Metal chelates as biological probes

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Metal chelates as biological probes preparation and properties
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Thesis (Ph. D.)--University of Florida, 1990.
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Includes bibliographical references (leaves 152-158).
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by Frances Elizabeth Armitage.
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METAL CHELATES AS BIOLOGICAL PROBES:
PREPARATION AND PROPERTIES






By


FRANCES ELIZABETH ARMITAGE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


yl af giu0i' LiARIJES


1990





















































Copyright 1990

by

Frances Elizabeth Armitage












ACKNOWLEDGMENTS


Partial support for the contrast agent project was

provided by the Florida Division of the American Cancer

Society (Hazel C. M. Kerr Research Grant) and the Division

of Sponsored Research of the University of Florida. Imaging

studies were funded by internal grants from the Department

of Radiology, University of Florida College of Medicine.

Grateful acknowledgment is appropriate for the collaboration

of many colleagues: Dr. King Li, Dr. Ron Quisling, and Dr.

Chris Mladinich, D.V.M., of the Department of Radiology

(College of Medicine, University of Florida) who provided

time, expertise, and thoughtful insight for the imaging

studies required in the contrast agent project; the many

associates in the Department of Chemistry whose feedback

enhanced the direction of the projects; and past and present

mentors whose expressed enthusiasm toward exploring the

basics of chemistry gave me the confidence to pursue this

endeavor.


iii














TABLE OF CONTENTS


page

ACKNOWLEDGMENTS..................................... iii

KEY TO SYMBOLS AND ABBREVIATIONS....................... vi

ABSTRACT ..............................................vii

CHAPTERS

1 INTRODUCTION................................... 1

2 SYNTHESIS AND ELECTROCHEMICAL PROPERTIES
OF EDTA COMPLEXES OF RUTHENIUM(III)..............6

Chapter Overview................................6
Synthesis and Characterization.................10
Synthesis of [RuIII(Hedta)H20]...............10
Synthesis of [RuIII(Hedta)L] Complexes.......13
Cyclic Voltammetry Measurements................16
Results and Discussion......................... 16
Evaluation of Synthetic Methods..............16
Properties of New Ru(Hedta)L Complexes.......23
Electrochemistry Results....................28
Chapter Summary................................ 31

3 SYNTHESIS AND CHARACTERIZATION OF
POLYSACCHARIDE ESTERS OF
POLYAMINOPOLYCARBOXYLIC ACIDS
AND THEIR GADOLINIUM(III) COMPLEXES............34

Chapter Overview............................... 34
Materials and Methods...........................39
Synthesis of DTPA-Polysaccharides.............39
Determining Reaction Success by HPLC.........44
Purification of DTPA-Polysaccharide Esters...46
Nature of Reacted Polysaccharide by HPLC.....48
Synthesis and Purification of Gd3+ Complexes.48
Characterization of Compounds................50
Results and Discussion.........................58
Evaluation of Synthetic Methods...............58
Compound Characterization Results.............74
Relaxivity Results...........................86
Chapter Summary...............................98







4 MAGNETIC RESONANCE IMAGING STUDIES
USING GADOLINIUM CONJUGATES
AS CONTRAST AGENTS.......................... 103

Chapter Overview...............................103
Materials and Methods..........................106
Phantom Studies..............................106
Animal Imaging Studies......................107
Results and Discussion........................109
Phantom Results..............................109
Animal Imaging Results...................... 119
Chapter Summary...............................143

5 SUMMARY.........................................145

APPENDIX.............................. ................150

REFERENCES........ .....................................152

BIOGRAPHICAL SKETCH...................................159












KEY TO SYMBOLS AND ABBREVIATIONS


NMR = Nuclear magnetic resonance.

MRI = Magnetic resonance imaging.

T1 = Spin lattice (longitudinal) nuclear relaxation time.

T2 = Spin spin (transverse) nuclear relaxation time.

R1 = Spin lattice relaxivity.

TR = Time between pulse repetitions in MRI.

TE = Time waited between pulse and data acquisition in MRI.

ROI = Region of interest in MRI image, used to obtain signal
intensity data with 95% confidence limit.

EDTA = Ethylenediaminetetraacetic acid.

EDTA-R = EDTA with functional group modification on
ethylene backbone.

Hedta3- = EDTA ligand with 3 deprotonated carboxylate
functions.

H2edta2- = EDTA ligand with 2 deprotonated
carboxylate functions.

DTPA = Diethylenetriaminepentaacetic acid.

TTHA = Triethylenetetraaminehexaacetic acid.

GdDTPA = DTPA complex of gadolinium(III).

GdTTHA = TTHA complex of gadolinium(III).

GdDTPA-polysaccharide = GdDTPA linked by ester bond to
either dextran or inulin.

Ef = Formal reduction potential, mV.

A Ep = Difference between anodic and cathodic peak
potential.












Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


METAL CHELATES AS BIOLOGICAL PROBES:
PREPARATION AND PROPERTIES

By

FRANCES ELIZABETH ARMITAGE

August 1990

Chairman: David E. Richardson
Major Department: Chemistry

Aqua ethylenediaminetetraacetato ruthenium(III) was

prepared by a variety of methods. Improvement in product

yield relative to prior art methods was obtained, and the

synthesis results are discussed in terms of product and

method suitability for future application in protein

conjugate preparations. Substitution of the aqua ligand by

a variety of nitrogen containing ligands was carried out,

and two new complexes are reported. Reversible cyclic

voltammograms were obtained, and a range in reduction

potentials (versus NHE) from 0.0 to 0.5 volts was observed.

The synthesis and characterization of polysaccharides

esterified with diethylenetriaminepentaacetato

gadolinium(III), GdDTPA-dextrans and GdDTPA-inulin, are

described. The synthetic method resulted in products

labeled with an average of 0.4 mole GdDTPA per mole of

vii







glucopyranose unit in dextrans of up to 70800 average

molecular weight and 0.5 mole of GdDTPA per mole of

fructofuranose unit of inulin. Chromatographic and

potentiometric evidence supporting the absence of chelate

induced crosslinking of the polysaccharides is presented.

The thermodynamic stability, log K (Gd3+ + L4- GdL-), of

the complexes was 18.02 0.13 based on an independent

chelate model. The in vitro ester hydrolysis half-life of

GdDTPA-dextran 70800 (370C, pH=7.4) was one-fourth that of

the nonmetallated compound.

The conjugates exhibited T1 relaxivities ranging from

1.5 to 2.3 times that of GdDTPA at 100 MHz, and decreasing

in vitro relaxivity with increasing molecular weight of

dextran carriers was observed. Phantom MRI studies indicate

that the T1 and T2 effects of the complexes differ from

GdDTPA, with the dextran and inulin-bound complexes

exhibiting a considerably faster drop in relative signal

intensity with increased concentration in T1 and T2 weighted

pulse sequences. Tissue enhancement in normal rabbits using

GdDTPA labeled dextran of 9400 and 70800 average molecular

weight persists in excess of 60 minutes for liver, kidney,

and brain microvascular regions. No toxic effects for the

complexes were noted. These macromolecular agents have

potential application in blood-pool persistent MRI contrast

enhancement of organ abnormalities and blood flow studies.


viii












CHAPTER 1

INTRODUCTION



Polyaminopolycarboxylates, chelating molecules that

contain two or more amine and carboxyl functional groups,

are ligands that generally form metal complexes of high

thermodynamic stability.1-4 Provided the metal ion can

accommodate all of the potential donor groups of the ligand

(example ligands are shown in Figure 1.1), as the number of

donor groups in the ligand increases the thermodynamic

stability of the metal complex generally increases.


