclic monomers under the action of these macroinitiators could be easily envisioned taking into account previous studies with other aluminum alkoxides.^^ It should be mentioned that Hult-type monodendrons in combination with stannous(II)-2-ethylhexanoate were also able to initiate the ring-opening polymerization of e-caprolactone producing DL, LDL (Fig. 23, 24) and DLD copolymers (structures A-C, Fig. 1).^^ These interesting macromolecules with both blocks being composed of aliphatic polyesters could be regarded as another fascinating example of the "architectural" copolymers discussed earlier."^^ The unique capability of the reactive groups at the "focal" points of convergent-growth monodendrons to participate in numerous chemical transformations facilitated the preparation of new versatile initiators for mediated radical polymerizations. (For guidelines in the classification and description of nitroxyl-mediated radical polymerization and metal-mediated (atom-transfer) radical polymerization see Howell^"^.) In a series of elegant studies, Frechet and coworkers exploited the fact that the active centers in these processes are quite tolerant to other functional groups present in the molecules and produced series of initiating systems with different structure and chemical composition (Fig. 24).65 Monodendrons modified at the "focal" point and at the periphery were used as stable macroradicals,^^^ unimolecular macroinitiators^^^ or simultaneously
76
IVAN GITSOV
-
?}j 25 Figure 24.
in both functions^^^ in the radical polymerization of vinyl monomers mediated by TEMPO (2,2,6,6-r£traMethylPiperidine-l-(9xy) radicals. The initial expectations that the voluminous dendritic fragments will favorably complement the stable TEMPO radicals and improve control over initiation and propagation steps were not accomplished. It was found that the molecular weight characteristics of the synthesized poly(styrene) that constituted the linear portion of the LDBC were in close resemblance to those obtained with the conventional TEMPO systems: at molecular weights below 24,000 the polymer polydispersity is relatively low for this type of process (M^/M^ 1.14-1.28),^^^ but rapidly deteriorates to 1.3-1.5 with the increase of the targeted molecular weight. The observed behavior of dendritic macroinitiators is not surprising considering the fact that the active centers are relatively far from the "focal" point of the monodendrons to experience any steric influence therefrom. Besides styrene, vinyl acetate^^^ and p-acetoxystyrene^^^ were successfully polymerized. It should be noted that the copolymerization of styrene and methyl methacrylate (9/1 ratio) enabled for the first time the formation of LDBC where the linear block is a random copolymer.^^^ The Cu(I)-mediated radical polymerization of styrene initiated by Frechet-type monodendrons with a benzyl halide at the "focal" point yields copolymers with similar results.^^^"^ This method provides also easy access to surface functionalized DL- and DLD-type copolymers.^^^"^ A brief comparison between the two main processes that use monodendritic
Linear-Dendritic Block Copolymers
77
initiators could reveal their specific advantages and useful application range. The anionic polymerization is the superior technique when the desired molecular weights are high and rigorous control over the molecular weight characteristics is required. It is operational for both vinyl- and heterocyclic monomers and produces DL-type copolymers with a larger variety of end groups in the linear block. The mediated radical polymerization is the method of choice when large quantities of LDBCs are needed, the size of the linear segment could be small and very low polydispersity is not required. In distinction to the anionic systems, mediated radical reactions do not require high vacuum and can tolerate a variety of unprotected functional groups both in the initiator and monomer. Naturally, divergent dendrimers could also be applied as macroinitiators after appropriate modification of their surface groups. The resulting products are most commonly copolymers of D(L)„ type (Fig. 3, G; see Note c). In a recent study carbosilane dendrimers of the second and third generations were lithiated with 5^c-butyllithium and the resulting multifunctional anionic species (26, Fig. 25) were used in the construction of star-branched LDBCs with poly(styrene), poly(dimethylsiloxane) and poly(oxyethylene) arms.^^ The products formed have a monomodal distribution, but their molecular weights deviate from the predicted theoretical values indicating possible intramolecular association of lithium counterions or incomplete and slow initiation. Star-branched poly(oxyethylene)s were also prepared from carbosilane dendrimers with alcoholate active centers.^^ In another interesting example PAMAM dendrimers of generations 3 and 4 were able to initiate the polymerization of a highly polar acetylenic monomer (2-ethynylpyridinium trifluoromethane sulfonate) in DMSO and produced hydrophilic D(L)„-type LDBCs with highly ionic outer shell of linear polyacetylenes.^^ The degrees of polymerization of the linear blocks were reported to vary between 1 and more than 16, but no molecular weight distributions were given. A third-generation PAMAM dendrimer was also applied as an initiator and grafting platform for short-chain peptides based on y-benzyl-L-glutamate A^-carboxyanhydride.^^ In a similar fashion sarcosine A^-carboxyanhydride polymerized under the action of the 64 primary amino groups placed at the periphery of a fourth-generation poly(propylenimine) dendrimer.''^ The LDBCs had very narrow molecular weight distributions and were obtained in nearly quantitative yields. The polymerizations initiated by Hult-type dendrimers based on the bis-MPA branching unit (bis-Methylol Propionic Acid) deserve special attention. In several interesting studies, Hedrick and coworkers were able to "switch" the initiating mechanism of these compounds by simple chemical transformation of the peripheral groups.^^ Unique multi-layered block copolymers with predictable molecular weights and relatively narrow molecular weight distributions could be obtained by consecutive polymerization of e-caprolactone and addition of protected bis-MPA monodendrons.^^^ Using the schematic description
78
IVAN GITSOV
;i
p
Si
0
WM
26 Figure 25.
of LDBCs discussed in this review, these macromolecules could be assigned to the D[(LD)„L^] block copolymer type, where m > n. Regrettably, no data are currently available on the initiation and coupling efficiencies. It was shown in a following study that a second-generation tridendron containing 12 2-bromo-/5'(9-butyryl reactive groups (Fig. 26, 27) could initiate copolymerization of methyl methacrylate and tert-huiyl acrylate under typical ATRP conditions.^^^'^ The sequence of co-monomer addition could be reversed enabling the construction of amphiphilic star-block LDBCs where the hydrophilic block (generated quantitatively after hydrolysis of the tert-huiyl groups) could be placed either as the outer shell of the star or in the interior. A detailed study of the initiation mechanism of these systems revealed that the process is kinetically controlled thus broadening the molecular weight
Linear-Dendritic
Block Copolymers
79
27 Figure 26.
distribution of the individual arms in the star7^ It should be noted that the surface steric crowding is a common problem for all multi-site initiators and must be considered in designing the specific synthetic strategy for the targeted linear-dendritic copolymer.
III. CHARACTERIZATION TECHNIQUES The favorable outcome of any synthetic strategy depends not only on the successful formation and isolation of the desired product, but on the analytical evidence for its chemical structure and purity as well. A wide array of analytical tools and techniques are available and were employed to explore and verify the composition and macromolecular organization of the LDBCs formed by the different methods. Their detailed discussion is beyond the limits and the scope of this review and the reader is advised to consult the specialized literature for the particular analytical methodology. A more detailed description of some of the methods could be found for example in specialty monographs.^"^ It should be noted that no single procedure is currently available that would provide "universal" structural information in a single analysis. Therefore the combination of chromatographic and spectroscopic techniques is an imperative requirement for the collecting of quantitative information for the purity, the molecular weight characteristics, and the chemical composition of the copolymers formed.
80
IVAN GITSOV
A. Size Exclusion Chromatography The size-exclusion chromatography (SEC) is used extensively to monitor the progress of copolymer formation, presence of unreacted reagents (monomers) and finally to verify the purity of the isolated LDBCs. This is a nondestructive liquid chromatography method that is based on the separation of the dissolved (co)polymer molecules according to their hydrodynamic volume — a "size exclusion". In distinction to the conventional affinity chromatography, the solute does not interact with the stationary phase and is distributed in the mobile phase between the individual beads or in their numerous pores. Depending on the size of these pores and the hydrodynamic volume of the copolymer, it can elute faster (size of the macromolecule equal or larger than the pore size) or slower from the SEC column (size of the macromolecule two times smaller than the pore size). This distribution of macromolecules between the stationary and mobile phase is described by the distribution coefficient K^ in the operating equation of the method Ve = Vo + ^d X Vi
(1)
where Ve is the elution volume of the species, VQ is the interstitial (mobile phase) volume, and Vi the volume inside the pores. The relation between elution volume and molecular weight is made through calibration with standards of known molecular weight. The size fractionation in the column yields the molecular weight distribution. Because of the traditional usage of SEC in the determination of these molecular weight characteristics for various polymers and copolymers, it is a widespread belief that the method can also be used in the quantitative molecular weight analysis of linear-dendritic materials. Numerous papers provide LDBC molecular weights in poly(styrene) equivalents. Other linear standards have also been used. Certainly, the linear portions of the copolymers could be subjected to analysis of this type. Dendrimers, however, have been long known to appear smaller in the SEC data^^ because the highly branched dendritic globule would reside longer in the pores than the statistical coil of a linear polymer with the same molecular weight. Combination of these two blocks in a single macromolecule only complicates correct data analyses. It was shown, for example, that the incorporation of two Frechet-type monodendrons in a poly(styrene) chain yields materials (Fig. 1, B) whose molecular weights are underestimated below and overestimated above a certain molecular weight.^ Even a single monodendron in the copolymer (Fig. 1, C) causes substantial deviation from the predicted values."^ Similar results were reported for other LDBCs as well.^^'^^ The molecular weight distributions are also affected. The presence of the virtually monodisperse dendritic fragment will decrease overall polydispersity of the copolymer especially when the linear block is short. Thus for star-like copolymers (Fig. 3, G) constructed of large
Linear-Dendritic Block Copolymers
81
Frechet Monodendron, t=0 h
61
DLD Copolymer, t=3 h
uv
/ O
dRI
PEG,t=Oh /
Ql
I ■-'^
10^
r
Mol Mass, g/mol Figure 27.
poly(ethyleneimine) dendrimer and short poly(sarcosine) arms/^ values for M^/M^ between 1.01 and 1.04 were reported and the claim was made that all 64 amino groups initiate the polymerization simultaneously. A close examination of the published SEC traces for these materials, however, reveals the presence of lower molecular weight fractions in noticeable amounts (reference 74, fig. 1), a clear evidence for the dominant influence of the dendritic core in these copolymers. It was confirmed lately that the linear arms in star-like LDBCs with low overall polydispersity could have broad molecular distributions.^^ The effective application range of SEC is in the monitoring of the process of LDBC formation. Especially useful is this method when the synthesis is carried out with preformed blocks. Their molecular weight characteristics could be previously determined and their positions in the eluograms could be verified in advance. The copolymers formed after the coupling of the fragments often have significantly higher molecular weights and could be easily identified in the chromatograms of reaction mixtures. The addition of UV (photodiode array or single wavelength) detectors and precise calibration of their response substantially improve the accuracy in the determination of the copolymer compositions.^'^'^^^ An example is presented in Fig. 27. The combination of SEC with different light scattering modules provides more precise information for the molecular weights of the copolymers formed.^'^^'^^ Lack of adequate resolution and mass sensitivity prevents the meaningful application of SEC in those cases when single or multiple dendrons are grown divergently on the linear block. The creation of each additional generation causes only
82
IVAN GITSOV
Mol mass, g/mol Figure 28,
a minor increase in the overall molecular weight of the copolymers (Fig. 28"^^^^) and prevents the quantitative detection of any branching defects. A technique that is particularly useful in this respect is discussed briefly below. B. MALDI-TOF Mass Spectrometry
The Matrix-Assisted Laser Desorption/Zonization Time-Of-flight (MALDITOF) mass spectrometry is gaining increasing popularity in the compositional analysis of natural and synthetic polymers due to the high precision, resolution and lack of macromolecular fragmentation.^^ Molecular weight determination with unparalleled accuracy and end-group analysis are also possible.^^ The method is based on the preferential adsorption/co-crystallization of the analyzed polymer and a selected low molecular weight matrix. After their subsequent desorption, volatilization, and ionization under the action of soft laser pulses, the polymer species are detected in a typical mass-spectrometry mode. The calibration of the instrument is typically performed with a monodisperse natural macromolecule with known molecular weight. The first MALDI-TOF analyses of LDBCs were reported by Frechet and coworkers.^^ In the initial report and few following studies, they analyzed the coupling products of poly(ethylene glycol) and reactive poly(benzyl ether) monodendrons. The analyses reconfirmed previous data from SEC with double detection for the complete attachment of the monodendrons to the PEG — a synchronous shift of all individual peaks was observed in the mass-spectra after the coupling, but the mass difference of 44 between them (corresponding to the mass of the individual repeating unit in the PEG block) was preserved. In other studies, the method was instrumental in the unambiguous proof of the perfect dendrimer growth on PEG chains of various length^^'^^ and the polycondensation of dendritic macromonomers.^^ Despite its obvious advantages, the MALDI-TOF mass spectrometry has also some shortcomings. Several matrices like dihydroxybenzoic acid, derivatives
Linear-Dendritic Block Copolymers
83
of hydroxycinnamic acid, dithranol and others were used, but their selection is rather empirical. On many occasions, the molecular weight distribution for complex mixtures of polymerized dendritic macromonomers containing homologues with different degree of polymerization could not be precisely determined because of the different volatility of low and high molecular weight compounds. The resolution deteriorates rapidly with molecular weights above 10,000 and the crucial benefit of end group and structural defects detection is lost. An emerging new microscopy technique that might prove helpful for the analysis of LDBCs in the high molecular weight range will be discussed in the following section. C. Atomic Force Microscopy The atomic force microscopy (AFM) was used with spectacular success for the analysis of nanoscale structures at different surfaces and interfaces. The method is based on the deflection of a sensitive cantilever caused by electronic interactions of its sharp tip with the surface underneath. The "atomic force" generated during the microscopic movement of the tip both in contact and taping mode can be measured and used to generate a topographical picture of the investigated surface. Under favorable conditions, the resolution can be improved to atomic level; therefore, the direct measurement of macromolecular sizes and volumes becomes possible. In a ground-breaking work Percec, Moller, Sheiko and others were able to visualize and measure the dimensions of spherical and cylindrical LDBCs that were difficult to measure by SEC with multi-angle laser light scattering detection.^^ It is, however, too early to evaluate the capabilities of this method and its full potential for the precise determination of molecular weight characteristics. D. Nuclear Magnetic Resonance Undoubtedly the nuclear magnetic resonance (NMR) is the most widely used method for identification and structure elucidation in the modem synthetic organic chemistry. The unique orientation of the nuclear magnetic dipoles of atoms in a homogeneous external magnetic field that is affected by their electronic (i.e., chemical) environment is the basic operating principle of this absorption spectroscopy. This environmental sensitivity facilitates the recognition of most groups placed in the interior or at the periphery of monodendrons or dendrimers. In the LDBC synthesis, the method was utilized to monitor the disappearance or appearance of specific moieties after the coupling of preformed blocks,^'^ polymerization of dendritic monomers,^^'^^^ divergent dendrimer growth on linear chains"^^'^^'^^ and polymerization by dendritic macroinitiators.^^'^^'^^'^^ The NMR was frequently used to compute M^ of the LDBC formed typically from
84
IVAN GITSOV
Structure 17, Fig. 17
-CH2- PEG star
-CH3 Protecting groups
Branching units -CH2- M 1
.
o, ppm
1
1
1
1
1—
6 Figure 29,
the ratio of protons in the dendritic termini to the protons in the repeating units of the linear block.^^'^^'^^ It should be noted, however, that the method has a detection limit of few percent and the purity of the obtained copolymers should be additionally verified with a chromatographic technique such as SEC with double detection (Fig. 29)."^^ Moreover, the disappearance of a particular group in the NMR spectra should not always be interpreted as a coupling or initiation act because of the diminished sensitivity at higher molecular weights^^
NOTES ^The terminology of the highly branched polymers is not firmly established. It is widely accepted that "(niono)dendron" is a perfectly branched convergently or divergently grown polymer with multiple surface groups and a single functional group at its "focal" point. "Dendrimer" is typically a polymer formed by the coupling (growth) of several monodendrons to (from) a multifunctional core molecule. ^The term "architectural copolymers" was first utilized by Tomalia et al.^^, but for LDBCs that contain linear and dendritic blocks with chemically different structures. ^ The first reports on divergent dendritic initiators are beyond the classification limits of this review because of imperfect structure (hyperbranched polyphenylenes^^^) and low generation (first-generation polyethylenimine^^^). The same restriction ought to be applied to some excellent recent studies.^^'^"^
ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Professor Jean Frechet at the University of California, Berkeley for the initial introduction to the wonderful world of dendritic macromolecules and his continuous inspiration throughout all these years. Thanks are also due to all coworkers and colleagues who contributed experimentally or theoretically to the investigations I was personally involved in. These projects were funded entirely or in part by grants from the William and Mary Greve Foundation, Inc., the National Science Foundation, the Twinning Program of the National Research Council, the Research Corporation, Rohm and Haas, Allied Signal and Rhodia.
Linear-Dendritic Block Copolymers
85
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34. 35. 36. 37.
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38. 39. 40. 41. 42. 43.
44.
45. 46. 47.
48. 49. 50. 51. 52. 53.
54. 55. 56. 57. 58. 59. 60. 61.
Linear-Dendritic Block Copolymers 62. 63. 64. 65.
66.
67. 68. 69. 70. 71. 72.
73. 74.
75. 76.
77.
78.
87
Mecerreyes, D.; Dubois, P.; Jerome, R.; Hedrick, J. L.; Hawker, C. J. J. Polym. Set., Part A: Polym. Chem. 1999, 37, 1923. MoUer, M.; Lim, L. S.; Hedrick, J. L. Polym. Prepr. 2000, 41(1), 52. Howell, B. A. Polym. Mater. Sci. Eng. 2000, 83, 578. (a) Matyjaszewski, K.; Shigemoto, T.; Frechet, J. M. J.; Leduc, M. Macromolecules 1996, 29, 4167; (b) Leduc, M.; Hawker, C. J.; Dao, J.; Frechet, J. M. J. /. Am. Chem. Soc. 1996, 118, 11111; (c) Leduc, M.; Hayes, W.; Leon, J. W.; Frechet, J. M. J. Polym. Mater. Sci. Eng. 1997, 77, 105; (d) Hayes, W.; Leduc, M.; Leon, J. W.; Frechet, J. M. J. Polym. Mater. Sci. Eng. 1997, 77, 143; (e) Leduc, M.; Hayes, W.; Frechet, J. M. J. /. Polym. Sci., Part A: Polym. Chem. 1998, 36, 1; (f) Emrick, T.; Hayes, W.; Frechet, J. M. J. / Polym. Sci., Part A: Polym. Chem. 1999, 37, 3748. (a) Kim, Y. H.; Webster, O. W. Macromolecules 1992, 25, 5561; (b) Warakomski, J. M. Chem Mater. 1992, 4, 1000; (c) Heise, A.; Hedrick, J. L.; Trollsas, M.; Miller, R. D.; Frank, C. W. Polym. Prepr. 1998, 39(2), 627; (d) Heise, A.; Trollsas, M.; Magbitang, T.; Hedrick, J. L.; Frank, C. W.; Miller, R. D. Polym. Prepr. 1999, 40(2), 376; (e) Hovestad, N. J.; Jastrzebski, J. T. B. H.; van Koten, G.; Bon, S. A. F ; Waterson, C ; Haddleton, D. M. Polym. Prepr. 1999, 40(2), 393. Vasilenko, N. G.; Rebrov, E. A.; Muzafarov, A. M.; EBwein, B.; Striegel, B.; MoUer, M. Macromol. Chem. Phys. 1998, 799, 889. Comanita, B.; Noren, B.; Roovers, J. Macromolecules 1999, 32, 1069. Balogh, L.; de Leuze-Jallouli, A.; Dvomic, R; Kunugi, Y.; Blumstein, A.; Tomalia, D. A. Macromolecules 1999, 32, 1036. Higashi, N.; Koga, T.; Niwa, N.; Niwa, M. Chem. Commun. 2000, 361. Aoi, K.; Hatanaka, T.; Tsutsumiuchi, K.; Okada, M.; Imae, T. Macromol. Rapid Commun. 1999, 20, 378. (a) Claesson, H.; Trollsas, M.; Atthoff, B.; Hedrick, J. L. Polym. Prepr. 1998, 39(2), 511; (b) Heise, A.; Hedrick, J. L.; Trollsas, M.; Hilbom, J. G.; Frank, C. W.; Miller, R. D. Polym. Prepr. 1999, 40(1), 452; (c) Heise, A.; Hedrick, J. L.; Frank, C. W.; Miller, R. D. J. Am. Chem. Soc. 1999, 121, 8647. Miller, R. D.; Heise, A.; Diamanti, S. Polym. Prepr. 2000, 41(1), 48. Janca, J. Steric Exclusion Liquid Chromatography of Polymers, Marcel Dekker: New York, 1984; Silverstein, R. M.; Webster, F. Spectrometric Identification of Organic Compounds, 4th ed., J. Wiley: New York, 1997; Sheiko, S. S. Imaging of Polymers Using Scanning Force Microscopy: From Superstructures to Individual Molecules, in Advances in Polymer Science, Schmidt, M., Ed., Springer: Berlin, 2000, Vol. 151, p. 61. (a) Hillenkamp, F ; Karas, M.; Beavis, R. C ; Chait, B. T. Anal. Chem. 1991, 63, 1193A; (b) Barr, U.; Deppe, A.; Karas, M.; Hillenkamp, F.; Glessmann, U. Anal. Chem. 1992, 64, 2866. (a) Montaudo, G.; Montaudo, M. S.; PugUsi, C.; Samperi, F. Macromolecules 1995, 28, 4562; (b) Montaudo, G.; Montaudo, M. S.; Puglisi, C ; Samperi, F. Rapid Commun. Mass Spectrom. 1995, 9, 453; (c) Weidner, S.; Kiihn, G. Rapid Commun. Mass Spectrom. 1996, 10, 942. (a) Leon, J. W; Frechet, J. M. J. Polym. Bull. 1995, 35, 449; (b) Yu, D.; Vladimirov, N. G.; Frechet, J. M. J. Polym. Mater Sci. Eng. 1999, 80, 219; (c) Yu, D.; Vladimirov, N.G.; Frechet, J. M. J. Macromolecules 1999, 32, 5186. (a) Percec, V; Ahn, C.-H.; Ungar, G.; Yeardley, D. J. P; Moller, M.; Sheiko, S. Nature (London) 1998, 391, 161; (b) Prokhorova, S. A.; Sheiko, S.; Moller, M.; Ahn, C. H.; Percec, V. Macromol. Rapid Commun. 1998, 19, 359; (c) Percec, V; Ahn, C.-H.; Cho, W.-D.; Jamieson, A. M.; Kim, J.; Leman, T.; Schmidt, M.; Gerle, M.; Moller, M.; Prokhorova, S. A.; Sheiko, S. S.; Cheng, S. Z. D.; Zhang, A.; Ungar, G.; Yeardley, D. J. P /. Am. Chem. Soc. 1998, 120, 8619.
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DENDRIMERS CONTAINING FERROCENYL OR OTHER TRANSITION-METAL SANDWICH GROUPS Beatriz Alonso, Ester Alonso, Didier Astruc, Jean-Claude Blais, Laurent Djakovitch, Jean-Luc Fillaut, Sylvain Niate, Frangoise Moulines, Stephane Rigaut, Jaime Ruiz, and Christine Valerio Abstract Introduction Metallostars: The CsHsFe"^ Induced Hexa-Alkylation or Hexafunctionalization of Hexamethylbenzene III. Metallodendrimers: The CsHsFe^ Induced OctafunctionaUzation of Durene IV. A Route to NonametalUc and Higher Dendrimers Using the Triple Branching: CsHsFe^ Induced Tri-Allylation of Toluene and Nona-Allylation of Mesitylene V. Synthesis of a Phenol Dendron and of Organometallic Dendrons: One-Pot CsHsFe"^ Induced Activation of Ethoxytoluene VI. Convergent and Divergent Syntheses of Large Ferrocenyl Dendrimers . . VII. Polyferrocenium Dendrimers: Molecular Batteries VIII. Amidoferrocenyl Dendrimers as Sensors for the Recognition of H2PO4 and HSO^: I. II.
Advances in Dendritic Macromolecules Volume 5, pages 89-127 © 2002 Elsevier Science Ltd. Allrightsreserved ISBN: 0-7623-0839-7
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90 90 91 95
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90 IX. X. XL
ALONSO ET AL. Cationic N-Alkylaniline Iron-Sandwich Dendrimers as Sensors for the Recognition of Chloride and Bromide Anions Other Ferrocene Dendrimers Conclusion Acknowledgements References
117 118 122 123 123
ABSTRACT Thefirstorganometallic stars and dendrimers were those containing either ferrocene or an isolobal [FeCp(Ti^-arene)][PF6)] sandwich. They were made by CpFe"*" induced polyfunctionalization of the polymethylbenzene. These metal-sandwich groups were located either at the center or at the periphery of the star or dendritic frame and contained reversible redox centers. This allowed their use as redox catalysts and electrode modifiers. Recently, large ferrocene dendrimers have been isolated and characterized in two stable redox forms which indicates that these metallodendrimers can behave as molecular batteries. This review also includes works by other research groups on ferrocene dendrimers till the end of the second millennium.
1. INTRODUCTION Metallodendrimers^"^ constitute a fascinating aspect of dendrimer chemistry^"^^ because of their potential in catalysis,-^^ molecular recognition (sensors),^^ molecular electronics^"^ and molecular biology or medicine.^^ In most metal complexes, however, the redox systems are not chemically reversible. We have been interested for some time in electron-reservoir complexes, i.e., systems which are fully reversible.^^ The complexes [FeCp(T]^-arene)][PF6)], isolobal to ferrocene^^ and cobaltocenium salts, belong to this category. These complexes are also proton reservoirs, because the cationic charge of the complexes considerably enhances the acidity of the numerous benzylic protons in the polymethylbenzene ligands. This later property has led us to exploit the CpFe"^ induced polyfunctionalization of polymethylbenzenes to synthesize iron-centered stars and iron-centered dendritic cores according to iterative processes since the late 1970s. The first paper appeared a few months after Vogtle's first iteration paper, both works being reported independently (Scheme 1) The polyfunctional cores and dendrimers made in this way have been decorated with ferrocene, cobaltocenium and [FeCp(Ti^-arene)][PF6)] units at the periphery in the early 1990s. In the mid-1990s, other research groups have also reported a variety of other ferrocene dendrimers which we will briefly discuss in the last section of this review.^^ Finally, it should be stated that metallocene dendrimers, which we will review here, are not the only redox-reversible systems. In particular, Bryce and Becher's groups^^'^^ have developed many syntheses of dendrimers, which contain tetrathialfulvalene
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
91
ELECTRON AND PROTON RESERVOIR COMPLEXES]
E° = - 2V vs. Cp2Fe in DMSO
H'
p/Ca = 29.2 DMSO
^ ■H-
C-H Bond Dissociation Energy: AG° = 65.7 Iccal/mol in DMSO 19e Redox Sensors Molecular Batteries Molecular Electronics
Molecular Engineering
-<
>-
18e
Dendritic Architectures Nanosciences
Scheme 1. Concept of electron and proton reservoir using permethylated late transition-metal sandwiches. Electron reservoir: both redox forms are easily available in large scale, robust, and the redox potential is very negative. Proton reservoir: both the protonated and deprotonated forms are easily available in large scale and robust, the pK^ is not high, making deprotonation easy by common bases such as KOH or t-BuOK, and there are many protons which can be removed and replaced by functional chains (see Scheme 2).
units at the periphery, and Gorman^^ ^^ has reported iron cluster-centered dendrimers and interesting electron-transfer studies.
IL METALLOSTARS: THE C5H5Fe+ INDUCED HEXA-ALKYLATION OR HEXAFUNCTIONALIZATION OF HEXAMETHYLBENZENE Thus, reaction of [FeCp(C6Me6)][PF6)],^^"^^ with excess KOH (or r-BuOK) in THF or DME and excess methyl iodide, alkyl iodide, allyl bromide or benzylbromide leads to the one-pot hexasubstitution (Scheme 2)^^~^^. With allyl bromide (or iodide) in DME, the extremely bulky dodeca-allylation^^ product can be reached when the reaction is prolonged for two weeks at 40°C. With alkyl iodides, the reaction using r-BuOK only leads to dehalogenation of the alkyl iodide giving the terminal olefin. Thus, one must use KOH, and the reactions with various alkyl iodides (even long-chain ones) were shown to work very well with this reagent to give the hexa-alkylated Fe(II)-centered complexes. The yellow 18-electron complexes can be reduced to the dark-green 19-electron complexes, but the stabilities of these organo-iron radicals decrease as the chain
92
^n: Fe
PFe"
ALONSOETAL.
RX, ^BuOK ou KOH
^n-
THFouDME
R = CH3, (CH2)nCH3, (CH2)4Fc (X = I), CH2Ph,p-CH2PhOR', CH2CH=CH2,(X = Br) Scheme 2. The peralkylation or perfunctionalization reaction: this reaction is made possible by the proton-reservoir property of the starting organometallic cation (see Scheme 1) and spontaneously reproduced by iteration many times until a steric limit is reached.
^
^n*
:
^
'
- ^ ^
Scheme 3, High-yield and high-scale synthesis of metal-centered stars with tentacles: application of the peralkylation reaction to long-chain alkyl iodides under mild conditions.
length of the alkyl group is increased. Nevertheless, it was possible to isolate the 19-electron complexes up to the hexahexylbenzene complex at low temperature and to record their temperature-dependent Mossbauer spectra between 4.2 and 200 K. The rhombic distortion, observed initially for [FeCp(C6Me6)] and due to the Jahn-Teller effect, is now weaker and milder (Scheme 3).^^ The hexa-alkylation was also performed with alkyl iodides containing functional groups at the alkyl chain termini. For instance, alkyl iodide bearing methoxy^^ or olefinic ending"^^ were prepared. Finally, 1-ferrocenylbutyl iodide reacts nicely to give the hexaferrocene star containing the CpFe"^ center (Scheme 4).
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups 93
KOH, DME Fc (CH2)4l Fc = ferrocenyl counter-anion: PFe
Scheme 4. Branching out: synthesis of hexaferrocenylalkyi metal-centered redox stars.
^
^^y^=^^^^^ ^^
A^
^y:=^^'=^r^ ^^
_re^'
_re_
^^7
Fe""
Fe
-Fe_
^ ^
_Fe_
PFe
hepta-Fe"
Scheme 5, Oxidation or reduction of metallostars to localized mixed-valence complexes. Fe centers for which the oxidation state is not indicated on the scheme are Fe(ll).
This heptanuclear complex is of interest because of its various redox states. The six-ferrocene units are independent as can be seen from the single cyclovoltammetry wave. They could be oxidized to give the mixed-valence heptacation in which the central iron group withstands oxidation and retains its Fe(II) state, because it is protected by the positive charge'*^ ^ (this protection can be estimated to approximately 1.5 V (Scheme 5)."^^^) The central iron serves as an internal reference to count the number of peripheral ferrocenes using intensity ratio either in the cyclovoltammogram or in the Mossbauer spectrum of the oxidized Fe(II)/Fe(III) complex.^^^ Both the hexa- and dodeca-allylation reactions are well controlled. On the other hand, the reaction with excess benzylbromide"^^ or p-alkoxybenzylbrom-
ALONSO ET AL.
94
c^^> I . Fe
<^T^ I Fe
Chart 1. Examples of redox active hexa- and hepta-metallic stars synthesized by reactions of the iron-centered hexaphenate or metal-free hexaphenate with organometallic electrophiles such as ferrocenoyi chloride or [FeCp(Ti^-p-CH3-C6H4F)][PF6].
jjg43,44 Q^Yy gives the hexabenzylated or hexa-alkoxybenzylated complex as the ultimate reaction product. Cleavage of the methyl group in the /7-methoxybenzyl derivatives synthesized in this way yields the hexaphenolate stars which could be combined with halogen containing Fe-sandwich electrophilic compounds such as chlorocarbonylferrocene or [FeCp(T]^-C6H5Cl)][PF6].'^^''^ Alkynyl halides cannot be used in the CpFe+ induced hexafunctionalization reaction, but alkynyl substituents can be introduced from the hexa-alkene derivative by bromination followed by dehydrohalogenation of the dodecabromo compound."^^ The hexa-alkene is also an excellent starting point for further syntheses, especially using hydroelementation reactions. Hydrosilylation reactions catalyzed by Speir's reagent led to long-chain hexasilanes"^^ and hydrometallations were also achieved using [ZrCp2(H)(Cl)].'^^ The hexazirconium compound obtained is an intermediate for the synthesis of the hexa-iodo derivative (Scheme 6).^^ One of most useful hydroelementation reactions is the hydroboration using 9-BBN and leading to the hexaborane, which is oxidized to the hexol using H2O2 under basic conditions'^. This chemistry can be carried out on the iron complex or alternatively on the free hexa-alkene which may be liberated from the metal by photolysis in CH2CI2 or MeCN using visible light.'^ Williamson coupling reactions between the hexol and 4-bromomethylpyridine or -polypyridine led to hexapyridine and hexapolypyridine and to their ruthenium complexes."^^'"^^ The hexa-ol is indeed the best source of hexa-iodo derivative either using HI in acetic acid or even better by trimethylsilylation using SiMcsCl followed by iodation using Nal.^^ This hexa-iodo star was condensed with
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups ZrCp2(CI)
SiMe2R RMegSi
RMe2Si-
/
95
^SiMegR
(CI)Cp2Zrv
(CI)Cp2Zr
>
^ZrCp2(CI)
-^T^. ZrCp2(CI)
(COCpsZ/
Scheme 6. Hydroelementation for the functionalization of hexa-olefin stars: hydrosilylation, hydrozirconation and hydroboration.
/7-hydroxybenzaldehyde to give an hexabenzaldehyde star, which could further react with substrates bearing a primary amino group. Indeed, this reaction yielded a water-soluble hexametallic redox catalyst which was active in the electroreduction of nitrate and nitrite to ammonia on a Hg cathode in basic aqueous solution, vide infra (Scheme 7).^^
III. METALLODENDRIMERS: THE C5H5Fe+ INDUCED OCTAFUNCTIONALIZATION OF DURENE If the hexafunctionalization of hexamethylbenzene leads to stars, the octafunctionalization of durene leads to dendritic cores (Scheme 8). The first of these octa-alkylation reactions was reported as early as 1982, and led to a primitive dendritic core containing a metal-sandwich unit.^^ Thus, as the hexafunctionalization, this reaction is very specific. Two hydrogen atoms of each methyl group are now replaced by two methyl, allyl or benzyl groups.^^ This feature is due to the fact that each methyl group has only one methyl neighbor in durene instead of two such neighbors in hexamethylbenzene. It is remarkable that the consequence of this difference is so clear-cut. We believe that this is due to the respective rates of the organometallic reaction on one hand and the reaction between the base and the halide on the other hand. Later, the octabenzylation reaction has been used for further dendritic construction. Indeed, the p-chlorocarbonylation of the octabenzyl core is re-
-
SiMe3CI, Nal
HN
I
Q P
n ur "I I"
CHO
Scheme 7. Hexafunctionalization of aromatic stars with the heterodifunctional, water soluble organometallic redox catalyst (bottom) for the cathodic reduction of nitrates and nitrites to ammonia in water.
1
e
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
97
Kf-BuO or KOH (excess) RBr or Rl (excess) THF or DME R = alkyl, allyl, benzyl, etc.
Scheme 8, Octa-alkylation, -allylation, or -benzylation of durene by a series of 8 deprotonation/alkylation (or allylation or benzylation) sequences induced by the 12-electron activating group CpFe"*" in a one-pot reaction under mild conditions and in high yields.
markably regioselective and has been used to undergo reactions with amines of interest such as Newkome's tripodal amine terminated by nitrile groups.^^ This reaction provides a rapid route to the 24-nitrile dendrimer.^^ Another reaction of interest in the present context is that of the octachlorocarbonylbenzyl core with 1-ferrocenylpentylamine providing the expected octaferrocenyl compound (Scheme 9).^^ The reaction of the octa-iodomethylbenzyl core with [Fe(Ti^-C5H5)(r]^C6Me5CH2)], i.e., the deprotonated form of [Fe(Ti^-C5H5)(Ti^-C6Me6)][PF6], lead to the yellow octa-sandwich C6H2-l,2,4,5-[CH(CH2CH2/?-C6H4CH2CH2T]^-C6Me5FeCp"^I~)2]4, which was almost insoluble (Scheme 10a). Its structure was indicated by the yellow color and the Mossbauer spectrum, both being characteristic of the FeCp(arene)"^ frame.^^ A very interesting series of dendrimers containing 24-transition-metal sandwich units has been synthesized from the 24-nitrile dendrimer by reduction of the nitrile groups to primary amines using BH3Me2S in THF followed by reaction of the 24-amine dendrimer with chlorocarbonylferrocene or with [Fe(Ti^-C5Me5)(ri^-C6H5F)][PF6] (Scheme lOb).^^ Both 24-branch metallodendrimers proved very useful and complementary for molecular recognition, as will be discussed later in this chapter. Double branching, i.e., replacement of two out of three hydrogen atoms by two groups on each methyl substituent of an aromatic ligand coordinated to an activating cationic group CpM"^ in an 18-electron complex is not reserved to the durene case. It is encountered as well in the o-xylene ligand,^^'^^ in the pentamethylcyclopentadienyl ligand (in pentamethyl cobaltocenium^^ and in penta-^^ and deca-methylrhodocenium^^) and even in the hexamethylbenzene ligand. With this latter ligand, only the allyl group could be introduced in double branching, and this reaction required two weeks at 40°C,^^ whereas single branching was complete after only one day at 40°C. The extremely bulky dodeca-allyl complex formed is chiral, but its directionality is completely blocked (no interconversion between the clockwise and counterclockwise directionalities)^^ contrary to the directionality of decafunctionalized ligands coordinated to CpCo+ or CpRh+, whose interconversion could be observed by
ALONSO ET AL.
98 —I ^CO> '
+ _
DPhCHsBr KOH,DME
CI
Q
)0C 0
Cl
NH2CH2)4FC
NH2C CH2CH2CH2CN)3
•^^ Hti^~°''
Mr-J
S NC
/ NC
K ^
NC
NC
Scheme 9. Synthetic scheme for 24-CN and octaferrocenyl dendrimers.
^H NMR (at least for the deca-isopropyl and deca-isopentyl cyclopentadienyl cobalt and rhodium complexes^^"^^) (Schemes 11-13). IV. A ROUTE TO NONAMETALLIC AND HIGHER DENDRIMERS USING THE TRIPLE BRANCHING: C5H5Fe+ INDUCED TRI-ALLYLATION OF TOLUENE AND NONA-ALLYLATION OF MESITYLENE In all the above examples, the polybranching reaction of arene ligands was limited by the steric bulk. In the toluene and mesitylene ligands, the deprotonationallylation reactions are no longer restricted by the neighborhood of other alkyl groups. All the benzylic protons, i.e., three per benzylic carbon, can be replaced
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
99
(i) CH3OH; (iJ) UAIH/THF; (tH) Nal, BF3.a20/CH3CN
^>-—^
;H2
(a)
— < ^
^^
^
Scheme 10a. Synthesis of a small dendrimer terminated by 8 CpFe"^(Ti^-pentamethylbenzene) moieties starting from CpFe''"(T]^-1,2,4,5-tetramethylbenzene). The octanuclear compound obtained is almost insoluble in all the solvents.
by methyl or allyl groups in the one-pot iterative methylation or allylation reactions. Thus, the toluene complex can be triallylated and the mesitylene complex can be nona-allylated, these reactions being carried out smoothly at room temperature in the presence of excess KOH and allyl bromide. The nona-allyl complex was photolyzed using visible light to remove the metal group CpFe+, then hydroborated using 9-BBN, and the nonaborane was oxidized using H202/OH~ to the nonol. Reaction of this nonol with [FeCp(r]6-p-Me-C6H4F)][PF6] in the presence of K2CO3 in DMF yielded the nona-sandwich complex via CpFe"^ induced nucleophilic substitution of the halogen by the alkoxy branch, and the metallodendrimer was purified by column chromatography (Scheme 14). The cyclovoltammogram of the metallodendritic complex shows that the nine redox Fe(II) centers are reduced at seemingly the same potential. At —30°C, this wave is reversible and the number of redox centers is found to be 8 ib 1 using the Bard-Anson formula.^^'^^ Alternatively, the Michael reaction of the nonol with acrylonitrile yields a nonanitrile, which can be reduced to the nona-amine. This nona-amine was allowed to react with chlorocarbonylmetallocenes and other chlorocarbonyl sandwich complexes to yield nona-amidometallocenes^^~^^ and nona-amidosandwich compounds^^ (Scheme 15).
ALONSO ET AL.
100
O
HjN
o
HjN
0
[FeCp(Ti5-C5H4COCI)]/
NH
.[FeCp*(Ti«-C6H5F)][PF6]
oC^^
0
N '
N
O
^
o
2:
O
NH CO
^
OC ^;=;
(PF6")24
(b)
24-Fc
24-FeAr
Scheme 10b. Construction of dendrimers with 24 ferrocenyl or cationic iron-sandwich groups.
