Advances in Dendritic Macromolecules, Volume 5, Volume 5

ADVANCES IN DENDRlTlC MACROMOLECULES ADVANCES IN DENDRlTlC MACROMOLECULES Editor: GEORGE R. NEWKOME Volumes 1-4 were...

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ADVANCES IN DENDRlTlC MACROMOLECULES


Editor: GEORGE R. NEWKOME Volumes 1-4 were published by:

JAl PRESS INC. 100 Prospect Street Stamford, Connecticut 06904-0811 USA Copies are available from Elsevier Science. Please contact your nearest Regional Sales Off ice.


Editor: GEORGE R. NEWKOME Departments of Polymer Sciences and Chemistry The University of Akron Akron, Ohio, USA

VOLUME5

2002

2002

JAl An Imprint of Elsevier Science Amsterdam - London - New York - Oxford - Shannon -Tokyo

ELSEVIER SCIENCE Ltd. The Boulevard, Langford Lane Kidlington, Oxford OX5 ICB, UK

0 2002 Elsevier Science Ltd. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier Science Global Rights Department, PO Box 800, Oxford OX5 lDX, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also contact Global Rights directly through Elsevier's home page (http://www.elsevier.com), by selecting 'Obtaining Permissions'. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: ( + 1 ) (978) 7508400, fax: ( + 1 ) (978) 7504744, and in the UK through the Copyright LicensingAgency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WlPOLF: UK; phone: (+44) 207 631 5555, fax: (+44) 207 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permissionof the Publisher is required for all other derivative works, includingcompilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, includingany chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Global Rights Department, at the mail, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligenceor otherwise, or from any use or operation of any methods, products, instructionsor ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drugs dosages should be made.

First edition 2002 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. British Library Cataloguing in Publication Data A catalogue record from the British Library has been applied for. ISBN:

0-7623-0839-7

8 The paper used in this publication meets the requirements of ANSI/NISO 239.48-1992 (Permanence of Paper). Printed in The Netherlands.

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

This Page Intentionally Left Blank

LIST OF CONTRIBUTORS Beatriz Alonso

Laboratoire de Chimie et Organometallique UMRCNRSN°5802 Universite Bordeaux I 33405 Talence Cedex, France

Ester Alonso


Didier Astruc


Jean-Claude Blais

Laboratoire de Chimie Organique Structurale et Biologique EPCNRSN°103 Universite Paris VI 75252 Paris Cedex, France

Laurent Djakovitch


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


Stephane Rigaut


Jaime Ruiz


Christine Valeria


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


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

^—^

'^yi**i«

7.8

7.6

7.4

7.2

6.8

7.0

6.6

SîTAK^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 (âct) of these dendrimers indicates no strong steric influence on the Z -> £" thermal isomerization. The activation energy (âct) 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^Ê 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


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^

/cô (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.-^^


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


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


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


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


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


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


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 ô 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


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


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


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,


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


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.


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


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


^ 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

^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"Î~)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


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).


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;

â ^

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

—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.


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.


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


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-allyl dendrimer 8

' FcSi(Me)2H

81-allyl dendrimer 9

Karstedt cat

>


f

FcSi(Me)2H ^

243-allyl dendrimer 10


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


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


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


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


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^/

-^î?

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,


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 Franôise 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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22. 23. 24. 25. 26. 27. 28.

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.

Dendrimers Containing Ferrocenyl or Other Transition-Metal Sandwich Groups 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72.

73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88.

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89. 90. 91.

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111


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

129

130 130 131 134 135 137 139 139 139 139 144 149 149 150 165 167 188 189 191 194

130

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.

132


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-^^/î

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


(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


Figures. Return of the longitudinal magnetization to its equilibrium value, M^ = Mo(l-e~^/î). 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.


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


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


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.


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


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, (Tî 2)M. and the mole fraction of the metal ion (Pm), Eq. 9.