9 9
HOC- --- p-COH
N N
HO-J \- OH
o 0

9 9
HOC ---- ---- /FCOH
N N N
HOg LpOH \-,OH
o 0 0

9 9
HOClr--- --- r---s\COH
N N N N
HOCJ LFOH LOH LqOH
8 0 0 0


Ethylenediaminetetraacetic
acid (EDTA), 6 donor groups



Diethylenetriaminepentaacetic
acid (DTPA), 8 donor groups



Triethylenetetraaminehexaacetic
acid (TTHA), 10 donor groups


Figure 1.1. Some polyaminopolycarboxylic acids and
their number of potential lighting donor groups.

1









The stabilization of potentially toxic metal ions by

these chelates prompted their medical application in toxic

metal mobilization and control of endogenous metal metabolic

problems.5 In addition to these therapeutic applications,

there is considerable potential for the metal complexes of

polyaminopolycarboxylates as biological probes.6 The

ligands can be modified to contain a functional group which

is reactive toward functional groups of proteins or other

types of biologically important molecules. In this fashion,

the chelating agent becomes bifunctional in that it serves

to ligate a metal ion and to covalently link the completed

metal ion to a carrier molecule in the formation of the

conjugate.

Shown in Figure 1.2 are several potential biological

probe applications for the metal chelates. Considerable

research activity has been directed toward all of the areas

represented in Figure 1.2, however, the development of

radiopharmaceuticals has been the primary focus of

bifunctional chelating agents in biomedical applications.6,7

These radiopharmaceuticals may incorporate covalent linkage

between the bifunctional chelate and a tumor specific

antibody (or a tumor localizing carrier molecule), or the

antibody may be both tumor and metal chelate specific such

that no covalent linkage between the antibody and metal

chelate is necessary.

















































Carrier Chelating
Molecule Agent


Metal Ion
(NMR or radioactive)


Figure 1.2. Schematic representation of biological
probe applications for metal chelates.









Also shown in Figure 1.2 is the application of metal

chelates in studies concerning electron transfer of

metalloproteins.8,9 In this type of application, a redox

tunable metal center is linked by a bifunctional chelator to

a metalloprotein. The binding of NMR active or radioactive

metals to macromolecular carriers by bifunctional chelates

has been developed so that abnormal tissue can be more

easily distinguished from normal tissue when instruments

currently available in noninvasive diagnostic medicine are

used.10,11 These applications of metal chelates are

directly related to the two areas of study undertaken in the

present work.

The contents of Chapter 2, "Synthesis and

Electrochemical Properties of EDTA Complexes of Ruthenium

(III)," are potentially useful for future investigations

concerning electron transfer between redox tuned ruthenium

centers and metal centers of metalloproteins. By covalently

linking the Ru3+ metal chelate, in which there is one inner

sphere coordinated water molecule, to metalloproteins and

substituting other ligands for the aqua ligand, the

difference between the potentials of the metal center of the

metalloprotein and the ruthenium center can, in principle,

be easily manipulated.

Chapter 3 describes the synthesis and characterization

of gadolinium polyaminopolycarboxylate chelates which have

been covalently linked by ester bond to polysaccharide








carrier molecules. These studies were undertaken so that

the new gadolinium conjugates could be assessed for their

potential application as contrast agents when used in

magnetic resonance imaging (MRI), a noninvasive biomedical

diagnostic tool. The investigation of the in vitro and in

vivo behaviors of the new compounds when used as contrast

agents in MRI is presented in Chapter 4.

Several techniques were used to characterize the new

gadolinium conjugates in an attempt to ascertain the effect

of covalent attachment of the polysaccharide on the various

properties of the metal chelate unit. Additionally, some of

the evaluations and suggested future evaluations represent

studies that are often neglected when new compounds become

candidates for potential biological applications. The final

chapter (Chapter 5) includes a summary of the results of the

two different areas of study and a brief discussion

pertaining to the predicted directions of each project.












CHAPTER 2

SYNTHESIS AND ELECTROCHEMICAL PROPERTIES
OF EDTA COMPLEXES OF RUTHENIUM(III)



Chapter Overview



The complex [RuII(NH3)5H20]3+ undergoes substantially

slower exchange of the aqua ligand with w-acid ligands than

the corresponding ruthenium(II) complex.12 The aqua complex

of ruthenium(III) ethylenediaminetetraacetic acid,

[RuIII(Hedta)H20], however, exhibits a liability 7 orders of

magnitude greater than that of [RuIII(NH3)5H20]3+.13 This

unusual liability is such that the water in [RuIII(Hedta)H20]

undergoes r-acid ligand substitution more rapidly than

[RuII (Hedta)H20]- and represents an anomaly in the general

inertness of ruthenium(III) complexes toward substitution.

Investigations which involved the chromium(III) complex

of EDTA suggest that the high degree of liability of the aqua

complex is attributable to the interaction between the

coordinated water molecule and an uncoordinated acetate

group of EDTA.14 The structure of the ruthenium(III)

complex of EDTA in solution has an uncoordinated acetate

group. Through the modification of electrode surfaces with








[RuIII(Hedta)H20] by linkage of the uncoordinated acetate

function, Oyama and Anson15,16 observed that aqua

substitution by pyridine and isonicotinamide occurred much

faster in the RuII complex rather than the RuIII complex.

This reversal in order of reactivity toward substitution,

however, was not observed when [RuIII(Hedta)H20] was

directly attached to the electrode surface through the metal

center. These results tend to support the water acetate

interaction mode of liability for the [Ru(III)(Hedta)H20]

complex.

The liability of [RuIII(Hedta)H20] is well suited for

potential application in redox investigations of

metalloproteins in that, following covalent attachment to

the protein using a modified EDTA chelate, the inner sphere

coordinated water molecule may be easily substituted by w-

acid ligands. The increasing n donor ability of the

substituting ligands would be expected to result in more

positive reduction potentials while facile substitution

would provide a means by which changes in the formal

reduction potential on the ruthenium(III) center could be

readily achieved.

Recent work by Matsubara and Creutz17 involved the

investigation of [Ru I(Hedta)L] complexes which were

prepared by substitution of water in the aqua complex by

thiocyanate, pyridine, imidazole, isonicotinamide, pyrazine,

and acetonitrile. The complexes, including the aqua







8

complex, ranged in formal reduction potentials from -0.01 to

+0.26 V versus NHE in pH = 5.5 acetate buffer. Their

results also indicate that, in the pH range from 0.8 to 8.5,

the [RuIII(edta)H20]- species was the only reactive form of

the complex involved in the ligand substitution reaction

even though the pKa of the free -CO2H function is 2.37 and

the pKa of the coordinated water is 7.63.

The mode of covalent attachment of EDTA to proteins may

occur through one of the EDTA acetate groups or through a

function which has been linked to the ethylene backbone of

EDTA. Since the liability of the aqua complex is very likely

dependent on the presence of an uncoordinated acetate

function of EDTA in [RuIII(Hedta)H20], the use of one of the

acetate groups in covalent linkage to the protein is not

desirable. A number of modified EDTA ligands have been

prepared such that covalent attachment to proteins can be

achieved by a variety of methods without a reduction in the

denticity of the ligand.18 Although the reduction

potentials for [RuII(Hedta-R)L] in which the EDTA has been

modified at the ethylene backbone may be altered relative to

those of the unmodified EDTA complexes, the range of

reduction potentials should be comparable.

A goal of this work was to synthesize a series of

[RuIII(Hedta)L] complexes in which the EDTA is pentadentate

and the variable ligand, L, occupies the sixth coordination

site. Ideally, the series of complexes would exhibit a wide








range of reduction potentials such that they may, following

covalent linkage to proteins or other macromolecules, be

useful in subsequent investigations concerning antibody

production or biological electron transfer. In order to

accomplish this goal, an investigation of synthetic methods

to produce high yields of [RuIII(Hedta)H20] was necessary.

The use of the high liability of the aqua complex to

explore many of the r-acid ligands investigated by Matsubara

and Creutz as well as an assessment of the redox tuning

ability of other t-acid ligands needed to be conducted so

that a wide range of reduction potentials could be obtained.