KOH, RX DME
M = Co, Rh
R = Me, Et, CHgPh, CH2-CH=CH2
Scheme 11, Deca-alkylation, -allylation or -benzylation of 1,2,3,4,5-pentamethylcobaltocenium or 1,2,3,4,5-pentamethylrhodocenium in a one-pot reaction consisting of 10 deprotonationalkylation sequences. Steric constraints inhibit further reaction, and the 10 groups introduced are self-organized according to a single directionality (see Scheme 12).
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
101
H H
H
groups R = Me ommited for clarity
M = Co, AG* = 71.13 kJ.mol''' (17.0±0.2 kcal.mol"'') Rh, AG* = 70.21 kJ.mol"'' (16.8±0.2 kcal.mol''') Scheme 12, Interconversion between the clockwise and counterclockwise directionalities of the deca-alkylated or decafunctionalized 18-electron cationic metallocenes (from the reactions of Scheme 11) observed by ^H NMR. The coalescence temperature is all the higher as the R group introduced is large. 1) CH2=CHCH2Br KOH, DME,RT
PF.
►
2)hv
Scheme 13. One-pot CpFe+ induced triallylation of toluene.
1)CH2=CHCH2Br KOH, DME.RT 2)hv
1)HB(siamyl)2 THF
2) H2O2 NaOH
K2CO3, Bu4NBr THF/DMSO, RT
(PF6")9
Scheme 14. One-pot, large-scale, high-yield CpFe+ induced nona-allylation of mesitylene and subsequent functionalization of the olefinic branches leading to redox-active nonametallic dendrimers.
102
ALONSO ET AL.
CO NHj
CO^
0
/
NHCO
NEtg, CH3CN, RT
NHa
(a)
NHz
NH
NH
^ >
9-amine
o-r^W^
NH ^ C O (CH3)n^
NH C O ^
9-Co
^X. ^,„^^^ (CH3)n
(PF6')9
Scheme 15. Synthesis of a nona-amidoferrocenyl dendrimer using the core designed according to the CpFe+ induced nona-allylation of mesitylene.
These metallodendrimers also give only one cyclovoltammetry wave whose intensity corresponds to approximately 9 electrons using the technique indicated above (the solvent was CH2CI2 for the nona-amidoferrocene and MeCN for the polycationic dendrimers). The chemical reversibility was observed at room temperature, although some adsorption was noted. The nona-amine was the starting point for the construction of dendrimers according to the iteration first reported by Vogtle, then developed by de Brabander-van den Berg and Meijer^^ and Womer and Miilhaupt.^^ Thus reaction of the
Dendrimers Containing Ferrocenyl or Otiier Transition-Metal Sandwich Groups
jp-NHa NHj
CH2=CH-CN "2O,80-C 80%
0^NH2
K^^
4&
103
^^^
CHoCI;
^a ^
CO f^"
ob
^
^
^
H2N
I8-NH2
"^^
Scheme 76. Syntheses of cationic nona-amido-sandwich complexes of iron and cobalt (18electron forms) using the same core as that used in Scheme 15.
nona-amine with acrylonitrile gave a 18-nitrile dendrimer which was reduced to the 18-amine dendrimer. The dendritic construction was continued in this way until the 144-nitrile dendrimer. Purification of these dendrimers was achieved at each generation by column chromatography of the polynitrile dendrimer.^^ Each polyamine dendrimer reacted with chlorocarbonylferrocene, but the 36and 72-amidoferrocene dendrimers obtained were found to be insoluble in all the solvents. The last soluble amidoferrocene dendrimer of this series is the 18-amidoferrocene (Scheme 16).^^'^^ This dendrimer later proved very efficient for anionic recognition (vide infra).
V. SYNTHESIS OF A PHENOL DENDRON AND OF ORGANOMETALLIC DENDRONS: ONE-POT CsHsFe^ INDUCED ACTIVATION OF ETHOXYTOLUENE The triple branching reaction of Scheme 13 being very straightforward, we sought a more sophisticated version compatible with a functional group in the para position of the tripod in order to open the access to a functional dendron.
104
ALONSOETAL
<^5y
I
"•
nBu4NPFg
^--T--^
THF
^ -
\ Scheme 17, Heterolytic C-O cleavage reaction in aryl ether complexes by t-BuOK or KOH induced by the activating 12-electron fragments CpFe+. This reaction is very useful as it v^as applied to the convenient one-pot synthesis of the phenoltriallyl dendron (see Scheme 18).
Serendipitously, we found that KOH or f-BuOK under very mild conditions easily cleaved the iron complexes of aromatic ethers. The activating CpFe"^ group again induces this reaction, which is very general for a variety of aromatic ether complexes (Scheme 17).^^'^^ Since this cleavage reaction is carried out with the same reagent and solvent as the one used in the trialkylation reaction (ideally f-BuOK in THF), we have attempted to perform both reactions in a well-defined order (triallylation before ether cleavage) in a one-pot reaction. Indeed, this works out well and the Fe"^ complex of the phenol tripod was made in 50% yield in this way. This complex can be photolyzed in the usual way using visible light, which yields the free phenol tripod. However, we have also further investigated the possibility to obtain the cleavage of the arene ligand in situ at the end of the phenol tripod construction. ^BuOK is a reductant when it cannot perform other reactions. Since the two important reactions are over, then comes the third role of ^BuOK:
-n+ y
CH2=CHCH2Br f-BuOK F© P^' ► CH3--^^^>—OEt THF
Scheme 18. One-pot syntheses of the phenol-triallyl iron complex and metal-free dendron by variation of the experimental condition. The iron complex can be demetallated by visiblephotolysis or the metal-free phenol-triallyl can be more rapidly obtained directly from the pethoxytoluene-iron complex.
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
single-electron reductant. Reasoning in this way turned out to be correct. Indeed, the cleavage of the arene intervenes rapidly at the 19-electron stage, because 19-electron complexes of this kind are not stable with a heteroatom located in exocyclic position (most probably because the heteroatom coordinates to the metal from the labile 19-electron structure). After optimizing the reaction conditions, a 50% yield of free phenol dendron from the ethoxytoluene complex could be reproducibly obtained,^^'^^ and this reaction is now regularly used in our lab to synthesize this very useful dendron as a starting material (Scheme 18). The functionalization of the three allyl chains of the phenol dendron could be achieved by hydrosilylation reaction catalyzed by the Karsted catalyst.^^ Indeed, it is very interesting that there is no need to protect the phenol group before performing these reactions. For instance, catalyzed hydrosilylation using ferrocenyldimethylsilane gives a high yield of the triferrocenyl dendron HOp-C6H4C(CH2CH2CH2SiMe2Fc)3 that is easily purified by column chromatography.^^'^^
VI. CONVERGENT AND DIVERGENT SYNTHESES OF LARGE FERROCENYL DENDRIMERS The protection of the phenol dendron using propionyl iodide gave the phenolate ester, which was hydroborated. Then, oxidation of the triborane using H202/OH~ gave the triol, reaction with SiMcsCl gave the tris-silyl derivative, reaction with Nal yielded the tri-iodo compound, and reaction with the tri-ferrocenyl dendron provided the nona-ferrocenyl dendron which was deprotected using K2CO3 in DMF. The nona-ferrocenyl dendron was allowed to react with hexakis(bromomethyl)benzene, which gave the 54-ferrocenyl dendrimer. This convergent synthesis is clean and the 54-ferrocenyl dendrimer gave correct analytical data, although a mass spectrum could not be obtained (Scheme 19). This approach is somewhat limited, however, since larger dendrons, which one would like to synthesize in this way, cannot be made because dehydrohalogenation becomes faster than nucleophilic substitution of the iodo by phenolate for bulkier higher generations of dendrons. Although this problem might be overcome by modifying the iodo branch in such a way that there would be no hydrogens in P positions, the condensation of higher dendrons onto a core would become tedious or impossible for steric reasons. This well-known inconvenience is intrinsic to the convergent dendritic synthesis. On the other hand, divergent syntheses are not marred by such a problem since additional generations and terminal groups are added at the periphery of the dendrimer.,The limit is that indicated by De Gennes, i.e., the steric congestion encountered at a generation where the peripheral branches can no longer by divided. Another obvious limit intervenes if the molecular objects added onto the termini of the branches are
105
106
X -co-
I
o o Cvl
_
CD
♦6
o o o LU
—CD
^.
^^^
^P^^-w'
0
in
0
+
LA
ALONSO ET AL.
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
large and interfere with one another. We have developed a divergent synthesis of polyallyl dendrimers indicated in Scheme 20 whereby each generation consists in hydroboration, oxidation of the borane to the alcohol, formation of the mesylate, and reaction of the phenol dendron with the mesylate. This strategy has allowed us to synthesize dendrimers of generations 0, 1, 2 and 3 with respectively 9 (Go), 27 ( d ) , 81 (G2) and 243 branches (G3) (Scheme 20). The MALDI-TOF mass spectrum of the 27-allyl dendrimer only shows the molecular peak with only traces of side products. That of the 81-ally 1 shows a dominant molecular peak, but also important side products resulting from incomplete branching. That of the 243-allyl could not be obtained, possibly signifying that this dendrimer is polydisperse (correct ^H and ^^C NMR spectra were obtained, however, indicating that the ultimate reactions had proceeded to completion). This dendrimer was soluble which indicated that this generation is not the last one, which might be reached. The ferrocenylsilylation of all these polyallyl dendrimers was carried out using ferrocenyldimethylsilane and was catalyzed by the Karsted catalyst^^ at room temperature or 40°C. The reactions were complete after a day except for the ferrocenylsilylation of 243-allyl which required a reaction time of two weeks indicating some degree of steric congestion (Schemes 20-23). The ^H and ^^C spectra indicate the absence of regioisomer. The solubility in pentane decreased from good for the 9-Fc dendrimer to low for the 27-Fc dendrimer and nil for the superior dendrimers, but the solubility in ether remained good for all the ferrocenyl dendrimers. Likewise, the retention times on plate or column chromatography increased with generation and no migration was observed for the "243-Fc" dendrimer. This silane, reported by Pannel and Sharma,^^ was already used by Jutzi et al.^"^ to synthesize the decaferrocenyl dendrimer [Fe(CCH2CH2SiMe2Fc)io] (with Fc = ferrocenyl) from deca-allylferrocene. The cyclic voltammetry of all the ferrocenyl dendrimers on Pt anode shows that all the ferrocenyl centers are equivalent and only one wave was observed. It was possible to avoid adsorption using even CH2CI2 for the small ferrocenyl dendrimers, but it was required to use MeCN for the medium size ones (27-Fc, 54-Fc and 81-Fc). Finally, adsorption was not avoided even with MeCN for the "243-Fc" dendrimer. From the intensity of the wave, the number of ferrocenyl units could be estimated using the Anson-Bard equation,^^ and the
Scheme 19, Convergent strategy for the clean synthesis of a 54-ferrocenyi dendrimer. This scheme also illustrates the property of molecular batteries of such large ferrocenyl dendrimers. The deep-blue ferrocenium form can be synthesized quantitatively, characterized and reduced back to the orange ferrocenyl dendrimer. The overall cycle proceeds without any decomposition with all these silylferrocenyl dendrimers including the 243-ferrocenyl dendrimer.
107
108
ALONSO ET AL. 1) mesitylene AICI3
1) p-chlorotoluene AICI3 < c ^ f^
2)aq.HPFs
2) aq. HPFe PF6"
1
^
^ 1)CH2=CHCH2Br I 2) hv KOH, DME, 20°C I
EtOH IK^.2CO3
t^
"
PF6"
Et0-
-
"one-pot"
CH2=CHCH2Br f-BuOK, THF, -SOX --> 20°C
dendron A
^^
R = BR2
H2O2 CIS(0)2Me »► OH ► NaOH
R = 0S(0)2Me
27-allyl dendrimer iteration dendron A 81-allyl dendrimer iteration f dendron A 243-allyl dendrimer
Scheme 20.
l\V)ll\\|l\\i\\\ll Chart 2. Third-generation polyallyl dendrimer with a theoretical number of 243 allyl branches synthesized according to Scheme 20. This "243-allyr' dendrimer (as the 9-allyl, 27-allyl, and 81allyl dendrimers of generations 0, 1 and 2, respectively) was hyd rosily I ated using dimethylferrocenylsilane Fc(Me)2SiH to "243-Fc", a molecular-battery dendrimer with a theoretical number of 243 peripheral ferrocenyl unit (see characterizations in Figs. 1 and 2).
number found were within 5% of the branch numbers except for the "243-Fc" dendrimer, for which the experimental number was too high (250) because of the adsorption. VII. POLYFERROCENIUM DENDRIMERS: MOLECULAR BATTERIES The first polyferrocenium dendrimers reported and characterized inter alia by Mossbauer spectroscopy (a "quantitative" technique) by us in 1994 were mixed
110
ALONSO ET AL.
Fe
Me
KarstEdtcat
9-aI^ldeidrin er
Scheme 21,
valence Fe(II)/Fe(III) complexes. Since then, we have been seeking to find larger ferrocenyldendrimers, which could also withstand oxidation to their ferrocenium analogues. Amidoferrocene dendrimers were not the best candidates, although they give fully reversible cyclic voltammetry waves, since it is known that ferrocenium derivatives bearing an electron-withdrawing substituent are at least fragile, if stable at all. This inconvenience is probably enhanced in the dendritic structures because of the steric effect which forces ferrocenium groups to encounter more easily than as monomers. Thus, we have oxidized our silylferrocenyl dendrimers using [N0][PF6] in CH2CI2 and obtained stable polyferrocenium dendrimers as dark-blue precipitates, as expected from the known characteristic color of ferrocenium itself. These polyferrocenium dendrimers were reduced back to soluble orange polyferrocenyl dendrimers using decamethylferrocene as the reductant.^^ No decomposition was observed either in the oxidation or in the reduction reactions which were very clean, and this redox cycle could be achieved in quantitative yield even with the "243-ferrocenyl" dendrimer. The zero-field Mossbauer spectrum of the 243-ferrocenium dendrimer (Fig. 1) showed a single line corresponding to the expected spectrum known for ferrocenium itself,^^ confirming its electronic structure. Thus, these polyferrocenyl dendrimers are molecular batteries, which could be used in specific devices. Indeed, as large as they may be, they transfer a very large number of electrons rapidly and "simultaneously" with the electrode. By "simultaneously" we mean that, visually, the cyclic voltammogram looks as if it were that of a monoelectronic wave. One must question the notion of the isopotential for the many ferrocenyl units at the periphery of a dendrimer. In theory, all the standard potentials of the n ferrocenyl units of a single dendrimer are distinct even if all of them are equivalent and independent. This situation arises since the charge
I
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
S
111
112
ALONSO ET AL. 9-allyl dendrimer 7
FcSi(Me)2H ^ Karstedt cat
9-ferrocenyl dendrimer 11
FcSi(Me)2H > Karstedt cat
27-ferrocenyl dendrimer 12
\ 27-allyl dendrimer 8
' FcSi(Me)2H
81-allyl dendrimer 9
Karstedt cat
>
81-ferrocenyl dendrimer 13
f
FcSi(Me)2H ^
243-allyl dendrimer 10
243-ferrocenyl dendrimer 14
Karstedt cat Scheme 23.
100.0--1 ^
99.5
^
99.0
H
98.5H
% ^
98.0^ 97.5-2
0
Velocity (mm/s) Figure 1. Zero-field Mossbauer spectrum of the 243-ferrocenium dendrimer 14 at 4 K showing the single line, which corresponds to the classic almost zero quadrupole splitting known for ferrocenium itself. Isomer shift: 0.57 (1) mm s~^ vs. Fe; G = 100 (4);
of the overall dendrimer molecule increases by one unit of charge every time one of its ferrocenyl units is oxidized to ferrocenium. The next single-electron oxidation is more difficult than the preceding one since, the dendritic molecule
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
having one more unit of positive charge, it is more difficult to oxidize because of the increased electrostatic factor. Thus, the potentials of the n redox units are statistically distributed around an average standard potential centered at the average potential (gaussian distribution).^^ In practice, the situation is complicated by the fact that the dendritic molecule, as large as it may be, is rotating much more rapidly than the usual electrochemical time scales.^^'^^ Under these conditions, all the potentials are probably averaged. The fast rotation is also responsible for the fact that all the ferrocenyl units come close to the electrode within the electrochemical time scale. Consequently, there is no slowing down of the electron transfer due to long distance from the electrode even in large dendrimers. Indeed, the waves of the ferrocenyl dendrimers always appear fully electrochemically reversible indicating fast electron transfer. The ferrocenyl dendrimers also adsorb readily on electrodes, a phenomenon already well-known with all kinds of polymers.^^ When polymers contain redox centers, the adsorbed polymers have long been shown to disclose a redox wave for which the cathodic and anodic waves are located at exactly the same potential, and the intensity of each wave is proportional to scan rate. Continuous cycling shows the stability of the adsorption of the electrode modified in this way. The ferrocenyl dendrimers described show this phenomenon as expected. The stability of the electrode modified by soaking the Pt electrode in a CH2CI2 solution containing the ferrocenyl dendrimer and cyclic scanning between the ferrocenyl and ferrocenium regions is all the better as the ferrocenyl dendrimer is larger. For instance, in the case of the 9-ferrocenyl dendrimer, scanning twenty times is necessary before obtaining a constant intensity, and this intensity is weak. With the 27-, 54-, 81-, and 243-ferrocenyl dendrimers, only approximately ten cyclic scans are necessary before obtaining a constant wave, and the intensity is much larger. When such derivatized electrodes are washed with CH2CI2 and re-used with a fresh, dendrimer-free CH2CI2 solution, the cyclic voltammogram is obtained with A^p = 0. Other characteristic features are the linear relationship between the intensity and scan rate and the constant stability after cycling many times with no sign of diminished intensity (Fig. 2). Under these conditions, one may note that the argument of the fast rotation of the dendritic molecule to bring all the redox centers in turn close to the electrode does not hold for modified electrodes. Some redox centers must be close to the electrode and some must be far. It is probable that a hoping mechanism in the solid state is responsible for fast electron transfer and for averaging all the potentials of the different ferrocenyl groups of a single dendritic molecule around a mean value. The proximity of the ferrocenyl groups at the periphery of the dendrimer is a key factor allowing this hoping to occur since it is known that electron transfer with redox sites which are remote or buried inside a molecular framework is slow, if at all observable.^^"^^
113
114
ALONSO ET AL.
V - 0.05 V s v = O.IOVs v^0.20Vs v = 0.30Vs v = 0,40Vs
-03
0.5
V vs FeCp2
0:5
Figure 2, Cyclic voltammogram of the 243-ferrocenyl dendrimer ("243-Fc") in CH2CI2 solution containing 0.1 M [n-Bu4N](PF6): (a) in solution (10-"^ M) at 100 mV s"'' on Pt anode; (b) Pt anode modified with "243-Fc" at various scan rates, dendrimer-free clear CH2CI2 solution (inset: intensity as a function of scan rate; the linearity shows the expected behavior of a modified electrode with a fully adsorbed dendrimer).
Vm. AMIDOFERROCENYL DENDRIMERS AS SENSORS FOR THE RECOGNITION OF H2PO7 AND HSOj The recognition of anions is a challenging problem which has been addressed for a long tirne.^^"^^ The first use of dendrimers as sensors was reported in 1996 when it was found that amidoferrocene dendrimers were able to recognize oxo-anions such as H2PO4 and HSO4 .^^'^^ Mononuclear, tripodal and dendritic (9- and 18-ferrocenyl) compounds containing amidoferrocenyl termini were compared. Electrochemistry proved to be a better technique than ^H NMR for the purpose of sensing. Titration was carried out in an electrochemical cell containing the sensor molecule, and the cyclic voltammetry wave was recorded as the tetra-A^-butylammonium salt of the anion was added. In the case of HSO4 , a progressive shift of the single Fe(II)/Fe(III) wave towards less positive potentials was observed until one equivalent of anion per dendritic branch was added. In the case of H2P0^, a new wave grew at a less positive potential upon addition of the salt. The intensity of this wave increased proportionally to the amount of anion added until one equivalent of anion was added. Concomitantly, the intensity of the initial wave decreased as the new wave increased, and the initial wave completely disappeared when one equivalent of anion per
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
115
Table / . Titration of the amidoferrocene dendrimers by various n-Bu4N"^ salts monitored by the variation AE° ( ± 2 0 , in mV for one equivalent of anion per branch) of the standard redox potential E"" of the redox couple in cyclic voltammetry
H2P0;
HSOJ
1-Fc
3-Fc
9-Fc
18-Fc
45 e
110 30
220 65
315 130
For HSO4 , the variation AE° is represented in Ref. 3 for the various dendrimers.
dendritic branch was added. It clearly appears that the shifts A^"" of potentials observed after addition of one equivalent anion per dendrimer branch (Table 1) considerably increase in the seriesi 1-Fc -^ 3-Fc —> 9-Fc —> 18 Fc. These experiments show a dramatic dendritic effect represented in Fig. 3 for the titration with the HSOJ anion. The magnitude of interaction with the anion increases as follows: H2PO4 > HSO4 > c r > N o ^ In fact, the interactions with Cl~ and NO^ appear to be weak with these particular metallodendrimers. Studies using other dendrimers are underway. Both situations upon titration — appearance of a new wave and shift of the initial wave — have already been analyzed from the thermodynamic standpoint.^^ In the first situation in which H2P0^ is concerned, Eq. 1 applies: A£°(V) - 0.0591og[ir(+)//^(0)]
(1)
at 25°C. Measurement of A^"" leads to K(-\-)/K(0). The determination of K(-\-) requires the determination of ^ ( 0 ) , the binding constant ^(0) between the neutral ferrocene form of the dendrimer and H2P04^, in the present case by ^H NMR using Hynes' EQ NMR program.^^ Indeed the shift of the amide proton also shows that the equivalence point is reached after addition of one equivalent H2POJ per dendrimer branch (from (5NH = 6.82 ppm before titration to 6.65 ppm after this addition). In the second situation concerning the other anions, this apparent binding constant ^ ( 0 ) between the neutral ferrocene dendrimer and the anionic substrate is very small (> 1) and does not intervene in the expression of AE° (Eq. 2): AE^Y)
= 0.0591og[cir(+)]
(2)
at 25°C, were c is the concentration of added anion. Thus, K(-\-) is directly accessible by measurement of AE° only (Tables 1 and 2). The ^H NMR monitoring of the titration is best achieved using Hyne's program.^^ At this time, it is not as useful in the case of H2P0^ as in the case
ALONSO ET AL.
116
IS-Fc
number of equivalents of n-Bu4N'^ HSO4" per ferrocenic unit
2
^ ; ^
CO
3-Fc
N_:_#^ CO
^
oC V
9-Fc
Fe
18-Fc
Figure 3. Titration of 1-Fc (1-Fc = [FeCp(Ti5-C5H4CONHCH2CH20Ph)]), 3-Fc, 9-Fc and 18Fc (10~^ M) by [n-Bu4N[[BF4] 0.1 M at 20°C in CH2CI2 using cyclic voltammetry (reference electrode: SCE; working electrode: Pt; sweep rate: 100 mV/s).
of the other anions (vide infra) because the interaction is weak between H2PO4 and the neutral amidoferrocene form. Indeed, equivalent points are very variable and very far from corresponding to one equivalent anion per branch, whereas they do so for the ferrocenium form which more strongly binds the different anions. In general, the ferrocenium form of the tripod or dendrimer binds the anions relatively strongly. The reason is the synergy between the electrostatic attraction
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
117
Table 2. Apparent association constants /((+) (±10%) determined in CH2CI2 by cyclic voltammetry for the amidoferrocene dendrimer series from the shift of standard redox potentials using Eqs. 1 and 2
H2PO4 HSO~
1-Fc
9-Fc
18-Fc
9390 544
216.00 8530
61400
For 9-Fc, KM was determined from the combination of K{0) determined by ^ H NMR in CD2CI2 and the K{+)/K{0) ratio obtained from the cyclic voltammogram using Eq. 1. For 18-Fc, the /C(+)//C(0) ratio was found to be 219000.
and the intermolecular hydrogen bonding formed between the acidic amide H atom and the anionic substrate through an oxygen atom of an oxo-anion or the halogen anion. Both factors are important and, if one of them is absent, the interaction becomes loose and cannot be used for sensing (except in the case of H2P0^ for the dendrimers). This effect has previously been recognized by Beer and others and used.^^"^^ Of special interest here is the dramatic dendritic effect observed for the anions. Even when the synergy between the electrostatic and H-bonding is fulfilled, the A£° value is unobservable or small when the amidoferrocene used is monometallic (1-Fc) or trimetallic (3-Fc). The shape selectivity designed in the dendrimer is crucial and its effect is much more marked for 18-Fc than for 9-Fc as the ferrocene termini are closer to each other when the dendritic generation increases. This dendritic effect is thus maximum for the generation (18-Fc), which precedes steric saturation by ferrocene groups on the dendrimer surface (36-Fc). It can be understood insofar as the insolubility of sterically saturated ferrocene dendrimers is complete in all the solvents for the following generation. In the amidoferrocene dendrimers, the amide H atom is located on the branch located behind the ferrocene unit providing the surface bulk. Thus, the anion must reach the inside of the microcavity formed by the amidoferrocene units at the surface of the dendrimer. These conditions become optimal for redox sensing and recognition by the close ferrocene units at the 18-Fc generation, since the channels allowing the entry of the anions into the surface microcavity to reach the amide H atom are as narrow as possible.
IX. CATIONICN-ALKYLANILINE IRON-SANDWICH DENDRIMERS AS SENSORS FOR THE RECOGNITION OF CHLORIDE AND BROMIDE ANIONS The polycationic 24-FeAr dendrimer of Scheme 10b was also useful for the recognition of Cl~ and Br~ whereas the results with the oxo-anions were not good. The best method appeared to be the shift of ^NH monitored in the ^H
118
ALONSOETAL
Table 3, Apparent association constants /((+) (dm^ mol~^) (±10%) determined from the variation of the 8NH signal in DMSO-de at 20°C with the EQ-NMR program^^
ciBrHSO-
1-FeAr
3-FeAr
24-FeAr
10 2 14
118 129 461
1221 431 6
Small values of the order of 10 (without a physical meaning) reflect the lack of equivalence point and underline the dendritic effects. For HSO4 , a negative dendritic effect is observed by comparing the values obtained for the 3-FeAr and 24-FeAr complexes.
NMR spectra upon addition of the tetra-A^-butyl ammonium salts of the halides. The titration with the monometaUic, tripodal trimetallic and 24-FeAr dendritic complexes was compared in order to investigate the dendritic effect. Whereas the results of the titration of the mono- and tripodal trimetallic complexes are poor and do not provide an equivalence point, titration of the 24-FeAr dendrimer yields very nice results with equivalence points for both Cl~ and Br~. Very interestingly, however, the equivalence point for Cl~ corresponds to one equiv. Cl~ for three dendritic branches (one dendritic tripod), whereas the equivalence point for Br" corresponds to one equivalent Br~ per dendritic branch (Fig. 4, Table 3). In these A^^-alkylaniline complexes, the NH group is acidic because of the electron-withdrawing properties of the CpFe"^ unit, and one hydrogen bond (N...H...X~) can be formed. The size of the halide turns out to be a key factor in the interactions with the dendritic termini and exo-cavities. The larger Br~ anion also interacts less strongly than Cl~ with the NH group because the electrostatic interaction between NH and the halide anion is weaker with Br~ than with Cl~. In summary, these two series of metallodendrimers are useful and complementary in anionic recognition, the amidometallocene dendrimers being best suitable for sensing the oxo-anions, but not the halides, and the polycationic Fe-A^-alkylaniline dendrimers being most useful to recognize the halides.
X. OTHER FERROCENE DENDRIMERS The research of references has been carried out with Chemical Abstract till nearly the end of 2000 using ferrocene(a)dendrimer and ferrocenyl(a)dendrimers with up to two words between ferrocene or ferrocenyl and dendrimer. The Cuadrado-Moran group has reported dendrimers containing from 4 to 16 equivalent SiMciRFc groups (R = CH2CH2 or NHCH2CH2)^'^ which can form derivatized electrodes.^^ Amperometric biosensors have been developed for the titration of glucose in blood.^"^ The Madrid group investigated the extension of ferrocene mediators to ferrocene dendrimers in order to circumvent the problem of the instability of ferrocenium in solution.^^ Recently, this group
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
0
1
2
3
4
119
5
Number of ec|uiv. of Ch added per branch
0
1
3
2
4
5
Number of equiv. of Br* ud4ed per branch
*
lOCl-Fe)
Fe-^-
llO-Fe)
Figure 4. Variation of 8NH for the exocyclic amine proton measured by ^ H NMR spectroscopy upon addition of n-Bu4NCI (a) and n-Bu4Br (b), given in number of equivalents n per branch to 1-FeAr, 3-FeAr and 24-FeAr
120
ALONSOETAL.
\J^/
-^^i?
C/iarf 3. 64-Amidoferrocenyl dendrimer synthesized by B, Alonso from Meijer's commercial 64-DAB dendrimer and ferrocenoyi chloride FcCOCI in the presence of NEt3.^^
reported recognition of H2PO4 and HSO^ with dendrimers containing 4, 8 or 16 SiNHCH2CH2Fc groups.^^'^^ The effect of these anions on the ferrocenyl wave was comparable with the previously reported amidoferrocenyl dendrimers described in the preceding section. This group also reported dendrimers terminated with 4 or 8 mixed-valence Si-bridged biferrocene group SiFcJ.^^ Finally, this group has reported the branching of carbonyl ferrocene groups onto the commercial DAB polyamine dendrimers, providing polyamidoferrocenyl dendrimers up to the 64-amidoferrocenyl dendrimer (Chart 3). Cyclic voltammograms showed reversible waves in CH2CI2, but not in DMF due to the instability of the ferrocenium form in DMF. These metallodendrimers adsorb on electrodes. The thermodynamic and kinetics of adsorption of these dendrimers has been studied using electrochemical quartz crystal microbalance (EQCM) by Abruna's group who also recorded molecularly re-
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwicii Groups
solved images on Pt (1,1,1) single crystal electrode using non-contact AFM (AFM Tapping mode).^^ Kaifer's group has studied these 4-Fc, 8-Fc and 16-Fc polyamidoferrocenyl dendrimers as guests for inclusion complexation by the hosts P-cyclodextrin and dimethyl-P-cyclodextrin providing aqueous solubility of the hydrophobic dendrimers, increasing with dendritic generation. Only one cyclic voltammetry wave was observed for the 4-Fc and 8-Fc. On the other hand, two waves for complexed and uncomplexed ferrocenyl groups were found for the 16-Fc dendrimer, which indicated that steric effects at the periphery inhibited the complexation of all the ferrocenyl units. ^^^ The DAB polyamine dendrimers were also sources of polycobaltocenium dendrimers^^^ in analogy to the cobaltocenium dendrimers already reported previously by our group.^^ With these cobaltocenium dendrimers, Kaifer's group studied the P-cyclodextrine: dendrimer assembly leading to solubilization of the neutral cobaltocene form in water which was monitored by the removal of the adsorption peak observed by cyclic voltammetry. ^^^ The DAB polyamine dendrimers were complexed by the Madrid group with a mixture of ferrocenyl and cobaltocenyl carbonyl chlorides giving statistical mixtures of ferrocenyl and cobaltocenyl branching. ^^^ Kaifer's group has synthesized asynmietric dendrimers of different sizes containing a single amidoferrocenyl group^^^ by reaction of Newkome and Behera's dendritic amine. ^^^ The heterogeneous electron transfer rate constant, the apparent diffusion coefficient and the standard redox potential E"" (from £1/2) decrease with increasing dendritic generation. This shows that buried redox centers transfer electron at lower rates than surface ones and that the dendritic structure shields the redox center. In addition, the electron transfer rate depends on the orientation of the dendrimer, i.e., how close the redox site is from the electrode surface.^^"^'^^^ The voltammetric response of the one-electron oxidation of ferrocene dendrimers depends on the molecular orientation effects which are controlled by the presence of carboxylic vs. cystamine termini. For instance, at high pH, the negatively charged carboxylate dendrimers approach the positively charged monolayer, and the ferrocene-electrode distance is kept maximum, resulting in a decreased electron-transfer rate.^^^ Ferrocenes have multiple properties which can be adapted to the dendritic structures.^^^ For instance, Togni's group reported dendrimers containing chiral ferrocenyl diphosphines which are efficient for highly enantioselective hydrogenation reactions.^^^^'^ The catalytic activity is very similar to that obtained with monomeric catalysts, but dendritic catalysts can be easily removed using commercial nanofiltration membranes due to their nanoscopic size. Ferrocenyldiphosphine ligands at the core of dendrimers having carbosilane tethers were used in Pd-catalyzed allylic alkylation.^^^^ Shu et al. synthesized Frechet-type poly (arylether) dendrimers with ferrocenyl groups at the periphery and showed that these groups were independent. ^^^
121
122
ALONSOETAL.
Catalano et al. synthesized heterometallic dendrimers containing 4 Pt(IV)-l,2bipyridine units and 8 ferrocenyl groups. ^^^ Deschenaux et al. reported the first examples of liquid-crystalline ferrocene dendrimers. These dendrimers were synthesized using l,3,5-tris(chlorocarbonyl)benzene as a core and cholesterol dendrons containing ferrocenyl units.^^^ Phosphorus-containing dendrimers have been synthesized with ferrocenyl units on the core, within the branches and at the periphery. Coulometry showed that up to 1354 ferrocenyl units were found at the ninth generation.^^^ Ferrocene dendrimers have been used as mediators with microelectrodes for the determination of analytes in clinical and environmental issues including drug screening. The ferrocene dendrimers are tagged with affinity ligands such as biotin, haptens, carbohydrates, DNA fragments etc. The system is applied in conjunction with amperometry, chronocoulometry and voltammetry.^^^ PAMAM dendrimers have been modified with ferrocenyl groups by reaction of ferrocenecarboxaldehyde in various amounts, and these redox-active dendrimers were applied to the construction of a reagentless enzyme electrode. A multilayer assembly of enzyme was constructed by alternate layer-by-layer deposition of ferrocenyl-tethered dendrimers with periodate-oxidized glucose oxidase. The optimum level of modification of surface amines was found to be 32%. The bioelectrocatalytic signal was shown to be directly correlated to the number of bilayers.^^^ An affinity biosensor system based on avidin-biotin interaction on a gold electrode was developed using this strategy. ^^"^ Pulse-field gradient spin-echo (PGSE) measurements on three different ferrocenyl phosphine dendrimers provided a practical alternative to classical methods used in organometallic chemistry for the determination of molecular size.^^^ Unsymmetrical dendrimers were analyzed by MALDI-TOF mass spectrometry with both conventional and electron-transfer matrixes. ^^^ Dendrimers containing a single ferrocenyl unit located "off-center" were submitted to interaction with P-cyclodextrin, and the dendritic groups were found to hamper the formation of inclusion complexes.^^^ Ferrocenyl dendrimers were synthesized using the formation of quinodimethane intermediates.^^^ Cyclic siloxanes were used as cores and frameworks for the construction of ferrocenyl dendrimers containing up to 16 ferrocenyl groups; these dendrimers were mediators for the oxidation of ascorbic acid.^^^ Ferrocenyl dendrimers were adsorbed on an Au surface, and electrochemical and ac-impedance were measured in order to investigate the porosity towards the redox couple. ^^^
XI. CONCLUSION In conclusion, the field of ferrocenyl- and metal-sandwich dendrimers has been exploding in the last two years. Applications are searched in biology, medicine,
Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups
material science and molecular electronics. Indeed the molecular electronics aspect interacts with the various other fields.^^ An interdisciplinary approach using these nanoscopic materials as parts of more sophisticated devices will obviously be a driving force at the beginning of this third millennium.
ACKNOWLEDGEMENTS We thank Dr Fran^oise Chardac (Universite Bordeaux I) and Professor Francois Varret (University of Versailles) for their kind assistance with the bibliography and Mossbauer spectroscopy, respectively. Financial support from the Institut Universitaire de France (DA), the CNRS, the universities Bordeaux I and Paris VI, the Ministere de I'Enseignement et de la Recherche Scientifique and the Region Aquitaine is gratefully acknowledged.
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MAGNETIC RESONANCE IMAGING CONTRAST AGENTS: THEORY AND THE ROLE OF DENDRIMERS Erik Wiener and Venkatraj V. Narayanan
I. II. III.
IV.
V.
Abstract Introduction MRI Concepts Related to Image Contrast Pharmaceutical Requirements for MRI Contrast Agents A. Efficacy B. Relaxivity C. Safety Physical Chemistry of MRI Contrast Agents A. Theory B. Inner Sphere Contribution C. The Assumptions in SBM Theory D. Predictions of SBM Theory E. Number of Inner Sphere Water Molecules F. Inner Sphere Proton Relaxation Time G. Outer Sphere H. Experimental Results Biological Issues A. Toxicity B. Stability C. Targeting Mechanisms
Advances in Dendritic Macromolecules Volume 5, pages 129-247 © 2002 Published by Elsevier Science Ltd. ISBN: 0-7623-0839-7
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130 130 131 134 135 137 139 139 139 139 144 149 149 150 165 167 188 189 191 194
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ERIK WIENER and VENKATRAJ V NARAYANAN
D. Contrast Agent Delivery E. Enzymatically and Physiologically Activated Contrast Agents . . . . F. Genetic Engineering of Endogenous Contrast Acknowledgments References
195 229 231 233 234
ABSTRACT Macromolecular MRJ contrast agents have the potential to improve diagnosis and monitor therapy in vivo. Dendrimers have been used extensively, and much research has gone into their design for various applications. Below we outline current developments and provide the basic concepts, both biological and chemical, for future innovations with these agents as molecular and cellular imaging agents. The design of these agents depends on a number of parameters. We show that the choice of dendrimer family, paramagnetic ion, ion-chelate, and method of attaching the ion-chelate complex to the dendrimer all influence the parameters that control the agents efficiency as an MRI contrast agent. We provide the basic theory for how contrast is achieved in MRI by reviewing basic MRI theory. We then discuss the chemical and biological principles behind MRI contrast agent design, and review many of the basic MRI contrast agent classes or applications with an emphasis on how dendrimers might play a role.
I. INTRODUCTION In this article you will find a review of the potential role dendrimers have in diagnostic imaging as magnetic resonance imaging (MRI) contrast agents. It outlines developments that already exist, and the authors hope to illuminate questions and areas for future research in this field. We try to keep this article self contained, and therefore provide the basic knowledge and examples required to design MRI contrast agents and point out where dendrimers may have a role. To this end we present a review of the chemical and biological principles that one must consider in developing MRI contrast agents, and apply them to the rational design of dendritic polymer-based MRI contrast agents. We start with some background information that explains how contrast is achieved in an MR image. This is followed by reviewing basic MRI theory and both the chemical and biological principles behind MRI contrast agent design, even though the readers can find plenty of exceptional review articles in the literature. If the readers have the time to examine other articles, many excellent reviews on the physical chemistry of MRI contrast agents exist in the literature. ^""^ Dendrimers are considered a technology in this review. Different families and different generations within a family have potential applications in the many classes of MRI contrast agents. Thus you will find a brief review of the different MRJ contrast agent classes and their applications followed by a brief description of how dendrimers might fit in. The field of MRI contrast agents extends over a vast literature, and we do not try to give a complete review of every small
Magnetic Resonance Imaging Contrast Agents
131
chelate, but present examples of how the principles apply to the different design criteria and applications. The hope is that with this the reader can apply these principles to dendrimer-based systems. For specific agents we refer you to the reviews listed above. Magnetic resonance imaging is a powerful diagnostic tool. Part of its strengths is derived from the modality's flexibility. This flexibility arises from its ability to collect both multiple and identical views as a function of many different parameters. Thus, with multiple views one sees different orientations of the object with contributions from different tissues. Imaging identical views with different parameters results in seeing the same tissues but with differing contrast. For example, two tissues which were isointense and indistinguishable in one image may have quite different contrast in the other image. Even with such flexibility, it is often desirable and advantageous to use exogenous contrast agents.
IL MRI CONCEPTS RELATED TO IMAGE CONTRAST Many good books describing nuclear magnetic resonance and magnetic resonance imaging (MRI) techniques exist, so we will only provide a brief review.^'^ In nuclear magnetic resonance a sample is placed in a static magnetic field BQ and a net excess of spins align along the direction of this static field (Fig. 1). If, in a frame of reference rotating at the same frequency as the individual magnetic
z
iMo
Figure 7. Development of net magnetization along a static magnetic field. In the presence of a magnetic field nuclei with spin quantum number equal to 0.5 will align either with or against the field. Based on the Boltzman distribution, more nuclei align with the static field than against it. The sum of the components of the magnetic moments projected onto the axis of the field is greater for the components with the field than against. This results in a net longitudinal magnetization, Mo, along the axis of the static magnetic field, z. The summation of the components in the x-y plane all cancel out, and the system has no net transverse magnetization, Mx,y. This system is said to be dephased.