1

Pmq

(T{)i

Pmq

141

TIM

hu

+

(9)

^M

^m/

\T2M

(T2)i \ T2M

"^m /

+ 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


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 Tê

^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

143


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.

144


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


145

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


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.

147


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.

148


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


149

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

150


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

151


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+.


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-


153

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^.

154

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.^^


155

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

156


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


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


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


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


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


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 vîth 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


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


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


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, Aâ» 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


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


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 «. /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 ^Ô 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.

ÔH

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 iÔNMR 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-


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


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.


175

ou -

♦

25 -

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20-

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■ >

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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

Q.*

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


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


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

1

1

1

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1

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2

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3

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4

I

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8

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 Tâve = V^ll * ^-L-

(ns)

(ns)

^11 (ns)

(ns)

0.40 0.64

0.67 1.5

0.15 0.21

3 10

âve

181

Magnetic Resonance Imaging Contrast Agents 1

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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

âve

1.5 2.8

182


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

^ÔNMR ^Ô NMR and EPR and ^ÔNMR iÔNMR ^ÔNMR iÔ NMR and EPR and NMRD NMRD and iÔ NMR NMRD and ^Ô NMR NMRD and ^ô NMR ^Ô NMR and EPR ^Ô NMR and EPR ^Ô NMR and EPR NMRD ^ÔNMR ^ÔNMR NMRD ^Ô NMR and EPR and ^ÔNMR 1^0 NMR ^Ô NMR and EPR and ^Ô NMR and EPR and ^ÔNMR ^ÔNMR ^ÔNMR ^ÔNMR

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


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^ÔH

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 HOÔ

MO'

NH ^

O

/

\

o

f''L7\

0«

HOÔ

QAOH

HO''^

Ô

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 ^ Ô" '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


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


HU -

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PROTON LARMOUR FREQUENCY (MHz)

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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.

188


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. Âctual 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

190


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


191

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]

192

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 âLla] 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.^"^^

Magnetic Resonance Innaging Contrast Agents

193

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

194

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


195

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

196


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


197

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.

198


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.^^^"^^^


199

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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Advances in Dendritic Macromolecules, Volume 5, Volume 5

Advances in Dendritic Macromolecules. Volume 3

Advances in Dendritic Macromolecules, Volume 2

ADVANCES IN CATALYSIS VOLUME 5, Volume 5

Advances in Dendritic Macromolecules : 1995 (Advances in Dendritic Macromolecules) (Advances in Dendritic Macromolecules)

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Central Burma, Volume 5

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Longsal Teachings: Volume 5

Advances in Dendritic Macromolecules, Volume 5, Volume 5

Advances in Dendritic Macromolecules. Volume 3

Advances in Dendritic Macromolecules, Volume 2

ADVANCES IN CATALYSIS VOLUME 5, Volume 5

Advances in Dendritic Macromolecules : 1995 (Advances in Dendritic Macromolecules) (Advances in Dendritic Macromolecules)

Advances in Geophysics, Volume 5

Advances in Immunology Volume 5

Advances in Computers, Volume 5

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Advances in Mergers and Acquisitions, Volume 5

Advances in Chemical Engineering, Volume 5

Advances in Atomic Spectroscopy, Volume 5

Advances in Applied Microbiology, Volume 5

Advances in Carbohydrate Chemistry, Volume 5

Advances in Botanical Research, Volume 5

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Advances in Carbohydrate Chemistry, Volume 5

Advances in Developmental Biochemistry, Volume 5

Advances in Applied Mechanics, Volume 5

Advances in Clinical Chemistry Volume 5

Advances in Antiviral Drug Design, Volume 5

Advances in Carbohydrate Chemistry, Volume 5

Advances in Hospitality and Leisure, Volume 5

Advances in Medicinal Chemistry, Volume 5 (Advances in Medicinal Chemistry)

Advances in Atomic Spectroscopy, Volume 5 (Advances in Atomic Spectroscopy)

Central Burma, Volume 5

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