An evaluation of the effects of pH on the reduction

potentials of the complexes was also necessary, particularly

in the range of physiological pH wherein relevant reduction

potential data could be generated for subsequent biological

studies.

Finally, the reactions required for the high yield

synthesis of [RuI I(Hedta)H20] may or may not be appropriate

for use with protein-modified EDTA ligands. An adjunct

study required the assessment of alternative methods of

[RuI (Hedta)H20] production wherein the protein-

[Ru II(Hedta-R)H20] conjugate could possibly be synthesized

without protein degradation.









Synthesis and Characterization



Synthesis of RuIII (Hedta)H20l

All glassware was washed with 1 M EDTA to remove trace

metals followed by thorough rinsing with deionized water.

Ruthenium trichloride hydrate (Aldrich, hydrate content was

not analyzed) was used in all preparations.

Reaction of ruthenium trichloride with H2edta2- without

silver ion present. The procedure for the synthesis of

[Ru(Hedta)H20] was modified from that outlined by

Scherzer.19 Following dissolution in 20 mL of deionized

water, 4 mmole of ruthenium trichloride was added to 20 mL

of an aqueous solution which contained 4 mmole of Na2H2edta

(Mallinckrodt). The solution was stirred while heating at

750 800C.

The dark brown precipitate was filtered while hot and

washed with hot water followed by room temperature methanol.

The product was dried under vacuum in a desiccator.

Approximately 60 mL of methanol was added to the filtrate

and the solution was undisturbed for 24 hours at room

temperature. The green-yellow precipitate was filtered,

washed with cold methanol, and air dried.

Reaction of ruthenium trichloride with H2edta2- with

silver ion present. Fifteen mmole of RuC13 was added to 100

mL deionized water and heated at 600C while stirring for 1

hour. Thirty mmole of AgBF4 (Ozark Mahoney) was added and








stirring and heating continued for 1/2 hour. The solution

was filtered, allowed to stand at room temperature and

filtered again. This process was repeated until no silver

chloride precipitated from solution and no precipitate

formed on addition of a few drops of solution to 1 M HC1.20

A total of 15 mmole of Na2H2EDTA (Mallinckrodt) was added to

the clear filtrate. The solution was stirred while heating

at 80C for 2 hours. After cooling to room temperature a

brown precipitate was filtered, washed with methanol, and

air dried. The filtrate was rotary-evaporated to

approximately 40 mL and ethanol added until precipitation

began. The solution was allowed to stand at room

temperature for 48 hours, filtered and solid washed with

methanol and dried in vacuum desiccator for 1 hour.

Elemental analysis for RuCIOH15N209"2H20: calculated %C =

27.03, %H = 4.31, %N = 6.30; observed %C = 26.89, %H = 4.10,

%N = 6.26.

Reaction of [Ru(Hedta)Cl]-: process 1. The

[Ru(Hedta)Cl]- was used as obtained from the direct reaction

of RuC13 with H2edta2- without silver ion present (above).

Conversion to the aqua complex was accomplished by method

outlined by Mukaido.21

Reaction of [Ru(Hedta)C11-: process 2. Solid

[Ru(Hedta)Cl]-, prepared as above with no silver ion

present, was placed in deionized water and stirred at room

temperature. Zinc amalgam was prepared from mossy zinc








which had been washed in 1 M perchloric acid, amalgamated

with 0.5 M Hg(C104)2 in 0.5 M HClO4, and rinsed with water.

The amalgam was placed in the dark brown heterogeneous

[Ru(Hedta)Cl]- solution and stirred at room temperature for

12 hours. The olive-green mixture was filtered and the

filtrate was purged with oxygen for 1/2 hour. This resulted

in an intensely yellow solution. The solution was rotary-

evaporated to approximately 25 mL and pH adjusted to 2 with

perchloric acid. Ethanol was added until precipitation

began and solution was allowed to sit at room temperature

for 12 hours. The product was filtered and washed with

ethanol.

Reaction of ruthenium oxide. Ruthenium oxide was

prepared by modification of methods of Cady22 and Kallen.23

A flask containing 75 mL concentrated sulfuric acid and 3.15

g RuC13 hydrate was heated at 800C while stirring until the

solution tested negative for chloride ion with silver

nitrate solution. A solid mixture containing 16.0 g K2S208

(Mallinckrodt) and 1.0 g AgCO3 (Mallinckrodt) was added to

the solution and the solution was heated at 800C for 1 hour.

The solution was cooled to 50C and oxalic acid (Aldrich)

was slowly added to the brown solution until the solution

color turned yellow. The flask was equipped with a side arm

and a collection trap immersed in an ethanol/dry ice bath.

All glassware between the reaction flask and the cold trap

was heated to approximately 500C. Deionized water was added








over a 4-hour period by dropping funnel while the solution

was heated at 750C, and a flow of nitrogen gas from the

solution surface to the collection trap was used to transfer

the RuO4 vapor to the trap.

The RuO4 was dissolved in 50 mL of 2.0 M HBF4 (Fisher)

and 5 grams of 30 mesh tin was added. The mixture was

stirred at room temperature for 5 hours. The pink solution,

after being purged with oxygen for 1 hour, turned yellow.

To this solution, 2 grams of H4edta (Mallinckrodt) was added

and dissolved by the addition of NaHCO3 (Fisher). The

solution was stirred at room temperature for 10 hours, then

heated for 4 hours at 800C. The solution was cooled to 400C

and methanol added to precipitate the solid. The mixture

was left undisturbed at room temperature for 12 hours then

filtered and washed with methanol.



Synthesis of FRuII(Hedta)L1 Complexes

Solutions which were 4 mM in [RuIII(Hedta)H2] were

prepared in pH = 5.56 acetate, 7.09 phosphate, and 9.12

borate buffers. To separate containers of 2 mL of the

solution, an 8-fold molar excess of ligand was added. The

ligand reagents used were pyridine, pyrazine, 2-

methylpyrazine, 2-nitrobenzonitrile, 4-nitrobenzonitrile,

isonicotinamide (all obtained from Aldrich), sodium

thiocyanate (Fisher), and acetonitrile (Kodak). The







14

complexes were not isolated. The solutions were immediately

evaluated by cyclic voltammetry.

Synthesis of Ru(Hedta)1,10-phenanthroline. To 5 mL of

deionized water, 0.13 gram of 1,10-phenanthroline (Aldrich

gold label) was added with sufficient methanol to dissolve

the solid. To the stirring solution, 32 mg of the prepared

RuIII(Hedta)H20"2H20 was added and resulted in a blood-red

solution within 5 minutes. The solution was stirred at room

temperature for 48 hours and rotary-evaporated to dryness.

The purple solid was found to be only sparingly soluble in

acetone while the 1,10-phenanthroline was very soluble. The

excess ligand was removed from the solid by repeated washing

and decantation with acetone and the solid was dried in a

desiccator under vacuum. The calculated and observed

elemental analysis of the complex RuC22H21N408'5.5H20 are as

follows: calculated %C = 39.46, %H = 4.82, %N = 8.37;

observed %C = 38.58, %H = 4.42, %N = 8.46.

Synthesis of [RuIII(Hedta)(pyridine)1,21 In a minimum

amount of deionized water, 51 mg of the prepared

RuII(Hedta)H20"2H20 was dissolved. To the solution, 12 mL

of pyridine (Aldrich gold label) was added and solution

stirred for 1 hour. The solution was then heated at 55C

for 2 1/2 hours, cooled to room temperature, and allowed to

stir at room temperature for 72 hours. The light-orange

solution was rotary-evaporated to dryness and washed by

filtering with anhydrous ethyl ether. The dark yellow






15

powder was dried in a desiccator under vacuum. Based on the

relative areas for each reversible voltammogram, it was

calculated that the solid contained a mixture of 34.8%

Ru(Hedta)(pyridine)2 and 65.2% Ru(Hedta)pyridine. The

calculated and observed elemental analysis for the mixture,

with 5 moles of water associated per mole of ruthenium were

calculated %C = 34.28, %H = 5.10, %N = 7.99; observed %C =

34.34, %H = 3.88, %N = 7.66.