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ERIK WIENER and VENKATRAJ V NARAYANAN
moments about the direction of the static field, we excite the system with a transient magnetic field, perpendicular to the static field, the net magnetization will precess or rotate around the transient field. Once this field is turned off the net magnetization freely precesses around the static field at the Larmour frequency, coo = —yBo. This precession is directly proportional to the static magnetic field strength, and this oscillating net magnetization induces an electric current in a coil of wire. Ructuations in the local magnetic field at the Larmour frequency result in the decay of the precessing magnetization. The fact that the precession frequency is proportional to the static magnetic field strength means that we can use a linear gradient to spatially encode the nuclei under investigation. Conventional MRI uses linear magnetic field gradients to spatially encode water protons as a function of proton Larmour frequency and phase. Computer algorithms are then used to reconstruct the image. The contrast between two compartments, or tissues, in that image is defined as the difference in signal intensities between two tissues scaled to a reference signal intensity.^ The reference signal intensity can be the signal intensity of either tissue, the mean signal intensity of the two tissues, the signal intensity of a third tissue, that of an exogenous standard solution, or the image background noise. This definition for image contrast implies that altering the signal intensity of one tissue relative to another alters the contrast of those tissues. In magnetic resonance imaging, the signal intensity depends on both instrument/machine and biological parameters. Although the machine parameters are specific for a particular pulse sequence, the biological parameters are the same. Equations for the functional dependence of the signal intensity of spin echo (^SSE), inversion recovery (5IR), and fast low-angle shot (5flash) pulse sequences are presented in Eqs. 1 through 3, respectively, and they demonstrate the main instrumental and biological parameters that determine the signal intensity. SsE = KN(U) * (1 - 2e-t™-TE/2]/r, ^ ^-TR/T, ) ^ ^-TE/T,
^^
5iR = KNiH) * (1 - 2e-T'/^' + le-t™-'^^/^'/^' - e"™/^') * e-'r^/^' (2) sin(a) (1 - e"™''^') 1 —Q-^^/^i
XC/T.
*COS(Qf)
The instrumental parameters are TR, TE, TI, and a. They vary depending on the pulse sequence used. The sample or biological parameters are A^(H), Ti, and T2. TR is the repetition time and is the time between successive activations of the transient magnetic field or excitation pulse. The echo time, TE, is the time between the excitation pulse and a refocusing pulse. The flip angle, a, is the angle between the net magnetization and the axis parallel to the static magnetic
Magnetic Resonance Imaging Contrast Agents
(a)
Bi
(C)
(b)
▲z
Br
133
Az
M.
Mxy
■^^
y
►
(d)
M yxy v = =0 Figure 2. Precession of the net magnetization around a secondary field, B i , in a rotating frame of reference. While the secondary or transient field is on, the net magnetization will rotate around it the simplified manner being depicted in (a), assuming a rotating frame of reference. A 90° pulse tips the net magnetization into the x'-y' plane. The transverse relaxation time, Ti, characterizes the loss of magnetization in the x'-y' plane, or the dephasing of the system (b,c). The re-equilibration of the net magnetization along the z axis is characterized by the longitudinal relaxation time, T\.
field following an excitation pulse. The biological parameter A^(H) is called the spin density, and is merely the number of detectable nuclei per unit volume. For conventional proton MRI it is the number of water protons per unit volume of tissue. The sample or biological parameters Ti and T2 are the intrinsic relaxation times of the protons associated with the tissue. Relaxation mechanisms prevent the system from saturating following a few excitation pulses and results from local magnetic fields fluctuating at the Larmour frequency. During an excitation pulse the net magnetization, whose component is aligned parallel to the applied static magnetic field, is tipped into the plane perpendicular to the applied static field (Fig. 2), given that a is a 90° pulse. The system will return to its thermal equilibrium configuration following an exponential time course (Fig. 3). The time constant for this return to the original net magnetization along the axis parallel to the applied magnetic field is the longitudinal relaxation time, T\. It is simply the time it takes for the magnetization along the axis parallel to the applied static magnetic field
134
ERIK WIENER and VENKATRAJ V NARAYANAN
Figures. Return of the longitudinal magnetization to its equilibrium value, M^ = Mo(l-e~^/^i). The longitudinal relaxation time, Ti, is the time required for the longitudinal magnetization to recover to 63% of its equilibrium value, M^ = 0.63Mo.
to return to 63% of its original value. It is a measure of the time it takes for the excited spins to reach equilibrium with their surroundings. The transverse relaxation time, T2, is the time constant for the decay of the magnetization perpendicular to the applied magnetic field. It is simply the time for the detected signal to decay by 63%. The intrinsic tissue relaxation characterized by the longitudinal and transverse relaxation times results from fluctuations in the local magnetic field that occur at the proton resonance frequency. This means that selectively inducing fluctuating local magnetic fields at the appropriate frequency can induce changes in contrast.
III. PHARMACEUTICAL REQUIREMENTS FOR MRI CONTRAST AGENTS The dependence of the signal intensity and therefore contrast on the three biological properties described above implies that one can induce contrast by selectively altering any of the three properties. One can achieve this end with either direct or indirect agents. Direct contrast agents are similar to those used in CT or radio imaging where the imaging modality directly detects the presence of the compound. For CT this is by the attenuation of the X-rays resulting from their absorption by the contrast agent. For radio imaging, this is achieved by the detection of the radiation emitted by the contrast agent. For direct agents in magnetic resonance imaging this is achieved by altering the spin density, N(x), that is the number of observable nuclei per unit volume. One can increase the signal, as is the case with using fluorine agents with ^^F-MRI^~^^ or by using D2O in ^H-MRI or spectroscopy.^^"^'* Agents that alter the two relaxation properties of tissue are indirectly detected. One measures their effect on the water proton relaxation times.
Magnetic Resonance Imaging Contrast Agents
135
Like any pharmaceutical agent, either diagnostic or therapeutic, MRJ contrast agents must be efficacious and safe. Contrast agents by their very nature alter image contrast. Thus an efficacious agent must induce contrast between two tissues, or a tissue and a pathology. This constraint imposes both chemical and biological requirements. A. Efficacy Chemical Requirements of MRI Contrast Agents
The chemical requirements for direct and indirect agents significantly differ. Direct agents must contain the nuclei that the particular MR imaging protocol is sensitive to. These agents are essentially density replacement agents. The efficiency of these agents depends on the number of detectable nuclei per mole of molecule. For example in ^^F-MRI, the more MR identical fluorines attached to the contrast agent, the easier it is to detect. High amplification systems can achieve this end, like fluorodendrimers. Agents that alter Ti and T2 are not detected directly and have different chemical requirements. These agents are indirectly detected by their effect on the respective water proton relaxation times (ri 2) or relaxation rates (1/ Ti 2). Thus, indirect MRI contrast agents must induce local magnetic field fluctuations that occur at the absorption frequency. This means that any interaction that induces such magnetic field fluctuations can be used to design MRJ contrast agents. Magnetic dipole-dipole, electric quadrupole, chemical shift anisotropy, scalar coupling, and spin rotation interactions all induce such fluctuating magnetic fields. Most MRJ contrast agents induce fluctuating magnetic fields through magnetic dipole-dipole interactions between magnetic moments of paramagnetic ions and water protons. Paramagnetic ions are so effective because the magnetic moment of an unpaired electron is approximately 650 times larger than that of a proton. Ions with large numbers of unpaired electrons have very large magnetic moments, and can induce very large magnetic field fluctuations (Table 1). The total magnetic field seen by nuclei equals the sum of the static magnetic field plus the local magnetic field, Eq. 4. Bj^Bo
+ Bi
(4)
The local magnetic field seen by one magnetic moment depends on the size of the other magnetic moment (/X5), the magnitude of the vector connecting the two magnetic moments |J/,5|, and the angle between the vector connecting the two moments and the static field (0) (Fig. 4). Thus anything that alters these parameters alters the local magnetic field. In liquids, random thermal motions affect the rotational, vibrational, and translational degrees of freedom. Rotation of the metal ion-water complex alters 0. Stretching vibrations along the
136 Table 1,
ERIK WIENER and VENKATRAJ V NARAYANAN Large numbers of unpaired electrons create large magnetic moments
Ion
Number of unpaired electrons (S)
Biologically important paramagnetic ions Fe2+ 4/2 Fe3+ 5/2 Co2+ 3/2 Co3+ 4/2 Cu2+ 1/2 Mn2+ 5/2 Other paramagnetic ions Cr3+ Cr2+ Gd3+ Eu2+ Tb3+ Dy3+ Organic free radicals
Magnetic moment, fx (BM) 5.1 5.9 4.1 5.0
5.9 3.8 4.9 7.6
3/2 4/2 7/2 7/2 6/2 5/2 1/2
9.5 10.6
Bloc
±jUs(3cos20-1) 'IS
Figure 4. Fluctuations in the local magnetic fields at the Larmour frequency induce relaxation. Rotational, vibrational, and translational degrees of freedom alter the local magnetic fields. The magnitude of these fields is related to the size of the magnetic moment that is interacting with the proton, the distance between the proton magnetic moment and magnetic moment of the interacting nuclei, and the relative rotation of these two moments.
water-ion bond or the proton-oxygen bond alters the magnitude of the vector connecting the two magnetic moments |d/,5|, as does the relative translation or diffusion of the two magnetic moments, |J/,5|. This model of the causes of
Magnetic Resonance Imaging Contrast Agents
137
fluctuating magnetic fields illustrates some of the chemical properties that go into the fluctuating local magnetic fields, and therefore contrast agent theory. It shows that the size of the magnetic moment inducing the relaxation, the number of unpaired electrons, the distance between the magnetic moments, the molecular tumbling of the complexes, and the relative diffusion of the two magnetic moments all influence the fluctuations in the local magnetic field. This means that protons can experience fluctuating magnetic fields when they are either directly bound to or near the metal ion center. We classify these sources of local magnetic field fluctuations as inner and outer sphere contributions. B. Relaxivity The ability of an agent to induce changes in water proton relaxation rates is measured by the relaxivity of that agent. Relaxivity is defined as the change in relaxation rate per unit concentration of paramagnetic ion. The addition of paramagnetic ions to a solution or tissue increases both the longitudinal and transverse relaxation rates. The observed relaxation rate, (l/ri,2)obs. after the addition of the paramagnetic ion is the sum of the native relaxation rate of the solution or tissue, (1/TI^2)N^ plus any contribution from the paramagnetic species, (l/ri,2)p, Eq. 5. 7'(l,2)obs
'
^(1,2)N
+ ^
^(l,2)p
(5)
In dilute solutions of paramagnetic species, the observed relaxation rate is linearly dependent on the metal ion concentration. The slope of the dependence is the relaxivity and the y-intercept is the native relaxation rate of the sample or tissue prior to the addition of the paramagnetic ion or contrast agent, Eq. 6. ^(l,2)obs
= r(i,2) * [M] +
^(1,2)N
(6)
The relaxivity is the incremental increase in relaxation rate per unit concentration of paramagnetic ion. The units are conmionly given as mM~^ s~^. Protons can experience fluctuating magnetic fields when they are either bound to the metal ion center or in the vicinity of the paramagnetic species. The ability to divide these contributions into outer and inner sphere contributions to the fluctuating magnetic fields means that the paramagnetic contribution to the observed relaxation rate can also be divided into outer and inner sphere contributions, Eq. 7 (Fig. 5, inner and outer sphere mechanisms). This means that the observed relaxation rate is a function of the inner and outer sphere contributions to the relaxivity. This implies that we can separate the relaxivity into outer sphere, (ri,2)o. and inner sphere components, (ri 2)1, Eq. 8. 1 1 1 + 7:;r— (7) (Tl,2)p (T,,2)i (Tl,2)o
138
ERIK WIENER and VENKATRAJ V NARAYANAN
Figure 5. Inner and outer sphere mechanisms determine the paramagnetic contributions to the relaxation rate. The inner sphere contribution stems from the interaction of the metal magnetic dipole with the dipole of the proton attached to the water molecule directly coordinated to the metal ion. Outer sphere contributions occur as a result of the metal ion magnetic moment interacting with the magnetic moments of protons on the water molecules in the second coordination sphere or bulk solvent molecules translationally diffusing by the solute.
1 7'(l,2)obs
1 ^(l,2)p
+
1
1
^(1,2)N
(Tl,2)i
= [(ri,2)i + (n,2)o] * [M] +
1 (^'1,2)0
1 '(1,2)N
1 ^(1,2)N
(8)
Biological Requirements ofMRI Contrast Agents The need to change the contrast of one tissue or pathology relative to adjacent tissue also imposes biological constraints. The signal intensity of one tissue must preferentially change relative to the signal intensity of the other, for a period of time that is long relative to the duration of the diagnostic procedure. This can be achieved by either the preferential distribution of a compound between two tissues or a differential effect on a biophysical property. For direct agents one must preferentially deliver the agent to one tissue relative to another. That is, the agent must accumulate in one tissue more than another to induce regional concentration differences. For indirect agents we must only selectively alter the relaxation rates of one tissue relative to another. This can be accomplished by either concentration differences or by tissue specific differences in the relaxivity. For example, the tissue specific or pathology specific enzymatic activation of a contrast agent.
Magnetic Resonance Imaging Contrast Agents
139
C. Safety The safety criteria for diagnostic agents in general are more stringent than those for therapeutic agents. Diagnostic agents must be nontoxic, but they must also have few if any serious side effects. Doses of transition metals and lanthanide ions used in MRI are relatively high, and at these concentrations the free metals and ions are quite toxic and many of the ligands that are used to reduce the toxicity of the ions through chelation are toxic when free of ions.^^~^^ This means that one source of toxic effects from paramagnetic MR contrast agents result from the dissociation of the ion-chelate complex into the free ion and ligand, or from the transmetallation and loss of the ion from the complex.^^'^^ The toxicity of an ion-chelate complex partly depends on the complexes stability relative to its rate of excretion from the body. That is, the complex must stay together longer than it stays in the body. This means that both kinetic and thermodynamic stability are important, in addition to selectivity. Thermodynamic stability determines how much free ion goes into solution, while kinetic stability determines how fast the free ion is formed. Selectivity is a measure of stability in the presence of other ions that can compete with Gd(III) for the chelate.
IV. PHYSICAL CHEMISTRY OF MRI CONTRAST AGENTS A. Theory The two contributions from inner and outer sphere sources to the relaxivity allow us to conveniently divide the theory of relaxation into two parts. In depth reviews on the subjects of inner and outer sphere relaxation exist.^^~^^ In this section we present the concepts that illustrate which parameters affect both the outer and inner sphere relaxivities. We explain why the efficiency of macromolecular or dendrimer-based MRI contrast agents depend on the choice of paramagnetic ion, the chelate, the macromolecule or dendrimer, and how the chelate is linked to the dendrimer or macromolecule. B. Inner Sphere Contribution The Solomon-Bloembergen and Bloembergen-Morgan (SBM) equations describe the inner sphere contribution of the dipole-dipole interaction between the magnetic moments of paramagnetic ions and protons along with the scalar contribution quite well.^^"^^ This theory provides insights into the parameters that a chemist can modify in the hopes of improving the efficiency of MRI contrast agents. However, two major problems develop in the quantitative application of this theory to bulk solvent relaxation data, especially to macromolecular agents.
140
ERIK WIENER and VENKATRAJ V NARAYANAN
Bulk Water Figure 6. Bound water is responsible for transferring the inner sphere contribution to the bulk solvent. The water proton residence life time of the bound water molecule governs the fraction of the bulk solvent that experiences an inner sphere dipole-dipole interaction. The inner sphere contribution depends on the amount of the paramagnetic Ions, the number of water protons in the inner sphere, their relaxation time, and how long they experience the interaction.
The first problem arises from the multiparameter curve fitting techniques used to derive the parameters from experimental data. The solutions obtained by the fitting procedures are not unique.^^'^^ This means that other data in addition to bulk solvent relaxation data are required. Koenig has indicated a need for independently derived values of the water residence time.^^~^^ The second problem arises from the fact that many of the assumptions used to derive SBM theory are not valid for macromolecular agents. Many theories were developed to take these new restrictions into account.^^'^^ This leads to an uncertainty about which theory to use. In this section we will first explain what chemical properties influence the inner-sphere contribution to the relaxation rate, and how the invalid assumptions affect the quantitative predictions of the relaxation rates. We show that even though quantitative analysis is difficult, SBM theory provides an accurate qualitative description of the inner-sphere contribution to the relaxation rate with enormous predictive value. From this theory comes an understanding of how the choice of paramagnetic ion, the chelate, the macromolecule, and how the chelate is linked to the macromolecule affect the relaxivity. For dilute solutions of paramagnetic ions Swift and Connick^^ and Luz and Meiboom^^ showed that the inner sphere contribution to the relaxation rate of the bulk solvent is governed by the chemical exchange of water molecules between the bulk solvent and the inner coordination sphere of the paramagnetic ion (Fig. 6). The relaxation rate, l/(Ti)i or 1/(72)1, depends on the number of exchangeable water molecules in the metal ion inner coordination sphere (q), the life time of the water protons in the inner coordination sphere (TM), the relaxation time of the protons from the water molecules bound to the paramagnetic metal ion, (T^i 2)M. and the mole fraction of the metal ion (Pm), Eq. 9.
Magnetic Resonance Imaging Contrast Agents
1
Pmq
(T{)i
Pmq
141
TIM
hu
+
(9)
^M
^m/
\T2M
(T2)i \ T2M
"^m /
+ A
+ Aw2
The mole fraction equals the molar concentration of the paramagnetic ion divided by the sum of the molar concentration of water and the paramagnetic ion, P„ = [M]/([H20] + [M]) = [M]/(55,500 mM + [M]). The initial condition of dilute solutions of paramagnetic ions imply that the concentration of water is more than 100 times larger than the concentration of ion and that we can approximate the mole fraction by P^ = [M]/55,500 mM. We can then rearrange Eq. 9a to show that the inner sphere relaxivity associated with Ti depends on the number of detectable exchanging inner sphere water molecules, the time spent in that inner sphere coordination site by the water protons, and the relaxation time of the bound water protons, Eq. 10. 1
[M]
q
(10) = (n)i * [M] (Ti)i TIM + TM 55,500 SBM theory provides the expression for the bound water proton relaxation rates 1/T(I^2)M, Eqs. 11a and lib. The theory divides the relaxation rate into dipolar and scalar interactions. ^ TIM
^ 2 y , V 5 ( 5 + l)p^ 15
r
7re
3re
+
4
+ 1
1
T2M
15
S(S + 1) *
(,)'
y,'g'S(S+l)p'
4 + 3UJ
4TC +
(11a)
1 + a>?r2 "S'-e
13re l+a)ir2
S(5 + l ) * re +
3rc
+ l + « , 2.r2 \^
l+coWl
(lib)
The dipolar interactions occur over a distance "through space" and the scalar interactions occur as a result of orbital overlap, i.e., "through bonds" or "contact". The first part of the sum in Eq. 11a models the dipolar term and the second part of the term accounts for the scalar contribution. The theory predicts that the electronic g factor (g), the spin quantum number (5), the distance between
142
ERIK WIENER and VENKATRAJ V NARAYANAN
the proton magnetic moment and the magnetic moment of the paramagnetic ion (dis), a measure of the overall interaction or correlation time of the two moments (TC), and the absorption frequencies (ct)s and coi) affects the longitudinal relaxation rate of the bound metal. Both the proton gyromagnetic ratio (YI), and the Bohr magneton (^) are physical constants. The overall correlation time, tc, is a measure of how long it takes fluctuations in the local magnetic fields of the bound protons to occur. For the dipolar interactions these fluctuations may arise from rotation of the ion-chelate-water complex, exchange of the water molecule with the bulk solvent, or changes induced by fluctuations of the electron magnetic moment, each with its own time scale, Eq. 12. 1 1 1 1 - = - + - + — tc
Ts
tr
(12)
TM
The rate of chemical exchange between the bulk solvent and the coordinated waters is I/TM- The longitudinal relaxation rate of the electron magnetic moments is given by 1/TS, and the rate of molecular tumbling for the ionchelate-water complex is given by 1/rr. For the scalar interactions the magnetic fluctuations arise from exchange of the water molecule with the bulk solvent, or changes induced by fluctuations of the electron magnetic moment, Eq. 13.
(.3)
i =i + l T^e
^S
^M
The general theory of magnetic resonance applies to magnetic moments regardless of whether they are generated by electrons or nuclei. The relatively fast electron spin relaxation imposes a time dependence on the magnetic moment of the electron spin. This dynamic time dependence creates fluctuating magnetic fields, and implies that both the electronic relaxation time, rs, and therefore the nuclear relaxation induced by the electronic relaxation may be frequency dependent, Eq. 14. 4rv
(14) ts Ll+^s^v 1 Electronic relaxation may result from several processes including rotation of the ion-chelate complex or symmetry distortions of the complex induced by collisions with solvent molecules. For paramagnetic ions with more than one unpaired electron the dominant relaxation mechanism in solution is from the modulation of the zero field splitting. Zero field splitting (ZFS) results from electrostatic interactions between the ligand electrons and the unpaired electrons of the paramagnetic ion, and from the development of covalent-like bonds between the ligand and the paramagnetic ion as a result of the overlap of the ligand electron orbitals with the orbitals of the unpaired electrons of the paramagnetic ions. ZFS is the creation of an energy difference between atomic
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X"
X"
d^2
X'
dxy
d,2.y2
X"
X"
dyz
^ZX
Figure 7. Electron density and directionality of d orbitals. In the absence of ligands the energy levels of the d orbitals are degenerate. A single unpaired electron outside of closed shells will have an equal probability of being in any one of the orbitals. Adapted from Cotton, F.A. and Wilkinson, G. Advanced Inorganic Chemistry: A Comprehensive Text, 3rd edition, WileyInterscience, p. 557.
orbitals of the paramagnetic ion that had the same energies prior to coordination with the Hgand; it is a spUtting of the energy levels in the absence of an applied field. The magnetic moments of the ligand electrons lift the degeneracy of the magnetic energy levels associated with the paramagnetic ions. This imposes both a directionality and an effect of ligand field distance. This separation of energy levels arises because electrons prefer to occupy orbitals in which they can maximize the distance from the ligand electrons. For example, in the absence of an applied field or ligand the sole unpaired electron in a paramagnetic ion in a d orbital will occupy any d orbital (Fig. 7) with equal preference. That is the energy levels of the d orbitals are equal (Fig. 8). If ligands coordinate along the ±Z direction, then the electron would prefer to occupy those orbitals without any Z directionality. That is, a difference in the energy levels between the d^2 and other orbitals develops as a result of ligand coordination. Similarly if identical ligands coordinate along the ±X and Y axes, then the energy level of the dx2-y2 orbital rises to the same value as the d^z. This results in a static ZFS. If the ligand field is highly symmetric then the ZFS averages to zero as a result of rotational motions.
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ERIK WIENER and VENKATRAJ V NARAYANAN
Octahedral complex Figure 8. Coordination of ligands by metal ions lifts the orbital degeneracy of the metal ion. Octahedral complexes have d^ and d^2_^2 orbitals with higher energy than the d;c>'/ dy^, and dzx- This results from the directionality of the orbitals and repulsion of like electronic charges that results from putting the electron density of the ligands along the six axes. Adapted from Cotton, F.A. and Wilkinson, G. Advanced Inorganic Chemistry: A Comprehensive Text, 3rd edition, Wiley-lnterscience, p. 558.
Zero field splitting also results from the overlap of the ligand atomic orbitals with those of the paramagnetic ion to create molecular orbitals or covalent-like bonds. The greatest contribution to this effect results from spin-orbit coupling which modulates how much the ligand and paramagnetic ion share the electrons. Spin-orbit coupling significantly affects the magnetic properties of the ionchelate complex. It explains deviations of the measured magnetic moments of a paramagnetic ion from those calculated using only spin values. It also explains the inherent temperature dependence of some magnetic moments. Regardless of whether an ion-chelate complex has a static ZFS or if the ZFS averages to zero, collisions of solvent molecules with the ligands can result in a transient ZFS. The rate or frequency of the collision-induced fluctuations seen by the paramagnetic ion is described by l/ty. The constant B contains the electronic and ZFS parameters and is related to the magnitude of the transient ZFS. The time dependence or modulation of the zero field splitting results from collisions of solvent molecules with the ligands and causes a transient ZFS in systems with high symmetry. These collisions randomly alter the ligand-metal bond length or direction and their associated orbital overlap and electric fields. This in turn alters the magnitude and direction^^ or just the direction^^ of the electronic energy level splittings. C. The Assumptions in SBM Theory
SBM theory assumes that the ZFS is equal to zero. However SBM theory is still valid if the magnitude of the ZFS is smaller than the inverse of the relevant
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correlation time, l/tc. If this occurs then the static ZFS averages to zero. In the case of macromolecular agents, the static ZFS is often greater than l/tr, and SBM theory is then only valid if the ZFS is smaller than the electron Larmour frequency, cos. For Mn(II) and Gd(III) ions this means that SBM theory is invalid at low fields, below 3 to 8 MHz proton Larmour frequency (0.07 to 0.19 T). For ions with larger ZFS, e.g., Ni(II), SBM theory is invalid below proton Larmour frequencies of 60 MHz or 1.4 T. In general, for systems with large ZFS, SBM theory predicts greater relaxivities than are actually measured. The presence of large values for the ZFS results in lower relaxivities of the ions at low field strengths, relative to cos, than those predicted by SBM theory. If a paramagnetic ion has nuclear spin it can interact with the spin of the unpaired electrons. The SBM equations are invalid if this interaction is of the same order as the electron Larmour frequency, cos. For Mn(II) ion-chelate complexes this occurs at a proton Larmour frequency of about 1 MHz. This means that based on initial assumptions SBM theory would predict relaxivities that are too high in the low field region for Mn(II) complexes as a result of nuclear electron spin interactions of the paramagnetic ion. However, the theory actually applies under less stringent conditions. Pfeiffer et al.^^ proved that if the water residence time, Tm, were much greater than the electron relaxation time, Ts, then SBM theory is valid. In the case of Gd(III) the electron and nuclear spin interactions are very small. Furthermore the ion-chelate complexes used for MRI have tm values vary from 200 ns to more than 1000 ns while the rs values are less than 10 ns, although chelates have been prepared with tm = 10 to 15 ns. SBM theory also makes assumptions about the nature of the dipole on the metal ion. The dipolar term was derived assuming that the electron spin was a point dipole centered at the metal ion. This neglects any distribution into the electron orbitals (Fig. 7) and consequently any electrostatic interactions from the ligands. It also neglects any bonds formed between the ligands and the metal ions. Any distribution of the dipole around the metal ion will be perceived as an error in either the distance between the magnetic moments of the proton and the metal ion or as an error in the number of inner sphere water molecules. Waysbort and Navon^^ demonstrated that for Mn(II) hexahydrate r is underestimated by less than 2% and q is overestimated by 13%. More recently Kowalewski et al.^^ developed a more general treatment which measures an rgff. They found that the point dipole approximation was valid for the protons on the water molecule but not the oxygen which was the coordinating atom. The proton intemuclear distances and r^ff differed by less than 1%. They found in general that significant deviation from the point dipole approximation occurred only for the atoms directly coordinated to the metal ion."^^ The electronic g-factor is the value that when multiplied by the Bohr magneton and the square root of 5(5 + 1) gives the average value of the
146
ERIK WIENER and VENKATRAJ V NARAYANAN
magnetic moment for the unpaired electrons of the paramagnetic ion, and it is derived from the ^-tensor. It measures the directionality of the magnetic properties for the entire coordination complex. This directionality depends on the electrostatic field imposed on the electron by the ligands. SBM theory assumes that the ^-tensor is isotropic. For S'-state ions like Gd(III) and Mn(II) it is believed that the g-factors are isotropic. The development of SBM theory used the assumption that the electronic relaxation had only one relaxation time. This assumption is not accurate. Like nuclear magnetic relaxation, electron magnetic relaxation also exhibits longitudinal and transverse relaxation mechanisms, i.e., tsi and rs2, and their values may depend on the strength of the magnetic field too. Similar to NMR, the electron longitudinal relaxation time is greater than or equal to the electron transverse relaxation time. More than one relaxation mechanism may contribute to each relaxation time, and therefore they may have more than one correlation time. For example, in addition to the ZFS caused by distortions of the ligand field symmetry induced by collisions with solvent molecules described above, rotation may also influence electron relaxation. If this occurs then the electron relaxation time and the overall rotational correlation time are correlated and we cannot separate the two effects, i.e., TS and Zr from TC. We have also only talked about dilute solutions, if however we have concentrated solutions or the ion-chelate complexes are close to each other, then solute-solute interactions can occur via dipole-dipole mechanisms of two paramagnetic ions. This would introduce a translational component to the electronic relaxation in addition to rotation. Interactions between the electron spin and nuclear spin of the paramagnetic ion results in hyperfine splitting which can also create more than one relaxation time. For paramagnetic ions with more than one unpaired electron, several energy levels develop in the presence of a static magnetic field, and this causes more than one electron longitudinal and transverse relaxation times. Most paramagnetic ions used for MRI have more than one unpaired electron, for example Fe(II), Mn(II), and Gd(III). For these ions the assumption of a single electron relaxation time is invalid. The situation simplifies for Mn(II) and Gd(III). Rubinstein et al."^^ and Luckhurst^^ showed that only one tsi and rs2 were important when COST^W < 1. and that they are equal. Three longitudinal and three transverse relaxation times were found for Mn(II) when COST^W > 1» but only one of the longitudinal relaxation times dominates the relaxation mechanism at any one electron Larmour frequency. Similarly, although Gd(III) also has many longitudinal and transverse relaxation times, only one longitudinal relaxation time dominates the relaxation mechanisms. A number of research groups have modified the SBM equations to account for different values of tsi and tsi-^'^''^^''^^ The last two assumptions deal with the very nature of molecular tumbling by macromolecular systems. In order for SBM theory to be quantitatively accurate.
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bulk H2O
Figure 9. Multiple rotational correlation times can result from attaching an ion-chelate complex to macromolecules. Overall tumbling, segmental motions, and rotation about the metal-wateroxygen axis can result in multiple rotational correlation times and anisotropic rotation.
molecular tumbling must be isotropic, that is it must have only one rotational correlation or rotate with only one degree of freedom. For many backbones used to make macromolecular MRI contrast agents molecular tumbling occurs with multiple rotational correlation times, that is they are anisotropic and rotation occurs about different axes. For example, a water molecule in the inner coordination sphere of an ion-chelate complex attached to a macromolecule may undergo rotation about a number of axes (Fig. 9). Rotation may occur about the bond linking the water molecule to the paramagnetic ion, the linker connecting the ion-chelate complex to the macromolecule, or as a function of the tumbling of the entire ion-chelate-macromolecule complex. Woessner^^'^ showed that if all the rotational rates were smaller than the proton Larmour frequency, then the longest rotational correlation time dominates T^. The other faster motions only reduce the magnitude of the dipole-dipole interaction between the magnetic moments of the water proton and the paramagnetic ion. That is it depended on the frequency and amplitude of rotation. The theory proposed by Woessner predicts that the angle between the axis of rotation and the vector connecting the proton to the paramagnetic ion significantly influences the reduction of the dipole-dipole interaction. Thus the relative position of the nucleus and the axis of rotation affects the magnitude of the relaxivity. This has potentially important implications for the placement of the linker molecule relative to the water molecule. The range of reduction can vary from 0 to 75% for an axis that is 180° vs. one that is 90°. This reduction would only occur as long as the rotational correlation time dominated tc, that is for as long as the rotational correlation time remained smaller than the electronic relaxation time, Ts, and the water residence time, tm. Once the rotational correlation time exceeds the electronic relaxation time then the effects of anisotropic motion are reduced.
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The last assumption has the greatest potential to impact the quantitative validity of SBM theory. The theory was derived assuming the Redfield limit of fast exchange for electron relaxation. This means that it was assumed that the rotational correlation time was much smaller than the electronic relaxation time, Tr < Ts. This is not the case for macromolecular agents in which rs < T^. This is called the "slow motion" regime or non-extreme narrowing condition. Benetis et al.^^ showed that even in the slow motion regime SBM theory might be valid if the motion of the electron spin is negligible relative to molecular tumbling or water exchange. Note that the relaxation rates that are calculated for systems in the "slow motion" regime using Redfield theory are less than the actual values. The above discussion showed that a number of assumptions used to derive SBM theory are invalid and that errors arise in the use of this system to quantitatively derive physical parameters from relaxation data of ion-chelate complexes. This is especially true for macromolecular or dendrimer-based systems. Many new theories or modifications of SBM appear in the literature that account for the presence of ZFS, multiple electronic relaxation times, anisotropic rotation, and slow motion relative to electronic relaxation.^'^'^^'^^ In addition, many good reviews of the older theories exist.^^""^^"^^ Below we discuss some of the newer theories and their implications. Abernathy and Sharp developed a spin dynamics method to calculate electron and nuclear relaxation times."^^ In this technique both intermolecular and intramolecular relaxation effects are calculated using a non-Redfield limit approach and taking into account ZFS interactions and rotationally dependent electron spin relaxation, rsr- The theory assumes isotropic rotation and uses the Redfield theory to calculate tsv The spin-dynamic simulations accurately calculate paramagnetic enhancements of nuclear relaxation rates for systems with spin quantum numbers greater than 1. Bertini et al.^"^ developed a theory for enhancement of nuclear relaxation by paramagnetic ions that accounted for ZFS, anisotropic g and spin-orbit coupling in the "slow motion" regime. The method uses Bloembergen-Morgan equations to calculate rsi,2, and thus the actual field dependence of the electronic relaxation time in the presence of ZFS may differ from the calculated value. The theory also only accounts for a single longitudinal and transverse relaxation time, but the authors claim this has negligible impact on the calculations. Strandberg and Westlund"^^ developed a modified SBM theory which accounts for transient ZFS with two electronic relaxation times and the "slow motion" regime for the electronic relaxation, i.e., ts < Tr. The use of two correlation times for electronic relaxation allows for different relaxation mechanisms to be considered as a function of environments. The modified theory adds a correction term to the dipolar interaction and an order parameter to the SBM theory. They specifically analyzed Gd(III) systems. The theory simulated data for Gd(III)(H20)g"^, four low molecular weight ionchelate complexes with tr < ts, and a macromolecular complex with rs < tr
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fit quite well. Although the correction term could theoretically reach values as high as 15%, the corrections for the systems tested reached a maximum of less than 3%. Introduction of the order parameter improved the nuclear relaxation dispersion profiles. The above results indicate that application of unmodified SBM theory to a study of inner sphere proton relaxation enhancement by paramagnetic ions results in small errors. The unmodified theory cannot be used for accurate quantitative analysis of the physical parameters relevant to the relaxation enhancement of water protons by macromolecular complexes, but demonstrate qualitatively the physical parameters that effect the inner sphere relaxivity of even macromolecular or dendrimer-based agents. D. Predictions of SBM Theory
Although the assumptions used to derive the SBM theory are invalid and cause difficulty in making quantitative analysis from relaxation data alone, the theory allows us to make very powerful qualitative predictions about the chemical parameters that influence the relaxivity and how to modify them. In this section we discuss the predictions made by SBM theory of what chemical and physical properties are important to the optimization of MRI contrast agents. We also present simulations of how the different parameters affect the relaxivities of MRI contrast agents. A look at Eq. 10 shows that the relaxivity depends on the number of water molecules (q), the relaxation time of the protons on the water molecules bound to the paramagnetic ion (riM), and the time that the water molecules stay coordinated to the paramagnetic ion (TM). E. Number of Inner Sphere Water Molecules
The value of q is the number of inner sphere water molecules that exchange with the bulk solvent rapidly enough to be observed. The relaxivity is proportional to q, all else being equal. Although free Gd^+ and Mn^+ have higher relaxivities than chelated complexes of the same ions, the free ions are toxic. Thus the biological need for safety and stability can result in a reduction of the number of inner sphere water molecules, and a concomitant reduction in the relaxivity. For example, the inner sphere relaxivity of Gd^"^, Gd(III)-EDTA, and Gd(III)-DTPA at 3TC and 20 MHz are approximately 7.0, 4.6, and 2.0, and the number of inner sphere waters are approximately 8 or 9, 2 or 3, and 1, respectively. While a clear trend between q and ri exists it is apparently not linear. This occurs because TIM and r^ are not the same for each system and the exact number of water molecules is not known. What these results imply is that the choice of chelate drastically affects the relaxivity. One of the mechanisms of this effect on the relaxivity is from the differences in q. The safety and stability of the complex often overrides the relaxivity issues, but
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ERIK WIENER and VENKATRAJ V NARAYANAN
improvements in relaxivity, safety, and stability are not necessarily mutually exclusive. Different chelates may exist with the same q value, but with different stabilities and toxicities of the ion chelate complex, or systems with one inner sphere water molecule might have higher kinetic stabilities than systems with no inner sphere water molecule. F. Inner Sphere Proton Relaxation Time
The inner sphere proton relaxation time, TIM, has the greatest number of chemical and physical parameters that affect the relaxivity (see Eqs. 11 and 12). Many of these parameters can be adjusted to optimize the relaxivity. SBM theory predicts that the electron spin angular momentum (5), which is related to the number of unpaired electrons, the distance between the magnetic moments of the protons and paramagnetic ion (J), and the overall correlation time (TC) influence the dipolar contribution to TIM and that the overall correlation time and an electronic correlation time influence the scalar contribution. Both correlation times are affected by two or more relaxation mechanisms associated with rotation, electronic relaxation, or chemical exchange, each of which has its own correlation time tr, rs, and TM, respectively. The choice of ion, chelate, macromolecule, and linker can all be used to optimize the relaxivity of an agent. The choice of paramagnetic ion influences the number of unpaired electrons, the electronic relaxation time, and the maximum number of inner sphere ligands through its ligand field symmetry. The number of unpaired electrons influences the electron spin angular momentum, S. The nature of the \/T\u dependence on S, i.e., the factor S^ -\- S, is depicted in Fig. 10. ff all else is equal then those paramagnetic ions with 7 unpaired electrons and S = 7/2 (e.g., Gd^"^) have a 21 times higher I / T I M than paramagnetic ions with one unpaired electron and S = 1/2 (e.g., Cu^^), and a 2 fold higher value than paramagnetic ions with 5 unpaired electrons and S = 5/2 (e.g., Mn^+ and high spin Fe^+). In addition to the number of unpaired electrons, the choice of ion also affects the electron relaxation time. Fast electron relaxation times reduce the relaxivity through their dominance of tc and tg. Thus any mechanism that induces electronic relaxation will reduce the relaxivity. This dominance of TC only arises when ts < Tr and TM, and it allows us to divide paramagnetic ions into two classes: those with fast electronic relaxation times and those with long electronic relaxation times relative to tr of ion-chelate complexes. For low molecular weight ion-chelate complexes, Tr is approximately 10~^^ to 10~^^ s. Table 2 is organized by increasing electronic relaxation time, and within each time interval or range of TS, it is organized by increasing spin quantum number. Those ion-chelate complexes with electron relaxation in the range of 10~^^ to 10~^^ have relaxivities that are approximately 20 times smaller than those
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Magnetic Resonance Imaging Contrast Agents
Effect of Spin Quantum Number
CO
1.5 2.0 2.5 Spin Quantum Number (S) Figure 10, Effect of the spin quantum number, S, on the multiplication factor 5"^ + 5 in I / T I M If all else is equal, then S'^ -\- S acts as a multiplicative factor in determining I / T I M - This means that ions with S = 7/2, Gd(lll), will have a twofold higher value than ions with S = 5/2, Mn(ll) and Fe(lll), and a 21 fold higher value than ions with S = 1/2. In reality all the other parameters are not the same, but Gd(lll) still has much higher values than Mn(ll) and Fe(lll).
Table 2. Ion
Electronic relaxation, r^
Spin quantum
Dy3+ Tb3+ Eu2+
10-^2^0 10-^3 10-12 10-12 to 10-13
5/2 6/2 7/2
8.75 12
Cr2+
10-11 to 10-12
Fe2+
10-1° to 10-11
4/2 4/2
6.0 6.0
#, S
5*
(5+1)
15.75
Cr3+
10"^ to 10-10
3/2
3.75
Co2+ Co3+ Fe3+
1 0 - ^ to 10-10
3/2 4/2
3.75
1 0 - ^ to 10-10
Mn2+
10-^ to 1 0 " ^ 10-«to10-^
5/2 5/2 7/2 1/2
8.75 8.75 15.75
Gd3+ Organic free radicals
1 0 - ^ to 1 0 - ^
6.0
0.25
Only a few paramagnetic ions have relatively long electronic relaxation times. Gadolinium ions have the best combination of r^, /x, and 5 * (5 + 1) making it the ion of choice for most contrast agents. Iron has ferro- and superparamagnetic properties making iron oxide particles very useful too. The next ion of choice is Mn2+.
ERIK WIENER and VENKATRAJ V NARAYANAN
152
0.01
0.1
1
10
100
Proton Larmor Frequency (MHz) Figure 11. Increases in the rotational correlation time increase relaxivity. The parameters used are for DOTA at 25°C with r = 3.14, q = I, TSO = 4.73 x IQ-i^ s, tv = 1.1 x IQ-ii s, TM = 244 ns. The rotational correlation time, tr, is varied at 0.077, 0.15, 0.31, 0.62,1.24, 2.48, 4.96, 7.5, and 10 ns.
with electronic relaxation times greater than tr, i.e., 10"^^ to 10~^^ (e.g., for a good compilation of early data see Lauffer'^^).^^'^'^^ From the standpoint of large S and long TS S B M theory predicts that contrast agents prepared from Gd^"^, Mn^+, and Fe^"^ will in general have higher relaxivities than those prepared from other ions. Having chosen ions with long electronic relaxation times relative to tr, SBM theory predicts that increasing the molecular tumbling time, Tr, should increase the relaxivity of an ion-chelate complex. This assumes that the water residence time is long relative to tr and ts, which is a valid assumption for most ion-chelate complexes used (see below). Fig. 11 shows the effect of increasing the rotational correlation time on the nuclear magnetic relaxation dispersion (NMRD) profile of Gd(III)-DOTA predicted by the dipolar contribution to the SBM theory. The scalar contribution for Gd(III) is extremely small and negligible. The parameters used in the simulation are given in the figure legend, and are typical of those found for Gd(III)-DOTA complexes. Increasing the rotational correlation time increases the relaxivity with a peak in the relaxivity appearing in the high field region. As tr increases from 0.077 to 10 ns the electronic relaxation time will dominate TQ. That is ts will become less than ij, and the frequency dependence of ts described by the Bloembergen-Morgan equations, Eq. 14, becomes evident as TS now dominates tc and contributes to I/TIM-
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35
2
3
4
5
6
7
8
9
Rotational Correlation Time (ns) Figure 12. The peak value in the relaxivity levels off as the rotational correlation time becomes relatively long.
In addition to the peak that develops in the high field relaxivity, the gains in relaxivity associated with increasing the rotational correlation time levels off at around 4 to 6 ns, for the parameters used. This is better shown in Fig. 12. Most of the gain in relaxivity, about 90%, occurs by the time r^ reaches about 3 ns. This value will differ depending on the chelate as both TS and TM will differ. The rotational correlation time for a hard sphere is given by Eq. 15. ^'
6Aot
kT
(15)
The viscosity of the medium is given by r;, Vh is the hydration volume of the molecule (4/37rr^ with r the radius of hydration), k is the Boltzman constant, and T the absolute temperature in K. For globular proteins or spherical macromolecules in water at 20°C, the overall rotational correlation time increases about 1 ns for every 2400 Da."^^ Thus a spherical macromolecular system whose only rotational correlation time is derived from the overall molecular tumbling should have a rotational correlation time of approximately 4 to 5 ns at around 9600 to 12,000. Eq. 15 implies that one can increase the rotational correlation time by increasing the viscosity of the medium seen by the chelate or by increasing the hydration volume of the ion-chelate complex by attaching it to a macromolecule. Maximum increases in relaxivity are achieved when tr becomes much larger than r^.
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ERIK WIENER and VENKATRAJ V. NARAYANAN
Recall that MRI contrast agents must only selectively alter Ti^. This can be achieved by selectively altering the relaxivity of an agent in one compartment relative to another compartment or by creating a concentration difference between the two compartments. Viscosity differences are found within biological systems, and may be taken advantage of. Lauffer^"^ points out that agents targeted to the cellular cytosol may have higher relaxivities than those found in the extracellular fluid space, as the cytosol has a higher viscosity.^^'^^ The microviscosity within a cell varies with position and intracellular organelles.^^"^"* This means one might achieve differing relaxivities depending on where the agent goes within the cell. Even different cell types have differing microviscosities. For example erythrocytes have a higher viscosity than myocytes. The rotational correlation time of hemoglobin is more than two times that of myoglobin.^^ Rat fast-twitch and slow-twitch muscle fibers have different viscosities^^ and SV40 transformed fibroblasts have a 30% lower apparent viscosity than their normal counter parts.^^ The viscosity of tissue or interstitial space can also vary as a function of tissue and/or pathology. Chondroitin sulphate and aggrecan significantly alter the viscosity of hyaluronan solutions which might affect the viscoelastic properties of extracellular fluids and matrices.^^ Bleomycin-treated rat lung parenchyma show changes in the viscoelastic properties.^^ Alternatively, agents that distribute throughout mucinous tumors might experience the higher viscosities associated with mucous,^^"^^ and may have higher relaxivities than the same agent in neighboring tissue. More intriguing is the targeting of enzymes specific for a cell type, tissue, organ, or pathology that alters the relaxivity of a contrast agent. This is an area of much current interest. Attachment of ion-chelate complexes to macromolecules may be achieved either through direct covalent linkage or by noncovalent interactions like the hydrophobic effect. Binding of paramagnetic ions to macromolecules and the resultant change in relaxivity is called the proton relaxation enhancement effect, and was first observed by Eisinger et al.^^ with DNA and with proteins by Cohn and Leigh.^ Rigidly attaching paramagnetic ions to macromolecules would provide extremely large proton relaxation enhancements, as shown by Fig. 11. Any motion of the chelate separate from the overall molecular tumbling could significantly alter the magnitude of the enhancement effect. Such segmental motions would reduce the relaxivity as described earlier for anisotropic motion. If the rotational rates are not large relative to the proton Larmour frequency, then the protons see an average rotational correlation time, and this may dictate the optimal choice of macromolecule used as a backbone for macromolecular contrast agents. Such segmental motions were identified early for linear polymers. A number of reports indicate that these segmental motions dominate the rotational correlation time of linear polymers which become independent of molecular weight after 10,000 Da.^^~^^ Kellar et al.^^
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have prepared a possible exception to the rule for linear polymers. They prepared a series of linear copolymers from a,co-alkyldiamines and Gd(III)-DTPA to form a nonionic bisamide derivative with the chelate in the backbone structure. This polymeric system showed an increase in relaxivity associated with an increase in the number of methylene carbons and subsequent molecular weight. They hypothesize that intramolecular hydrophobic interactions between the methylene groups make the polymer more rigid and reduce the local segmental motions. Toth et al.^^ demonstrated that the water residence time of these polymers is independent of the alkyl chain size. For globular proteins with lysine residues available for conjugating ion-chelate complexes one must worry about rotations of the methylene groups. Jardesky et al.^^ reported the rotational correlation time of the 8-CH2 group in lysine from a number of literature sources and found that it had values of 0.4 to 1.5 ns, depending on the protein. Metzler^^ examined the rotational correlation time of amine and hydroxyl terminated ammonia core polyamidoamine dendrimers and classified two types of carbons based on their rotational correlation times. Terminal carbons exhibited very rapid molecular dynamics with rotational correlation times increasing from 0.012 to 0.025 ns as the molecular weight increased from 360 to 87,300 g/mole. Internal carbons exhibited slower molecular motions with rotational correlation times of 0.09 to 2.8 ns over the same molecular weight range. That is, they observed continual increases in the rotational correlation times of interior carbons for dendrimers with molecular weights well above 10,000. This is quite different from what was observed with linear polymers. All the macromolecules used as backbones for macromolecular contrast agents have segmental motions which will effect the maximum obtainable relaxivities. In the case of dendrimers, these motions are adjustable by the appropriate choice of dendrimer family. Dendrimers also offer a second technique for increasing the rotational correlation time. Choosing the appropriate initiator core allows one to control the shape.^^ Cigar and ellipsoid shapes can be synthesized. Ellipsoid shapes result in longer rotational correlation times than predicted by Eq. 15. In general elongated shapes rotate more slowly as was shown for chymotrypsin.^^ Such a contribution from anisotropic rotation would effect the overall average rotational correlation time if the rotational rates, l/tr, were less than the proton Larmour frequency. In addition to covalently linking ion-chelate complexes to macromolecules, one can increase the rotational correlation time by noncovalent interactions of ion-chelate complexes with macromolecules. These interactions may result from ionic or hydrogen bonding or from hydrophobic or Vanderwall's forces. Potential target proteins with high concentrations in the blood or serum include serum albumins or globulins. In addition certain pathologies result in the secretion or shedding of large amounts of proteins or surface antigens into the blood or surrounding tissues which can be targeted.^^'^^ For example ovarian
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ERIK WIENER and VENKATRAJ V NARAYANAN
tumors secrete or shed the high affinity folate receptor into the tumor interstitium and serumJ^'^^ For macromolecular systems with Tr > tg, any mechanism that induces electronic relaxation would reduce the relaxivity. We showed earlier that ZFS is one mechanism of electronic relaxation, that this resulted from collisions of the ligands with solvent molecules, and from a lack of symmetry in the ligand field. This implies that any structure or chelate that increases the ligand field symmetry, and/or reduces the effect of a solvent molecule collision with a ligand, i.e., one that reduces the flexibility of the ligand or momentum transfer to the ligand, will reduce the ZFS and increase the electronic relaxation time. The correlation time characterizing the fluctuations in the ZFS, ty, and the magnitude of the ZFS, B, is given by Eq. 14. This equation implies that the electronic relaxation rate at zero field, l/tso, is proportional to the product of the correlation time characterizing the ZFS fluctuations and the magnitude of the ZFS, Eq. 16. — = SBXy, tso = j-r—
(16)
McLachlan describes the electronic relaxation rate from ZFS using a mean square zero energy term, Eq. 17.^^
/ ^ x ZFS ^ ^ ^
^
^^^ (—V2
+ , Jl\
2)
(17)
The zero field electronic relaxation rate depends on the spin quantum number (5), Ty, and the mean square of the energy separation imposed by the ligands in the absence of a magnetic field, which is a measure of the average magnitude of theZFS(A2),Eq. 18. / 1 \^^' [45(5+1)-3] . — = ^ * A^Tv
(18)
For ions with S = 7/2 then the corresponding zero field electronic relaxation time is inversely proportional to the mean square of the energy separation imposed by the ligands in the absence of a magnetic field and the correlation time of the fluctuations in the ZFS, Eq. 19. ^^0 = T
^
(^^)
Both expressions for the zero field electronic relaxation time imply that decreasing the product of ZFS magnitude and correlation time will make electronic relaxation at zero field less efficient, and increase the relaxivity in the low field region. One can accomplish this by either making B and A^ smaller and/or Ty smaller. Fig. 13 shows the effect of increasing the zero field
157
Magnetic Resonance Imaging Contrast Agents 'Cso(ns) 0 001
6 -
0.008
£ *
\.
0.07
2 .. ..
"N\
4 -
\\
■ >
x JO (D
0)
x: a.
2 -
"— ^'"y/^/
CO i_
•-.....-- / ^ / /
0)
c c
0 -
0.01
(
0.1
1
1
1
10
100
1000
Proton Larmour Frequency (MHz) Figure 13. Decreasing the average magnitude of the ZFS increases TSO and the relaxivity. In this simulation Zr = 0.077 ns, ty = 1.1 x 10"^^ s, TM = 244 ns, q = I, and r = 3.14 x 10"^. TSO is varied while holding ty constant, with TSO = 0.001, 0.004, 0.008, 0.02, 0.07, 0.2, and 2 ns. The highest value (2 ns) has the highest, and the lowest value (0.001 ns) has the lowest plateau value of the relaxivity.
electronic relaxation time on the relaxivity of rapidly rotating Gd based MRI contrast agents. In this simulation we use the parameters for DOTA defined in the figure legend. By holding ty constant and increasing TSO we are effectively reducing the magnitude, or its mean square, of the energy separation between the orbitals induced by the ligands following coordination of the paramagnetic ion. For very small TSO a peak appears in the high field region. This occurs because at these values of tso, TS dominates tc and its dependence on magnetic field strength manifests itself in the NMRD profile. For larger values of TSO the rotational correlation time dominates tc and the curve has a dispersion or inflection point where co^ = 1/tc. For rapidly rotating complexes increasing the zero field electronic relaxation time results in increasing the relaxivity of the ion-chelate complex in the low field region and only small increases occur in the high field region. For macromolecular systems with rotational correlation times longer than the electronic relaxation times, the characteristic peak in the relaxivity associated with the magnetic field dependence of the electronic relaxation time always appears in the NMRD profile. For contrast agents with long rotational correlation times, increasing the zero field electronic relaxation time results in higher relaxivities in both the low and high field regions. Although the largest percent
158
ERIK WIENER and VENKATRAJ V NARAYANAN
1000 Proton Larmour Frequency (MHz) Figure 14. Effect of increasing TV while holding TSO constant. Increasing TV while TSO remains constant shifts the peak in the NMRD profile and broadens it. The simulation parameters are q = l, Tso = 0.473 ns, r = 3.14 x 10"^ cm, Tr = 8 ns, and TM = 244 ns, with ty = 5 , 1 1 , 25, 50, 100, and 300 ps. The lowest value (5 ps solid line) has the lowest and the highest value (300 ps dash-dot-dot line) has the highest value of the peak relaxivity.
changes in the relaxivity occur in the low field region significant increases in the relaxivity are also predicted in the high field region too. The theory also predicts that the peak in the relaxivity shifts to lower field strengths as TSO increases. Thus the optimal or highest relaxivity in the high field region may occur at lower zero field electronic relaxation times, depending on what frequency one is optimizing for. In the previous section we held the correlation time for the ZFS fluctuations constant and altered the magnitude of the ZFS splitting. Altering the correlation time tv also modulates the relaxivity both through its effect on the zero field electronic relaxation time and on the frequency contribution to the overall electronic relaxation time, Eqs. 17 and 19. The simulation in Fig. 14 depicts the effects of increasing TV and holding TSO constant. This results in the simultaneous decrease in the magnitude of the ZFS. Fig. 14 shows that increasing ty and decreasing A^ raises, shifts, and broadens the peak in the NMRD profile. Alternatively one can increase the correlation time Ty and hold the magnitude of the ZFS constant, effectively decreasing TSQ. This results in reduced relaxivities in the low field region, but higher relaxivities in the high field region. Fig. 15. A shift of the peak in the NMRD profile also occurs. These simulations show that by tailoring ty, A^, and tso one can optimize the relaxivity of macromolecular
159
Magnetic Resonance Imaging Contrast Agents 70
Tv (ns)
60 50 -I 40 ] ^
30
■ >
*x
I
20 H
^
10 H
0.01
0.1
10
100
1000
Proton Larmour Frequency (MHz) Figure 15. Reducing tso by increasing tv and holding A^ constant increases the magnitude of the peak relaxivity, alters the peak position, and increases the ratio of the peak/low field relaxivity. The fitting parameters are ^ = 1, r = 3.14 x 10"^ cm, rr = 8 ns, TM = 244 ns, and A^ = 0.16 X 10^^, with Tv = 6,11, 25, 50, and 100 ps. The highest value (100 ps) has the lowest and the lowest value (6 ps) has the highest plateau value of the relaxivity, low field region of the NMRD profile.
contrast agents, and put the peak in the NMRD profile over a particular magnetic field strength. The simulations of SBM theory also show that designing the coordination complex for the largest zero field electronic relaxation time may not be optimal either. The important parameters are the magnitude of the ZFS and the correlation time of the fluctuations, and different combinations of these two parameters may yield different relaxivities in the zero field region, but similar values in the high field region. Fig. 16 shows a simulation of the inner sphere NMRD profiles for macromolecular contrast agents with Gd(III)-DTPA and Gd(III)-DOTA as the ion-chelate complex. Although the relaxivity in the low field region differs by 300 to 500%, it differs only by less than 10% in the high field region. This analysis predicts that the relaxivities at clinically relevant field strengths of macromolecular agents prepared from Gd(III)-DTPA or Gd(III)-DOTA should have very similar values. This may not be the case for their derivatives, as the contribution from TM will differ. The previous discussion of electronic relaxation only considers relaxation through a ZFS mechanism. If other mechanisms induce electronic relaxation, then other correlation times and multipliers must be introduced. If rotation
ERIK WIENER and VENKATRAJ V NARAYANAN
160
E
> x
0)
c o
1000 Proton Larmour Frequency (MHz) Figure 16. Theory predicts that the high field inner sphere contribution to the relaxivity of constrained DTPA (dotted line) and DOTA (solid line) complexes with Gd(lll) varies by less than 10%.
contributes to electronic relaxation then the rotational correlation time must be included. This makes separating the contributions of electronic relaxation and rotation to the relaxation rate of the bound water molecules difficult, as TS has a Tr component. SBM theory also assumed that dilute solutions of paramagnetic ions were used. This criterion prevented contributions from interactions of electron magnetic moments, that is electron-dipole electron-dipole interactions did not contribute to electron relaxation. While macromolecular agents may be used in dilute solutions, the local concentration of paramagnetic ions on the surface of the macromolecule may be high. Electron-dipole electron-dipole interactions begin at about 20 A, and start affecting electronic relaxation at about 6 A.^^ This means that the choice of dendrimer family and generation can affect these parameters as the number of surface groups per unit surface area increases. That is families where the dense packing limit occurs at smaller generations might have electron-electron dipole interactions that effect the relaxivity at earlier generations than those with the dense packing limit at higher generations. The water residence time, TM, of the ligand water molecules in the inner coordination sphere of the paramagnetic ion controls the length of time that the water molecule is exposed to the large magnetic moment of the unpaired electrons, and how many of the bulk solvent water molecules experience those magnetic moments up close. SBM theory predicts a parabolic response of the
Magnetic Resonance Imaging Contrast Agents
161
50
10000 Water Residence Time x^ (ns) Figure 17, The optimal water residence time is 10 < TM < 60 ns. Rotationally constrained ion-chelate complexes with water residence time of more than 60 ns, like Gd(lll)-DTPA and Gd(lll)-DOTA, will be TM limited.
relaxivity to the water residence time, Fig. 17. If the sampling time is too rapid then TM will be much less than tr and TS, and it will dominate l/tc. In this case the water molecules are not in the inner coordination sphere long enough for the electron magnetic moment to significantly influence the proton magnetic moment. On the other hand, if the sampling time, TM, is too long then TM is equivalent to or larger than TIM, and a low relaxivity results. That is, not enough of the bulk solvent water molecules experience the electron magnetic moments "up close and personal". In Fig. 18 we simulate the NMRD profiles of Gd(III)-chelates complexes attached to macromolecules and vary the water residence time. The parameters used in the simulation are those for DTPA with a rotational correlation time of 3.66 ns, approximately that found for an ion-chelate complex attached to a generation-6 ammonia core polyamidoamine dendrimer. Adjusting the water residence time has the largest percentage effect on the peak relaxivity in the high field region. Altering the water residence time from 240 to 20 ns results in a 44% increase in the peak relaxivity. These simulations show that SBM theory predicts that macromolecular contrast agents with water residence times longer than about 60 ns are limited by TM- It predicts that the effect of a limiting water residence time on macromolecular agents is both a reduction in the maximum obtainable relaxivity and a reduction in the rotational correlation times that will continue to produce increases in the relaxivities.
162
ERIK WIENER and VENKATRAJ V NARAYANAN
1000 Proton Larmour Frequency (MHz) Figure 18. Theory predicts that attaching ion-chelate complexes with shorter water residence times to dendrimers will improve the innersphere contribution to the total relaxivity. Simulation using DTPA constraints attached to a G = 6 dendrimer and varying the water residence time.
Longer water residence times reduce both the maximum relaxivity achievable and the rotational correlation time at which this maximum is achieved. Fig. 19 shows a simulation of the peak relaxivity as a function of the rotational correlation time at two different water residence times. In this figure we show the peak relaxivity as a function of rotational correlation times for a system with an optimal water residence time of 24 ns and the water residence time obtained for DTPA attached to macromolecules via one of the acetate arms with an amide linkage, TM = 620 ns. The first 90% of the increase in relaxivity associated with increasing the rotational correlation time for a system limited by the water residence time, TM = 620 ns, is predicted to occur at around 1.6 ns, whereas the same relative increase for a system independent of water residence time, TM = 24 ns, is predicted to occur at about 4 ns. The above analysis stems from simulations of SBM theory, and the quantitative value is not as great as the qualitative predictive value. These simulations show that SBM theory predicts that for dendrimer-based MRJ contrast agents the increases in the peak relaxivity associated with increasing generation will plateau as the rotational correlation time becomes very large relative to the electronic relaxation time, and that increasing the water residence time will reduce the maximum attainable relaxivity of a particular dendrimer family, and reduce the generation that this plateau starts at. For macromolecular agents based on other backbones, SBM theory predicts that increases in the peak relaxivity will
Magnetic Resonance Imaging Contrast Agents
163
Rotational Correlation Time (ns) Figure 19, Limitations by the water residence time reduce the maximum achievable relaxivity and the rotational correlation time that they occur at. Simulation of the peak inner sphere relaxivity as a function of rotational correlation times for systems v^ith water residence times of 24 ns (solid line), and 620 ns (dashed line).
reach a plateau as the molecular weight or hydrodynamic radius are increased, and that increasing the water residence time will reduce the molecular weight or hydrodynamic radius that the plateau starts at. This assumes that the overall rotational correlation time of these other backbones increases with molecular weight or hydrodynamic radius and is not governed by local segmental motions. The rotational correlation time at which one stops obtaining increases in the relaxivity of a macromolecular contrast agent depends on the water residence time of a TM limited system, and this will depend on the chelate and the linker. Like the rotational correlation time and the electronic relaxation time, the water residence time can be systematically modified. The water residence time depends on the mechanism of water exchange, the length of the water-ion coordination bond, and the relative crowding of the ligand field. An excellent review of this parameter and exchange mechanisms can be found in Reference 1. For Gd(III)-based systems there are two main exchange mechanisms. The fastest mechanism relies on the ability of the Gd(III) coordination sphere to expand from 8 to 9 or have more than one inner sphere water molecule. In this mechanism an additional water molecule associates with the Gd(III)-complex prior to the bond breaking of the initial water molecule in the inner coordination sphere. This mechanism is called an associative mechanism. Exchange rates are higher for this mechanism, making residence times (the inverse of the
164
ERIK WIENER and VENKATRAJ V NARAYANAN
exchange rate) shorter. The Gd(III)-chelate systems used in MRI already have nine coordinating Ugands, and cannot expand the coordination number. These systems use the second mechanism which does not involve the incoming water molecule. This mechanism consists of the dissociation of the bound water molecule prior to the binding of the new water molecule. It is called a dissociative mechanism and includes an octacoordinated activated transition state with ^ = 0 whose formation is rate limiting. This results in a lower exchange rate and higher or longer residence times relative to the associative mechanism.^^ Powell et al.^^ indicate that a chelate structure which favors the dissociative mechanism for a system that already has nine coordinating ligands and only one inner sphere water molecule will have a shorter residence time and a longer exchange rate than one with a less crowded structure. They conclude that the crowding of the water-binding site determines the mechanism and rate of the water exchange for the types of systems found in current MRI contrast agents. Increasing the crowding of the inner sphere water coordination site favors the dissociative mechanism. "This would favor a dissociative water exchange mechanism giving the observed increase in the water exchange rate."^^ "All these results indicate that the best strategy for increasing water exchange rates on Gd(III) complexes for use as contrast agents is to increase the crowding at the water-binding site and so favor the dissociative exchange mechanism, and that one way to achieve this may be to modify the strength of the ligating groups."^^ Tweedle et al.^^ found that crowding the water coordination site resulted in longer water-ion coordination bond lengths, and that this resulted in shorter water residence times. This results from steric constraints that favor one isomeric structure with a shorter water residence time over a second known structure with a longer water residence time. The relaxivity is inversely proportional to the sixth power of the distance between the proton magnetic moment and the magnetic moment of the paramagnetic ion, dis, and in theory any large change in dis could offset the gain obtained by shortening the water residence time. Fig. 20 shows a plot of l/(dis)^ vs. dis. The largest changes occur for the shorter bond lengths. An increase in the bond length of 0.2 A from 1.5 to 1.7 A results in a 53% decrease in l/(Jis)^ and consequently I / T I M , vs. only a 32% decrease in going from 3.0 to 3.2 A. This means that shortening of the water residence time should be accomplished with less than a 0.2 A increase in the proton ion bond length. Alternatively, design of a stabile chelate with q = 2 will have both a larger q and faster associative exchange mechanism. In summary, SBM theory predicts that the inner sphere component of the relaxivity depends on q, TM, S, JIS, TS, and r^. All of these parameters are adjustable, and can be altered to optimize the relaxivity of dendrimer and macromolecular MRI contrast agents. The correct choice of ion influences q, S, and Ts. Rational design of the coordination chemistry influences q, dis, ts, and
Magnetic Resonance Imaging Contrast Agents
165
0.005
0.000
Distance between Magnetic Moments of Protons and Paramagnetic Ion (A) Figure 20. Tiny changes in dis have smaller effects on the factor (1/Jis)^ in the expression for l/TiM' Altering dis, found in clinically approved agents, from 3.0 to 3.2 only affects multiplicative factor (l/dis)^ by 32%, but can affect the water residence time by more than an order of magnitude.
TM- The choice of dendrimer family and generation or macromolecule and the chelate linker chemistry effects z^ and TMG. Outer Sphere In the previous section we examined the chemical and physical properties that affected the inner sphere contribution to the relaxivity. For ion chelate complexes with only one inner sphere water molecule, the outer sphere contribution to the relaxivity is quite significant, it contributes approximately half the total relaxivity. Freed,^"^'^^ Hubbard,^^ Koenig,^^'^^ and Pfeifer^'^ have developed theories for outer sphere relaxation. Freed showed that for translational mechanisms, the outer sphere relaxation depended on the spin quantum number (S), a correlation time for diffusion (TD), the distance of closest approach that can be achieved by the paramagnetic ion-chelate complex and the water molecule (d), and the electron relaxation time (rs), Eq. 20.
X Ujicos, TD, TS) + 3j(a>i, TD, TS)] * [M] = (ri)o[M]
(20)
166
ERIK WIENER and VENKATRAJ V NARAYANAN
The constants yi, ys, K and S have the same meaning as they do in Eq. 11. The distance of closest approach, Avogadro's number, and the correlation times for diffusion are given by d, A^a» and TD. The diffusional correlation time depends on the distance of closest approach and the diffusion constants of the water molecule and the paramagnetic ion-chelate complex, Eq. 21. TD
(21)
= 3(Di + Ds)
j(co, TD,rs) =
-f
Re L
V
rsj
1/2
3\
Ts/
V3V
rsj
J (22)
The spectral density functions J((JO, TD, TS) are the real part of a complex function, Eq. 22. These equations demonstrate that the outer sphere contribution to the relaxivity is a function of the number of unpaired electrons as it contributes to the overall magnetic moment and spin quantum number, the closest distance that the magnetic moments of the water molecule and the paramagnetic ion-chelate complex can get to each other (J), the electronic relaxation time of the paramagnetic ions (rs), and the diffusion coefficients of the water molecule and the paramagnetic ion (Di and Ds). The translational diffusion coefficient depends of the hydrodynamic radius (a), the temperature in degrees Kelvin (T), and the viscosity of the solution (r]), Eq. 23. kT D= ^ (23) onarj Boltzman's constant is given by k. This theory then predicts that attaching a small ion chelate complex to a macromolecule will increase the outer sphere contribution to the relaxivity too. This increase will only occur to the extent that the diffusion constant of the ion-chelate complex contributes to l/D. The theory also predicts that the electronic relaxation time, and its field dependence also significantly contributes to the outer sphere relaxivity. Therefore, the above attachment of an ion-chelate complex to a macromolecule will also affect the outer sphere contribution of the relaxivity for any paramagnetic ion whose electronic systems relax through rotational mechanisms too, beyond that of the effect on l / D . The above mechanism and its mathematical formalism explain the outer sphere contribution to the relaxivity for a system dominated by translational diffusion. One can consider it a special case of outer sphere relaxation where the second coordination sphere has a residence time TJ^ that is rapid when compared
Magnetic Resonance Imaging Contrast Agents
167
to the relative diffusion time. The second coordination sphere is composed of those water molecules that are hydrogen bonded to the inner sphere ligands and contribute to the solvation of the complex. Two other cases which have varying contributions of rotational and translational diffusion to the outer sphere component of the relaxivity also exist. This will affect the relative contribution of the outer sphere relaxivity to the total relaxivity. If the water residence time of the second coordination sphere is long relative to the relative diffusion constant, ^M ^ ^D, then the contribution to the relaxivity of the second coordination sphere follows the theory described for the inner sphere contribution. That is rotational diffusion will dominate the outer sphere contribution to the relaxivity. Alternatively the residence time of the second coordination sphere waters may approximate the relative diffusion time, T^^ TD. In this case both rotational and translational diffusion mechanisms contribute to the outer sphere component of the relaxivity. This analysis predicts that increasing rj^ and/or the number of second coordination sphere water molecules should also increase the outer sphere contribution to the relaxivity. In addition, the electronic relaxation time contributes to the outer sphere relaxivity, especially for systems that are rotationally dependent. H. Experimental Results Inner Sphere Water Molecules, q In the previous sections, we discussed the theoretical descriptions of both the inner and outer contributions to the relaxivity. These theories allowed us to make predictions about what chemical and physical parameters can be modified to optimize the relaxivity of MRI contrast agents. In this section we present a review of the experimental data that support these predictions, and draw conclusions about the physical and chemical properties that limit the relaxivity of macromolecular MRI contrast agents. Plenty of experimental evidence exists demonstrating the relationship between the relaxivity and the number of exchangeable inner coordination sphere water molecules. Lauffer and Caravan et al. present excellent compilations of the data, therefore we will limit ourselves to the data for the commonly used ions in MRI contrast agents and early failures. Table 3. The outer sphere relaxivity is quite substantial, as demonstrated for systems with ^ = 0. It provides approximately 50% of the relaxivity for ion-chelate complexes containing only one exchangeable inner sphere coordinated water molecule. Early results indicated that a trade off between the number of exchangeable inner sphere water molecules and toxicity exists (see toxicity below). This coupled with the large outer sphere contribution led to clinical agents with at least one inner sphere water molecule, although
ERIK WIENER and VENKATRAJ V NARAYANAN
168 Table 3. lon-chelate
^
Cd(lll)-aqua ion EDTA
8-9 2-3
D03A HP-D03A BOPTA DTPA
1.8-1.9 1.3 1.2 1-1.2
DTPA-BMA DOTA
1 0.9-1.2
TETA TTHA Mn(ll)-aqua ion EDTA DTPA DOTA Fe(lll)-aqua ion EDTA DTPA EHPG
Role of the inner sphere water protons
-0.6 -0.2 7 1 0 0 6 1 0 0
n
Proton Larmour frequency (MHz)
-9.3 5.4 6.9 4.8 3.7 4.4 4.2 4.3 3.8 3.8 3.4 3.5 2.1 2.0 8.0 2.5 1.2 1.1 8.0 1.8 0.73 0.95
20 20 20 20 20 20
20 20
pH
Temperature
Ref.
6.4 6.4
35 35
22 88
7 7.4 7.3 7.3 6.4 7.4 7.4
40 37 39 35 35 37 37 37 92 37 37 35 35 35 37 35 37 37 37-40
89 92 90 91 88 92 92 93
7.4 20 20 20 20 20 20 20 20 20 20
6.4 6.4 <3.0 ~7 ~7 7.4
94 22 88 88 94 94 94 95
Regardless of the ion, the relaxivity increases with more inner sphere water molecules, but even one H2O provides significant relaxation effects. The values of q were estimated from results obtained with other lanthanide ions such as Eu(lll), Td(lll), or Dy and should be considered approximate.
some experimental agents with ^ = 0 were developed. More recently, stabile Gd(III)-chelates with q = 2 were developed ^^. Spin Quantum Number, S
The experimental demonstration of the contributions of the spin quantum number to the overall relaxivity of MRI contrast agents requires a comparison of different chelates in order to keep q constant. Even though the electronic relaxation times may differ from one ion to another or for different chelates, one can get an idea of the contribution of the spin quantum number to the overall relaxivity. Tweedle et al.^^ ran a number of early studies on the relaxivities of polyaminocarboxylate complexes with Gd(III), Mn(II), and Fe(II). Both Gd(III) and Mn(II) have a similar range of electronic relaxation times. The chelates Gd(III)-DTPA and Mn(II)-EDTA each have only one inner sphere water molecule, and are linear or straight chain multidentate ligands. The relaxivities of these complexes are 4.3 and 2.5 respectively, which is proportional to their
169
Magnetic Resonance Imaging Contrast Agents 0>«. /OH
o
^N
o HOHO. ^ ^ ^ ^ N ^ ^ ^ ^
N:
OH \ / ^ O HO
OH
.OH
OH DTPA
DOTA
Chart r. Structures of DOTA and DTPA.
spin quantum numbers. The ratio of their relaxivities is 1.72. This ratio is very close to 1.8 which is the ratio of the 5 * (5* + 1) products. Electronic Relaxation Time^ TS Much of the early experimental data on the contribution of the electronic relaxation time to the relaxivity was obtained on small rapidly rotating ionchelate complexes. For ion-chelate complexes with electronic relaxation times larger than the rotational correlation times, differences in the electronic relaxation time will manifest themselves in the low field region of the NMRD profiles. SBM theory predicted that more rigid and symmetric chelates will have longer electronic relaxation times. Geraldes et al.^^'^^ determined the zero field electronic relaxation times, tso, for Gd(III)-DOTA and Gd(III)-DTPA from NMRD data to be 850 and 85 ps, respectively. Disrupting the ligand field symmetry by converting a carboxylic acid ligand into a propyl amide reduced Tso of the Gd(III)-DOTA derivative to 170 ps. They reported that the complexes with longer zero field electronic relaxation times had higher relaxivities in the low field region. Many investigators have determined the electronic relaxation parameters, tso, A^, and ty, of different Gd(III)-chelate complexes with a variety of methods. The NMRD methods must make assumptions about the water residence times, q, and the distance between the magnetic moments of the paramagnetic ion and the proton. The work by Merbach's group used ^^O NMR, EPR, and NMRD to measure all of the variables. Table 4 lists these parameters for many different chelates. The values for the parameters of a given ion-chelate complex vary dramatically between the different methods used to derive them. However, restricting the comparison of chelates to the same method within a specific report shows that Gd(III)-DOTA has smaller A^ and ty relative to Gd(III)-DTPA. This results in a longer tso, see Eqs. 18 and 19. These results support the predictions that a more rigid and symmetric chelate will exhibit longer zero field electronic relaxation times which will result in higher relaxivities in the low field region of the NMRD profile. Alternatively, chelates with similar ligand field symmetry and flexibility should have similar electronic relaxation parameters. This
170
ERIK WIENER and VENKATRAJ V. NARAYANAN o
OH
HO, HO^
^0
N
NH
HO D03A
EDTA
0^;^^0H HO
°c )
HO «^-
- ^ N ^ ^ ^ ' ^ - - ^ N - ^ O
-^"^
Ov
OH
OH BOPTA
HP-D03A
HONH
^-s^
^-^
.N^
P
V ^ '
o,
OH
NH
OH
DTPA-BMA
O.
^OH
HO, N
N
N
N
° C J,?
OH
HO TETA
EHPG
Chart 2, Structures of EDTA, D03A, HP-D03A, BOPTA, DTPA-BMA, TTHA, TETA and EHPG.
is demonstrated with the linear chelates Gd(III)-DTPA, Gd(III)-DTPA-BMA, and Gd(III)-EOB-DTPA. These chelates show only small variations in tso with values of 72, 81, and 90 ps, respectively. Koenig's group obtained values of 85 ps for Gd(III)-DTPA and Gd(III)-DTPA-BMA.^^ For the three similar chelates both A^ and ty are similar. The Gd(III)-DOTA had a TSO roughly 5 times larger
Magnetic Resonance Imaging Contrast Agents Table 4,
171
The zero field electronic relaxation times and the parameters that affect its magnitude ^2,-2
Compound Gd(lll)-DTPA-BMA Gd(ill)-DTPA-PA2 Gd(lll)-DTPA-BA2 Gd(lli)-DTPA-MEA2 Gd(lll)-DTPA-MPEA2 Gd(lll)-DTPA-BHEA2 Gd(lll)-PC2A Gd(lll)-PCTA Gd(lll)-BP2A Gd(lll)-DTPA
Gd(lll)-EOB-DTPA Gd(III)-DTPA-MA Gd(III)-DTPA-BMA Gd(lll)-BOPTA Gd(lll)-DOTA
Gd(lll)-D03A
(ps)
(ps)
50 57 53 53 57 76 96 87 119 72 b 0.88 49 90 107 81 90 76 473 1.8 54 129
13 26 25 21 32 46 23 15 19 25 63 9.5 4 3.0 25 34 26 11 38 3.5 14
Method
Rei
37 37 37 37 37 37 37 37 37 25
NMRD NMRD NMRD NMRD NMRD NMRD NMRD NMRD NMRD Multiple
22 25 25 25
NMRD i^ONMR Multiple Multiple
25 25
NMRD Multiple
22 25
NMRD NMRD
102 102 102 102 102 102 103 103 103 82 104 99 105 105 82 105 106 82 104 99 107
Temperature
rc) 1.28 X 10^0 a 0.56 0.63 0.75 0.46 0.24 0.37 0.64 0.37 0.46 15 1.8 2.3 2.6 0.41 0.38 0.422 0.16 12 0.43 0.46
^ All values below this point and in this column were calculated from Eq. 17 until footnote b. •^ All values below this point and in this column were calculated from Eq. 18 or Eq. 19.
than the three linear chelates, and values of A^ and Xy that are 2.9 and 2.3 times smaller. Clarkson et al.^^ reported that the values of A^ and Ty for Gd(III)-DOTA were 4.2 and 2.7 times smaller than those for Gd(III)-DTPA. Recall that for rotationally constrained ion-chelate complexes with relatively long water residence times the zero field electronic relaxation time is not predictive of the peak high field relaxivity. In SBM simulations that held A^ constant and increased TV, TSO was reduced and the peak relaxivity shifted to slightly lower fields and increased. We presented data that tested this prediction by comparing Gd(III)-DOTA and Gd(III)-DTPA complexes constrained rotationally by attaching them to ammonia core polyamidoamine dendrimers via an isothiocyanate linker. ^^^ Although the low field relaxivities differed significantly, the peak relaxivities were approximately the same. Fig. 21. Tweedle et al.^^'^^^ also showed that in a rotationally constrained system Gd(III)-DTPA and Gd(III)-DOTA have similar relaxivities at 20 MHz, and ambient temperature. They reported values of 31 and 35 s'^ Gd(III)-DTPA-BMA mMr^ for Gd(III)-DTPA and Gd(III)-DOTA in a highly viscous sucrose/water solution. These values are quite close to those obtained for the dendrimer derivatives.
172
ERIK WIENER and VENKATRAJ V NARAYANAN 45 40
OO
OO
o
"fas *
o
^30-1 >
LU
25-^ 20
••
o
^285% •
o c» o
•• •
15 O
10
Q_
0.01
0.1
1
10
100
PROTON LARMOUR FREQUENCY (MHz) Figure 21. DTPA (dots) and DOTA (circles) chelates attached to a g = 6 ammonia core PAMAM via a thiourea bond and complexed w/ Gd(III) have similar high field values of the relaxivity.
Note that both these ion-chelate complexes have long water residence times, and rotationally constrained systems may be TM limited, see below. Rotational Correlation Time, Xr
The effect of increasing the rotational correlation time on the proton relaxation enhancement effect was known since the earliest observations of PRE. SBM theory predicts that for ions with long electronic relaxation times relative to the rotational correlation time, tr will dominate tc. This implies that increasing the rotational correlation time should result in sizable increases in the relaxivity. This was observed quite readily for ions binding to endogenous binding sites of DNA or proteins. Burton^^, Dwek^^ and Koenig^^ present comprehensive reviews of PRE studies on protein/enzyme systems. This review will therefore restrict itself to applications of PRE in the development of macromolecular MRI contrast agents. Many early studies of macromolecular MRI contrast agents supported the prediction that increasing the rotational correlation time of an ion-chelate complex by attaching it to a macromolecule would increase the ion relaxivity of that complex. Lauffer and Brady performed the first comprehensive analysis of attaching ion-chelate complexes to proteins. They attached Gd(III) and Mn(II) derivatives of EDTA and DTPA to bovine serum albumin (BSA) or bovine immunoglobulin. ^^^ They observed an increase in the relaxivities of the ionchelate complexes from 2.9 and 4.1 to 32 and 19 s~^ mM~^ after conjugat-
Magnetic Resonance Imaging Contrast Agents
OH
OH
OH
OH EOB-DTPA
R i = R 2 = NHC3H7
DTPA-PA2
Ri = R2 = NHC4H9
DTPA-BA2
Ri = Ro = NHCHoCHoOCH.