Synthesis of rRuIII(Hedta)(2.2'-dipyridine)]. To 10 mL

deionized water, 0.16 gram of the prepared

RuI (Hedta)H20"2H20 and 1.2 gram of 2,2'-dipyridyl (Fisher)

were added. Methanol was added drop-wise to the mixture

until all dipyridyl dissolved and the solution was stirred

at room temperature for 12 hours. The solution was rotary-

evaporated to dryness to produce dark purple crystals which

were not soluble in ethyl ether. The excess dipyridyl

(soluble in ethyl ether) was removed by stirring of the

solid with ethyl ether and decantation of the ether

solution. The washing process was repeated until three

successive decantations resulted in no visible white powder

upon evaporation of ethyl ether. The solid purple powder

was dried in a desiccator under vacuum. Calculated

elemental analysis results for RuC20H21N408 '12H20 were as

follows: calculated %C = 31.50, %H = 5.95, %N = 7.35;

observed %C = 31.78, %H = 3.52, %N = 7.21.









Cyclic Voltammetry Measurements

Buffer solutions of 0.3 M ionic strength (potassium

salt of trifluoromethanesulfonic acid) of pH = 5.56

(acetate), 7.09 (phosphate), and 9.12 (borate) were used.

All measurements were conducted using a silver / silver

chloride reference microelectrode (Bioanalytical Systems

model MF-2020), and 1/4" diameter platinum disks which had

been spot welded to platinum wire for working and auxiliary

electrodes. Cyclic voltammetric measurements were obtained

using a Princeton Applied Research model 173 potentiostat /

galvanostat and an EG&G Parc model 175 universal programmer.

Voltammograms were recorded using a Houston Instrument

Omnigraphic 2000 x-y recorder. A scan rate of 20 mV/sec was

used for all solutions. All solutions were purged with

argon prior to voltammetry. Formal reduction potentials

were calculated using the midpoint of the anodic and

cathodic peaks of the reversible voltammograms. Cyclic

voltammetry of each complex was repeated two or more times

and the deviation in formal reduction potential was less

than 10 mV.



Results and Discussion



Evaluation of RuIII(Hedta)H20"2H20 Synthetic Methods

The reaction of RuC13 with H2edta2- in aqueous solution

results in the formation of the monochloro complex and aqua








complexes of the ruthenium chelate19 and is indicated in

equation 2.1.


2RuC13 + 2H2edta2-(aq) + 2H20

H (aq) + 5Cl-(aq) + Ru(H2edta)Cl (s)
+ Ru(Hedta)H20 (s) (2.1)


The aqueous rate of precipitation of the chloro complex is

substantially faster than that of the aqua complex and this

difference was exploited in the synthetic method. The yield

was 24% [Ru(H2edta)C1] and 1% [Ru(Hedta)H20].

The presence of silver ion in solution did not alter

the yield of the chloro complex significantly (26%) but

markedly effected the yield of aqua complex (25%). Equation

2.2 represents the reaction of RuC13 with silver ion present

and equation 2.3 shows the subsequent reaction of the

pentaqua monochloro complex with H2edta2-


2H20 + RuC13 (aq) + 2 Ag+ (aq)

2AgCl (s) + RuCl(H20)52+ (aq) (2.2)


2RuCl(H20)52+ (aq) + 2H2edta2- (aq)

Ru(H2edta)Cl (s) + Ru(Hedta)H20 (s) (2.3)


Improvement in yield may be possible using a 3 to 1, rather

than 2 to 1, mole ratio of Ag+ to RuC13. The observed slow









formation of the silver chloride precipitate, however,

suggests that the use of greater than a 2 to 1 mole ratio of

reactants may require more time for the removal of Ag+

remaining in solution. The slow rate of precipitation from

solutions of chloro complexes of ruthenium(III) in the

presence of Ag+ was also observed by Connick and Fine24 and

the precipitate was reported as an aggregate containing Ag+,

Cl-, and Ru3+ of unknown composition.

In dilute solutions of [Ru(H2edta)Cl], ligand exchange

will slowly occur between the chloride ion and water,

represented in equation 2.4.



Ru(H2edta)Cl (aq) + H20 < > Ru(Hedta)H20

+ H+ + Cl- (2.4)



The most widely applied method for the conversion of the

monochloro complex to the aqua complex, that of Mukaido,21

involves the removal of H+ and Cl- from solution, shown in

equation 2.5. The method resulted in 48% yield of the aqua

complex from the monochloro complex starting material.



Ru(H2edta)Cl (aq) + H20 Ru(Hedta)H20

+ HC1 (g) (2.5)



Ruthenium(III) complexes normally undergo substitution

reactions slowly.25 By reducing the complexes to








ruthenium(II) and thereby producing a more labile species,

the rate of substitution of chloride ion by water in

[RuII(H2edta)Cl]" may be substantially greater than that of

the ruthenium(III) complex. The method for achieving this

is represented in equations 2.6a-b. The method resulted in

78% yield of the aqua complex and this result tends to

support the assumption that the liability of

[Ru T(H2edta)Cl]- complex is significantly greater than that

of [Ru (H2edta)Cl]. This method of synthesis represents
considerable improvement when compared with the yield

obtained with Mukaido's method (78% versus 48%).


Zn (s) + 2RuIII(H2edta)Cl (aq) --

2RuII(H2edta)H20 (aq) + 2C1- (aq) + Zn2+ (aq) (2.6a)

4RuII(H2edta)H20 (aq) + 02 (g)
4RuIII(Hedta)H20 (aq) + 2H20 (2.6b)


The preparation of [Ru(Hedta)H20] from ruthenium oxide

eliminated the problem encountered in reactions of RuC13

wherein the competitive reaction to produce the monochloro

complex took place. The reaction scheme involving RuO4,

represented by equation 2.7a-c, resulted in 16% yield.


RuO4 (s) + 8H30+ (aq) + 3Sn (s) -

Ru(H20)62+ (aq) + 6H20 + 3Sn2+ (aq) (2.7a)








4Ru(H20)62+ (aq) + 02 + 4H30+ (aq)

4Ru(H20)63+ (aq) + 6H20 (2.7b)


Ru(H20)63+ (aq) + H2edta2- (aq)

Ru(Hedta)H20 (s) + 4H20 + H30+ (aq) (2.7c)


Although the synthesis of [Ru(Hedta)H20] from ruthenium

oxide resulted in relatively low yield, the method is

potentially important in the development of processes

wherein the chelate is covalently attached to proteins or

other macromolecular carriers. For these processes, two

possible modes of synthesis, utilizing EDTA in which the

ethylene backbone has been modified (EDTA-R), are

represented in Schemes 2.1 and 2.2.


Protein + EDTA-R > (Protein-R-Hedta)3- + 3H+ a.

(Protein-R-Hedta)3- + Ru(H20)6 3

Ru(Protein-R-Hedta)H20 +5H20 b.

Scheme 2.1


Protein + Ru(Hedta-R)H20 --- Ru(Hedta-R-Protein)H20

Scheme 2.2


Based on the results from the various methods of

synthesis of [RuIII(Hedta)H20] in this work, a suitable

method for the production of the protein modified complex








outlined in Scheme 2.1 would involve the synthesis of the

EDTA modified protein followed by metallation using

[Ru(H20)613+ which had been produced by reduction of

ruthenium oxide. For application to Scheme 2.2, the most

suitable method would involve the synthesis of Ru(H2edta-

R)C1 followed by its reduction then oxidation in aqueous

media to produce Ru(Hedta-R)H20. The complex could then be

allowed to react with the protein to produce the protein

modified complex.

An important potential problem associated with Scheme

2.2, however, involves the known26 dimerization of

[RuIII(Hedta)H20] in aqueous solutions above pH = 8.5.