DTPA-MEA2
Ri = R2 = NHCH2CH2N(CH2CH20H)2 Ri = R2 =NHCH2CH2-N
173
O
DTPA-BHEA2 DTPA-MPEA2
HN-CH3
■O2C-
-C02' N
N
■O2C-
-COo' 0= HN-CH3
DTPA-MA
DTPA-MA. c
Chart3, Structures of DTPA-PA2, DTPA-BA2, DTPA-MEA2, DTPA-MPEA2, DTPA-BHEA2, PC2A, BP2 A, EOB-DTPA, DTPA-MA and DTPA-MA5.
ing Mn(II)-EDTA and Gd(III)-DTPA to BSA. Attaching the Gd(III)-DTPA to a macromolecule with an even larger molecular weight resulted in an even higher relaxivity of 26 s~^ mM~^ Similar effects were not observed with Mn(II)-EDTA. Attaching this chelate to immunoglobulins resulted in virtually no change in the relaxivity, 31 s~* niM"^ relative to BSA, 32 s~^ mM~^ Subsequently Curtet et al.^^^ labeled monoclonal antibodies with ion-chelate complexes and reported that the antibody ion-chelate complex modified the relaxation parameters of water much better than the free ion-chelate complex. Shreve and Aisen^^^ and Manabe et al.^^^ also observed that attaching Gd(III)-DTPA directly to antibodies increased the ion-relaxivity over that of Gd(III)-DTPA. These results are consistent with the PRE phenomena observed
174
ERIK WIENER and VENKATRAJ V NARAYANAN
when paramagnetic ions bound to endogenous metal binding sites of macromolecules and support the prediction that attaching a low molecular weight ion-chelate complex to a macromolecule will increase the relaxivity as a result of a longer rotational correlation time. The Deby, Einstein, Stokes equation for rotational diffusion predicts that the rotational correlation time is a function of the hydrodynamic radius, and thus the molecular weight of a protein or polymer. This implies that the relaxivity of macromolecular agents should depend on the molecular weight until the rotational correlation time greatly exceeds the electronic relaxation time. Lauffer's and Brady's results with Gd(III)-DTPA attached to BSA and immunoglobulin confirmed this prediction, but their results with Mn(II)-EDTA and Gd(III)-EDTA failed to support this prediction. Additional studies on many new macromolecular contrast agents with different polymeric backbones confirmed these findings. Similar derivatizations of linear polymers such as polylysine^^^"^^"^ and dextran^^^ with Gd(III)-DTPA resulted in macromolecular agents with 2-3 times the ion relaxivity of Gd(III). We reported that attaching a derivative of either DTPA or DOTA to a cascade polymer can increase the relaxivity 6 fold.^^^'^^^~^^^ These results support the qualitative prediction that increasing the molecular weight results in an increase in relaxivity. The results described above demonstrate the effect of attaching a low molecular weight ion-chelate complex to a macromolecule on the relaxivity. While this attachment was accompanied by an increase in relaxivity, the magnitude of this increase was smaller than that predicted by SBM theory for a rigid molecule of similar diameter. More recent studies on linear polymers indicate that the increase in relaxivity is independent of the macromolecule's molecular weight, so that attaching a chelate to the same polymer, but with different molecular weights greater than 10 kDa, has very little effect on the relaxivity.^^^"^^^'^^^"^^^ In many cases the relaxivity decreased with increasing molecular weight within a family of polymers.^^^'^^^'^^^ Armitage et al.^^^ reported that dextran-based polymers esterified with Gd(III)-DTPA had relaxivities of 8.7, 8.1, 7.1, and 5.8 for derivatives with molecular weights of 9.4, 40.2, 70.8, and 487 kDa. Vexler et al.^^^ reported the relaxivities of polylysine derivatives with molecular weights of 36, 44, 139, and 480 kDa, at 0.25 T and 37°C. They found that the relaxivity was independent of molecular weight, with values of 10.4, 11.9, 10.9, and 10.9 s~^ mM"^ respectively. Desser et al.^^"^ reported on macromolecular agents prepared from the dianhydride of DTPA and a,oo-diaminopolyethylene glycols. They also found that the relaxivity at 20 MHz and 37°C remained constant at 6 s-i mM-i for 10.8, 13.6, 18.5, 21.9, 31.5, 39.6, and 83.4 kDa derivatives. Fig. 22. The unique linear polymeric system reported by Kellar et al. defies this trend and have relaxivities of about 8, 9, 15, and 19 s~^ mM~^ for average molecular weights of 8, 8.3, 10.3, and 15.7 kDa and polydispersities of 1.41, 1.48, 1.52, and 1.8 at 35°C and 20 MHz.
Magnetic Resonance Imaging Contrast Agents
175
ou -
♦
25 -
^
E
20-
♦
*
■ >
'x
CO
^
0 10-
5 -
T
0
0 ▼
T-
V
•
o
o m
T
0 -
I—'—'—'—'—I—'
100000
200000
300000
400000
500000
Molecular Weight (Da) Figure 22. Most linear polymer systems with Gd(lll)-chelates attached have relaxivities that act independent of molecular weight. Polyethylene glycol derivatives (dots), Dextran based agents at 81 MHz 23°C (squares), 84 MHz 37°C (black triangles), 10 MHz 37°C (white triangles), 100 MHz 25°C (upside down black triangles), proteins (black diamonds), Polylysine based derivatives 10 MHz 37°C (black hexagons), 100 MHz 37°C (white hexagons).
The NMRD simulations shown in Fig. 11, show that at high frequencies such as 80 to 100 MHz, one might not expect higher relaxivities with longer rotational correlation times for certain parameters. This might explain the observations of decreasing relaxivity with larger molecular weight agents for some of the dextran-DTPA agents at 81, 84, and 100 MHz, and the polylysine-DTPA derivatives at 100 MHz. However, it fails to account for the lack of an effect of molecular weight on agents at 10 and 20 MHz. A number of other hypotheses have been proposed to reconcile these apparent anomalies with the predictions of SBM theory. Armitage et al.^^^ suggested that intramolecular hydroxyl groups in the dextran derivatives could more easily wrap around and displace the inner sphere water molecules of the ion-chelate complex as the molecule becomes larger. Muller^^^ presented an alternative hypothesis. He proposed that changes in the electronic relaxation time reduce the ion relaxivity. For contrast agents derived from linear polymeric backbones, in general, either the flexibility of the spacer arm linking the chelate to the polymer or the local segmental motions of the polymer may dominate the rotational correlation time. As mentioned earlier, a number of reports establish that for linear polymers, segmental motions dominate the rotational correlation time, and that for polymers larger than 10 kDa these segmental motions become independent of molecular weight.^^"^^
176 Table 5,
ERIK WIENER and VENKATRAJ V NARAYANAN Rigidity of the backbone and linker effect the relaxivity of ollgomeric complexes
Molecular weight 1201 1257 2259 2258 2597 4390
Tren backbone Cyclen backbone Methylene-ether linker No linker (mM-'' s-'') (mM-'' s-'') (mM''' s''') (mM-'' $-'') 6.6 5.4 8.5 8.8 10.2 11.2
This implies that increasing the molecular weight of the linear polymers may have no effect on the rotational correlation, and similarly no effect on the relaxivity, unless one restricts the local segmental motions with hydrophobic interactions, as observed in the agents reported above. The rigidity of the backbone and linker effect the relaxivity of oligomeric systems. Ranganathan et al.^^^ showed that both the rigidity of the backbone and linker effect the relaxivity of oligomeric agents even for chelates with relatively long water residence times. Two backbone systems with the same linker and chelate but differing flexibilities are cyclen and tren. The tren backbone is based on a linear branched system and is more flexible than the macrocyclic backbone based on cyclen. Derivatives with the tren backbone had relaxivities of 8.5 and 11.2 s~^ mM~^ for compounds with molecular weights of 2259 and 4390, respectively. The cyclen derivatives had relaxivities of 8.8 and 10.2 for molecular weights of 2285 and 2597, respectively. Clearly the more rigid backbone with a molecular weight of 2597 has only 10% less relaxivity than a more flexible backbone with 1.7 times its molecular weight, and a 20% higher relaxivity than the more flexible system, even though it has a 12% higher molecular weight. Comparison of a system with similar polyhydroxycyclohexane backbones but linkers of differing flexibilities showed that even though the molecular weight was slightly less for the agent with the more rigid linker, the relaxivity was 25% higher. The agent with a flexible methylene-ether linker had a molecular weight of 1257 and a relaxivity of 5.4 s"^ mM~^ relative to that of an agent with no linker, or one ligand arm built into the polyhydroxycyclohexane ring, which had a molecular weight of 1201 and a relaxivity of 6.6 s~^ mM~^ (Table 5). The long water residence time of the chelate did restrict the maximum achievable gain in relaxivity of these oligomers. This may also be true for some of the other polymeric chelate systems used which would limit the gain in relaxivity associated with increasing molecular weight. The effect of molecular weight on the relaxivity of dendrimer-based contrast agents differs from that observed with contrast agents prepared from most linear
177
Magnetic Resonance Imaging Contrast Agents H
\
/
N
H
u N:
\
/
H
Cyclen
Chart 4. Structures of cyclen and tren.
o E Ev.
140 120 L
•
J
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100
^^
§1
c3 -^
S o o
•
1
80
CO
DC O c o ^
I
60
40 0
1 2 3 4 5 6 7 8 9 10 Propionate-PAMAM Generation
Figure 23, Relaxivity of dendrimer-based agents increases with increasing molecular weight. The N,N-dipropionate PAMAM family of chelates have large relaxivities that increase with generation.
polymers. The relaxivity of dendrimer-based agents increases with increasing molecular weight, Fig. 23. For the MA^-dipropionate-PAMAM derivatives (Chart 5) the peak relaxivity at 45 MHz and 37°C increased linearly, and nearly doubled as the molecular weight increased from 2174, g = 1.5, to 307,240, g = 8.5. The relaxivity increased 20% as the molecular weight increased from 38,120 to 307,240. For PAMAM-TU-DTPA derivatives the relaxivity at 10 MHz and 37°C nearly doubled as the generation and molecular weight increased from 2 to 6 and 8508 to 139,000 kDa. It begins to level off at higher generations. More recent work confirms our earlier findings. Margerum et al.^^^ also observed an increase in the relaxivity for dendrimer-based agents. They reported that agents prepared from l-(4-isothiocyanatobenzyl)amido-4,7,10-triacetic acid-tetraazacyclododecane and polyamidoamine dendrimers (PAMAM-TU-D03A-MA) and complexed with Gd(III) had relaxivities of 14.6 and 18.8 s"^ mM"^ at 25 MHz and 37°C and molecular weights of 18.4 and 61.8 kDa, respectively. This increase in relaxivity with increasing molecular weight is consistent with the hypothesis that the rotational correlation time of dendrimers increase
ERIK WIENER and VENKATRAJ V NARAYANAN
178
PAMAM-N
N,N-Dipropionate PAMAM
PAMAM
NH
PAMAM-TU-BZ-DOTA
Chart 5. (Continued on next page.)
with increasing molecular weight. Recall that Meltzer et al7^'^^^ first examined the molecular dynamics of dendrimers. They reported two types of motions or sites based on the location and responses of the atoms to increasing molecular weight. The interior atoms occur before the last amide bond, and the terminal
Magnetic Resonance Imaging Contrast Agents
179
/ \
H N-Z
z
/
/ N
\
z« NH2
z
^z
III
III
.
N
/ "^
Z-N \ H
H2N
/
.N^
\
Z
N-H /
H-N
/ \ Z
N
^
N-Z / H
HoN
Cascade 24
Charts. Structures of N,N-Dipropionate PAMAM, PAMAM-TU-DTPA, PPI-TU-DTPA, PAMAMTU-BZ-DOTA, gadomer-17 and cascade-24.
atoms are on the last ethylenediamine added. Motions of the terminal sites are more rapid than motions of the interior sites. The rotational correlation times of atoms in both sites increase with increasing molecular size or generation of the dendrimer. The rotational correlation times of atoms in the terminal sites only increase twofold as the molecular weight increased from 0.36 to 87.3 kDa, while those of the interior sites increased more than 30 times. This difference in response of the rotational correlation times to increases in molecular size imposes significant constraints on the linkers used to attach the chelates to the dendrimers. Ideally, the flexibility of the linker should occur at interior sites. We reported studies on the molecular dynamics of the ion-chelate complexes attached to dendrimers via an isothiocyanatobenzyl linker. These studies were instigated to determine if the rotational correlation times resembled those of the terminal or interior sites. ^^^'^^^ The rotational correlation times of vanadyl complexes of PAMAM-TU-DTPA were similar to the rotational correlation times of the interior sites of the corresponding generations reported by Meltzer (Fig. 24). Toth et al.^^^ subsequently confirmed our results. They reported studies which included the molecular dynamics of the PAMAM-TU-D03 A-MA prepared by Margerum,^^^ which linked the macrocyclic chelate D03A-MA to the amine terminated PAMAM by the same isothiocyanatobenzyl linker used by
180
ERIK WIENER and VENKATRAJ V NARAYANAN 2.5 2.0 L (D
I
1.5
I
10 h
1
1
1
1
1
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1
1
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1
1
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2
1
3
1
4
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Generation Figure 24, The average rotational correlation times of PAMAM-TU-DTPA ion-chelate complexes (dots) match those of the interior carbon atoms (circles) in the corresponding generation.
us. Although they used a different chelate and a different generation, they used the same linker and found that the rotational correlation time resembled that of the interior sites for the corresponding generation reported by Meltzer. The data reported above for x^ uses isotropic tumbling models, and none of the values equal those predicted by the Debye formula. This implies that rapid segmental motions do contribute to lowering the observed rotational correlation times of dendrimers. Recent studies of PAMAM-TU-DTPA derivatives using an anisotropic tumbling model are consistent with two types of motion.^^^ One motion is rotation about the axis that connects the ion-chelate complex to the dendrimer central core. The rotational correlation time, x\\, of these motions only increased 40% at 37°C as the molecular weight 8.5 to 139 kDa. The other motion consisted of the overall reorientation of the PAMAM-TU-DTPA, i.e., molecular tumbling. The rotational correlation time, r_L, of these motions increased more than 3 times under the same conditions. Table 6. The observation that the overall tumbling of dendrimers contributes to the rotational correlation time implies that different dendrimer families may
7aWe 6.
Rotational correlation times of PAMAM-TU-DTPA complexes of V O determined with EPR at 37°C
MW, PAMAM-TU-DTPA 8.5 kDa, generation 2 139 kDa, generation 6 T^ave = V^ll * ^-L-
(ns)
(ns)
^11 (ns)
(ns)
0.40 0.64
0.67 1.5
0.15 0.21
3 10
^ave
181
Magnetic Resonance Imaging Contrast Agents 1
>30
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t
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Proton Larmor Frequency (MHz)
100
Figure 25. Different dendrimer families can have different relaxivities for the same generation, chelate, and linker. A generation-2 polypropyleneimine dendrimer (dots), with a DTPA chelate linked via a thiourea bond has a higher relaxivity than a generation-2 ammonia core PAMAM (triangles) with the same surface.
have different relaxivities as a result of differences in their overall shape. We previously reported data comparing dendrimer polychelates prepared from 1,4-diaminobutane core polypropyleneimine (PPI-TU-DTPA) and ammonia core polyamidoamine (PAMAM-TU-DTPA) dendrimers.^^^ The Gd(III) complexes of generation-2 PPI-TU-DTPA derivatives had a higher relaxivity than the same generation PAMAM-TU-DTPA derivative, Fig. 25. Studies on the molecular dynamics demonstrate that the ion-chelate complexes attached to PPI dendrimers have longer average rotational correlation times than those attached to PAMAM dendrimers. Consistent with a potential difference in shape, the rotational correlation time of the axial rotations were the same for the two derivatives, but the overall reorientational correlation time for the PPI-TU-DTPA derivative was three times greater than that of the PAMAM-TU-DTPA derivative, Table 7. These results demonstrate that the dendrimer generation within
Table 7, Rotational correlation times of V O complexes on g = 2 dendrimer-TU-DTPA from different families determined with EPR Compound g = 2 PAMAM-TU-DTPA PPI-TU-DTPA
^11 (ns)
(ns)
0.24 0.24
10 33
^ave
1.5 2.8
182
ERIK WIENER and VENKATRAJ V NARAYANAN
a particular family of dendrimers effects the relaxivity, and that for the same generation, the dendrimer family can effect the relaxivity. The interior rigidity also contributes to the overall correlation time, and families of dendrimers with more rigid interiors may have higher relaxivities than the same generation of families with moreflexibleinteriors. Water Residence Time, Jm
While the data clearly show the effect of increasing the rotational correlation time and molecular weight on dendrimer-based contrast agents, the water residence time of the agents already reported in the literature may limit any further gains in the relaxivity. Fortunately appropriate modifications of the chelate and the coordination chemistry can alter the water residence time and the exchange rate. The data in Table 8 clearly show that alterations of the chelate or ligand field can significantly affect the water residence time. Converting
Table 8.
One can fine-tune the water residence time by altering the chelate structure Temp. Technique
Agent Aqua ion, Gd Gd(!ll)-PDTA Gd(m)-TREN-Me-3,2-HOPO Gd(Ili)-DTPA
Gd(lll)-BOPTA (DTPA-BOM) Gd(III)-DTPA-(BOM)2 Gd(lll)-DTPA-(BOM)3 Gd(lll)-EOB-DTPA Gd(lll)-DTPA-MA Gd(lll)-DTPA-MAiso Gd(III)-DTPA-BMA Gd(lll)-DTPA-BBA Gd(lll)-DOTA Gd(III)-HP-D03A Pip-Gd(lll)-(D03A)2 bisoxa-Gd(III)-(D03A)2 Gd(lll)-DTMA Cy2-Gd(ill)-DOTA Gd(lll)-DOTMA
(ns)
rc)
1.2 1.24 9.8 16 244 303 340 290 260 180 278 526 769 810 2330 2222 2500 244 208 350 666 714 1000 19000 16 34
25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25
^^ONMR ^^O NMR and EPR and ^^ONMR i^ONMR ^^ONMR i^O NMR and EPR and NMRD NMRD and i^O NMR NMRD and ^^O NMR NMRD and ^^o NMR ^^O NMR and EPR ^^O NMR and EPR ^^O NMR and EPR NMRD ^^ONMR ^^ONMR NMRD ^^O NMR and EPR and ^^ONMR 1^0 NMR ^^O NMR and EPR and ^^O NMR and EPR and ^^ONMR ^^ONMR ^^ONMR ^^ONMR
Ref.
NMRD
NMRD
NMRD
NMRD NMRD
132 82 132 97 132 82 81 81 81 81 105 105 105 91 105,132 133 82 132 134 82 82 135 134 83 83
Magnetic Resonance Imaging Contrast Agents
183
carboxylate ligands to amides significantly increases the water residence time. Methyl amidation of one carboxylate on DTPA increased the water residence time of DTPA from between 244 and 303 ns to a range of 526 to 769. Methyl amidation of two carboxylates increased the water residence time by almost an order of magnitude to 2330 ns. Similar results were obtained for DOTA derivatives. Amidation of a single carboxylate group on DOTA increased the water residence time from between 208 and 244 to between 625 and 1000 ns. The results presented in Table 8 demonstrate that in theory one can manipulate or fine tune the water residence time through the appropriate coordination chemistry. Few comparisons on the effect of altering the electron density or availability at the same ligand appear in the literature. The conversion of a carboxylate to an amide, while significantly altering the electron density of the ligand, creates a completely different coordinating group. Tweedle et al. looked at DOTA derivatives while maintaining the carboxylate coordinating groups. Methylating each of the acetate arms of DOTA to make DOTMA (Chart 6) reduced the water residence time almost 7 fold from the range of 208-244 to 36 ns. Similarly, alkylating the backbone of the macrocycle next to the tertiary nitrogens by placing two cyclohexyl rings on DOTA to make a Cy2D0TA reduced the water residence time almost 15 fold to 16 ms. Inductive effects from such substitutions should result in greater electron densities on the carboxylate oxygens and tertiary amines. This would create stronger coordination groups, and the results are consistent with the hypothesis presented by Powell for steric crowding of the water coordination site. Tweedle suggests a simple steric effect from the methyl groups results in a longer 0 - M bond length and faster water exchange. Furthermore, Gd(III)-DOTA exists in two isomeric structures with one having a much shorter water residence time than the other, and steric hindrance of the type observed in DOTMA favors the form with the shorter water residence time. The methylene-substituted chelate arm derivatives of DOTA with the RRRR stereoisomer configuration have very rapid exchange rates and favor the twisted over the regular monocapped square antiprismatic isomer. The exchange rates of the various isomers are such that RRRR > RRRS > RSRS > RRSS, and this correlated with the amount of the twisted form. The idea of steric crowding is also presented by Aime et al.^^ They found that crowding the water coordination site of DTPA with the benzyloxymethyl (BOM) group decreased the water residence time monotonically as a function of the number of BOM groups attached to the methyl carbon of the coordinating acid group. Thus they measured a water residence time at 25°C of 340, 290, 260, and 180 ns for Gd(III)-DTPA, BOM = 0, Gd(III)-BOPTA, BOM = 1, Gd(III)DTPA-(B0M)2, and Gd(III)-DTPA-(B0M)3, respectively. The long water residence times of the monoamides are relevant to macromolecular contrast agents as these were the main linkages used to attach
184
ERIK WIENER and VENKATRAJ V NARAYANAN o
HO
O
O^^OH
I
I
OH
r:^^
HO^
^ . < ^ ^ NH ^-^
^.-.
OH
^N^ ^ ^
OH
PDTA
^ ^
NH
OH
DTPA-BBA
-ooc-^ ^^ N ■OOC—^ \
/ - ^ \_y Y ^ /—\ /--cooN / ^—COO"
°C D
N OOC—^ \
N / '"^—COO'
pip(D03A)2
/ \ o
o
''i«"'-(D03A)2
/VJ^'S HO^O
MO'
NH ^
O
/
\
o
f''L7\
0«
HO^O
QAOH
HO''^
^O
DOTMA DTMA
P 1^
O.^ / O H
^ N
N:
\ HO
OH CY2-DOTA
Chart 6. Structures of PDTA, DTPA-BBA, pip(D03A)2, bisoxa(D03A)2, DTMA, DOTMA and Cy2-DOTA.
185
Magnetic Resonance Imaging Contrast Agents o COO'
Gd-DOTA
Gd-DTPA O ^ ^O" 'O
a
N
N
N
N
OH
O Gd-HP-D03A
Chart 7. Structures of Gd-DTPA, Gd-DOTA and Gd-HP-D03A.
chelates to the macromolecules. Early macromolecular contrast agents using polylysine-linked DTPA^^^ or DOTA^^^ to the 8 amines via amide linkages. These derivatives had very low relaxivities. Similarly, dendrimer-based contrast agents prepared by linking the DTPA to the terminal amine via an amide bond had very low relaxivities^^^ relative to those prepared from the isothiocyanate of DTPA.^^^'^^^'^^^ Dendrimer-based agents prepared from the isothiocyanate for a monoamidated DOTA also had low relaxivities relative to those prepared from the isothiocyante of DTPA or DOTA, Table 9. SBM theory predicts that the water exchange rates on the order of those
Table 9, Dendrimer PAMAM PAMAM PAMAM PAMAM PAMAM PAMAM PAMAM PAMAM
The water/proton residence times of the chelates limits gains in relaxivity 8
Chelate
6 6 5 5 4 4 2 3
TU-Bz-DOTA TU-DTPA TU-Bz-DOSA TU-Bz-DOTA TU-DTPA TU-BZ-D03A TU-DTPA DTTA-MA
Frequency (MHz)
Temperature
n
rc)
(mM-'' s-'')
(ns)
25 25 25 20 20 20 20 20
35 35 37 23 35 37 35 40
31 34 ± 2 18.8 ± 0 . 2 30 30 16.9 ± 0 . 4 16.4 11.9
208-244 ^ 244-303 ^ 667^ 208-244 ^ 244-303 ^ 769^ 244-303 ^ 526-769^
^M
^ The water residence times of the free chelates at 25°C. ^The water residence times of the chelates attached to the dendrimer. Actual measured values.^^^ Toth reports that attaching the chelate to the dendrimer does not alter the water residence time.
186
ERIK WIENER and VENKATRAJ V NARAYANAN
reported for DTPA, DOTA, and their amide derivatives would limit the relaxivity when these ion-chelate complexes were rotationally constrained. The first evidence indicating that the relaxivity of macromolecular contrast agents was limited by the water exchange rate or residence time was presented by Lauffer et al.^^^ They linked DTPA to the e nitrogen of lysine of BSA via an amide bond, and measured the total peak relaxivity, ri, of a BSA-Gd(III)-DTPA derivative at 5° and 37°C. The relaxivity increased from approximately 14 to 18 s~^ mM~^ as the temperature increased. An increase in the total relaxivity with increasing temperature is consistent with the water residence time or exchange rate limiting the relaxivity. Recall that the total relaxivity is the sum of the inner and outer sphere contributions. We showed earlier that the outer sphere contribution depended on the relative diffusion coefficients and the electronic relaxation times. Increasing the temperature results in a decrease in the outer sphere contribution to the relaxivity. The total relaxivity can only increase or remain constant with increasing temperature if the inner sphere contribution to the overall relaxivity increases to offset the decrease in the outer sphere contribution. Increasing the temperature decreases both the electronic relaxation time and the rotational correlation time. For agents limited by these times increasing the temperature and resultant decrease in both TS and tr would reduce the inner sphere relaxivity. Increasing the temperature also reduces the water residence time or increases the exchange rate. For contrast agents limited by long water residence times or slow exchange rates, increasing the temperature would increase the inner sphere contribution to the relaxivity. Thus increases in the total relaxivity with increasing temperature are indicative of long water residence times which limit the relaxivity, provided the exchange mechanism is enthalpically driven. Like Lauffer's observation for BSA-based contrast agents, we observed that increasing the temperature resulted in either higher total relaxivities or no change. Fig. 26, for Gd(III) complexes of PAMAM-TU-DTPA and PAMAM-TU-Bz-DOTA derivatives. The results of Tweedle also support the conclusion that the relaxivity of rotationally constrained DTPA and DOTA derivatives are limited by the water residence times. They observed relaxivities of 31 and 35 for systems constrained in a highly viscous solution. These matched the times obtained by us following the attachment of the chelates to generation-6 PAMAMs. The corresponding Cy2D0TA and DOTMA derivatives with shorter water residence times had improved relaxivities, ri, of 54 and 58 under the same experimental conditions. The magnitudes of the relaxivities of dendrimer-based contrast agents are consistent with the relative length of the water residence times of the free chelates. The monoamides of DTPA and the nitrobenzylamides of DOTA both have water residence times longer than either DTPA or DOTA, and they have lower relaxivities when attached to macromolecules. A dendrimer-based contrast agent prepared by coupling the terminal amines of a generation-3 ammonia
Magnetic Resonance Imaging Contrast Agents
HU -
oo 00 35»C Gd(III)
O
^ L
S
35 -
S
3g O H §
0 0 0
pH=7.4
s^ 30 -
• 50c
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100
PROTON LARMOUR FREQUENCY (MHz)
B 34 32
1
1
1
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pH=7.4
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10(
PROTON LARMOR FREQUENCY (MHz) Figure 26. The relaxivities of higher-generation DTPA and DOTA derivatives may be limited by the water residence time. Increasing the temperature of generation-6 PAMAM-TU-Bz-DOTA (A) or PAMAM-TU-DTPA (B) from 5° (dots) to 35°C (circles) does not reduce the relaxivity.
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ERIK WIENER and VENKATRAJ V NARAYANAN
core PAMAM derivative to one carboxylic acid on DTPA to form a monoamide has a relaxivity of 11.9 s~^ mM"^ at 40°C and 20 MHz.^^^ A monoamide derivative of DOTA was linked to the terminal amines of the same type of dendrimer via a benzylisothiocyanate derivative of D03A. This chelate has a similar water residence time as that of the monoamide of DTPA, and the generation-3 derivative has a relaxivity of 14.8 ± 0.4 s'^ mM"^ at 20 MHz and 37°C.^2^ DTPA and DOTA have shorter water residence times, and even smaller dendrimer-based agents have higher relaxivities. A generation-2 derivative prepared by reacting a benzylisothiocyanate derivative of DTPA with an ammonia core PAMAM resulted in a PAMAM-TU-DTPA derivative with a relaxivity of 16.4 s~^ mM~^ at 20 MHz and 35°C. Generation-4 and -5 D03A derivatives have relaxivities of 16.9 ± 0.4 and 18.8 ib 0.2 at 25 MHz and 37°C, as compared with 30 (35°C) and 30 (23°C) for the corresponding generation-4 and -5 PAMAM-TU-DTPA and PAMAM-TU-Bz-DOTA derivatives, respectively. The chelates with the shortest water residence times prior to attachment result in dendrimer-based agents with the highest relaxivities after coupling to the dendrimer surface. In addition to the temperature studies and chelate comparisons mentioned above Toth et al. measured the exchange rate of the PAMAM-TU-D03A derivatives. They reported rates ranging from 1 to 1.5 x 10^ s~^ as the generation increased from 3 to 5. This corresponds to water residence times of 1000 to 667 ns. The only direct comparison of macromolecular contrast agents with similar chelates are for the monoamide DTPA type of linker. Contrast agents of molecular weights 36,000 and 18,000 prepared from the linear polymer polylysine and a generation-3 ammonia core PAMAM were prepared with this chelate and linkage. Vexler et al. reported a ri of 10.4 s~^ mM~^ for the polylysine derivative at 25 MHz and 37°C.^^^ This compares with a ri of 11.9 s~^ mM~^ obtained for a generation-3 PAMAM. Clearly the water residence time influences the relaxivity of the current macromolecular and dendrimer-based contrast agents. A number of different dendrimer-based agents varying in generation and core have been prepared. The relaxivities are highest for those with thiocyanatobenzyl linkages of DTPA- and DOTA-based chelates. These chelates also have the shortest water residence times (Table 10).
V. BIOLOGICAL ISSUES The previous discussions of the physicochemical parameters that influence the relaxivity of an agent pertain to the efficiency of the agent. The efficiency of a contrast agent at altering the water proton relaxation rate is only one factor influencing its efficacy. The efficacy of an agent depends on the ability to selectively alter the proton relaxation rate or time of one tissue relative to another, and this ability is also influenced by biological factors. In addition, the efficacy of an agent is subordinate to safety issues. An efficacious yet highly
Magnetic Resonance Imaging Contrast Agents Table 10, Dendrimer
The relaxivity of the different dendrimer-based agents
Gener- Clielate ation
PAMAM 10 PAMAM 9 7 PAMAM PAMAM 6 PAMAM 5 PAMAM 6 PAMAM 2 PPI 2 PAMAM 3 PAMAM 3 PAMAM 4 PAMAM 5 PAMAM 3 PAMAM 3 PAMAM 2 PAMAM 2 GADOMER- 1 PAMAM
189
Frequency Temp. ri ^M (MHz) (mM-'' s-'') (ns) rc)
TU-Bz-DOTA TU-BZ-DOTA TU-BZ-DOTA TU-Bz-DOTA TU-Bz-DOTA TU-DTPA TU-DTPA TU-DTPA DTPA-MA TU-BZ-D03A-MA TU-BZ-D03A-MA TU-BZ-D03A-MA TU-BZ-DO3A-MA-PEG5000 TU-BZ-DO3A-MA-PEG2000 TU-BZ-DO3A-MA-PEG5000 TU-BZ-DO3A-MA-PEG2000 U-D03A-MA
20 25 25 25 20 25 20 25 20 25 20 25 20 20 20 20 20
23 23 23 35 23 35 35 20 40 37 37 37 37 37 37 37
36 36 34 31 30 34 ± 2 16.4 31 ± 2 11.9 14.8 ± 0 . 4 16.9 ± 0 . 4 18.8 ± 0 . 2 13.8 ± 0 . 4 13.7 ± 0 . 4 12.4 ± 0 . 4 11.0 ± 0 . 4 18-20
208-244 ^ 208-244 ^ 208-244 ^ 208-244 ^ 208-244 ^ 244-303 ^ 244-303 ^ 244-303 ^ 526-769^ 1000^ 769^ 667 b NA NA NA NA NA
^ Value of free DTPA, DOTA, or DTPA-MA. ^Actual measured value.^^^ NA = not available.
toxic agent is worthless as a diagnostic tool. Physiological, pharmacological, and biological issues determine the safety of an agent. In this section we will examine these issues as they pertain to the safety, stability, and selective delivery of MR contrast agents. A. Toxicity Toxicity deals with the adverse effects of drugs. The relative duration and intensity of the drug exposure required to elicit an adverse response determines if the toxicity is acute or chronic. Acute toxicity refers to an adverse reaction that results from brief intense short-term exposures of high doses. Chronic toxicity refers to the development of adverse reactions from prolonged exposure to low doses of an agent. The toxicity of ion chelate complexes can arise from either the free ion or chelate that results when an ion-chelate complex dissociates. Alternatively, the ion-chelate complex may itself elicit adverse reactions. Common ions used in MRI contrast agents demonstrate both acute and chronic toxicity. For example, acute exposure to manganese ion induces cardiotoxicity, and chronic exposure induces neurotoxicity. The acute effect of manganese results from its blocking of the Ca^"^ channels found in heart muscle known as the sarcolemmal ion channels. Chronic exposure to manganese ion
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results in a syndrome similar to Parkinson's disease in humans. It reduces the number of neurons in the substantia nigra, globus pallidus, caudate nucleus, and putamen, along with a corresponding reduction in dopamine levels in the substantia nigra. Iron overload also results in cardiotoxicity and neurotoxicity. Oxidative stress induced by iron overload results in the destruction of dopanergic neurons in the substantia nigra and a syndrome like Parkinson's disease. Heavy metal neurologic toxicity by ions such as iron and manganese generally occur via the same mechanisms. Both ions produce lesions in the basal ganglia with a concomitant reduction in dopamine. Evidence that free radicals, produced from the metal-ion-induced autoxidation of dopamine and other catecholamines, may cause neurodegenerative disease was reported by a number of laboratories. Both iron and manganese stimulate the production of free radicals.^^^~^^^ Inhibition of endogenous synthesis of free radical scavengers such as glutathione potentiate the Mn-induced depletion of dopamine and noradrenaline, and the oxidation of neuronal ascorbic acid. The addition of antioxidants such as ascorbate antagonized the Mn-induced depletion of dopamine. ^"^^ Desole et al. reported that the response of the neuronal antioxidant system was also consistent with a Mn-induced increase in reactive oxygen species. Gadolinium toxicity results from a number of mechanisms. Like manganese, gadolinium can substitute for calcium in many calcium binding proteins and enzymes. They have similar crystal ionic radii. The ionic radii of Gd^+ and Ca^"^ are 0.938 and 0.99 A, respectively. This means that gadolinium easily fits into proteins and channels that have evolved to fit calcium. This binding is tighter as gadolinium has a higher charge-to-radius ratio. Calcium is a major regulator of cellular function, including cellular proliferation, and such binding of gadolinium could disrupt a number of cellular processes. Although the mechanism is unknown, Ishiyama showed that intravenous injection of 0.06 mmole/kg of GdCla stimulated the in-vivo proliferation of hepatocytes.^^^ Gadolinium stimulates cell proliferation in osteoblast, and acts as an agonist for the parathyroid/kidney/brain calcium-sensing receptor, PCaR,^"^^ or the cation sensing receptor CaR.^^^ Gadolinium is a well-known Ca^"^ channel blocker^"^ and blocks the delayed rectifier K"^ current in heart cells isolated from the ventricles. ^"^^ Gadolinium toxicity also results from the formation of colloids. Gadolinium forms highly insoluble colloids with phosphate, carbonate, and hydroxide ions at concentrations found in the blood. Injection of GdCls caused mineral deposition in the kidney and lung capillary beds, phagocytosis by the mononuclear phagocytic system, followed by hepatocellular and splenic necrosis. Precipitates of gadolinium, calcium, and phosphate were found in splenic macrophages, Kupffer cells, and hepatocytes.^"^^ Chelation of metal ions such as Gd(III) reduces the in-vivo retention and alters the biodistribution. While the biodistribution studies mentioned above
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use GdCls, Tweedle et al.^"^^ examined the biodistribution of Gd(III) following its dissociation from linear and macrocyclic chelates. Only a small fraction of the injected doses remained in the animals after 14 days. The GI tract, femur, kidneys, and liver accumulated the dissociated gadolinium. After 14 days more gadolinium remained in the animals injected with the gadolinium complexes prepared with linear chelates than those prepared with macrocyclic chelates. Animals injected with gadolinium complexes of the bismethylamide of DTPA retained the most gadolinium after 7 and 14 days. B. Stability As both the free ion and free chelate exhibit some degree of toxicity, the stability of the ion-chelate complex is an important factor in the safety of these agents. For biological systems three aspects of stability are important: thermodynamic, relative, and kinetic. Thermodynamic stability refers to the affinity of the ligand or chelate for the metal ion. The stability constant or thermodynamic association constant determines how much free ion and free chelate simultaneously exists in the presence of a given amount of ion-chelate complex. Its inverse, the dissociation constant, measures how much of the ionchelate complex falls apart to release the free ion and free chelate. The in-vitro stability or dissociation constant in the absence of biologically relevant ions fails to predict the relative toxicity of different ion chelate complexes.^^ This results from the competition for the ligand by the endogenous ions of the biological system, such as Zn^"^, Mn^"^, Fe^+, Co^"^, Ca^+, and Cu^+, and for the ion by endogenous chelates such as transferrin, citrate, albumin, and amino acids. Many biological compartments have low pH which can result in significant amounts of protons. These protons coupled with the endogenous ions and ligands can cause the rapid displacement of paramagnetic ions such as Gd(III) from quite stable ion-chelate complexes. Thus the in-vivo thermodynamic stability constant of an ion-chelate complex may significantly differ from the in-vitro thermodynamic stability constant, and it is characterized by the selectivity constant, ^s- The selectivity constant is the affinity of a particular ligand toward a metal ion in the presence of other metal ions at a specific pH. It is conceptually similar to the conditional thermodynamic stability constant, A'cond, except that it accounts for all the metal ions that associate with the ligand, in addition to the protons. The conditional stability constant, ^cond^ for a ligand with n protonation sites is: [ML] K^cond [M]([L] + [HL] + [H2L] + • • • + [H„L]) [ML] [L] [M] [L] + [HL] + [H2L] + . • • + [H„L]
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ERIK WIENER and VENKATRAJ V NARAYANAN _ „
f[L] + [HL] + [H2L] + • • • + [H„L] V '
= ^therai (1 + ^ H l [ H ] + KniKniU^
] H
+ ^H1^H2 • • • ^Hn[H
= i^thenn ( 1 + E 0 ^"^ ["^1" ) \
n
m=l
]")
^^^^
/
where n is the maximum number of protonation sites. For ions that form oneone complexes with the ligand, the selectivity constant, K^, is similar to the conditional stability constant: Ks = /iTtherm ( 1 + E \ n
0 ^ H m [ H + ] " + J^ ^ ' L W ) m=l i=a /
(25)
where a is the endogenous ion competing for the ligand, and ^aLla] is the product of the thermodynamic association constant of the competing ion-ligand complex and the competing ion (a) concentration. A comparison of the in-vitro stability constant and the selectivity constant for some representative chelates are presented by Watson et al.^^^ and in Table 11. Thus the presence of other ions result in a K^ much less than ^themiInterpretation of data comparing different formulations of agents must take into account the cause of toxicity. At the LD50 other issues besides dissociation and subsequent retention of the ion may be the primary cause of toxicity, for example osmolality. The above discussions deal with the degree to which an ion-chelate complex may fall apart to yield the toxic components consisting of the free ion and free ligand. All of this is made irrelevant if the biological system excretes the agent before it has time to fall apart. The kinetic stability of an agent indicates how rapidly the agent will dissociate and is given by the dissociation rate. The
Table 11. The selectivity constant is lower than the thermal stability constant Chelate
Log /sTtherm
Log Ks
(NMG)Gd(lll)-EDTA (NMG)2Gd(lll)-DTPA Gd(lll)-DTPA-BMA Gd(lll)-DOTA Gd(lll)-D03A Gd(Ili)-HP-D03A Gd(lll)-D03MA
17.3 22.5 16.8 25.3 21 23.8 25.3
4.23 7.04 9.04 8.3 4.1 6.95 8.3
Adapted from Watson et al.^"^^
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rate at which an ion-chelate complex dissociates,fed,is inversely related to the thermodynamic stability constant: h = - ^
^ therm
(26)
wherefedandfeaare the dissociation and association rate constants and A^therm is the thermodynamic association constant. These equations imply that even though the thermodynamic association/stability constants of two ion-chelate complexes may be similar, they can have vastly different rates of dissociation if their association rates significantly differ from each other. For example, the rate of association for Gd(III)-DTPA is very rapid whereas that of Gd(III)-DOTA is very slow. Their respective thermodynamic equilibrium association constants are 22.46 and 24.7, respectively, and this results in faster dissociation rates for Gd(III)-DTPA relative to Gd(III)-DOTA. In general, linear acyclic chelates like DTPA are less kinetically stable than macrocyclic chelates like DOTA. At lower pH the acyclic chelates dissociate very rapidly.^^ The half-life of Gd(III) release from Gd(III)-DOTA is 11 days,^"^^ or greater than 1 month^^ at pH 1.0, 21 days at pH 1.5;^^^ it is predicted to be 2000 years at pH 6.0, and greater than 10"^ years at pH 7.^^ For the nonionic macrocyclic Gd(III)-HP-D03A it is 3 h at pH 1.0.^^ The acyclic chelates have half-lives of release of 10 min for Gd(III)-DTPA, and 30 s for the nonionic Gd-DTPA-BMA at pH 1.0.^^ Depending on the intended use of the contrast agent, it may see many compartments in vivo. The first compartment seen is usually the vascular compartment, and serum stability as a function of time is used to evaluate the kinetic stability of the ion-chelate complex in the presence of endogenous competing ions and ligands. Magerstadt et al. examined the serum stability of acyclic and macrocyclic chelates at pH 7.2 for more than 5 days. They reported that very little of the Gd(III) was released from Gd(III)-DOTA, but approximately 15% was released from Gd(III)-DTPA and Gd(III)-Bz-DTPA over 5 days.^^^ This difference in kinetic stability has significant implications for the design of contrast agents with prolonged retention by the body. It suggests that for agents with long excretion half-lives, such as large agents like macromolecular or dendrimer-based agents intended for targeting specific pathologies or organs, macrocyclic chelates will have advantages. The above discussion demonstrates that the rate of dissociation of the ionchelate complex relative to the rate of excretion contribute to the in-vivo release of the ion from the ion-chelate complex. As the chelate controls the dissociation rate, and the polymer size and charge controls both the rate and route of excretion, they will also modulate the toxicity of macromolecular agents. This means that both the chelate and dendrimer generation will influence the amount of Gd(III) retained by the body. That is different dendrimer-chelates-based agents could have different toxicities and leave different amounts of Gd(III) in
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ERIK WIENER and VENKATRAJ V NARAYANAN Table 12.