Although no data is available at physiological pH (the

likely pH for reaction with proteins) there exists a

possibility that the complex will dimerize prior to

reaction. Evidence supporting this was found in the UV-

Visible spectra for pH = 7.34 solutions of [RuII(Hedta)H20]

which were obtained immediately following solution

preparation and oxidation to RuITT with 02 and 24 hours

after solution preparation (Figure 2.1). The appearance of

the absorption band with Amax =390 nm in the solution which

was allowed to stand 24 hours is consistent with the

reported formation of the dimer. The production of a strong

absorption band with Amax = 568 nm at pH = 11.3 which shifts

to lower wavelengths with decreasing pH was reported by Ni,

et al. in the same paper26 and the species undergoes






22

reduction at a potential very close to that of the monomer.
The observed formation of the dimer species in solution at
physiological pH suggests that Scheme 2.2 may only be

appropriate for use if the reaction is carried out very
quickly, however, product purity may be questionable. It is
concluded that the more suitable method for conjugate
synthesis would follow that of Scheme 2.1.


0.8



S0.6



0.4



0.2


Wavelength (nm)


Figure 2.1. UV-Visible spectra of (a) freshly prepared
[Ru I(Hedta)H20] in pH = 7.34 phosphate buffer and (b)
the same solution after 24 hours.








Properties of New rRu(Hedta)L] Complexes

[RuII(Hedta)l.10-phenanthrolinel. The cyclic

voltammogram of the complex dissolved in pH = 5.56 acetate

buffer is reversible and is shown in Figure 2.2. The formal

reduction potential was + 443 mV versus NHE, and the

difference between anodic and cathodic peaks was 75 mV.

[RuIII(Hedta)(pyridine)1,2"5H201. The cyclic

voltammogram of the product dissolved in pH = 5.56 acetate

buffer of 0.3M ionic strength is shown in Figure 2.3. Two

formal reduction potentials were obtained from the

reversible voltammograms. The first, representing the

reduction of [Ru(Hedta)pyridine], was + 93 mV versus NHE

with a difference in peak potentials of 88 mV. The second

represents the reduction of [Ru(Hedta)(pyridine)2] and

occurred at + 400 mV versus NHE with a difference between

anodic and cathodic peaks of 100 mV. Based on the relative

areas for each reversible scan, it was calculated that the

solid contained a mixture of 34.8% Ru(Hedta)(pyridine)2 and

65.2% Ru(Hedta)pyridine.

[Ru Ii(Hedta) (2,2'-dipyridine) 12H201. The reversible

cyclic voltammogram of the product dissolved in pH = 5.56

acetate buffer of 0.3M ionic strength is shown in Figure

2.4. The formal reduction potential was + 512 mV versus NHE

and the difference between anodic and cathodic peaks was 75

mV. Shown in Figure 2.5, the UV-visible spectrum of

RuII(Hedta)2,2'-dipyridine in phosphate buffer differs from







that of the oxidized form primarily in the region of 2,2'-
dipyridyl absorption.









30-


20-



10-



0



-10-



-20-
-800


-600 -400 -200 0o
Potential (mV vs Ag/AgCl)


Figure 2.2. Cyclic voltammogram for Ru(Hedta)1,10-
phenanthroline in pH = 5.56 acetate buffer, 0.3M ionic
strength versus Ag/AgCl reference electrode. 20 mV/sec
scan rate.


-















30-


20-


10-


0-


-10-


-20 -
-500


'-300 -10' 16o 360 '560
Potential (mV vs Ag/AgCl)


Figure 2.3. Cyclic voltammogram versus Ag/AgCl
reference electrode of Ru(Hedta)(pyridine) 2'5H20 in
pH = 5.56 acetate buffer of 0.3 M ionic strength. 20
mV/sec scan rate.















30-


20-


10-


0-


-10-


-201-
-700


-500
Potential


-ioo -ioo 10'
(-00 -100 100
(mV vs Ag/AgCI)


Figure 2.4. Cyclic voltammogram versus Ag/AgCl
reference electrode for Ru(Hedta)2,2'- dipyridyl'12H20
in pH = 5.56 acetate buffer of 0.3 M ionic strength.
Scan rate = 20 mV/sec.


_ ~ __________

















1.0 "



0.8




O, a


80 *
.0


0.4



0.2-



0.0 -. I
200 300 400 500 600
Wavelength (nm)






Figure 2.5. UV-Visible spectra for [RuIII(Hedta)2,2'-
dipyridyl] (solid line) and [Ru I(Hedta)2,2'-
dipyridyl]1- (dashed line) and 2.2'- dipyridyl (dotted
line) in pH = 7.36 phosphate buffer, 0.15 M ionic
strength.









Electrochemistry Results

Reversible voltammograms were obtained for all

complexes studied. Shown in Table 2.1, the formal reduction

potentials for [RuII(Hedta)L] (for L = water, thiocyanate,

acetonitrile, isonicotinamide, pyrazine and pyridine)

obtained in this work at pH = 5.56 are, with the exception

of thiocyanate, all within 20 mV of the pH = 5.5 values

reported by Matsubara and Creutz.17 The observed formal

reduction potential for [RuIII(Hedta)thiocyanate]- (+ 112

mV) was approximately 40 mV greater than that reported and

may be accounted for by the presence of more than one inner

sphere coordinated thiocyanate ion. In the same report,

Matsubara and Creutz reported resolvable spectral changes as

a result of the binding of one and two thiocyanate ions.

Since the conditions for the preparation of [RuIII(Hedta)L]

in the present work were such that thiocyanate was in

excess, it is probable that the species undergoing reduction

may have been bis-isothiocyanato.

The large deviation in differences between anodic and

cathodic peak potentials (AEp) from the Nernstian 59 mV may

be due in part to adsorption of the complexes on the working

electrode while reduction was occurring. Thorough washing

of the electrode prior to sample reduction did not alter

this difference in peak potentials. The use of a hanging

mercury drop electrode may provide for smaller peak

potential differences.















Table 2.1. Formal reduction potentials, in mV versus
NHE, for [RuII(Hedta)L] complexes.



pH=5.56 pH=7.30 pH=8.95
Ligand Ef AEp Ef AEp Ef AEp


water -8 100 +18 113 -70 150

2-nitrobenzonitrile +12 100 +8 106 -53 140

4-nitrobenzonitrile +52 100 +15 118 -67 131

thiocyanate +112 75 +102 95 +78 94

isonicotinamide +190 81 +168 100 +133 130

acetonitrile +249 75 +237 125 +225 140

pyrazine +212 75 +256 90 +225 135

2-methylpyrazine +249 75 +231 90 +213 160

pyridine (mono) +112 81 +95 80 +72 160
(bis) +402 81 +376 80 +363 85

1,10-phenanthroline +443 75 NA NA

2,2'-dipyridyl +512 75 NA NA


NHE =
Ep =
NA =


Normal Hydrogen Electrode.
Difference between anodic and cathodic peak
potentials (mV).
Not assessed.








The most notable effect of pH on formal reduction

potentials was observed for the aqua complex. At pH = 5.56,

7.30, and 8.95, the complex exhibited reduction potentials

of -8, +18, and -70 mV, respectively. At the lower two pH

values, the difference in reduction potentials are close to

experimental error, however, at high pH a significant shift

toward more negative reduction potential was observed and

supports that the hydroxo complex was undergoing reduction.

For the other complexes, with the exception of pyrazine, a

general trend of more negative reduction potentials with

increased pH is observed.

As shown in Table 2.1, at pH = 5.56 a potential range

from -8 to +512 mV versus NHE was obtained when the new

compounds [Ru(Hedta)1,10-phenanthroline] and

[Ru(Hedta)(2,2'-dipyridine)] are included in the series of

complexes. This represents a range which is 400 mV wider

than the series investigated by Matsubara and Creutz. The

inclusion of Ru(Hedta)(pyridine)2, possibly a seven

coordinate27 complex, in the series at pH = 7.30 and 8.95

results in potential ranges from +18 to +376 and -70 to +363

mV versus NHE, respectively. It is expected that the

inclusion of the 1,10-phenanthroline and 2,2'-dipyridyl

complexes in these series of higher pH would increase the

observed range in potentials by 200 mV.








Chapter Summary



The results of this work show that a number of

different methods can be used to synthesize

[RuIII(Hedta)H20]. Of these methods, greatest yields are

obtained from the conversion of [RuII(H2edta)Cl] to the

aqua complex by either the presence of silver ion or the

reduction of [RuII(H2edta)Cl] to form [RuII(H2edta)H20]. A

method of preparation involving the reaction of ruthenium

oxide provides an alternative wherein the chelate is allowed

to react directly with [Ru(H20)6]3+. This method resulted

in low yields but may be the most viable for future

application in modifying proteins with ruthenium chelates.

The nature of the bond between the protein and EDTA and

potential implications concerning the liability of the inner-

sphere coordinated water molecule when [RuT11(Hedta)H20] has

been covalently linked to the protein needs to be addressed

in future work. The electron withdrawing ability of the

linking function on EDTA, carrier hydrogen bonding with the

uncoordinated acetate function or steric crowding by the

carrier could adversely effect the rate of aqua ligand

substitution and may present potential problems for

synthesis and use of these new conjugates. It is important

that the liability of [Ru 11(Hedta)H20 be maintained

following conjugation so that the substitution reaction with

T-acids can be utilized to easily prepare a series of








protein-[edtaRuIIIL] complexes from the protein-

[edtaRuIIIH20] conjugate. Prior to evaluating these protein

conjugates, cyclic voltammetry should be conducted using

complexes which contain the modified EDTA and results

compared with the reduction potentials obtained in this

work.

The complexes [RuIII(Hedta) ,10-phenanthroline] and

[RuII (Hedta)2,2'-dipyridine] represent new compounds, and

their formal reduction potentials are so positive that their

inclusion in the series of complexes provides a range in

reduction potentials 400 mV wider than that obtained in

previous work involving [RuIII(Hedta)L] complexes. The

reduction potentials for both complexes suggest that both

nitrogen donor atoms in the ligands are coordinated to the

metal center. Whether a coordinating acetate function of

EDTA is displaced as a result of the substituting ligand's

apparent bidenticity or the metal center becomes seven

coordinate as a result of the substitution reaction has not

been evaluated. Further investigations of these complexes

should include nuclear magnetic resonance (proton and

carbon) and X-ray crystallography to provide information

concerning their structure.

Examination of frequency shifts of the acetate carbonyl

stretch for the series of [Ru II(Hedta)L] complexes does not

provide information concerning the relative electron density

on the metal as a function of the r-acidity of the ligand,








L, but frequency intensity data does provide information

concerning the absence or presence of a protonated free arm

of the EDTA ligand.28 An investigation of the intensities

of infrared carbonyl stretches for the 1,10-phenanthroline

and 2,2'-dipyridyl complexes may provide information

concerning the denticity of the EDTA ligand when these other

ligands are coordinated to Ru3+. These new complexes of

[RuIII)(Hedta)l,10-phenanthroline] and [RuIIIHedta)2,2'-

dipyridine] should provide protein conjugate analogs in

which the redox tuning ligand is less likely to be displaced

by r-acid ligands available in biological media than many of

the other ligands studied.












CHAPTER 3

SYNTHESIS AND CHARACTERIZATION OF
POLYSACCHARIDE ESTERS OF
POLYAMINOPOLYCARBOXYLIC ACIDS AND THEIR
GADOLINIUM(III) COMPLEXES



Chapter Overview



Magnetic resonance imaging (MRI) is a noninvasive

diagnostic aid that has rapidly become the method of choice

for the initial screening of patients suspected of a variety

of physiological disorders including certain types of

cancer.29-31 The method often benefits from the intravenous

injection of paramagnetic compounds called contrast agents

into patients immediately prior to data acquisition in MRI.

These agents serve to provide contrast between different

tissue types by enhancing the relaxation differences of the

associated water protons.

Gadolinium(III) diethylenetriaminepentaacetic acid

(GdDTPA)32-36 and other paramagnetic compounds37-39 have

been used successfully as contrast agents to enhance the

imaging of several types of carcinomas in non-clinical

evaluations. GdDTPA, the only agent currently approved for

human use, undergoes rapid renal excretion following









extracellular biodistribution with a blood concentration

half-life of only 20 minutes.40 The short persistence and

extravascular distributive properties of GdDTPA make the

contrast agent ineffective for situations where a lengthy

blood-pool persistence of the agent may be required for

specific organ and/or lesion uptake to occur and unsuitable

for use in blood flow evaluations.

Several alternatives to GdDTPA have been proposed,

including agents that contain different paramagnetic metal

ions and chelating ligands41'42 and magnetic cluster or

spherical compounds.43'44 GdDTPA, however, exhibits a large

spin-lattice relaxation (T1) effect,45'46 the DTPA chelate

affords the complex great (1022) thermodynamic stability3

and, relative to most of the proposed alternatives, these

aspects of GdDTPA make the compound superior. In the

development of new contrast agents it is advantageous to

retain these more desirable qualities of GdDTPA.

One approach toward overcoming the specific limitations

of GdDTPA involves the covalent attachment of the well

characterized and clinically evaluated GdDTPA on to

macromolecular carriers.47 The use of macromolecular

carriers may result in compounds that exert greater effect

on T1 relaxation of surrounding water molecules48 as well as

enhanced blood-pool persistence and possible tissue

specificity relative to GdDTPA. Although several of the

variables that must be considered in the design of new








contrast agents are addressed by using macromolecular

carriers, the biological effect and fate of the carrier and

carrier-complex must also be considered.

Carrier molecules have been used extensively in

pharmaceutical applications and the same requirements for

application to macromolecule carried drugs are applicable to

macromolecular based contrast agents. These requirements

are:49 (1) the agent must retain activity following

conjugation, (2) the carrier must be nontoxic,

nonimmunogenic, and nonantigenic, (3) the carrier must be

biodegradable, and (4) the desirable properties of the

carrier must be retained following conjugation.

At the inception of this study, prior art consisted of

the use of proteins or lipids as macromolecular carriers.

Proteins such as bovine serum albumin or immunoglobulin G,

which have been used as carrier molecules50,51 in GdDTPA

conjugates, would elicit an immune response when used in

vivo and the level of loading of the paramagnetic agent on

the protein is low. These properties, in addition to

compromised thermodynamic and/or kinetic stabilities of the

metal-chelate moiety, are also potential problems for the

recently reported GdDTPA-crosslinked polysaccharide

compounds52 (compounds in which the polysaccharide chains

were covalently linked together by the chelating agent).

The use of polysaccharides as carrier molecules, however,

specifically dextrans with average molecular weights less









than 90,000 or inulin (in which the products are not

crosslinked by the chelate), may result in products which do

not possess these undesirable properties.53

Dextran contains a large number of secondary and very

few primary alcohol functions and has 1-->6 linked a-D-

glucopyranose units with some 1--'4 side branching present.

Inulin is a linear chain containing a-D-fructofuranose units

with a large number of both primary and secondary alcohol

functions. For both of these polysaccharides, the large

number of alcohol functions available for chemical

modification is an attractive feature in terms of the

potential application of these compounds as macromolecular

carriers.

In addition to the possibility of high GdDTPA loading

relative to that attainable with proteins, dextran and

inulin polysaccharides are nontoxic and undergo normal

metabolism. Other desirable qualities of the

polysaccharides include low expense, hydrophilicity, pH

stability, precedence in drug delivery, and increased plasma

persistence of the conjugated moiety.49,54 Additionally,

the potential for lanthanide promoted decomposition of the

chelate to carrier ester linkage55 in these polysaccharide

based complexes may provide a mechanism for the timely

release of the metal chelate from the carrier. These

properties suggest that the compounds may be useful contrast

agents.