Dendrimer agents tested so far have outstanding toxicity profiles compared with clinically approved agents
Agent
Animal LD50 Effective dose (ED) LD50/ED Rei (mmole/kg) (mmole/kg)
(NMG)2-Gd-DTPA Gd-DTPA-BMA NMG-Gd-DOTA Gd(lll)-D03A Gd-HP-D03A Dendrimer cascade 24 Dendrimer gadomer 17
Mouse Mouse Mouse Mouse Mouse Mouse Mouse
8.2 34.4 11.2 7 12.0 30 30-34
0.1 0.1 0.1 0.1 0.1 0.025 0.025
82 344 112 70 120 1200 >1200
Tweed le^^ Van Wagoner^^^ Meyer^^
Adami36 Raduchel^^^
the body. Not much toxicity information is published in the literature. Studies on the toxicity of the Gd(III)-free generation-3 ammonia core MA^-dipropionate PAMAM demonstrate that this chelate is quite well tolerated in rats. Adam et al.^^^ reported on a Gd(III) complex with a generation-3 ammonia core PAMAM with DTPA linked to the surface amines via a carboxylate arm to form an amide bond. This agent has a molecular weight of about 18,000 which is less than 30,000 as described and had a LD50 of 30 mmole per kg body weight of rat, Table 12. This is over 1000 times more than the effective dose of 0.025 mmole Gd(III) per kg body weight and gives a therapeutic index that is quite large. More recently, Radiichel et al. reported on a benzene tricarbonyl core polyamidoamine dendrimer based on lysine and a nonionic macrocyclic chelate surface, gadomer-17. This agent also has a molecular weight of approximately 17,500, and it exhibited a LD50 of between 30 and 35 mmoles/kg. The retention of Gd after injection of gadomer-17 mimicked the monomeric chelates. Less than 1% of the injected dose was retained 1 week following injection and less than 0.5% was retained 14 days following injection.^^^ The high LD50 in conjunction with the high relaxivity, and therefore lower effective dose, of this dendrimer-based agent gives it one of the highest margins of safety reported for MRI contrast agents. Each family and generation will have its own retention profile and toxicity. C. Targeting Mechanisms Contrast by its definition is the difference in signal intensity of one tissue relative to another scaled to some reference. An effective contrast agent must therefore selectively alter the signal intensity of one tissue or pathology relative to another. This means that the contrast agent must either selectively accumulate in one tissue, organ, or pathology relative to the surrounding tissue, organs, or pathologies, or the contrast agent must have a different relaxivity in one tissue, organ, or pathology relative to another tissue, organ, or pathology. In
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this section we will present a general discussion of the principles involved in selectively targeting imaging agents, followed by specific examples with a focus on dendrimer-based agents whenever possible. Passive and active targeting are two mechanisms for selectively delivering imaging agents to tissues, organs, or pathologies. Passive targeting refers to the nonspecific delivery or accumulation of an agent to or in a particular tissue. Examples include the accumulation of Gd-DTPA in brain tumors or iron oxide particles by the reticuloendothelial system in the liver and spleen. Active targeting refers to the specific delivery of an agent to a cell, tissue, organ, or pathology such as the specific binding of agents to receptors expressed on the cell surface like the asialoglycoprotein receptor on hepatocytes or surface antigens found on tumors, and endogenous transport systems like the organic anion transporter found on hepatocytes, amino acid transporters on tumor cells, or the folate uptake system on tumors of epithelial origin. D. Contrast Agent Delivery
Regardless of the targeting mechanism used, an agent must leave the blood stream before it can either passively or actively accumulate in a cell, tissue, organ, or pathology. (Unless you are targeting the blood pool itself.) This accumulation depends on a number of biological properties such as capillary surface area, capillary permeability, the capillary blood flow, the plasma half-life of the agent, and the ability of the agent to move through the tissue. The capillary surface area controls the number of exits. Tissues or pathologies with higher capillary densities will also have higher capillary surface area. This provides more opportunity for an agent to extravasate or leave the vasculature, such as in malignant breast tumors.^^"^""^^^ The ability to leave the vasculature is controlled by the vascular permeability. A number of different types of capillaries exist with different permeabilities to water soluble agents, and not all organs, tissues, or pathologies have the same type of capillary or capillary permeability. There are four main types of capillary or exchange-vessel endothelium, the cells that make up the walls of the vessels.^^^ Continuous endothelium is found in the capillaries of skin, skeletal, smooth, and cardiac muscle in addition to lungs. Transport across these capillaries occurs through small gaps in the tight junctions between cells. This allows the passage of water and solute molecules smaller than plasma proteins. Diffusion of lipid soluble molecules such as water, O2, inert gasses and gaseous anesthetics directly through the cell membranes and cytoplasm also results in the transport of these molecules to areas of lower concentrations. The cytoplasmic vesicles formed by fluid phase uptake, pinocytosis, or endocytosis encapsulate plasma and can result in the transport of macromolecules between the two surfaces of the endothelial cells. These vesicles can also fuse to form temporary
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channels which can allow the transport of plasma proteins and macromolecules across the endothelium of transport vessels or capillaries. Fenestrated endothelium occurs in the peritubular, renal glomerular, gastrointestinal mucosa, and glandular capillaries. The transport mechanisms found in the continuous endothelium also work in fenestrated capillaries. The main difference is that fenestrated endothelium also have circular regions 50 to 60 nm in diameter called fenestrae. In the kidney glomeruli these fenestrae have openings larger than the gaps found between the junctions. This allows the more rapid passage of water, ions, and other small solute molecules while still preventing the passage of macromolecules. Size is the most important parameter, but the surface charge on the macromolecule also affects the permeability through glomerular capillaries. The highly anionic protein serum albumin has a hydrodynamic radius of 3.6 nm and very low permeability. Neutral and cationic molecules of the same size as serum albumin have approximately 8 and 17 times greater permeability, respectively. ^^^ The fenestrae of the gastrointestinal mucosa and peritubular capillaries are closed by a thin diaphragm. Discontinuous endothelium is found in the liver, bone marrow, and spleen. Two types of discontinuous endothelium exist as defined by the height of the cells. The low form is found in liver and bone marrow, and the high form with its taller cells is found in the spleen. Both types have discontinuities or gaps of similar size of about 1000 nm and high permeability between the endothelial cells. These gaps also occur in the basement membrane. Such large gaps in both the basement membrane and between endothelial cells results in the unhindered passage of serum proteins, macromolecules, and particulates from the capillaries into the tissue stroma. Tight-junction endothelium are found in the brain, spinal cord, and other parts of the central nervous system, in addition to the retina. These capillaries have tall endothelial cells that meet and connect in tight junctions that extend all around their borders, i.e., there are no gaps or interruptions between the cells. Few vesicles are found in these cells, and transport across tight-junction endothelium is limited to passive diffusion and active transport via carriers. That is transport is very selective. Water and small lipid-soluble molecules freely diffuse through these cells, but ions, proteins, and other hydrophilic species like glucose, amino acids, or the iron transport protein transferrin must be transported. A number of biological responses can also affect capillary permeability. Inflammatory responses result in the secretion of histamine and bradykinin. These agents can induce arteriolar vasodilation and endothelial cell contraction. This contraction produces gaps between endothelial cells of nonmuscular venules and sometimes capillaries with a size up to 1000 nm. This increase in permeability allows the extravasation of serum proteins and other macromolecules. Tumor vessels have hyperpermeable capillaries. Many rodent and human
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tumor cells secrete vascular endothelial growth factor, also termed vascular permeability factor or VPF.^^^ This tumor cell product greatly enhances vascular permeability, induces angiogenesis, and specifically activates the mitogenic process in endothelial cells.^^^ Yeo et al.^^^'^^^ reported that the effusions from 21 out of 32 malignant human tumors contained high levels of VPF compared with only 7 of 35 lesions lacking cytological evidence of malignancy. In addition, 6 of the 7 patients with high VPF levels but negative cytological assays had their cancers identified as malignant by other criteria. Human malignant gliomas secrete a vascular permeability factor,^^^ as do human colon adenocarcinoma cells.^^^ Senger et al.^^^ reported that five tumor cell lines secreted VPF and that the two tumorigenic lines secreted at least four times more than their non-tumorigenic counterparts. Secretion of VPF by tumor cells induces a hyperpermeability to macromolecules of the microvasculature supplying solid tumors.^^^ Very early studies demonstrate that macromolecules extravasate from the tumor vasculature and that solid tumors accumulate high levels of macromolecules such as serum albumin^^"^"^^^ andfibrin.^^^"^^^This occurred in both animal and human tumors. Busch and Greene^^ reported that rats bearing the Walker 256 carcinoma accumulated 3 times more radio-labeled serum albumin than did liver 3 h following administration. Brown et al.^^^ reported that fibrinogen entered solid tumors significantly faster than normal tissues. Dvorak^^^'^^^ reports that the tumor microvasculature of a number of solid transplantable tumors is hyperpermeable to macromolecules. He identifies the source of this hyperpermeability as well-differentiated venules and small veins composed of a continuous endothelium with mostly closed interendothelial junctions. He reports that the tumor-host interface and the bands of stroma between tumor nodules contain the leaky vessels. This leakiness does not result from structural defects in the vessels, interendothelial cell gaps, or vessel immaturity. Roberts and Palade report that VPF/VEGF increase the permeability of postcapillary venules, capillaries, and both muscular venules and capillaries.^^^ They show that the increased permeability occurs in conjunction with an opening of the endothelial junctions and the induction of fenestrae in both venular and capillary endothelia which normally lack fenestrae. The luminal and abluminal sides of the endothelium also exhibited transendothelial channels with diaphragms. These morphological changes occur within 10 min, and are specific for VPF/VEGF as the system reverts to normal following precipitation with anti-VEGF antibodies. Neither heat inactivated VEGF, histamine, or saline induce similar changes. The macromolecules that extravasate from leaky tumor microvasculature accumulate in the tumors at higher concentrations than in the blood or control tissues, and some examples that demonstrate this passive targeting to improve cancer therapy and diagnosis appear in the literature. ^^"^"^^^ Few studies on the physiological or physicochemical factors that modulate the passive accumulation of macromolecules by tumors appear in the literature.
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Matsumura and Maeda^^"^ used ^^Cr-labeled proteins, including bovine serum albumin (BSA), to study the effect of molecular weight on the passive accumulation of macromolecules by sarcoma 180 tumors in mice. The tumors accumulated all of the protein derivatives at different rates depending on their size. Nagy et al.^^^ studied the effect of macromolecular size on the influx and efflux of fluorescein-labeled dextrans from the peritoneal cavity of mice carrying either ovarian tumors or the TA3/St breast adenocarcinoma transplanted into the peritoneum. They reported that tumor accumulation of macromolecules resulted from both increased influx and retarded efflux. The increased tracer influx varied inversely with tracer size. Takakura et al.^^^ studied the effect of the size and charge on the tumor-specific accumulation of macromolecules. They compared cationic, neutral, and anionic dextrans of similar molecular weight, as well as cationic and anionic bovine serum albumins. Low molecular weight, 10,000 kDa, and positively charged dextran derivatives cleared rapidly from the plasma through urinary excretion and hepatic uptake, respectively. The administration of large negatively charged molecules, such as carboxymethyl dextran, and BSA resulted in the accumulation of approximately 11, and 8% of the injected dose per gram of tumor tissue as compared to 4 and 1 to 2% for the comparable cationic forms, respectively. They concluded that the passive targeting of macromolecular drugs to tumors depended on the plasma half-life and recommended the use of polyanionic moieties larger than 70,000 kDa in molecular weight. Maeda coined the phrase "enhanced permeability and retention effect" for this accumulation of macromolecules by tumors. It is simply the passive tumor targeting of soluble macromolecules resulting from tumor capillary hyperpermeability and retention resulting from poor lymphatic drainage of the tumors. Thus tumor cells secrete a factor which alters the vascular permeability or influx and affects another one of the parameters that governs the extravasation or efflux of a drug or contrast agent. The above discussion on capillary structure and permeability demonstrates that one can take advantage of these differences to passively target agents, and this also applies to active targeting. Before an agent can get to a cell, tissue, organ, or pathology for active targeting it must also leave the vasculature, unless you are targeting the blood pool or capillary endothelial cells (see below). However, by taking advantage of active transport mechanisms associated with particular capillary endothelium one can also actively induce the extravasation of an agent and target specific tissues. For example, the tight junctional capillary endothelium of the brain must transport iron. These endothelial cells have very high concentrations of receptors for the iron transport protein transferrin. Linking agents to antibodies of this receptor have been used successfully to transport agents across the blood-brain barrier via a mechanism called transcytosis.^^^"^^^
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The findings of Takakura et al. described above are consistent with the idea that the blood-to-tissue extraction of an agent also depends on the plasma half-life, i.e., the time it takes for the blood concentration of the drug or contrast agent to decrease by 50%. Although the rank order of capillary permeability is cationic > neutral > anionic for normal capillaries, the greater accumulation of the anionic agent must result from a gain in plasma half-life that more than offsets any differences in capillary permeability. This difference in the effect of charge on tumor uptake may also result from real differences in the charge selectivity between tumor and normal capillaries, or differences in basement membranes. Basement membranes of tumors are disrupted and have less of the negatively charged proteoglycan heparan-sulphate in areas undergoing neovascularization. None of the work in the literature distinguishes between these two possibilities. The plasma half-life provides a measure of the time that an agent resides in the blood and is available to leave the capillaries and enter a tissue, organ, or pathology. For extracellular fluid space (ECF) agents, the time dependence of the plasma concentration of an agent can be described by a biexponential model, Eq. 27, where [CA]p gives the plasma concentration, a and ^ are time constants for distribution and plasma clearance, and A -\- B is the hypothetical plasma concentration at time zero. The ECF agents are lipid insoluble and have low molecular weights. [CA]p = Ae""' + Bc-^'
(27)
0.693 Ti/2, = - y -
(28)
The concentration of an agent in plasma is a function of the volume of distribution and the clearance or elimination of the agent from the body. The volume of distribution is the apparent volume in which the agent is distributed in the body during a steady state when the concentration of the agent in that volume equals that in the plasma. ^^^ The first exponential describes this distribution phase and the mixing of the agent with the plasma. The second phase is the elimination phase from the body, and it is assumed to follow first-order kinetics, and is described by the second exponential. It is therefore characterized by an elimination or plasma half-life. This is generally a valid assumption providing that the agent does not saturate the elimination process. Saturation can occur when the elimination is governed by a membrane transport system, or a receptor-mediated process. Saturable processes can result in nonlinear pharmacokinetics when the concentrations of agent saturate the elimination pathways. This nonlinear model does not apply to the extracellular fluid space agents, but may apply to the next generation of agents that are targeted to the hepatobiliary system.
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There are two main elimination processes consisting of the kidneys and the hepatobiliary systems. Linear pharmacokinetics generally occurs with agents that clear through the kidneys, assuming healthy kidneys. Nonlinear pharmacokinetics can develop for systems that clear exclusively through the hepatobiliary system. Increasing the size of the agent and modifying the charge can alter the relative contribution of the two clearance mechanisms and alter the plasma half-life, as can the association of an agent with serum proteins. The main mechanism of elimination via the kidneys is through glomerular filtration. The fenestrated capillaries have diameters of 100 nm and readily allow serum proteins through. However, the basement membrane is made up of a very dense meshwork of collagen and negatively charged glycoproteins which can sieve molecules by charge. Thus both size and charge are important parameters. Shape is also an important parameter, elongated flexible polymers have higher filtration coefficients than globular polymers of the same size. This means that spherical dendrimer-based agents should have longer plasma half-lives than agents prepared from linear polymers like polylysine. This was indeed reported by Adam et al.^^^ who showed that a 59,000 Da polylysine based agent had the same plasma half-life as an 18,000 Da agent derived from an ammonia core polyamidoamine, 1.4 h in rats. Capillary blood flow also effects the transport of contrast agents out of the capillaries and into the tissue interstitium. Contrast agent transport across capillaries is flow limited and depends on blood supply when the product of the capillary permeability and surface area are high.^^^ Under these conditions diffusion between the plasma and interstitial fluid approaches equilibrium, and the concentration of contrast agent rapidly falls when the plasma travels from the arteries to the veins. The flux of the contrast agent away from the capillary decreases as the rate of blood flow decreases or as the difference in the arterial and interstitial contrast agent concentrations decreases. This generally applies to very small ion-chelates complexes, except in capillaries with tight junctions, like those found in the central nervous system. In addition to tight junctions, the continuous endothelium presents a significant permeability barrier to macromolecules like proteins or large polymers. If the permeability and capillary surface area are both small then their product is also small. This results in transport across the endothelium that is no longer flow dependent, but is diffusion limited. In diffusion-limited systems, the differences between the arteriole and venous contrast agent concentration is small. In many tumor systems the penetration of antibodies is limited. This may result from either higher tumor interstitial pressures,^^^ an antigen barrier,^^^ or both. Transport through normal tissue is usually via free diffusion, and depends on the mobility of the solute through the tissue as characterized by the diffusion coefficient. Tumors lack an organized lymphatic drainage system that results in the build-up of significant interstitial pressures, which causes an outward flow
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of liquid to the edges of the tumor. This means that macromolecules are no longer free to diffuse inward but now must also deal with convective forces, and this may contribute to the poor tumor penetration by some antibodies. ^^^ Alternatively, intratumoral binding sites on tumor cell surfaces would also restrict diffusion. Such an antigen barrier would effectively reduce the mobility of a targeted agent through the tissue. Higher concentrations of some antibodies may overcome these problems and result in better tumor penetration, and this was observed.^^^~^^^ The above discussion deals with the five basic biological properties that influence the passive transport of an agent across the capillary endothelium into a tissue or pathology. These principles hold regardless of the targeting mechanism used. In order to passively or actively target a contrast agent to a cell, tissue, organ, or pathology it must leave the vasculature, unless you are targeting the blood pool itself or a pathological phenotype that is manifest in the capillaries. The role of size, shape, and charge in the extravasation of an agent into a tumor give some indications on how to optimize dendrimer-based agents. Extracellular Two classes of passive targeting are represented by the extracellular fluid space and intravascular contrast agents. Extracellular fluid space agents were the first commercially developed agents. They are hydrophilic, lack specificity, and freely diffuse through leaky capillaries to accumulate in the tissue or tumor interstitium. Initial applications started with the detection of pathologies in the central nervous system, CNS. The accumulation in the tumor interstitium results from differences between the leaky tumor capillary endothelium and the adjacent normally impermeable tight-junctional capillary endothelium of the CNS. The use of bolus injections, rapid imaging techniques, slow water exchange in the brain, and the presence of tight junctions in the brain capillary endothelium make it possible to use these agents in the measurement of relative blood volume.^^^'^^^ Although the extravasation, from capillaries lacking tight junctions, of these agents is rapid, imaging techniques have improved, and become sufficiently rapid^^^ to allow the use of these agents in determining capillary permeability^^^~^^^ and magnetic resonance angiography.^^^~^^^ Five extracellular fluid space agents are approved for clinical use today. The first two are based on the ionic complexes of Gd(III)-DTPA and Gd(III)-DOTA. The subsequent three agents were prepared as nonionic derivatives of these ionic complexes. They are the bis-methylamide of Gd(III)-DTPA, the hydroxypropyl amide of Gd(III)D03A, and the trihydroxybutylamide of Gd(III)D03A.i^^ All of these agents behave similarly. They have approximately the same relaxivity and plasma half-life. The longitudinal relaxivity at 20 MHz ranges from 3.5 ± 0.1 for Gd(III)-DOTA to 3.8 ± 0.1 for both Gd(III)-DTPA-BMA and Gd(III)-DTPA.^2 The volume of distribution ranges from 0.2 to 0.3 1/kg, and
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the elimination half-life is 1.5 h in humans and 20 min in rats. All are predominantly excreted through the kidneys and less than 1% of the injected dose remains in mice 1 week after injection.^^^ The lack of a formal charge on either the Gd(III)-DTPA-BMA and Gd(III)-HP-DOTA allows these agents to be formulated with lower osmolalities and viscosities. This allows the injections of high concentrations of agents over shorter times. Second-generation ECF agents have been prepared by linking more than one chelate together.^^'^^^'^^^~^^^ This still maintains the high water solubility and lack of specificity characteristic of ECF agents, but it increases the rotational correlation time, and the relaxivity. The fact that dendrimers are a class of compound implies that different families and generations within a family can have different biological and pharmacokinetic properties. Thus the low molecular weight agents can act as extracellular fluid space agents. A generation-2 ammonia core polyamidoamine based agent^^^'^^^ has a higher relaxivity than both the classes of monomeric and oligomeric agents, clears through the kidneys, and readily penetrates rat mammary tumors. Fig. 27. The increased size results in longer clearance kinetics than Gd(III)-DTPA, and stretches out the time required to reach maximum enhancement. Fig. 28. Intravascular Intravascular or blood pool agents have the potential of measuring regional blood volume, tissue perfusion or blood flow, abnormal capillary permeability, and can be used in magnetic resonance angiography. Like the ECF agents described above, the use of passive approaches to develop intravascular agents also works. More recently, Lauffer et al. also demonstrated active targeting of the blood pool. Passive approaches rely on making the agent large enough to remain in the vasculature for a long time relative to the imaging time. The two main techniques consist of making macromolecular contrast agents or particulate contrast agents. The first macromolecular agent was prepared by Lauffer and Brady following a technique developed for the nuclear medicine field. They attached five DTPA molecules to serum albumin and complexed them with Gd(III).^^^ Using similar techniques macromolecular agents were prepared from linear polymers such as polylysine,^^^"^^"^'^^'^^^ and dendritic polymers such as the polyamidoamines.'^^^ Many other agents based on linear polymers such as polysaccharides! (dextrans), polyethyleneglycol, and different dendrimer families with various chelates were also prepared.
Figure 27. Dendrimers easily penetrate rat mammary tumors. Ethyl-nitrosourea-induced rat mammary tumors before (upper part) and 10 min after injection of 0.02 mmoles Gd(lll) (on a generation-4 ammonia core PAMAM-TU-DTPA agent)/kg rat (lower part).
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Brasch et al. extensively studied in-vivo applications of macromolecular intravascular contrast agents using serum albumin DTPA derivatives as a model agent. They showed that macromolecular intravascular contrast agents are useful in defining tissue ischemia, reperfusion, and measuring blood volume.^^^"^^^ They proved the efficacy of such agents in measuring capillary permeability associated with cancer,^^^""^^^ responses to chemotherapy,^^"^ inflanmiation, pulmonary edema,^^^ and vascular mapping.^^^^ Kent et al.^^^ demonstrated the usefulness of such agents in measuring cerebral blood volume and perfusion associated with cerebrovascular occlusive disease. Dendrimer-based contrast agents have been extensively studied as intravascular contrast agents. Early reports of macromolecular contrast agents based on dendritic polymers used anmionia core polyamidoamine dendrimers linked to the DTPA chelate via the dendrimer surface amines and one of the five carboxylate arms on the chelate by forming an amide bond creating a FAMAM-MA-DTTA.-^^^ Later this approach was used with a generation-3 PAMAM to produce an agent called Gd-DTPA-24-cascade polymer. ^^^'^^^ PAMAMs were also reacted with a bifunctional chelate of DTPA (PAMAM-TUDTPA) that left the five carboxylate Ugands unaltered^^^'^^^ and a bifunctional chelate of D03A.^^^ The earliest intravascular applications of dendrimer-based agents used Gd(III) complexes of the PAMAM-TU-DTPA derivative for MR
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angiography.^^^ This dendrimer family was also used in analyzing tumor capillary permeability, and it was demonstrated that different generations exhibited different tumor capillary permeabilities.^^^ Margerum demonstrated that dendrimer generation and surface coating affected the plasma half-life. Dendrimer-based agents with a polyethyleneglycol surface had longer plasma half-lives. ^^^ A number of different groups demonstrated the utility of the PAMAM-MA-[DTTA-Gd(III)]„ derivatives as intravascular agents. The generation-3 derivative (Gd-DTPA-24-cascade polymer) was compared with a macromolecular polylysine based agent, and even though the molecular weight was 1/3 that of the polylysine derivative, similar enhancement characteristics were observed.^^^'^^^ Schwickert et al.^^^ demonstrated the ability of Gd-DTPA-24cascade polymer to enhance MR angiography. This agent also helped differentiate between benign and malignant canine breast tumors,^^^ hypovascular liver tumors,^^^ and rapidly growing tumors from slowly growing ones.^^^ Roberts et ^j 223,224 demonstrated the use of this agent in measuring capillary permeability. Bourne et al.^^^ reported on an ammonia core polyamidoamine derivative coupled to a bifunctional macrocyclic chelate of DOS A. They reported that a generation-5 agent, with a molecular weight of 58,000 Da, effectively improved MR angiography techniques at doses that were 5 times lower than those used with monomeric chelates. More recently, the benzenetricarbonyl core polyamidoamine dendrimer, gadomer-17, developed by Radiichel et al.^^^ was examined for use as an intravascular agent. Nguyen-minh et al. demonstrated the superiority of this agent to ECF agents in the detection of recurrent herniated disks.^^^ Li et al.^^^ demonstrated the effectiveness of gadomer-17 at improving coronary magnetic resonance angiography, while Jo et al.^^^ and Clarke et al.^^^ demonstrated its utility in 3D and 2D time of flight MR angiography, respectively. Chung et al.^^^ showed that gadomer-17 improved the visualization, relative to ECF agents, of viable myocardium after acute myocardial infarction. Nalcioglu's group have used this dendrimeric contrast agent to monitor changes in tumor vasculature following chemotherapy^^^ and radiation therapy.^^^ They have also compared the use of this 17,000 Da compound with serum albumin based macromolecular agents, and low molecular weight ECF agents in differentiating benign, and malignant rat mammary tumors of different grades.^^^ The dendrimer-based agent is capable of differentiating different tumor grades. Use of a dendrimer backbone to construct an effective contrast agent has been developed by a group at Schering AG.^^"^ Gadomer-17 consists of a trimisenic acid core attached to second-generation lysine dendrons with 24 complexed gadolinium ions on the periphery (Fig. 29). Quantitative renal elimination and higher intravascular retention time provide an excellent signal-to-noise ratio making it a highly desired contrast agent. An alternative approach to intravascular agents includes the use of partic-
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Gadolinium Complex <^J^^> Qj
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Figure 29, Dendritic MRI contrast agent with 24 peripheral complexed gadolinium ions.
ulates like ultrasmall superparamagnetic iron oxide particles, monocrystalline iron oxide nanoparticles^^^ (MIONs), manganese hydroxylapatite, paramagnetic liposomes, and paramagnetic red blood cells. The blood half-life of superparamagnetic iron oxide particles depends on their size, the hydrophilicity of the particle coating, and the particles net charge.^^^'^^^ Shorter half-lives are achieved with larger particles, coatings of low hydrophilicity, and particles with net charge differing from zero. Extravasation of the particle from the vascular endothelium is also controlled by size, but is also affected by the coating and charge.
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The magnetic properties of iron oxide particulate agents depend on their size. Whereas ferromagnetic particles retain a net magnetization after their removal from a magnetic field, superparamagnetic particles lose their net magnetization following removal from a magnetic field. The superparamagnetic threshold occurs in the range of 30 to 50 nm, and particles with sizes less than 20 nm are used as superparamagnetic contrast agents. In general, the larger-size superparamagnetic agents have transverse relaxivities, r2, that are much larger than their longitudinal relaxivities, ri. This resulted in their characterization as negative enhancers, because their accumulation in tissue results in the loss of signal intensity in T2 weighted images for that tissue. As the size of these particles declines, the r2/ri ratio also declines, and it was observed that the smaller agents could also enhance the signal of tissue, which has accumulated the agent, in T\ weighted images. Chambon reported the use of iron oxide particles as both T2^^^ and Ti^^^ based blood pool agents for studying myocardial perfusion. This coincided with Hamberg et al.'s^^^ and Pederson et al.'s^"^^ use of iron oxide particles to measure cerebral blood volumes with steady state susceptibility MRI and cerebral blood flow, respectively. These agents have been shown to improve MR angiography in both animals and humans. In pigs, ultrasmall iron oxide particles enhance the portal vein of the liver.^'^^'^'^^ Abdominal, coronary, and renal vessels in rabbits are also enhanced by this particulate intravascular agent.^'^^'^'^'^ Tanimoto et al. showed that the use of USPIO also benefited 3D phase contrast MR angiography techniques. They reported that USPIO improved the visualization of the abdominal aorta, renal artery, inferior vena cava, and portal vein in rats.^"^^ In humans the use of USPIO as intravascular agents improves the visualization of coronary and peripheral arteries^^^ using 2D time of flight and 2D and 3D spoiled gradient echo angiography techniques.^"^^ This agent also improves renal and coronary vessel definition in humans when using 3D MRA techniques.-^^^ Similar to the results found in the animal studies, Mayosmith et al. showed that the use of USPIO as intravascular agents improved the visualization of the aorta, inferior vena cava, and portal vein in humans.^"^^ Adzamli et al. reported a new concept in particulate intravascular agents.^^^ This agent was prepared by substituting the paramagnetic ion Mn(II) for the diamagnetic Ca(II) ion in polyethyleneglycol (PEG) stabilized hydroxylapatite.^^^ The percent PEG controlled the particle size. Higher percentages of PEG resulted in smaller particles. The larger particles were aggregates that dissociated into smaller aggregates in plasma, but remained in the blood for longer periods than the small particles. Newer, more stable, particles were prepared with a diphosphonate terminated PEG. This produces a 8 nm particle when PEG surface densities of 35-40 mol% were used. These agents have a ri and r2 of 18.7 and 22.3 s"^ rnM"^ at 20 MHz. Their plasma clearance or elimination half-life in the rabbit is 49 min, and these agents are effective in MR angiography.^^^
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Liposomes have carried imaging agents for many years.^^^ Liposome-based MR contrast agents have been prepared by either encapsulating the paramagnetic agent into the interior of the liposome^^^'^^^ or by incorporating the paramagnetic complex onto the outer surface of the liposome.^^^"^^^ The relaxivity of these agents depends on many factors. For liposomal agents based on encapsulation, the relaxivity depends on the water permeability of the liposomes. The water permeabihty depends on the lipid composition,^^^'^^^ the liposome structure,^^^ and size.-^^^ The lipid composition determines the phase behavior as a function of temperature. Higher concentrations of cholesterol result in lower relaxivities.'^^^ The liposome structure also affects the relaxivity, with multilamellar liposomes having lower relaxivities than those composed of unilamellar vesicles.^^^ The size of the liposome entrapping the MR contrast agent controls the lipid surface-area-to-entrapment-volume ratio. Smaller liposomes have higher relaxivities.^^^ For liposomes with the paramagnetic complexes attached to the outer surface, the relaxivity depends on the motional correlation times. For larger vesicles, 50 to 400 nm, the motions of the complex in the plane of the membrane dominate, and the relaxivity becomes independent of liposome size above 50 nm.^^^ linger et al. demonstrated the use of liposomes encapsulating Gd(III)-DTPA as an intravascular MR contrast agent.^^"^'^^^ The liposome size affects the intravascular retention time. Small liposomes were retained in the blood pool longer, although the Gd(III) was retained by organs for a very long time.^^^ Attaching the chelate complex to the liposome surface can result in very high relaxivities. linger et al. prepared liposomes with membrane-bound manganese, called "memsomes", which have higher relaxivities than other nanoparticles of similar size.^^^ Storrs et al. developed a new type of liposomal contrast agent based on a derivative of Gd(III)-DTPA bound to the surface and the two hydrophobic acyl chains containing diacetylene groups. Irradiation with ultraviolet light induces the diacetylene groups to polymerize.^^^ This polymerization improves the stability of the liposome. This may even reduce the molecular motions of the Gd(III)-chelate complex within the membrane surface, and impose a particle size dependence on the relaxivity above 50 nm. These agents produced similar enhancement of the intravascular space as other liposome preparations, but the elimination phase half-life of this agent was markedly longer at 19 h. In the same way that lipid membranes of liposomes hold Gd(III)-chelates inside the vesicles, they can also keep hydrophilic Gd(III)-chelates inside of cells. This approach was used very early by Doucet and others^^"-^^^ who loaded red blood cells with Gd(III)-DOTA via a hypo-osmotic shock method. Red blood cells are designed to stay in the intravascular space, and cells loaded with Gd(III)-DOTA act as excellent blood pool agents. The circulating blood signal remained enhanced by 100% for 48 h after injection, and the myocardial wall was clearly enhanced by 10 to 40%. Johnson et al.^^^ also used Gd-chelate
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loaded red blood cells. They used a different technique to load the RBCs with Gd(III)-DTPA called the osmotic pulse technique. This technique is basically a hyperosmotic shock method. They also reported that RBCs with entrapped Gd-chelates are stable and enhance the intravascular space. The total plasma clearance time of these agents was 0.003 1 kg~^ h~^ and the biological half-life of gadolinium within the RBC fraction was 4.38 h. They report an order of the degree of tissue enhancement with the renal medula > aorta > renal cortex > spleen > liver > muscle. An alternative approach to developing intravascular MR contrast agents is to induce an association between the contrast agent and one of the constituents of blood like serum proteins or red blood cells. Serum albumins make up one of the largest fractions of serum proteins found in plasma, and they bind a diverse number of different molecules.^^^ These binding sites include bilirubin, ibuprofen or L-tryptophan, several fatty acid, and cation binding sites. Lauffer first articulated this approach^"^ and presented the prototypical agent iron-ethylenebis-A^,A^'-[2-hydroxyphenyl]-glycinate, Fe-EHPG,^^^ that acted as both a liver agent and a serum albumin binding agent. The structural properties that affect the binding of this agent to serum albumin were also investigated.^^^'^^^ FeEHPG binds to human serum albumin via the bilirubin binding site. Increasing the lipophylicity increases the binding affinity to HSA.^^ Both the racemic (RR + SS) forms of Fe-(5-Br-EHPG) and Fe-(EHPG) preferentially bind to the high affinity site on HSA relative to the meso form. This may result from structural similarities between bilirubin IX^ and Fe-(EHPG).^^^'^^^ Similarly porphyrin binding to plasma proteins and their use as MR contrast agents was also examined. Many authors characterized the binding of porphyrins to serum albumins.^^^'^^^ Lamola et al.^^'* demonstrated that protoporphyrin IX was at least 90% bound to serum albumin when dissolved in human plasma. Shortly after the report by Lamola, Jackson et al.^^^ reported the use of Mn-protoporphyrin IX as an MRI contrast agent. Thus while originally developed as tumor enhancing agents,^^^ they bind to serum albumin and should enhance the vasculature when injected intravenously. Lyon et al.^^^ reported that plasma solutions slightly increased the relaxivity of Mntetrakis(4-sulfonatophenyl)-porphyrin (Mn-TPPS) relative to water solutions of
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the compound. This smaller-than-expected increase may have resulted from the relative concentrations of Mn-TPPS and serum albumin. Yushmonav et al.^^^ reported that at excess concentrations of albumin, Mn-TPPS binds to albumin in a high spin state, and that the relaxivity of the bound form significantly exceeds that of the free form. Serum albumin also has many fatty acid binding sites that can be targeted. Wesbey et al.^^^ reported the use of albumin targeted nitroxides as blood pool or perfusion agents to measure reduced blood flow in infracted myocardium. Bennett et al.^^ characterized the effect of nitroxides binding to human serum albumin on the relaxivity of the nitroxides. They report an increase in relaxivity upon binding to serum albumin. Structure activity studies show that the relaxivity of the nitroxide albumin complex is independent of the nitroxide position on the fatty acid chain. Studies in plasma, blood, and with various plasma proteins demonstrate that the primary interaction with plasma proteins result from that of serum albumins. In blood, Bennett et al. found approximately a 1:1 distribution of the lipophylic nitroxide radicals between HSA and RBCs.^^^ The relaxivity of the nitroxide on the RBCs was lower than that for the nitroxides bound to HSA. Vallet et al.^^^'^^^ examined the effect of the acyl chain length on the relaxivity of an albumin complex with the stable nitroxide TEMPO. They report that the relaxivity of the TEMPO/albumin complex depends on the acyl chain length and has an optimal length of about 10 carbon atoms. The noncovalent interaction of the nitroxide with albumin increases the relaxivity by a factor of 7 at 10 MHz. They examined the noncovalent interaction with a number of different proteins and reported that it is specific for serum albumin. Gallez et al.^^^ also compared the relaxivity of lipophilic nitroxides with hydrophilic nitroxides in plasma and in the presence of albumin. They examined the effect of five- and six-member ring nitroxides on the stability of the nitroxide to reduction. The five-member rings were more stable. Regardless of the ring size, the
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presence of serum albumin increased the relaxivity of the lipophiUc derivatives. The high toxicity of most of the derivatives tested limited the in-vivo studies to methyl-12-(2,2,5,5-tetramethyl-l-pyrrolidinoxyl-3-carbonylamino)dodecanoate. The compounds discussed above all demonstrate the feasibility of designing intravascular blood pool agents that noncovalently bind to serum albumins. The low relaxivities or high toxicities have motivated the development of second-generation agents based on chelates of Mn(II) or Gd(III). The design of these agents have primarily consisted of Gd(III)-chelates with some lipophylic group attached.^^'*"^^^ These agents have much higher relaxivities than those reported above with values ranging from 30 to 55 s~^ mM~^ for serum albumin bound adducts. Lauffer et al. developed the chelate (2-(/?)-[(4,4-diphenylcyclohexyl)phosphonooxymethyl]-diethylenetriaminepentaacetate and complexed it with Gd(III), DPCPOM-DTPA-Gd(III), Angiomark™. The relaxivity increased from 6.6 ± 0.4, in PBS, to 42.0 ± 0.3 s'^ m M - \ in PBS with HSA, and 54 lb 4, in human plasma, at 20 MHz. Under the experimental conditions greater than 94% of the compound was bound. The relaxivity and percent bound in plasma depended on the plasma donor species. The relaxivity in rabbit plasma was only 32 ib 2 even though 96% of the agent was bound. Adzamli et al. also developed a Gd(III)-DTPA-based noncovalent binding agent, 4-pentylbicyclo[2.2.2]-octane-l-carboxyl-di-L-aspartyl-lysine derived DTPA-Gd(III), POCAL-DTPA-Gd(III). They also reported high relaxivities in the presence of albumin or blood. The relaxivity increases from 6.2 in vitro to 30.5 in blood ex vivo at 20 MHz and 40°C.^^^ Aime et al. have taken a similar approach using macrocyclic chelates. Interaction of the macrocyclic chelate with HSA was induced by attaching benzyl-oxy-methylethers (BOM) to the acetate arms of Mn(II)-D03A and Gd(III)-DOTA derivatives. They reported that the strength of the association with HSA depends on the number of BOM arms. The relaxivity of Mn(II)-D03A-(BOM)3 and Gd(III)-D0TA-(B0M)3 increased from 1.18 to 8.1 and from 4.5 to 53.2 s~^ mM~^ at 20 MHz and 39°C, respectively, upon binding to HSA. This interaction occurs at nearly two equivalent binding sites in the HSA subdomains IIA and IIIA.^^^'^^"^ The observation that the degree of BOM substitution affects the association of the Gd(III)-chelate with HSA may explain why the liver agent Gd(III)-EOB-DTPA only weekly binds to HSA.^^^'^^^ This compound has only one ethoxybenzyl substituent which results in the association of only 20% of the chelate in a 1 mM Gd(III)-EOB-DTPA and 5% HSA solution^^^ and a 1-4 mM dissociation constant.^^^ The binding of Gd(III)-EOB-DTPA also involves the subdomain IIA of HSA. Another reversible binding approach was developed by Aime et ^j 291,292 xhey modified DTPA by making the bis-boronic acid phenylamide. The boronic acid moiety allows the chelate complex to interact with sin-diols found on glycoproteins. Interaction with glycated albumin results in a ternary complex, and the relaxivity increases from 4.6 to 6.9 s"^ mM"^ at 20 MHz,
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ERIK WIENER and VENKATRAJ V NARAYANAN R
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25°C, and pH 7.3. This small increase in the relaxivity may result from either weak binding or extremely long water residence times that are common to bis-phenylamides. In addition to increasing the relaxivity, the noncovalent binding of low molecular weight ion-chelate complexes increases the elimination phase and plasma half-life and partially restricts the agents to the vasculature thus reducing the volume of distribution. These additional properties help overcome some of the problems associated with MR angiography using the ECF agents, and these new agents significantly improve MR angiograms.'^^^'^^^ The two best characterized agents are the DPCPOM-DTR\-Gd(III) and POCAL-DTPA-Gd(III). Parmelee et al. compared DTPA-Gd(III) and DPCPOM-DTPA-Gd(III) in cynomolgus monkeys. They reported that the elimination half-life for the noncovalent binder was 2.56 times longer than DTPA-Gd(III) and the volume of distribution was reduced by 33% relative to DTPA-Gd(III).29^ The DPCPOM-DTPA-Gd(III) at one fourth of the DTPA-Gd(III) dose enhanced the vasculature in MR images for more than 60 min, whereas the vessels in images of animals receiving DTPA-Gd(III) were barely visible 10 min after injection.^^^ Grist et al.^^^ demonstrated the effectiveness of DPCPOM-DTPA-Gd(III) in both steady state and dynamic MR angiography in humans. This agent significantly enhanced both peripheral and carotid vessels. They obtained both vascular and selective arterial enhancement with dynamic MR angiography, and excellent spacial resolution in steady-state images of the arteries and veins.^^^ Prasad et al.-^^^
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showed the usefulness of this agent in measuring renal perfusion and MR angiography in pigs. Dolen et al.^^^ reported on the use of this compound to study myocardial perfusion. Lin et al.^^^ showed that noncovalent binders to serum albumin reduced the problems in MR angiograms associated with slow flow. This enabled the detection of carotid artery occlusions in an atherosclerotic pig model. In humans the use of DPCPOM-DTR\-Gd(III) overcomes problems associated with slow flow that occur during end-systole, and is suitable for use with navigator pulse-based cardiac-imaging techniques.^^^ In cardiac imaging this agent improved blood-to-muscle contrast in cine acquisitions, and improved functional and anatomic images with high muscle-to-blood contrast.^^^ Adzamli et al.^^^ reported that POCAL-DTR\-Gd(III) had a 142 min elimination half-life in rabbits. This compound also enhanced 3D MR angiograms. The blood half-life was reported as 60 min compared with 39 min for DTPA-Gd(III), and this resulted in significant enhancement of 3D MR angiograms.^^^ The LD50 in mice was 3 mmole per kg. The potential application of dendrimers to the noncovalent binding type of agents could bring together the advantages of both macromolecular and noncovalent binding. The use of a dendrimer that is cleared through the kidneys, is larger than the monomeric agents, binds to serum albumin, but is large enough to have significantly smaller volumes of distribution, would in theory provide more accurate blood volume measurements than the monomeric agents with higher volume of distributions. The above review demonstrates that there are many different approaches to the development of intravascular agents. These agents may play a very important role in perfusion and cardiac imaging. Dendrimer-based agents are one very effective approach for making an intravascular agent. Liver
Targeting contrast agents to the liver aids in the detection of focal liver diseases such as tumor diagnosis, cysts, inflammatory lesions, and diffuse liver disease such as fatty liver, radiation-induced hepatitis, cirrhosis, and iron overload. It may also help evaluate hepatobiliary and reticuloendothelial system function. In the case of the hepatobiliary system it can provide the distribution and function of the hepatocytes, enhance the biliary tract, and provide evidence for bile flow or obstruction. As with the vasculature, the liver can be both passively and actively targeted. The three main strategies used for targeting contrast agents to the liver employ either the reticuloendothelial system (RES), receptors expressed on the hepatocyte surface, or carrier systems expressed on the hepatocyte surface membrane. Passive targeting to the liver uses the RES, and takes advantage of the very large gaps in the liver capillary endothelium which allows small particulate material to escape and enter the liver. Cells of the RES are primarily found in the liver, spleen, and bone marrow. This
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system is responsible for the removal of colloidal suspensions from the blood. Macrophages, such as the hepatic Kupffer cells, of the RES phagocytose particulate matter of an appropriate size. Colloids are between 0.05 and 5 microns in size, and those less than 0.1 micron are preferentially accumulated by the bone marrow whereas the liver predominantly accumulates those of intermediate size ranging from 0.3 to 1 micron. Kupffer cells in the normal liver parenchyma make up roughly 85% of the RES activity in the body, while making up only 15% of the liver cell population.^^^ Prior to phagocytosis, the particles must be opsonized by either antibodies or other opsonins.^^^ The use of polyethyleneglycol on the surface of particulates retards the opsonization process, and extends the blood half-life of particulate agents. Particulate contrast agents such as liposomes, magnetite, and Mn-hydroxyapatite all accumulate in the RES. The second method for targeting agents to the liver uses receptors expressed on the liver cell surfaces. The asialoglycoprotein receptor is uniquely expressed on the surface of hepatic parenchymal cells. The extremely large number of receptors expressed on the cell surface coupled with the rapid internalization and recycling of these receptors make them an outstanding target for delivering drugs or contrast agents to the liver. This receptor has an affinity for a large number of desialated serum proteins that have sugar attached. In particular it has a very high specificity for galactose-terminated glycoconjugates. The third alternative approach to cellular targeting of the liver uses endogenous transport or carrier systems. The liver expresses at least four carrier or transport systems that accumulate, either organic anions, organic cations, neutral compounds, or bile salt conjugates. These systems are located on the hepatocyte surface and transport red blood cell breakdown products such as bilirubin, and other molecules like bile acids, and fatty acids. In the case of bilirubin or other organic anions, intermediate binding and storage molecules such as glutathione-5-transferase alter or sequester the molecules in protein storage vesicles prior to their transport into the bile. The large number of agents and choices of targets provides great flexibility in liver imaging. Contrast agents can either enhance or diminish the signal intensity of the tissue which it accumulates in, and one can target either the pathology or the neighboring tissue. This provides two methods of either increasing the signal intensity of the pathology relative to the neighboring tissue or decreasing the signal intensity of the pathology relative to the neighboring tissue. As contrast agents are designed to make it easier to detect a given pathology the preference of a brighter or dimmer pathology relative to the neighboring tissue belongs to the radiologist. However, the biology may also impose limits. Liposome-based particulate MRI contrast agents for liver enhancement were introduced just prior to iron oxide-based particulates. Recall that when used as blood pool agents one could either encapsulate the paramagnetic ions inside
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the liposome or attach the ion-chelate complex to the exterior of the liposome surface. The earliest applications encapsulated known MRI contrast agents or ions inside of the liposome. Magin et al.^^^ used liposomes to encapsulate MnCl2, which reduced the cardiotoxicity as these agents and accumulate in the liver, spleen, and bone marrow. The effective dose of the liposome-entrapped MnCl2 was 7 to 10 times lower than the LD50 of free MnC^. The livers of mice with transplanted R3230 adenocarcinomas enhanced 2- to 3-fold after injection, while the tumors showed very little change in signal intensity.^^"^ While the liposomes accumulate in the Kupffer cells, no substantial changes in the relaxation rate of the majority of liver is observed when the Mn(II) remains confined to the Kupffer cells. Bacic et al.^^^ reported that once inside the Kupffer cells, the Mn(II) is released from the liposomes, is transported outside of the Kupffer cells, and accumulated in the neighboring hepatocytes. Right after the introduction of liposomes as particulate agents, passive targeting of MR contrast agents to the liver was done with particulate magnetite-based agents. These are colloidal suspensions of multiple iron oxide crystals surrounded by a biocompatible matrix. Unlike the normal liver, liver tumors lack the phagocytic activity of Kupfer cells and thus the tumor signal intensity remains unchanged following the injection of an RES agent. In the case of iron oxide, the surrounding normal tissue shows drastic reduction in signal intensity of T2 weighted images.^^^"^^^ Particle size controls the blood half-life with larger particles clearing from the blood faster. Saini et al.^^^ first demonstrated significant reductions in the transverse relaxation times of both liver and spleen. These reductions lasted for up to 3 weeks after injection. These particles enhance the delineation between normal liver and implanted mammary carcinomas in the rat.^^^ Kawamura et al. examined the utility of a colloidal suspension of magnetite in rats fed the carcinogen diethylnitrosoamine. The dextran-coated magnetite improved tumor detection from 10% before injection to 65% after injection. This included tumors as small as 2 mm in diameter.^^^ Kamba et al. used a chondroitin sulfate-coated iron colloid to examine hepatocellular carcinoma (HCC) in humans. This agent was initially developed for the treatment of asiderotic anemia. Despite the low relaxivities, T\ and T2 are 0.44 and 2.3 s"^ mM"^ the decrease in liver signal enhances the visualization of the HCC quite nicely.^^^ In HCC the reticuloendotheUal system is impaired, and this technique provides a functional measure of the RES. It also provides a technique for grading HCC tumors, as the less differentiated HCC have fewer liver RE cells than the well-differentiated HCC.^^^ Kalbaka et al.^^^ and others^^^ encapsulated paramagnetic ions in the form of Gd(III)-DTPA into liposomes, and they showed that these agents also enhance the liver and spleen of mice and rats. Recall that the relaxivity of liposomal agents encapsulating an ion-chelate complex depends on the surface-area-to-volume ratio and the water permeability. Smaller vesicles with
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high water permeability are best.^^^ In rats the Gd(III)-DTPA entrapped in liposomes significantly improved the detection of liver metastases, whereas the free Gd-DTPA in the absence of liposomes significantly reduced metastasis detection.^^^ Like the encapsulated MnCl2, the Gd(III)-DTPA must escape the liposome prior to enhancing the liver. In a perfused rat liver model, treatment with Gd(III) entrapped in the liposome reduced the longitudinal relaxation time of the P-phosphorus atom of intrahepatic ATP. As ATP does not enter liposomes, the Gd(III)-DTPA must escape.^^^ The size of these agents affects the biodistribution and the plasma half-life, linger et al.-^^^ showed that 50 nm particles entrapping Gd(III)-DTPA remained in the blood longer than 100 nm liposomes, and more of the Gd(III) went to the liver, spleen, and bone marrow. The 100 nm particles predominantly accumulated in the spleen followed by the liver when normalized per gram of tissue. Kalbaka et al.^^^~^^^ and others^^^'^^^'^^^ examined liposomal particulate contrast agents with the paramagnetic ion-chelate complex attached to the outer surface of the vesicle. These agents are generally prepared using a fatty acid with a chelating head group. For Gd(III)-based systems, this generally consisted of linking DTPA via one or two of the carboxylate head groups. Kalbaka et al. developed bisdistearyl-DTPA derivatives using two carboxylate arms with either amide, ester, or thioester linkers.^^^'^^^ They also examined the effect of the alkyl chain length on the in-vivo life times.^^^ Like the encapsulation-based agents, these agents also enhance the liver, spleen, and bone marrow.^^"^ The in-vivo stability is modulated by the type of chemical bond that links the hydrophobic alkyl chain to the hydrophilic Gd(III)-DTPA complex. The order of stability in serum is thioester < ester < amide.^^^ This results in faster clearance from the liver for the less stable derivatives. The chain length of the hydrophobic alkyl groups also influenced the in-vivo residence time. Derivatives with shorter alkyl chains cleared faster.^^^ Phospholipid-based Gd(III)-DTPA agents were also developed.^^^'^^^ These agents combine the amphiphylic phospholipid with a hydrophilic Gd(III)-DTPA complex by linking the phospholipid head group to DTPA via only one of the carboxylate arms. The combination of phosphatidylethanolamine with DTPA resulted in a compound that retained the basic features of the phospholipid: a polar head group and hydrophobic fatty acid side chains. The relaxivities of the liposome-based agents varied depending on the percent chelating phospholipid used to make the liposomes, and are 21, 15, and 12 s"^ mM~^ for 12.5, 25, and 50% chelating agent, respectively. These agents also accumulated in the liver and spleen, and were excreted via the hepatobiliary system. The gut showed significant accumulation 24 h after injection. As with the Gd(III)-DTPA amphiphiles described above, liposomes with amphiphylic Mn(II)-chelates have also been prepared.^^^'^^^'^^^-^^^ Unger et al.^^^ examined the effect of liposome diameter and chain length on the
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relaxivity and ability to enhance liver metastases. Liposomes prepared with a 10 carbon alkyl chain amphiphile had higher relaxivities and showed better improvement in detecting liver metastases than those prepared with an 18 carbon alkyl chain amphiphile. The relaxivities were quite high and like the Gd(III)-DTPA systems, the smaller liposome had the higher relaxivities. Those of the 10 carbon long chain derivatives were 5.73, 24.0, and 30.16 for 400, 100, and 30 nm diameter liposomes.^^^ A comparison of liposomes containing trapped Mn(II) or surface bound Mn(II) showed that the derivatives containing the surface-bound Mn(II) resulted in greater and more specific enhancement.^^^ The above results show that the particulate agents used as intravascular agents also enhanced the RES system. This is a quite general application. Although Adzamli et al. did not examine enhancement of liver metastases with the Mn-hydroxyapatite agents, they did show that the liver enhanced following injection of these particles.^^^'^^^ Mn-hydroxyapatites accumulate in the RES system.^^^ These agents accumulate in the liver as a result of nonspecific phagocytosis of particles by the RES. They represent examples of these types of MRJ agents. Many other particulate agents can be conceived. The use of bifunctional chelates with the hyperbranched structures called combursts, dendrimers grafted on to linear polymers, should result in RES targeting as the combursts are extremely large. They can have molecular weights of 10 million daltons. Targeting the RES with particulates is a passive approach to liver targeting. These agents lack specificity for either the hepatocytes or tumors. Active targeting of tumors or hepatocytes can easily be accomplished by targeting cell surface receptors. The development of MRI contrast agents has followed that of nuclear medicine-based agents. This is also true of the liver-based agents, and like the hepatocyte nuclear medicine agents, MRI contrast agents targeted to hepatocyte receptors were prepared by targeting the asialoglycoprotein receptor. Vera et al.^^"^ demonstrated the targeting of radioisotopes to the asialoglycoprotein receptor, and showed that galactosyl-labeled proteins resulted in receptor-specific accumulation of the agent in liver hepatocytes. Josephson et ^j 325 ^j.g^ jnodified this approach to MRI contrast agents by targeting iron oxide particles to the asialoglycoprotein receptor. This was followed by Weissleder et al.^^^ who coupled arabinogalactan to ultrasmall superparamagnetic iron oxide particles (USPIO). These agents specifically accumulate in hepatocytes that express the asialoglycoprotein receptor. In rats, injection of low doses of the agent reduced the signal intensity of liver, but that of the spleen remained virtually constant. Reimer et al.^^^ demonstrated the use of these agents in the differentiation of normal liver, acute hepatitis, chronic hepatitis, and cirrhosis of the liver. These agents also differentiated normal liver and hepatocellular carcinoma at lower doses than RES agents.^^^ Dynamic contrast enhancement studies comparing the signal intensity curves of the livers in rats injected with either
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USPIO, arabinogalactan-USPIO, or a RES-based magnetite agent revealed significantly different kinetics.^^^ Competitive inhibition studies with asialofetuin or galactose inhibits hepatocyte accumulation of the asialoglycoprotein receptor targeted agents. Two paramagnetic-based agents were also targeted to the asialoglycoprotein receptor. Gallez et al.^^^ targeted a nitroxide spin label by conjugating pyrrolidinoxyl radicals to arabinogalactan. Livers in mice injected with the targeted agent show significantly more enhancement than those in mice injected with the unconjugated control radical. Vera et al.^^^ targeted a Gd(III)-DTPA-based agent to hepatocytes via the asialoglycoprotein receptor by linking DTPA to polylysine via one of the carboxylate arms. They conjugated a mixed anhydride of DTPA and 3-oxopropyl-l-thio-beta-D-galactopyranoside to polylysine making a polyDTPA-polyneogalactosyl-polylysine derivative with 858 chelators and 2284 galactose groups. Injection of 0.017 nmioles Gd(III) per kg rat enhanced normal hepatocytes or hepatic tissue, but not a liver-implanted mammary adenocarcinoma. Hepatocytes exhibit a number of transporter systems, outlined above. These systems exhibit transport maxima for agents specific to these sites, and their transport is competitively inhibited by compounds that are transported by the same carrier system. The primary goal of these agents is to examine the function of the hepatobiliary system, enhance bile ducts, illuminate biliary obstruction, and provide information on hepatocyte distribution which helps identify focal liver disease such as liver metastases. The best characterized carrier system is the organic anion transporter. Hepatobiliary studies with agents targeted to this transporter began with radiolabeled rose bengal. Structure activity studies using ^^"^Tc-iminodiacetic acid-based agents revealed four conmion structural properties to agents specific for the organic anion carrier. Organic anion carrier specific agents were organic anions with molecular weights between 300 and 1000, contained a lipophylic group with at least two planer substituents (like phenyl groups) that were far removed from the polar anion group, and they bound to serum albumin.^^^"^^^ Three transporters of sulfobromophthalein (BSP) have been identified in liver by classical methods.^^^ The BSP-bilirubin binding proteins have a molecular weight of 55 kDa^^^'^"^^ and the bilitranslocase is a 37 kDa protein.^^^ Antibodies to these proteins inhibit BSP uptake. The organic anion binding protein is a 55 kDa integral membrane protein located on the sinusoidal liver membrane.^^^ Recent studies using an expression technique identified a 71 kDa organic anion transporting polypeptide.^"^^ In addition to a chloride-independent BSP transporting mechanism with a K^ in the micromolar range, the presence of BSA results in a high affinity, A'm < 1 |xM, chloride-dependent extracellular BSP-facilitated transport mechanism. Transport is of the BSP only. The BSA remains extracellular. Expression of this protein in Xenopus oocytes also
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results in the chloride-dependent uptake of bile acids, taurocholate, and cholate. Immunohistochemical studies demonstrate that this protein is located on the basolateral side of rat hepatocytes. The observations reported by Lauffer et al. for a prototypical MRI hepatobiliary agent are consistent with the conclusions outlined above for radiolabeled hepatobiliary agents. The iron ethylene-bis(2-hydroxyphenyl)glycine Fe(EHPG)~ complex has two benzyl groups, a negative polar area, binds to serum albumin, and has a molecular weight between 300 and 1000. They prepared four derivatives differing in their lipophylicities as measured by the octanol: water partition coefficient. The percentage of the compound that bound to serum albumin coincided with the order of lipophilicity. The iron in Fe(EHPG)~ lacks an inner sphere water molecule, and as a result its relaxivity stems from outer sphere mechanisms only. Lauffer found that the longitudinal relaxivity equals 1 s~^ mM~^ and that the relaxivity of Fe(EHPG)"^ or its analogs increases upon binding to serum albumin by 2 to 4 times.^^^ This means that the in-vivo relaxivity can be much higher than the 1 s~^ mM~^ reported for an aqueous buffer solution. The signal intensity of rat livers increased by more than 200% in images obtained with a Ti weighted inversion recovery pulse sequence.^^^ Thirty minutes after injection, more than 9 and 15% of the injected dose accumulated in the liver and small intestines of rats, respectively.^^ In dogs, both the liver and gall bladder were enhanced. Analogs with different lipophylicities demonstrated that the compounds with an intermediate lipophylicity had optimal uptake from the blood, and biliary excretion from the hepatocytes. The most lipophylic derivative had the highest percentage of the injected dose remain in the blood and liver but the lowest in the small intestines.^^ These findings demonstrate that an optimal lipophylicity exists for targeting the hepatobiliary system and are in good agreement with previous studies.^"^"^'^"^^ Recently, Sheu et al.^^^ demonstrated that normal livers in mice enhance more than pathologic livers with biliary obstruction, or acute hepatitis after the administration of Fe(III)(EHPG)~. Subsequent hepatobiliary MRI agents used paramagnetic analogs to the radioscintography hepatobiliary agents. Either chemical instability or RES uptake rendered the gadolinium analogs unsuitable. Engelstad et al.^"^^ examined gadolinium derivatives of diethyl and diisopropyl IDA. They reported both urinary and hepatobiliary excretion along with a reduction in the proton longitudinal relaxation time of excised rat liver. Prolonged retention of the gadolinium was observed in rats treated with these agents, and the distribution resembled results obtained with GdCh. Golman et al.^"^^ tried a chromium derivative of HIDA. They reported similar pharmacokinetics to iodinated hepatobiliary contrast agents and significant enhancement of the gall bladder 10 min after the injection of 0.01 mmol/kg into rats and rabbits. Liver enhancement required the use of higher doses.
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chrfP OH /
/ H O
CO2H
CO2H
N, N'-bis(2-hyciroxybenzyl)ethylenediamine-N, N'-diacetic acid
Chart 11, Structure of N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid.
Hoener et al.^"^^'^^^ have developed two other iron chelates targeted to the liver. They first prepared an iron complex of phenylglutaryl-desferrioxamine B which accumulated in the bile of bile-duct cannulated rats. Bromosulfophalein inhibited the accumulation in the bile and blocked uptake by the hepatocytes. No enterohepatic recirculation was observed.^^^ The phenyl group was required for accumulation in the bile. Iron complexes of succinyl and glutaryl derivatives of desferrioxamine B and the parent compound itself enhanced the kidneys, but not the bile ducts and small intestines. An iron complex of phenylsuccinyl-desferrioxamine B had a similar enhancement profile to that of the iron phenylglutaryl-desferrioxamine B derivative.^^^ They also showed that an Fe(III) complex with A^,A^^-bis(2-hydroxybenzyl)ethylenediamine-A^,A/^^diacetic acid was primarily excreted by the hepatobiliary system. In bile-duct cannulated rats 52 ib 8% of a 0.05 mmole/kg dose was excreted into the bile after 90 min.^'*^ Derivatives of Gd(III)-chelate-based nonspecific extracellular fluid space agents have also been prepared to target the hepatobiliary system. The chelate Gd(III) - 3,6,9 - triaza - 3,6,9 - tris(carboxymethyl) - 4 - (4 - ethothybenzyl) - undecandicarboxylic acid (Gd(III)-EOB-DTPA) described earlier was designed as a hepatobiliary specific agent. Although the molecular weight of Gd(III)-EOB-DTPA fits the parameters described for the hepatocyte organic anion transporter, it has only one planar substituent which is not far removed from the polar anion group, and it only weakly binds to serum albumin. In spite of these characteristics, Clement et al. report that hepatocytes accumulate Gd(III)-EOB-DTPA via an organic anion transporter-dependent mechanism.^^^ Weinmann et al. reported that this compound has a longitudinal relaxivity at 37°C and 0.47 T of 5.3 ib 0.13, 8.7 ± 0.5, and 16.9 ib 2.2 s~^ mM~^ in water, plasma, and excised liver, respectively. Note that in plasma and liver the relaxivity is a weighted average from free Gd(III)-EOB-DTPA and that bound to macromolecules. Vander Elst et al.^^^ characterized this agent and from the binding constant and NMRD profile calculated that the relaxivity at 37°C and 0.47 T was 62 s~^ mM~^ The theoretical volume of distribution in rats is 0.020 ± 0.01 1/kg. The distribution and elimination half-lives in rats are 1.8 ib 2 min and 10.2 ± 1 . 9 min, respectively. The total clearance is 36.7 ib 3.6 ml min~^ kg~^. This agent exhibits both hepatobiliary and renal excretion with the majority of the agent passing through the hepatobiliary system of rats (70%). One week after injection, the liver and
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kidney each retained 1 |xg Gd/g tissue while the brain, fat, skeletal muscle, and femur retained, respectively, 0.08, 0.6, 0.2, and 0.1 \xg Gd/g tissue. In rats the use of this agent enhances the liver relative to tumors. The contrast-to-noise ratio of Gd-EOB-DTPA-treated rats with primary,^^^ transplanted,^^"^ or chemically induced^^^ liver tumors exceeded that of Gd-DTPA. The distribution of enhancement by Gd-EOB-DTPA also differed from 99ni'p^_ IDA. Although both agents are taken up by the organic anion carrier, they showed a different enhancement pattern between differentiated hepatocellular carcinoma and the neighboring liver. In the Gd-EOB-DTPA-treated rats, the liver enhanced more than the tumors. In contrast, the differentiated hepatocellular carcinomas accumulated more ^^"^Tc-IDA than the neighboring liver 30 min after treatment.^^"^ Clement et al.^^^ reported that Gd-EOB-DTPA enhanced the liver more than it enhanced a transplanted adenocarcinoma, and that the pattern of enhancement was the opposite of that seen with Gd-DTPA. Treatment with Gd-EOB-DTPA increased the contrast-to-noise ratio more than 5 fold for up to 50 min after injection. Ni et al.^^^ studied both primary and transplanted liver tumor models. They also reported that Gd-EOB-DTPA enhanced liver more than tumor giving significant improvements in the contrast-to-noise ratio of grade II to IV differentiated and undifferentiated hepatocellular carcinomas. Similar findings were observed in humans. Vogl et al.^^^ compared Gd-EOB-DTPA with Gd-DTPA and reported that at 0.25 to 0.5 times the dose of Gd-DTPA, Gd-EOB-DTPA exhibited similar dynamic enhancement characteristics during the dynamic phase. During the uptake phase, up to 4 h after injection, Gd-EOB-DTPA significantly improved the detection of additional metastases, hepatocellular carcinomas, and hemangiomas. Reimer et al.^^^ demonstrated the use of Gd-EOB-DTPA in characterizing hepatocellular carcinomas, metastases, and hemangiomas relative to one another. With dynamic contrast enhancement methods, hepatocellular carcinomas showed prolonged enhancement relative to metastases and hemangiomas. Metastases exhibited a heterogeneous uptake followed by a washout, and hemangiomas showed a delayed washout relative to metastases. Like Gd(III)-EOB-DTPA, dihydrogen [4-carboxy-5,8,ll-tris(carboxymethyl)l-phenyl-2-oxa-5,8,ll-triazatridecan-13-oato(5-)gadolinate(2-), also known as Gd(III)-BOPTA and Gadobenate, is a liver-targeted derivative of Gd(III)-DTPA with a single benzylether group.^^^ It has the requisite molecular weight for transport by the hepatocyte organic anion transporter, but similar to Gd(III)-EOB-DTPA it has only one planer substituent which is close to the polar anion group. It only weakly binds to serum albumin.^^^ This weak binding still results, in vitro, in a 142% increase in the signal intensity of rat blood relative to the parent compound Gd(III)"DTPA, and a 182% improvement in relaxation enhancement of blood in vivo relative to Gd(III)-DTPA 5 min after injection. In rats this agent has a high rate of biliary excretion and provides ex-
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cellent liver enhancement.^^^ Six hours following injection the urine contained 55% and the bile 39% of the injected dose. In humans 95% of the injected dose is excreted in the urine, but it still provides long and significant enhancement of the normal liver parenchyma. Inside the hepatocytes this agent binds to intracellular proteins increasing the relaxivity. This high intracellular relaxivity makes up for the low percentage of the injected dose that is excreted through the biliary pathway and sufficiently enhances the liver.^^^ This agent is excreted through the same pathway as bilirubin and BSP. De Haen^^^ reported that both bilirubin and BSP inhibit the accumulation of Gd(III)-BOPTA in the bile of rats. They conclude that the common transporter is located on the sinusoidal plasma membrane. The step limiting biliary excretion occurs on the bile-canalicular side of the hepatocytes. The Gd(III)-BOPTA successfully enhances the contrast between normal liver and a number of different liver diseases. In rats, Kreft et al.^^^ reported significant enhancement of tumor to liver contrast. This study included both infiltrating and noninfiltrating tumor models. Cavagna et al.^^^ also reported the ability of Gd(III)-BOPTA to differentiate focal liver disease from normal tissue. Both implanted liver tumors and those resulting from blood-borne metastases showed significant enhancement in the contrast-to-noise ratio. These findings were confirmed in humans. Injection of Gd(III)-BOPTA increased the contrast-to-noise ratio of metastases and resulted in the detection of many more small primary metastases.^^^ A number of studies have investigated the utility of Mn-dipyridoxaldiphosphate, Mn-DPDP, as a liver-specific contrast agent. The temporal hepatic enhancement provided a means of differentiating severely impaired livers from normal ones, and the gallbladder enhancement pattern distinguished between more subtle differences in liver function.^^^ Hepatocellular carcinomas have significantly reduced tumor-liver contrast following injection of Mn-DPDP, but other nonhepatomatous masses do not. This allows the distinction between primary HCC and secondary metastasis.^^^'^^^ This agent was designed to target the vitamin B6 carrier. Although the agent has a high stability constant (>10^^) its biodistribution is similar to free Mn^+. Gallez et al.^^^ demonstrated that MnDPDP deposited significant amounts of Mn^~^ in the brains of male Wistar rats. The maximum areas of uptake were the hippocampal region, thalamus, colliculi, amygdala, olfactory nuclei, and cerebellum. The injection of Mn-DPDP was also demonstrated to be teratogenic in rats.^^^ Colet et al.^^^ demonstrated that uptake of the agent by hepatocytes and perfused liver was not competed off by pyridoxine and therefore not via the vitamin B6 carrier as intended. They also demonstrated that the liver uptake is of the free dissociated Mn^+ via nifedipine inhibited calcium channels. In theory, dendrimer-based macromolecular agents can easily be targeted to the asialoglycoprotein receptor. The appropriate choice of macrocyclic
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Gd(III)-chelate and either arabinogalactan or other galactosyl moieties should hypothetically result in the same successful targeting as seen with polylysinebased derivatives. Pancreas
The liver agent Mn-DPDP also can act as a pancreas-specific contrast agent and significantly improve tumor to normal pancreas contrast.^^^"^^"^ Ahlstrom and GehP^^ demonstrated that Mn-DPDP increased the signal intensity of normal pancreatic parenchyma in Ti weighted images by 100%, but no enhancement of tumors was observed. The contrast-to-noise ratio increased by more than 200%. Targeting specific pancreatic receptors was achieved by the Weissleder group. They used two different methods consisting of either attaching secretin or cholecystokinin to monocrystalline iron oxide particles.^^^'^^^ The pancreatic cell targeted agents accumulated in the pancreas but not tumor. Lymph nodes
The Weissleder group has developed both iron oxide ^78-380 ^^^ Gd(III)^^^'^^^ based agents for MR lymphography. These iron oxide agents work following the same principles as the radiolabeled colloidal systems for radiolymphography. Once the agent extravasates it is trapped by lymph node macrophages. These macrophages have clean access to normal lymph nodes, but are blocked from entering metastatic lymph nodes. This allows the signal intensity of normal lymph nodes to decrease in T2 weighted images but not metastatic lymph nodes. They hypothesized that the dextran coat on the MION allowed the time for macrophage uptake. As a result of the hypothesized role of the dextran, they developed a gadolinium-based agent consisting of Gd(III)-DTPA-polylysine conjugated to dextran (Gd-DTPA-PGM). Subcutaneous injection of the agent increases lymph node signal intensity in Ti images of normal animals but not metastatic lymph nodes. Tumor Targeting
The flexibility of MRI provided by both the indirect detection of the contrast agent and the many biological parameters that influence the contrast provides a large number of approaches in the design of contrast agents that enhance a particular pathology or metabolic function relative to other tissue. In the case of targeting tumors we have already seen both passive and active targeting techniques for liver tumors. We have also seen techniques that left the tumor either bright or dark relative to the neighboring tissue. The passive targeting of iron oxide particles to the RES or the active targeting of coated iron oxide particles to hepatocytes expressing the asialoglycoprotein resulted in a brighter tumor relative to healthy liver. The active targeting of contrast agents to the organic anion carrier resulted in a darker tumor relative to the healthy liver.
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Recall that regardless of the use of passive or active targeting techniques, the agent must leave the blood stream and enter the tumor, and five basic biological properties control the extravasation of a contrast agent or therapeutic from the tumor capillaries into the tumor. The tumor capillary surface area defines the size of the leakage sight, and is related to the capillary density. The magnitude of the capillary blood flow determines the types of limitations placed on the extravasation, i.e., diffusion or flow limited. The capillary permeability determines if the agent can even leak out of the vasculature and enter the tumor. The plasma half-life of the agent determines the duration that the leakage is possible. The ability of the agent to traverse the tumor interstitium controls the degree of tumor penetration by the agent, and this parameter is affected by both a binding sink and the interstitial pressure. Passive. The first passive tumor targeting took advantage of differences in the permeability of brain and brain-tumor capillaries. Normal brain capillaries have tight junctions that restrict the movement of ionized or polar molecules. Tumor capillaries do not. Thus ionic or polar contrast agents generally do not pass into healthy brain, but do pass through the leaky capillaries of brain tumors thus permeating tumor interstitium. An analogous approach was developed for other parts of the body by Brasch who hypothesized that macromolecular contrast agents that were too large to extravasate from normal capillaries would highlight tumors as a result of the enhanced tumor capillary permeability. Tumors secrete factors that enhance the permeability of tumor capillaries to macromolecules, and the lack of any organized lymphatic system results in the retention of these macromolecules.^^^ A second approach that takes advantage of differences in capillary density instead of permeability uses dynamic contrast enhancement with low molecular weight contrast agents. Tumors secrete factors that stimulate capillary growth, and malignant tumors have areas with much higher capillary densities than benign tumors.^^^'^^^ The concept is that malignant tumors will demonstrate faster rises to peak enhancement and a quicker washout phase than benign tumors. Experiments in human breast cancers have demonstrated an improved specificity of diagnosis with this technique. Dendrimers have the potential to improve both types of passive techniques, and give the potential to control the plasma half-life. The use of dynamic contrast enhanced techniques with Gd(III)-DTPA in the differentiation of breast tumors requires high temporal resolution as malignant tumors reach their peak enhancement within minutes. In competition are the techniques of Schnall which require high spacial resolution to mark structural features that indicate malignancy. These techniques take more than 2 min. In theory the use of dendrimers or slightly larger molecules would prolong the time it takes to reach the peak enhancement and allow a combination of both techniques. The increased time to
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peak enhancement is demonstrated in Fig. 28. This figure shows a comparison of a 4th-generation ammonia core PAMAM-TU-DTPA-Gd(III) derivative with Gd(III)-DTPA. Other groups have used dendrimers to study tumor permeability and differentiate benign from malignant tumors. Schwickert et al.^^^ examined the angiographic properties of Gd-DTPA-24-cascade polymer in the peritumoral vessels of rats. This agent is a third-generation ammonia core PAMAM with DTPA linked to the surface amines via an amide bond through one of the acid groups DTPA, that is a PAMAM-MA-DTTA. It has a molecular weight of less than 18 kDa, which is less than the <30 kDa they stipulate, and a relaxivity at 3TC that is more than 2 times that of Gd(III)-DTPA. They compared this agent with both Gd(III)-DTPA and albumin-(MA-DTTA-Gd(III))3o which have molecular weights of 547 and 92,000 Da, respectively. The dendrimer-based agent combined the advantages of both the low molecular weight agent and the high molecular weight blood pool agent. It enhanced the tumor rim as well as Gd(III)-DTPA and produced excellent vessel delineation, although less than that of the albumin agent. Subsequently, Adam et al.^^^ compared the ability of Gd(III)-DTPA and the PAMAM-MA-DTTA to differentiate benign from malignant spontaneous canine mammary tumors. The two agents demonstrated significantly different enhancement kinetic profiles. Injection with Gd(III)-DTPA resulted in a rapid signal increase followed by subsequent decay of all the canine tumors over the 30 min observation period with no difference between the three tumor types. Injection of the dendrimer-based agent resulted in similar enhancement profiles for the two types of benign tumors, but the malignant carcinomas demonstrated slower delivery, delayed peak enhancement, and slower tumor clearance or a plateau over the same 30 min observation time. The dendrimer exhibited a washout half-life of 120 ± 66 min in the malignant tumors. This was significantly different, P = 0.0122, from the washout half-life of the combined benign tumors, 28 ± 22 min and 27 ± 9 min for the adenomas and mixed-tissue tumors, respectively. This resulted in higher uptake of the dendrimer-based agent by the carcinomas at 20 min after injection. Tacke et al.^^^ demonstrated that the PAMAM-MA-DTTA-based agent enhanced the contrast between liver tissue and hypovascularized liver tumors better than Gd(III)-DTPA. Two minutes following the injection of contrast media, the contrast-to-noise ratio of the tumor and liver for the dendrimer-based agent was significantly better than that of Gd(III)-DTPA. The dendrimer increased the signal intensity of the hypovascularized liver tumor, but to a much smaller degree than Gd(III)-DTPA, and the time to peak enhancement was also prolonged. Su et al.^^^ compared the enhancement profiles of PAMAM-MA-DTTA-Gd(III) with Gd(III)-DTPA and a polylysine-MA-DTTA-Gd(III), having an average molecular weight of 50 kDa, in three tumor models. They reported that both the dendrimer-based agent and the polylysine-based agent exhibited a prolonged time to peak enhancement of
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the interstitial space, and slower washout kinetics relative to Gd(III)-DTPA in the faster-growing tumor model. Daldrup et al.^^^ demonstrated that microvascular permeability to macromolecular MR contrast agents correlates with histologic tumor grade in ENU-induced rat mammary tumors. Su et al.^^^ followed with the inclusion of a dendrimer-based agent. They compared the ability of Gd(III)-DTPA, gadomer-17, and albumin-(MA-DTTA-Gd(III))3o to differentiate benign from malignant breast tumors. Recall that gadomer-17 is a second-generation dendrimer with a trimesinic acid linked to three diethylenetriamine molecules core conjugated to lysine monomer units with the terminal amines linked to a nonionic macrocyclic chelate. This gives a macromolecular contrast agent with 24 Gd(III) ions and a molecular weight of 17.5 kDa. As with the other macromolecular agents, the gadomer-17 exhibits quite different enhancement profiles relative to the low molecular agent Gd(III)-DTPA. The time to peak enhancement is prolonged as is the efflux from malignant tumors. The macromolecular agents demonstrated an ability to differentiate between different infiltrating ductal carcinoma tumor grades, but not between fibroadenoma and infiltrating ductal carcinoma. The use of macromolecular blood pool agents to measure relative blood volume also allowed the differentiation of tumor subtypes and showed significant differences between tumor periphery and center.^^^ Active. Initial tumor targeting of MRI contrast agents started with the direct attachment of Gd(III) to antibodies. Burton et al. first labeled immunoglobulins with Gd(III) using endogenous metal-ion binding sites.^^^ Lauffer and Brady then attached Gd(III)-chelate complexes to general antibodies and described the effect on the relaxivity of the Gd(III).^^^ This was followed by Curtet et al. who made a tumor-specific agent by coupling DTPA to a monoclonal antibody against human colon adenocarcinoma. ^^^ This agent specifically accumulates in a human colorectal adenocarcinoma xenograft in nude mice, and ^^^Gd images demarcate the tumor. Analysis of the tumor longitudinal relaxation rate ex vivo demonstrated significant changes relative to the unlabeled antibody, GdCla, and Gd(III)-DTPA. Although the antibody altered the relaxation rate ex vivo, it was realized that the relaxivities achieved with these agents were so low that each cellular receptor would require about 100 to 2000 Gd(III)-chelate complexes.^^^ Directly attaching this many ion-chelate complexes would alter the binding specificity and immunoreactivity of the antibody. The use of polymeric carrier systems like polylysine or proteins like BSA were developed to overcome this problem.^^^'^^^'^^^ These systems were then attached to the antibodies, but mixed results were obtained. The main problem was that the relaxivities obtained with the particular carriers and chelate were still too low. There are four techniques for overcoming the problems outlined above. One is by brute force. Develop a system or carrier that uses the low relaxivity
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ion-chelate complexes but has thousands of the Gd(III)-chelates. The second is to develop a system with high Gd(III) ion relaxivities and attach enough of these ion-chelate complexes to a carrier such that the carrier chelate system has a high molecular relaxivity. Alternatively, one can devise an agent that accumulates in cells, but is enzymatically transformed thus both increasing the relaxivity and trapping the agent. Lastly, one can genetically alter the cell to stimulate the uptake of endogenous or exogenous contrast agents. This latter approach results in MRI-sensitive reporter genes for gene therapy. The use of large particulate agents as carriers limits the location of the agent within the tumor to the perivascular or vascular space but allows the attachment of thousands of ions at a single site. Agents such as liposomes do not readily traverse the tumor stroma, thus one must consider antigens associated with either the perivascular or vascular space of the tumor. One very elegant approach reported by Sipkins et al.^^^ was to target antigens associated with tumor angiogenesis. This allowed the design of an agent that needed only to stay in the blood pool or vasculature. They used a polymerized liposome, developed in the same laboratory,^^^ with Gd(III)-chelates and biotin expressed on the surface and coupled it with avidin to a biotinilated antibody against the integrin a^P^. The rabbit V2 squamous cell carcinoma model specifically accumulated the agent in angiogenic areas. This agent provided a measure of angiogenic hot spots. Dendrimers offer the perfect carrier and amplification system. The linear increase in diameter and geometric increase in surface groups results in a larger fraction of the total mass coming from the ion-chelate complex when compared with linear polymers such as polylysine. In polylysine-based systems only 30% of the weight comes from the drug, but in the ammonia core PAMAM systems more than 70% of the weight comes from the Gd(III)-chelate. One can achieve even higher fractions by using building blocks with higher multiplicities. In addition to the sheer numbers of ion-chelate complexes per unit size, the appropriate choice of chelate can result in extremely high ion relaxivities. We first demonstrated this in 1990 when we reported relaxivities of 120 s~^ mM~^ for the A^,A^-dipropionate PAMAMs. These agents lacked the necessary biological ion-chelate stability so we then prepared the PAMAM-TU-DTPA and PAMAM-TU-Bz-DOTA derivatives. The first demonstration of targeting dendrimer-based MRI contrast agents to tumor cells was accomplished by conjugating folic acid to the dendrimers.^^^ This conjugation successfully targets dendrimers to cells that express the high affinity folate receptor in vitro and a generation-4 derivative can deliver enough Gd to detectably alter the cellular longitudinal relaxation rate.^^^ Recently, this system successfully targeted ovarian tumor xenografts in nude mice that express the hFR. The nature of the r\ and r2 NMRD profiles suggest that at high field strengths
228 Table 13,
ERIK WIENER and VENKATRAJ V NARAYANAN Folate-PAMAM-TU-DTPA dendrimers specifically accumulate in tumors that express the high affinity folate receptor
Contrast agent
Xenograft model
Folate-dendrimer (n = 5) Folate-dendrimer {n = 5) Gd(lll)-HP-D03A Competitive inhibition
hFR positive HFR negative hFR positive hFR positive
Post-PCE -2.6 +5.8 -1.1 -0.4
± 14.0 ± 5.66 ± 6.0 ±1.6
24 h after PCE -33.1 +5.9 +8.3 +6.0
± ± ± ±
52.0 16.3 18.6 7.7
PCE = percent contrast enhancement. (See text for statistical significance.) Using a planned comparison the PCE 24 h post injection for receptor positive tumors treated with folate-dendrimer differed significantly from the three controls at better than p < 0.05.