The goals of the present study included the synthesis

of dextrans ranging in average molecular weight from 9,400

to 487,000 and inulin (5,000 average molecular weight) that

have been extensively labeled with GdDTPA by one ester bond

per GdDTPA moiety. The purpose of the work was to develop a

means by which new compounds, which may exhibit properties

superior to the commercially available contrast agent

(GdDTPA), could be easily produced. The repetitive

structure of the dextrans and inulin allows for the

treatment of the DTPA-polysaccharides and GdDTPA-

polysaccharides as monomer units in some of the evaluations

required for product characterization. Characterization of

the esters was intended to evaluate the degree of GdDTPA

loading on to the polysaccharide (i.e. the number of GdDTPA

molecules covalently attached to the polysaccharide molecule

per repeating saccharide unit), provide viable evidence for

products that are not crosslinked by the chelate, determine

the thermodynamic stability of the new compounds, evaluate

the relative rates of metal promoted ester hydrolysis in the

gadolinium complexes and the apo-conjugates, and to

investigate the in vitro solvent T1 relaxation effects by

the gadolinium complexes using nuclear magnetic resonance

(NMR). In order to accomplish these goals, it was necessary

to devise a method of synthesis that would: (1) provide

reproducibly high levels of loading of the chelate on to the

polysaccharide carrier, (2) result in products that are not








crosslinked by the chelate (in which only one carboxyl

function of the chelate is required in ester bond

formation), and (3) result in no degradation of the

polysaccharide carrier.


Materials and Methods



Synthesis of DTPA-Polvsaccharides

Synthesis of anhydrides of DTPA. The isobutyl and

ethyl mixed anhydrides of DTPA were prepared by method of

Krejcarek and Tucker56. A 1:5 mole ratio of DTPA (Aldrich)

to triethylamine (Kodak) in anhydrous acetonitrile (Aldrich)

were used. Isobutylchloroformate (Aldrich) or ethyl-

chloroformate (Kodak) was reacted with the triethylamine

salt. Following precipitation of solid triethylamine-

hydrochloride, the cold mixture was filtered and solid

washed by cold filtering with acetonitrile. The washings

were added to the filtrate and the solution was rotary-

evaporated to form a white paste. Repeated washing and

decantation with anhydrous ethyl ether resulted in a white

powder following vacuum drying in a desiccator.

The dicyclic anhydride of DTPA was prepared, isolated,

and washed by method of Eckelman57. Acetic anhydride

(Fisher) was reacted in a 4 to 1 mole ratio with DTPA in ACS

grade pyridine (Aldrich). It was found that heating at 650C

for 24 hours resulted in significant product discoloration.






40

Heating at 45C did not result in the formation of the dark

brown solid/solution and this temperature was used in all

subsequent Eckelman based cyclic anhydride preparations.

Modifications in Eckelman's method57 were used to

produce the monocyclic and mixtures of mono- and dicyclic

anhydrides of DTPA. Reaction mixtures containing mole

ratios of DTPA to acetic anhydride of 1.0:1.0, 1.0:1.0 with

a trace amount of trifluoroacetic anhydride (Fisher)

present, 1.0:2.0, and 1.0:3.2 were used.

Halpern's method58 of synthesis and isolation was also

used to produce the reported monocyclic anhydride. Thionyl

chloride (Fisher) was reacted with DTPA in the presence of

excess trifluoroacetic anhydride (Fisher).

Synthesis of acid esters of polysaccharides. All

glassware was oven dried prior to use. All reactions were

conducted in flasks equipped with a condensing or vigreaux

column topped with a calcium chloride drying tube. All

preparations which required the use of trifluoroacetic

anhydride or acetic anhydride were, following the reaction

heating time, heated sufficiently to distill off their

respective acids. Damp litmus held above the heated flask

contents was used to ascertain the completeness of removal

of these acids. All polysaccharides were lyophilized for 3

hours prior to use. Acetone was used to precipitate

products and hydrated cyclic anhydrides (DTPA) in all

preparations involving the reaction of mono- or di- cyclic








anhydrides of DTPA. Methanol or ethanol was used to

precipitate products and DTPA in all preparations in which

the mixed anhydrides were used. In all reaction conditions

tried, a ratio of one mole of mixed or cyclic anhydride to

one mole of alcohol function per mole repeating

glucopyranose or fructofuranose unit of the polysaccharide

was used.

The reactions of the mixed anhydrides with

polysaccharides were carried out using the following three

general reaction conditions:

1.) Refluxing in pyridine (or pyridine and dimethyl-

sulfoxide sufficient to solubilize the dextrans) for from

one to two hours.

2.) Stirring at room temperature from 15 minutes to 12

hours in dimethylsulfoxide or benzene following activation

of the polysaccharide alcohol functions. This activation

was accomplished by the addition of one mole of n-butyl

lithium (in hexane, Aldrich) with one mole of alcohol

function per mole of repeating unit on the polysaccharide.

3.) Heating at 600C in a one to one volume ratio of

pyridine (Aldrich) to formamide (Kodak) solvent system for

four hours with and without the presence of anhydrous zinc

chloride.

The reactions which involved the dicyclic anhydride

reacting with the polysaccharides were conducted using the

following two general reaction conditions:








1.) Refluxing in toluene (Fisher) in the presence of a

trace quantity of trifluoroacetic anhydride (Fisher) for one

to four hours.

2.) Heating at 60C in 1 to 1 volume ratio of pyridine and

formamide with and without the presence of anhydrous zinc

chloride.

The reactions involving the prepared monocyclic

anhydride or mixture of mono- and dicyclic anhydrides

reacting with the polysaccharides were conducted under the

following two general reaction conditions:

1.) The monocyclic anhydride prepared by Halpern's58 method

was refluxed with the polysaccharide in 1 to 1 pyridine and

formamide for four hours in the presence of zinc chloride.

2.) The monocyclic anhydrides or mixture of mono- and

dicyclic anhydrides prepared by stoichiometric control of

the ratio of DTPA to acetic anhydride from 1:1 to 1:3.2 were

conducted with and without a trace quantity of

trifluoroacetic anhydride present in 1 to 1 pyridine and

formamide. The reactions were carried out at either 60C or

45C for 4 to 5 hours in the presence of zinc chloride (when

no trifluoroacetic anhydride was used).

Optimized carrier-liqand synthesis. The optimized

method of synthesis for the DTPA esters of the

polysaccharides involved the synthesis of a mixture of the

mono- and di-cyclic anhydrides of the acid and the sub-






43

sequent reaction of this mixture with the polysaccharide. A

detailed method for synthesis is provided as follows.

Diethylenetriaminepentaacetic acid (0.1 mole) was

placed in 200 milliliters of anhydrous pyridine and heated

at 450C while stirring for 24 hours with 3.2 mole acetic

anhydride. During the process, all of the solid dissolved

and near the end of the reaction time a solid precipitated.

The solid was filtered, washed with acetic anhydride

followed by anhydrous ethyl ether, and dried under vacuum in

a desiccator.

The polysaccharide (either inulin or dextrans with

average molecular weights of 9400, 40200, 78000, or 487000,

all obtained from Sigma) was placed in a flask containing a

1 to 1 volume mixture of pyridine and formamide59 which had

been previously dried over molecular sieves. The weight

ratio of mixed solvent to polysaccharide was approximately

250 to 1. The flask contents were stirred while heating at

400C for 1/2 hour to dissolve the polysaccharide. For each

gram of polysaccharide used, either 1/2 milliliter of

anhydrous pyridine made turbid with zinc chloride or 0.1

milligram of anhydrous zinc chloride was added to the

reaction flask and heating continued for 1/2 hour. One mole

of the prepared cyclic anhydride per mole of repeating

glucopyranose or fructofuranose unit of the polysaccharide

was placed in the flask and the contents were stirred while

heating at 500C for four hours under a small reflux








condenser equipped with a calcium chloride drying tube.