images that are effected by r2 will have a greater change in the signal intensity for the same amount of contrast agent. Although the g = 4 PAMAM-TU-DTPA derivative has a peak ri of 30 s"^ mM~^ at 0.5 T, it falls off at higher fields. This is not the case for r2. At 4.7 T the rx and ri of the g = 4 folate conjugated PAMAM-TU-DTPA derivative are 9.32±0.08 and 41 ± 3 s"^ m M - \ respectively. We therefore monitored T2 weighted images at 4.7 T to see if the g = 4 folate conjugated PAMAM-TU-DTPA derivative altered the tumor signal intensity and tumor contrast.^^^ The targeted dendrimer-based agent selectively alters the tumor contrast of ovarian tumors that express the hFR. Table 13 shows data comparing the percent contrast enhancement (PCE) in T2 weighted images as a function of contrast agent, time after injection, and tumor model. The T2 weighted images of the human ovarian tumor xenografts that express the hFR in nude mice showed a —33 di 52% enhancement in the contrast 24 h after injection of the folate conjugated dendrimer-based contrast agent. This is consistent with an r2 agent that accumulates in the target tissue. The signal intensity of the target decreases while that of the reference remains constant. This decline in the PCE compares with only a -h8.3 ± 1 8 % change in the same tumor model 24 h following the injection of a low molecular weight nonspecific extracellular agent (Gd(III)-HP-D03A). These results differ significantly at better than P < 0.05. This comparison does not address specificity; it only indicates that the larger agent accumulates in the tumor and that the smaller agent does not. One mechanism that may account for this accumulation in a nonspecific manner is an increased plasma half-life or blood pool effect. Proof of specificity and elimination of a blood pool effect comes from the results of two control experiments. The first examines the contrast of a human ovarian tumor xenograft lacking the hFR in nude mice, an hFR-negative control, after injection of the folate conjugated dendrimer-based contrast agent. Any nonspecific blood pool effect should occur regardless of receptor expression, and thus would also arise in this tumor model. The tumor PCE 24 h after the injection of the folate conjugated dendrimer-based contrast agents into mice
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with tumors that lack the hFR, i.e., the receptor-negative tumor model, was +5.9 lb 16.3. This PCE significantly differs from the PCE obtained with the same agent in the hFR-positive model at better than P < 0.05. It also means that accumulation of the folate conjugated agent requires expression of the hFR and is not the result of a blood pool effect. Competitive inhibition studies also attest to the specificity of the interaction or accumulation and can rule out a blood pool effect. In the event of a blood pool effect the PCE of tumor should remain constant regardless of the presence of free folate. The average PCE of tumors expressing the hFR 24 h after the coinjection of 0.3 mmoles/kg of free folate and 0.06 mmole Gd(III)/kg, on the folate conjugated dendrimer agent, was +6 lb 8. This differed significantly from the PCE of the same experiment obtained in the absence of any free folate, P < 0.05. None of the values of the PCE for the three control experiments significantly differed from one another. E. Enzymatically and Physiologically Activated Contrast Agents As an alternative to selectively altering the concentration of a contrast agent within a tissue or pathology relative to the neighboring tissue, one can alter the contrast by selectively altering the relaxivity of the contrast agent. One technique uses the amplification inherent in enzymatic systems. By choosing the appropriate enzymatic substrate-chelate complex one can in theory alter q, ^r» ^m» ^s. ^» or S. The first reported example of enzymatic activation of a conventional MRI contrast agent was made by Moates et al.^^"^ in an MRI-sensitive reporter gene system. This used a "caged" Gd(III) ion such that enzymatic activation increased the number of inner sphere water molecules, q, from 0.7 to 1.2. It provides a means of selectively altering the relaxivity of one tissue relative to another based on differences in enzyme expression. The agent consists of a caged Gd(III) complex which is of a macrocyclic chelate capped with a galactopyranose residue. They attached a galactopyranosyl residue onto a D03A chelate such that it blocked the water binding site. The 4,7,10triaceticacid-1 -(eth-2-oxy-P-galactopyranosyl)-1,4,7,10-tetraazacyclododecane (EGAD) when treated with P-galactosidase loses the galactopyranosyl cap to produce an MRI contrast agent with a larger value of q and a higher relaxivity. McMurry et al.^^^ developed an alternative enzymatically labile system that results in longer rotational correlation times instead of higher q values. They modified DTPA by attaching 4,4^-dihydroxybiphenyl via one of the hydroxy groups and converted the other to a phosphomonoester. The negative charge on the phosphomonoester prevents the agent from noncovalently binding to the hydrophobic binding site of serum albumins. The agent has a fast/short rotational correlation time, low binding to serum albumin, and a relatively low relaxivity. Activation with alkaline phosphatase converts the ionic phosphomonoester into a nonionic hydroxyl group. This unmasking of
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ERIK WIENER and VENKATRAJ V NARAYANAN OH
CH2OH
"OOC
"OOC-^^^
V^COO" EGAD
Enzyme Activated Phosphomonoester
Chart 12, Structures of enzymatically activated agents.
the hydrophobic group allows the agent to bind to the hydrophobic binding region of serum albumin. This increases the rotational correlation time and relaxivity of the agent. Li et al.^^^ demonstrated a third method of selectively altering the relaxivity that uses changes in the physiological status of the cell. The relaxivity of the prototype they developed changes as a function of the cellular Ca^"*" concentration. This agent attaches two D03A molecules to the fluorescent calcium indicator BAPTA such that in the absence of Ca^"^ one each of the iminoacetate groups attached to the BAPTA core associate with each of the Gd(III) ions coordinated to the DOS A molecule blocking the inner sphere water binding site and reducing the q value. The presence of Ca^"^ causes the iminoacetate groups to swing away from the Gd(III) ion to coordinate the calcium ion with the resultant increase in q and the relaxivity. Two other physical properties, reviewed earlier, that influence the relaxivity and have the potential for use in the design of functional or molecular MRI contrast agents are r^ and S. Sherry et al. recently reported that the appropriate placement of phosphonate groups on bisamide derivatives of DTPA can dramatically alter the relaxivity, and hypothesizes that this results from an increase in the water proton exchange rate of the inner sphere water molecules with the bulk solvent. He also demonstrates that ester groups block this increase in relaxivity. Thus the appropriate choice of masking group and enzyme could allow the enzymatic activation of the phosphonate to increase the proton exchange rate and thus increase in relaxivity. All of the above techniques suffer from the fact that the outer sphere component to the relaxivity still contributes to the base line relaxivity, so that the relaxivity goes from on to more on. One possible technique that in theory would go from a very low value of the relaxivity, basically a diamagnetic value, to an extremely high value takes advantage of different spin states of an ion depending on the ligand field. For example, Fe^"^ has a low and high spin state depending on the strength of the ligands. The low spin state is diamagnetic.
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and the high spin state is paramagnetic with S = 4/2. In theory, if one has the appropriate enzymatically labile functional groups that interfere with the strength of the ligand field one can go from a strong field to a weak field with a resultant low to high spin state conversion. That is a diamagnetic to a paramagnetic shift. Enzymatic activation would alter the functional group such that it weakened the ligand field. One might also use the paramagnetic low spin state of Fe^+ (S = 1/2) and shift it to a high spin state (S = 5/2). The 5 * (5 + 1) dependence of the inner sphere and outer sphere relaxivity means that the high spin state should have a relaxivity approximately 11.7 times higher than the low spin state, if all else was equal. In theory, this is better than the 1.4 times improvement observed with altering q. One physiological and natural example of this approach is the conversion of low spin Fe^"^ in oxyhemoglobin, no unpaired electrons, to high spin Fe^"^ in hemoglobin, 4 unpaired electrons. All of the techniques described above, both realized and hypothetical, take advantage of only one physical property. Tweedle^^^ suggested that one could combine systems by placing enzymatically or physiologically labile chelates on macromolecules like dendrimers giving the ion chelate complexes long rotational correlation times plus the enzymatically induced changes. This in theory would provide larger dynamic ranges in the relaxivity. F. Genetic Engineering of Endogenous Contrast The enzymatic systems described above can be used in reporter gene systems, especially as P-galactosidase is used as a reporter gene already in non-MRI applications. These systems, however, require two steps, the first being the successful transfection while the second requires the delivery of the enzymatically labile contrast agent to the transfected cells. Such systems are excellent for ex-vivo applications or applications using large cells that can have the contrast agent injected into it, but much more complicated than approaches that genetically alter the accumulation of endogenous contrast agents or paramagnetic ions. Systems that alter the endogenous concentrations of paramagnetic ions and contrast agents offer a simplified MRI-sensitive reporter gene system. The first example of a MRI reporter gene system was introduced by Koretsky et al.^^^ This group transfected cells with the gene encoding the human transferrin receptor. Transferrin is an iron transport protein found in the blood. After the iron is internalized the cell stores it in an iron storage protein known as ferritin which has both high and low iron loaded forms. This endogenous protein has significant T2 effects as a result of the iron. That is the r2, or transverse relaxivity, is much higher than most other proteins. Transfection of cells with the gene encoding the transferrin receptor resulted in increased expression of the receptor and a 2-3 fold increase in iron content. Expression of
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the receptor in tumor xenografts, generated with transfection-positive cells, was readily detected. T2 weighted images clearly delimited transferrin-expressing tumors by the decrease in signal intensity. These tumors showed a 20% decrease in T2 relative to tumors generated with nontransfected cells. Moore et al.^^^ introduce a unique modification on detecting transferrin receptor expression. They prepared an iron oxide probe targeted to the transferrin receptor. They showed that the contrast agent entered the cells but did not result in iron-induced down-regulation of the receptor, and that MRI could monitor receptor expression and regulation. Melanomas have relatively high water proton longitudinal relaxation rates caused by high melanin content, and melanin synthesis can be used in MRJsensitive reporter gene systems. Melanomas have high amounts of copper and iron,"^^^ and melanosomes have high concentrations of Fe^"^, Fe^"^, and Cu^"^."^^^ Melanin itself, when complexed with paramagnetic ions, acts as an endogenous macromolecular contrast agent. Okazaki et al.'*^^ demonstrated that melanincontaining tissue has significant amounts of Fe^"^ and Mn^"^ coordinated to the melanin. Enoch et al.^^^ reported that the NMRD profiles of Fe^"^, Mn^"^, and Cu^+ melanin complexes were consistent with the aggregation of the ionmelanin complexes into macromolecular particles which increased the relaxivity of the agents. This was also observed by Aime et al."^^"^ who reported a marked increase in the longitudinal relaxation rate and that the NMRD profile of Mn^"^-melanin indicated the formation of a macromolecular metal complex. Tosk et al."^^^ reported that melanin influenced the T\ and T2 only in the presence of paramagnetic ions, in the absence of Fe^+ melanin had no effect. Enochs et al."^^^ also reported that melanin increased the signal intensities in MR images of phantoms in the presence of iron that scaled with the iron concentrations, but that the relaxivities of different synthetic melanins complexed with iron varied. They also showed that the melanin in melanic melanoma cells scavenged paramagnetic ions. Zecca and Swartz'*^^ demonstrated that in-vivo neuromelanin coordinates large amounts of paramagnetic ions including Fe^+, Cu^"^, and Mn^+. All of these results support the conclusion that macromolecular aggregates of melanin with bound paramagnetic ions can act as an endogenous contrast agent. This conclusion is consistent with the findings that correlate melanoma signal intensity with melanin content but not total iron content."^^^'^^^ These findings are consistent with the concept that the paramagnetic ionmelanin aggregates act as the contrast agent. Measurements of the total ion and total paramagnetic ion content cannot tell you the role of iron or other paramagnetic species bond to melanin as this may make up only a small fraction of the ion content, but the relaxivity of the macromolecular complex can be quite high relative to the other ions. The fact that melanin binds iron, copper, and manganese in vivo would suggest that as there is more melanin in the cells there should be more of the paramagnetic ion-melanin aggregates too.
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The amount of the endogenous macromolecular contrast agent composed of paramagnetic ions coordinated to melanin aggregates found in cells is controlled by a number of enzymes. Weissleder et al.^^^ took advantage of the role that the enzyme tyrosinase has in melanin biosynthesis as a potential MRI-sensitive reporter gene. They transfected both mouse fibroblasts and human kidney cells with a gene containing a human tyrosinase. Transfection resulted in increased tyrosinase messenger RNA, melanin production, and ion scavenging relative to nontransfected cells. Transfected cells also showed higher signal intensities relative to nontransfected cells in MR images. The potential roles of dendrimers in MRI-sensitive reporter gene transfection is indirect. Macromolecular polymeric systems can act as gene vectors, and folate can even target polylysine-DNA complexes to cells that express the high affinity folate receptor. Baker et al. have used dendrimers as synthetic vectors for gene therapy or transfection. A review of the use of dendrimers as synthetic vectors goes beyond the scope of this article. One could even use a combination of the contrast agent and vector approaches of dendrimers. Linear polymers like polylysine also transfect cells and are used as backbones for MRI contrast agents. Kayyem et al."^^^ was first to demonstrate the feasibility of this approach. They showed that DNA encoding a marker gene could electrostatically bind polylysine molecules conjugated to transferrin and polylysine molecules containing covalently bound Gd(III)-DTTAMA complexes. These particles were cotransported into cells expressing the transferrin receptor. Work by de Marco et al."^^^ performed a similar experiment by coupling an aminated polylysine conjugated dextran wrapped around an iron oxide superparamagnetic core. The amine content controlled the DNA loading capacities. These agents transfected human cells in culture with an efficiency that varied from 0.3 to 4.1%. This DNA-contrast agent system produced signal intensity changes that colocalized with a radioactive label for the plasmid. This dextran-based synthetic vector transfected cells in vivo and was visualized with MR imaging. These systems demonstrate the potential for similar applications with dendrimers and bring together the advantages of dendrimer-based systems both as MRI contrast agents and synthetic vectors.
ACKNOWLEDGMENTS This work was supported in part by the National Institutes of Health PHS awards 1R29CA61918 and P41RR05964. We also thank the Beckman Institute. We thank Dr. Paul Lauterbur for his insightful comments over the years. Dr. Sheela Konda, Steven Wang, Mike Aref. Finally, we thank Dr. Randall Lauffer for his inspiration of this work. At the time we started this project no update to his seminal review article of 1987 was available, although during the initial phases of this project one appeared in Chemical Reviews. We therefore
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thought it appropriate to update the material following his original outline, and to provide simulations of Gd(III)-DOTA complexes for comparisons with the ones he provided for Gd(III)-DTPA, especially as no literature simulations for Gd(III)-DOTA complexes or its derivatives were available, and both Gd(III)-DTPA and -DOTA derivatives were the two main chelate backbones used clinically and experimentally. REFERENCES 1. Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. Rev., 1999, 99, 2293. 2. Lauffer, R. B. Magn. Reson. Q. 6, 1990, 65. 3. Gore, J. C. In MRI of Brain and Spine, Scott W. Atlas, Ed.; Raven Press: New York, 1991, p. 69. 4. Watson, A. D.; Rocklage, S. M.; Carvlin, M. J. In D. D. Stark, W. G. Bradley, Eds.; Magnetic Resonance Imaging, Mosby Yearbook: St. Louis, MO, 1992, p. 372. 5. Morris, P. G. Nuclear Magnetic Resonance Imaging in Medicine and Biology, Clarendon Press, Oxford Press, New York, 1990; Mansfield, P.; Morris, P. G. NMR Imaging in Biomedicine, Academic Press: PL, 1982; Stark, D. D.; Bradley, W. G. Eds. Magnetic Resonance Imaging (2nd ed.), Mosby Year Book: St. Louis, MO, 1992, 1, 253 pp. 6. Liang Z.-R; Lauterbur, P. C. Principles of Magnetic Resonance Imaging: A Signal Processing Perspective, IEEE Press Series in Biomedical Engineering, New York, 2000. 7. Hendrick, R. E.; Raff, U. In Magnetic Resonance Imaging (2nd ed.), D. D. Stark, W. G. Bradley, Eds.; Mosby Year Book: St. Louis, MO, 1992, Vol. 1, p. 113. 8. Mehta, V. D.; Sivasubramanian, A.; Kulkami, P. V.; Mason, R. P.; Antich, P. P. Bioconjug. Chem. 1996, 7(5), 536. 9. Venkatasubramanian, P. N.; Shen, Y J.; Wyrwicz, A. M. Magn. Reson. Med. 1996, 35(4), 626. 10. Lutz, J.; Noth, U.; Morrissey, S. P.; Adolf, H.; Deichmann, R.; Haase, A. Adv. Exp. Med. Biol. 1996, 388, 53. 11. Neil, J. J.; Song, S.-K.; Ackerman, J. J. Magn. Reson. Med. 1992, 25, 56. 12. Kovar, D. A.; Lewis, M. Z.; Lipton, N. J.; Karczmar, G. S. Magn. Reson. Med. 1997, 38(2), 259. 13. Obata, T.; Ikehira, H.; Shishido, F.; Fukuda, N.; Ueshima, Y; Koga, M.; Kato, H.; Kimura, R; Tateno, Y Acta Radiol. 1995, 36(5), 552. 14. Wirestam, R.; Larsen, V. A.; Stbgaard, M.; Thomsen, C ; Vikhoff, B.; Larsson, H. B.; Stahlberg, R; Henriksen, O. Acta Radiol. 1995, 36(1), 85. 15. Wolf, G. L.; Baum, L. Am. J. Roentgenol. 1983, 141, 193. 16. Weinmann, H. J.; Brasch, R. C ; Press, R. C ; Wesbey, G. E. Am. J. Roentgenol. 1984, 142, 619. 17. Tweedle, M. R; Gaughan, G. T.; Hagan, J.; Wedeking, P W.; Sibley, R; Wilson, L. J.; Lee, D. W. Int. J. Radiat. Appl. Instrum.[B] 1988, 75, 31. 18. Cacheris, W. P; Quay, S. C ; Rocklage, S. M. Magn. Reson. Imaging 1990, 8, 467. 19. Burton, D. R.; Forsen, S.; Karlstrom, G.; Dwek, R. A. Prog. NMR Spectrosc. 1979, 13, 1. 20. Bertini, I.; Luchinat, C. NMR of Paramagnetic Molecules in Biological Systems, BenjaminCummings: Menlo Park, CA, 1986. 21. Dwek, R. A. Nuclear Magnetic Resonance in Biochemistry: Applications to Enzyme Systems, Clarendon Press, Oxford Press: New York, 1973, p. 174. 22. Koenig, S. H.; Brown, R. D. Magn. Reson. Med. 1984, 1, 478-495. 23. Kowalewski, J.; Nordenskiold, L.; Benetis, N.; Westlund, R-0. Prog. NMR Spectrosc. 1985, 17, 141.
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INDEX
absorption spectra, 16, 20, 36 absorption spectroscopy, 15, 17 p-acetoxystyrene, 76 activated contrast agent, 138 activation energy, 17, 18, 20, 21 active, 195, 196, 198, 202, 223 adamantane, 32 aggregates, 32 aggregation, 33-35 albumin, 172, 191, 196-198, 204, 205, 209-211,219,225,229 alkylation, 6, 9, 11 allyl group, 11 amphiphilic dendrimers, 32-34 amphiphilic dendrons, 31 angiogenesis, 197, 227 angiography, 201, 202, 205, 207, 212, 213 Angiomark™, 211 anionic, 196, 198, 199 anisotropic, 147, 148, 154, 155, 180 antenna, 39, 40 antibodies, 173, 197, 198, 200, 214, 226 antibody, 226, 227 antigens, 155, 195, 227 arabinogalactan, 217, 218, 223 arachidic acid, 31 asialoglycoprotein, 223 asialoglycoprotein receptor, 195, 214, 217, 218, 222 atomic force microscopy, 83 azobenzene, 14 249
azobenzene core, 5, 7, 8 azobenzene fluorescence, 33 azobenzene photochromism, 14, 15, 17, 24 azobenzenes, 2, 3, 18, 20, 28, 35 benign, 205, 224-226 1,3,5-benzenetricarbonyl trichloride, 9, 12 benzyl amine, 56 benzyl aryl ether, 2, 3, 11, 29, 30 benzyl-oxy-methylether (BOM), 183, 211 bilayer, 33, 34 biodistribution, 190, 216, 222 bis-methylol propionic acid, 77 blood flow, 200, 202, 207, 210 blood pool, 195, 201, 202, 208, 210, 214, 227, 228 blood volume, 201, 202, 204, 207, 213, 226 blue-shift, 8, 32, 33 r-Boc-glycine, 67 BSA-Gd(III)-DTPA, 186 butyllithium, 50, 77 C3-symmetric, 9, 18 calix[4]arene, 38 capillary, 195, 198, 200 capillary blood flow, 195, 200, 224 capiUary permeability, 195, 196, 199202, 204, 205, 224 capillary surface area, 195, 224
250 e-caprolactone, 72, 74, 75, 77 cascade polymer, 174, 204, 205, 225 cascade-24, 179 cationic, 196, 198, 199 chelate, 131, 139, 140, 142, 144, 145, 147, 149, 150 chelates, 168-170, 177, 186, 188, 191, 193,220,231 chemical exchange, 140, 142, 150 chemotherapy, 204, 205 cholecystokinin, 223 chromatographic behavior, 2, 25, 28 chronic toxicity, 189 circularly polarized, 41 cirrhosis, 213, 217 conditional thermodynamic stability constant, 191 conformational flexibility, 32 continuous endothelium, 196, 197, 200 contrast, 132, 134, 138, 213, 221-224, 228, 229 contrast agent, 135, 137, 139, 147, 149, 152, 154, 157, 159, 162, 167, 172, 176, 186, 188, 193, 194, 199, 200, 204, 205, 208, 209, 213, 217, 223, 224, 228, 229, 232, 233 contrast agents, 130, 134 convergent method, 9 core-shell morphology, 27 correlation time, 142, 145, 146, 150, 156, 159, 208 covalently, 155, 233 crystallization, 35 Cy2D0TA, 183, 186 cysts, 213 dendrimer, 130, 131, 139, 148, 155, 161, 162, 164, 165, 171, 176, 177, 179182, 185, 186, 188, 189, 193-195, 200-202, 204, 205, 213, 217, 222, 224-229, 231, 233 dendrimer end groups, 34 dendritic, 46, 49, 50, 54, 57-60, 62, 63, 66-68, 71-73, 76, 80-84 dendritic bromide, 4-6 dextran, 174, 175, 198, 202, 215, 223, 233
INDEX diethyl aluminum alkoxide, 74 diffraction grating, 41 diffusion, 136, 137, 165-167, 174, 196, 200, 201 diffusional correlation time, 165, 166 dihydroxybenzoic acid, 82 dimethyl 5-hydroxyisophthalate, 68 1,1 -diphenylethylene, 50 iV,A^-dipropionate-PAMAM, 177, 179, 194, 227 direct, 134, 135, 138, 226 discontinuous endothelium, 196 dissociation, 139, 164, 191, 192 dissociation rate, 192, 193 dithranol, 83 DMAP, 5, 9, 12 donor/acceptor azobenzenes, 31 DOTA, 152, 157, 159, 161, 169-171, 174, 183, 185-188, 193, 201, 208, 211,234 DOTMA, 183, 184, 186 dynamic light scattering (DLS), 33, 39
EjZ photoisomerization, 2 echo time (TE), 132 elastic modulus, 35 electron paramagnetic resonance (EPR), 169, 180 electron relaxation time, 145, 165 electronic g factor, 141, 145 electronic correlation time, 150 electronic relaxation time, 142, 146148, 150, 152, 156, 157, 162, 166, 168, 169, 174, 175, 186 electrons, 135, 137, 142, 144, 146, 150, 160, 166, 231 encapsulation, 3 endogenous contrast agent, 227, 2 3 1 233 endotheUal junctions, 197 energy harvesting, 39 energy transfer, 40 enzymatic, 138, 229-231 eosin Y, 36-38 2-ethyl-2-oxazoline, 65, 66 ethylene oxide, 74
Index 2-ethynylpyridinium trifluoromethane sulfonate, 77 extracellular, 154, 199, 201, 202, 218, 220, 228 Fe(II), 146, 168 Fe-EHPG, 209 fenestrae, 196, 197 fenestrated endothelium, 196 ferrocene, 90, 93, 115, 117, 118, 122 flexibility, 131, 156, 169, 175, 179, 214, 223 fluorescence depolarization, 33 fluorescence intensity, 35, 37 fluorescence quenching, 37 folate, 195, 228, 229, 233 foHc acid, 227 gadolinium, 139, 145, 146, 148, 151, 152, 163, 164, 168, 172, 174, 177, 181, 186, 190, 191, 193, 194, 202, 204-206, 208, 209, 211, 216, 218, 219, 223, 226, 229, 230 gadomer-17, 179, 194, 205, 226 galactopyranose, 229 galactosidase, 39 Gd(III)-BOPTA, 183, 221, 222 Gd(III)-DTPA, 149, 155, 159, 161, 168171, 173, 174, 183, 193, 201, 202, 208, 209, 215, 216, 221, 224-226, 234 Gd(III)-EOB-DTPA, 170, 211, 220, 221 gel permeation chromatography (GPC), 28, 29, 42 gradient, 132, 207 hepatitis, 213, 217, 219 hepatobiliary, 199, 200, 213, 216, 218220 hepatocellular, 190, 215, 217, 221 hexakis(azobenzene) dendrimers, 11-13, 21, 27, 42 hexamethylcyclotrisiloxane, 73 high affinity folate receptor, 156, 227, 228, 233 holography, 41 host-guest, 3, 35
251 hydrodynamic radius, 29, 30, 42 hydrodynamic volume, 2, 28-30, 42 hydrophobic, 31, 32 hydroxycinnamic acid, 83 hyper-Rayleigh scattering (HRS), 32 hyperpermeable, 196, 197 indirect, 134, 135, 138, 223, 233 inflammation, 204 infrared irradiation, 39, 40 inner sphere, 137, 139-141, 149, 150, 160, 164, 165, 167, 186, 230, 231 inner sphere water molecules, 141, 145, 149, 164, 167, 168, 175, 219, 229, 230 intermoment distance, 142, 164, 165 intravascular, 201, 202, 204, 205, 207209,211,213,217 4,4'-iodoazobenzene, 7 iron, 190, 195, 196, 198, 213, 215, 219, 220,231,232 isosbestic point, 20 isotropic, 146-148, 180 kinetic stability, 139, 192, 193 kinetics, 15, 17, 20 Kupffer cells, 190, 214, 215 Langmuir monolayers, 35 Larmour frequency, 132, 133, 136, 145147, 155 ligand, 139, 142-146, 150, 156, 157, 160, 164, 167-169, 176, 183, 191193, 230 ligand field, 143, 146, 150, 156, 163, 169, 182, 230, 231 linear polymer, 154, 155, 174, 175, 177; 188, 200, 202 linearly polarized, 32 linear-dendritic, 58, 64, 80 linear-dendritic copolymers, 46, 48, 50, 79 linker, 147, 150, 163, 165, 171, 176, 216 liposome, 206, 208, 214-217, 227 liquid crystal, 3 liver, 191, 195-197, 205, 207, 209, 211, 213-223, 225
252 longitudinal, 131, 137, 142, 146, 201, 207, 219, 220, 227, 232 longitudinal relaxation time, 133, 146, 148, 216, 219 lymphography, 223 lysine, 39, 155, 186, 194, 205, 226 macromolecular, 139, 140, 145-149,153164, 167, 172, 174, 183, 185, 186, 188, 193, 198, 202, 204, 205, 213, 222, 224, 226, 232, 233 macromolecular isomer, 9, 11 magnetic moment, 131, 135-137, 139, 142-147, 150, 160, 161, 164, 166, 169 magnetic resonance imaging (MRI), 130, 131, 132, 134 MALDIMS, 11, 12 malignant, 195, 197, 205, 224-226 manganese, 189, 190, 206, 208, 232 mapping, 204 Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight, 82 metallocene, 90, 101 metallodendrimers, 90, 95, 97, 99, 102, 118, 120 metallostars, 91, 93 MION, 223 Mn(II), 145, 146, 151, 168, 172, 207, 211,215,217 Mn-DPDP, 222, 223 Mn-TPPS, 209, 210 mole fraction, 140, 141 molecular tumbling, 142, 147, 148, 152154 mono(azobenzene) dendrimers, 4, 5, 7, 8, 14 monocrystalline iron oxide nanoparticle (MION), 206 MRI, 133, 139, 145, 146, 164, 207, 219, 223, 232 neutral, 198, 199, 214 nitroxides, 210 non-centrosymmetric, 31, 32 noncovalent, 154, 155, 210-213 nonlinear optics, 3
INDEX nuclear magnetic relaxation dispersion (NMRD), 152, 157-159, 161, 169, 175, 220, 227, 232 nuclear magnetic resonance, 83 i^ONMR, 169, 171, 182 optical storage, 3, 18 organic anion carrier, 221, 223 organo-iron radicals, 91 organometallic, 92, 94-96, 122 osmotic behavior, 33 outer sphere, 137, 139, 165-167, 186, 219,231 A^-oxysuccinimide, 56 palmitoyl, 32, 33, 35 PAMAM, 172, 179, 181, 188, 225 PAMAM-MA-DTTA, 204, 225 PAMAM-TU-BZ-DOTA, 179 PAMAM-TU-Bz-DOTA, 186-188, 227 PAMAM-TU-D03A-MA, 177, 179 PAMAM-TU-DTPA, 177, 179-181, 186188, 202, 204, 227, 228 pancreas, 223 paramagnetic ion, 135, 137, 139-142, 144-147, 149, 150, 154, 157, 160, 166, 191, 215, 232 passive, 196-198, 201, 202, 215, 217, 223, 224 pCHllO, 39 Pd(0)-catalyzed coupling, 8 pentaerythritol, 68, 71 permeability, 195-198, 200, 208, 215, 224-226 pH, 33, 35 pharmacokinetics, 199, 200, 219 phenylacetylene, 2, 3, 7-9, 17, 29, 30 phenylene diboronic acid, 60 photochemical stability, 2 photochromic dendrimers, 5, 7, 14, 28 photochromic molecular systems, 2 photoisomerization, 2, 15, 20, 36 photomodulation of dendrimer polarity, 24,27 photomodulation of dendrimer properties, 3, 4, 24, 41 photomodulation of dendrimer size, 28
Index photomodulation of hydrodynamic volume, 28, 29 photon flux, 40 photon harvesting, 40 photoresponsive behavior, 2, 14, 20, 24 photoswitchable host, 36 plasma half-life, 195, 198-201, 205, 212, 216, 224, 228 plasmid DNA, 39 polarized, 3 polyamidoamine, 155, 161, 171, 177, 181, 194, 200, 202, 204, 205 polybutadienyllithium, 54 polycationic dendrimers, 102, 117, 118 poly(dimethylsiloxane), 73, 77 poly(ethylene glycol), 53, 54, 82 poly(ethylene imine), 65, 81 polyethyleneglycol, 202, 205, 207, 214 polylysine, 174, 188, 200, 202, 205, 218, 226, 227, 233 polylysine-MA-DTTA-Gd(III), 225 polymer networks, 49 polymerized liposome, 227 poly(p-phenylene), 55 poly(propylene imine), 32, 33, 36 polysaccharides, 202 poly(sarcosine) arms, 81 poly(styrene), 50, 51, 70, 71, 76, 77, 80 polystyryllithium, 50 porphyrin, 209 PPI-TU-DTPA, 179, 181 pressure-area isotherm, 32 [1.1.1] propelane, 62 pulmonary edema, 204 quenching, 37 receptor, 190, 198, 214, 217, 223 recognition, 90, 97, 103, 114, 117, 118, 120 red blood cells, 206, 208, 209, 214 red-shift, 8 Redfield limit, 148 redox catalysts, 95, 96 reductive animation, 52 relaxation rate, 135, 137, 138, 140-142, 148, 156, 188, 226, 232
253 relaxation time, 133-135, 140, 141, 146, 149, 150, 158 relaxivity, 137-139, 141, 149, 150, 152154, 156-158, 160-162, 164, 165, 167, 174, 176, 177, 182, 186, 188, 194, 208, 210, 215, 226, 227, 229231 repetition time, 132 retention, 190, 192-194, 198, 205, 208, 219, 224 reticuloendothelial system (RES), 213215, 217, 219, 223 rotational correlation time, 146, 147, 152155, 160, 162, 172, 202, 229 safety, 139, 149, 150, 188, 189, 191, 194 second harmonic generation (SHG), 31, 32 secretin, 223 selectivity, 139, 199 selectivity constant, 191, 192 shape persistent, 17, 29 signal intensity, 132, 134, 138, 194, 215, 217, 223, 228 size-exclusion chromatography (SEC), 28, 80 Solomon-Bloembergen and BloembergenMorgan (SBM), 139, 141, 144, 149 Sonagashira conditions, 7 spin density, 133, 134 spin quantum number, 148, 150, 166, 168 spin relaxation, 39 spleen, 195, 196, 209, 213, 215-217 splenic, 190 stability, 139, 149, 150, 189, 191, 208, 210, 216, 227 star-branched, 48, 53, 54, 68, 77 surface rehef gratings, 41 symmetry, 142, 144, 146, 150, 156, 169 targeting, 154, 193, 195, 197, 198, 201, 202, 213-215, 217, 219, 223, 224, 227 TEM, 33 terephthaloyl chloride, 61 2,2,6,6-tetramethylpiperidine-1 -oxy, 76
INDEX
254 thermal isomerization, 2, 17, 18, 20, 21, 36, 39, 41 thermodynamic stability, 139, 191, 193 thin layer chromatography (TLC), 24, 25, 27, 28 tight-junction endothelium, 196 tissue perfusion, 202 toxicity, 139, 167, 189-192, 194, 211 transcytosis, 198 transesterification, 51, 53, 54, 73 transverse, 131 transverse relaxation time, 133,134, 146, 148, 215 trimethylsilylacetylene, 7 tris(azobenzene) dendrimers, 9, 13, 18, 24, 25, 29 tumor targeting, 198, 224, 226
ultrasmall superparamagnetic iron oxide particles (USPIO), 206, 207, 217, 218
vascular endothelial growth factor, 197 VEGF, 197 vesicles, 33-35 vinyl acetate, 76 viscosity, 153, 154, 166 VPF, 197 water, 135, 140, 141, 147-149, 153, 163, 164, 173, 183, 195, 196, 201, 220 water residence time, 140, 145, 147, 152, 155, 160-162, 164, 167, 169, 172, 176, 182, 212 Williamson reaction, 51, 55 XRD, 33 Z -^ E isomerization, 2, 3, 39, 40 zero field electronic relaxation time, 156, 157, 159, 169 zero field splitting (ZFS), 142, 144 ZFS, 142-145, 148, 156, 158, 159
Advances in Dendritic Macromolecules Edited by George R. Newkome, Departments of Polymer Sciences and Chemistry, The University of Akron, Ohio, USA
Volume 1, 1994, 198 pp. ISBN 1-55938-696-7 CONTENTS: Introduction to the Series: An Editor's Foreword, Albert Padwa. Preface, George R. Newkome. A Review of Dendritic Macromolecules, George R. Newkome and Charles N Moorefield. Stiff Dendritic Macromolecules Based on Phenylacetylenes, Zhifu Xu, Benjamin Kyan andJeffery S. Moore. Preparation and Properties of Monodisperse Aromatic Dendritic Macromolecules, Thomas X. Neenan, Timothy M. Miller, Elizabeth W. Kwock and Harvey E. Bair. HighSpin Polyarylmethyl Polyradicals, Andrzej Rajca. A Systematic Nomenclature for Cascade (Dendritic) Polymers, Gregory R. Baker and James K. Young. Index. Volume 2, 1995, 204 pp. ISBN: 1-55938-939-7 CONTENTS: Preface, George R. Newkome. The Convergent-Growth Approach to Dendritic Macromolecules, Craig J. Hawker and Karen L. Wooley. Cascade Molecules: Building Blocks, Multiple FunctionaHzation, Complexing Units, Photo-switching, Rolf Moors and Fritz Vogtle. Ionic Dendrimers and Related Materials, Robert Engel. Silicon-Based Stars, Dendrimers, and Hyperbranched Polymers, Lon J. Mathais and Terrell W. Carothers. Highly Branched Aromatic Polymers: Their Preparation and Applications, Young H. Kim. Dendritic Bolaamphiphiles and Related Molecules, Gregory H Escamilla. Index.
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Volume 3,1996, 214 pp. ISBN: 0-7623-0069-8 CONTENTS: Preface, George R. Newkome. Dendrimers and Hyperbranched Aliphatic Polyesters Based on 2,2-Bis(Hydroxymethyl)Propionic Acid (BisMPA), Henrik Ihre, Mats Johansson, Eva Malmstrom and Anders Hult. Consequences of the Fractal Character of Dendritic High-Spin Macromolecules on Their Physiochemcial Properties, Nora Ventosa, Daniel Ruiz, Concepcio Rovira andJaume Veciana. Dendrimers Based on Metal Complexes, Scolastica Serroni, Sebastiano Campagna, Gianfranco Denti, Alberto Juris, Margherita Venturi and Vincenzo Balzani. Redox-Active Dendrimers, Related Building Blocks, and Oligomers, Martin R. Bryce and Wayne Devonport. Organometallic Dendritic Macromolecules: Organosilicon and Organometallic Entities as Cores or Building Blocks, Isabel Cuadrado, Moises Mordn, Jose Losada, Carmen M. Casado, Carmen Pascual, Beatriz Alonso and Francisco Lobete. Index. Volume 4,1999, 208 pp. ISBN: 0-7623-0347-6 CONTENTS: Introduction, George R. Newkome. Organometallic Dendrimers: Synthesis, Structural Aspects, and Applications in Catalysis, Meredith A. Hearshaw, Alan T. Hutton, John R. Moss and Kevin J. Naidoo. Poly(Propylene Imine) Dendrimers, Marcel H.R van Genderen, Ellen M.M. de Brabander-van den Berg andE.W. Meijer. Chiral Dendrimers, Hak-Fun Chow, Tony K.-K. Mong, Chi-Wai Wan and Z.-Y. Wang. Molecular Topology of Dendrimers, Mircea V. Diudea and Gabriel Katona. Index.
Volumes 1-4 were published by: JAI PRESS INC. 100 Prospect Street Stamford, Connecticut 06905-0811 USA and are available from Elsevier Science. Please contact your nearest Elsevier Regional Sales Office.