Glucopyranose repeating unit concentrations in all

preparations were 0.1 M. In a typical procedure, 4.7 g

(0.50 mmol polysaccharide, 26 mmol glucopyranose) dextran

9400 was placed in a flask with 250 mL of 1:1 volume mixture

of formamide and pyridine. The flask contents were stirred

while heating at 400C for 1/2 h to dissolve the

polysaccharide. Following the addition of 0.5 mg of

anhydrous zinc chloride (Aldrich), the heating was continued

for 1/2 h. To the solution, 10.2 g (26 mmol) of the

prepared cyclic anhydride was added. The flask contents

were stirred while heating at 500C for 4 h under anhydrous

conditions. After cooling to room temperature, the

solution was rotary-evaporated to approximately 1/2 the

original volume. The solution was cooled in an ice bath and

acetone was added to precipitate the solid. The solid was

filtered, washed with cold acetone followed by anhydrous

ethyl ether, and dried under vacuum in a desiccator.



Determining Reaction Success by HPLC

All HPLC experimental work was conducted in pH = 7.0

phosphate buffer solution of 0.10 M ionic strength. A

Waters Model 6000A solvent delivery system was used with a

standard silica pre-column and Waters Protein-Pak 125

column. Detection was at 230 nm using an Instrumentation






45

Specialties Company model 1840 absorbance monitor. A flow

rate of 0.5 mL/minute was used for all evaluations.

Following the isolation of solid esterified poly-

saccharides and the unreacted aminopolycarboxylates, approx-

imately 0.5 mg of the solid was dissolved in 1.0 mL of the

eluting buffer. Between 10 and 30 microliters of the

solution was injected into the HPLC port. The resulting

elution profiles were obtained using a detector sensitivity

of 0.2 and a Scientific Products Quantigraph recorder.

Traces of the elution peaks were obtained and the mass of

each peak was used to calculate the approximate mass percent

of DTPA reacted. It was assumed that the extinction

coefficient of the products) was the same as that of DTPA

at 230 nm.

Fractions of the eluting solution were collected over

one minute intervals using an LKB Bromma Redirac model 2112

fraction collector. These fractions were subjected to sugar

assay.60 A mixture of 300 microliters of each fraction with

20 microliters of 80% phenol (in water) was vortexed for 10

seconds followed by the addition of 1.0 milliliter of

concentrated sulfuric acid to the center of the vortexing

solution. Following 5 additional seconds of vortexing, the

solutions were cooled to room temperature and absorbances at

490 nm (IBM Model 9430 UV-Visible spectrophotometer) were

recorded within one hour of solution preparation.









The sugar assay absorbances were superimposed on the

HPLC elution profiles for each sample. The results were

compared with : the standard elution profiles obtained by

sugar assays for the unmodified polysaccharides; the

elution profiles for the unmodified polysaccharides after

undergoing the identical esterification reaction but without

the reactant anhydride present; the elution profile for the

unmodified polyaminopolycarboxylic acid.



Purification of DTPA-Polysaccharide Esters

Spectrapor molecular porous membrane tubing (Spectrum

Medical Industries) with a globular protein molecular weight

cut-off range from 6,000 to 8,000 average molecular weight

was used. The dialysis tubing was soaked in an 800C

solution of 1.0 M EDTA for 1 hour and rinsed six times with

deionized water prior to use. Sugar assay (method given in

HPLC section above) of the wash solutions was used to

ascertain the suitability of the protein cut off range of

the tubing for the polysaccharides. The isolated solid

products were dissolved in deionized water (approximately 1

g /3 mL), placed in the dialysis tubing, and dialyzed

against deionized water at room temperature for 72 hours.

Wash solutions were changed every 12 hours. Dialysis was

terminated when the pH of the wash solutions became

constant. The solid polysaccharide esters were isolated by







47

rotary-evaporation of the dialysis bag contents followed by

lyophilization for 24 hours.

Elemental analyses of typical products obtained from

the general method of synthesis were calculated based on the

general formula (attached DTPA unit) (average esterified

polysaccharide unit)1. In all cases, the unmodified

polysaccharide was analyzed independently so the effect of

polysaccharide branching on the average repeating

glucopyranose or fructofuranose could be accounted for.

DTPA-Inulin, calculated for

(C14H22N309)0.53(C5.5H9.0605.11): C, 43.54; H, 5.86; N,

6.25. Found: C, 43.82; H, 6.05; N, 6.07. DTPA-Dextran

9400, calculated for (C14H22N309)0.42 (C6H10.9506.04): C,

42.24; H, 6.02; N, 5.22. Found: C, 42.93; H, 6.22; N, 5.28.

DTPA-Dextran 40200, calculated for

(C14H22N309)0.38(C6H11.0405.85): C, 41.01; H, 6.27; N, 5.12.
Found: C, 41.92; H, 6.04; N, 4.92. DTPA-Dextran 70800,

calculated for (C14H22N309)0.36(C6H10.8305.75): C, 42.71; H,

6.09; N, 4.87. Found: C, 42.64; H, 6.20; N, 4.86. DTPA-

Dextran 487000, calculated for (C14H22N309)0.06

(C6H11.2306.05) C, 40.52; H, 6.24; N, 1.24. Found: C,

41.12; H, 6.57; N, 1.25. For dextran 487000, the product

was also prepared by the general method but the reaction

solution was allowed to stir at room temperature for 30 h

prior to precipitation and isolation of product. The

calculated mass percent for a product prepared in this








manner was (C14H22N309)0.82 (C6H10.4706.05): C, 43.02; H,

5.89; N, 7.06. Found: C, 42.94; H, 6.05; N,7.08.



Nature of Reacted Polysaccharide by HPLC

The isolated solids (approximately 0.5 mg) were each

dissolved in pH = 7.0 phosphate buffer. Approximately 20

microliters of each solution was injected into the HPLC port

and an elution profile was obtained. Eluent fractions were

collected at one minute intervals and subjected to sugar

assay. The resulting elution profiles and superimposed

sugar assays were compared with the elution profiles of the

unmodified aminopolycarboxylic acids.

The HPLC results following purification (above) were

compared with the sugar assays of the unmodified poly-

saccharides. The elution profiles were also compared with

those of the polysaccharides following their subjection to

conditions identical to those of the reaction but without

the prepared cyclic anhydride present.



Synthesis and Purification of Gadolinium Complexes

The gadolinium complexes of EDTA, DTPA, and TTHA were

prepared by a method outlined for H2GdDTPA by Wenzel.61

Ethylenediaminetetraacetic acid (Mallinckrodt), di-

ethylenetriaminepentaacetic acid (Sigma), and triethyl-

enetetraaminehexaacetic acid (Sigma) were used as supplied.

The hexahydrate of GdCl3 (Aldrich) was used instead of Gd203






49

in the preparations and acetone was used to precipitate the

solids.

Metallation of the esterified polysaccharides was

achieved by dissolving the solids in deionized water

followed by the addition of a 0.2 molar stoichiometric

excess of the hexahydrate of GdC13 (Aldrich). The solutions

were stirred at room temperature for 1 hour, placed in

dialysis tubing, and exhaustively dialyzed against water at

room temperature until the wash solutions tested negative

for gadolinium ion with xylenol orange indicator (Aldrich).

The dialysis bag contents were rotary-evaporated to dryness

and the solids were lyophilized for 24 hours. A portion of

each of the solids was retained for elemental analysis and

the remainder was dissolved in deionized water and dialyzed

for 48 hours against a 0.1 M solution of EDTA at room

temperature followed by dialysis against water for 48 hours.

The dialysis bag contents were then rotary-evaporated to

dryness and lyophilized for 24 hours.

Elemental analysis and ICP analysis were conducted on

the products. Some calculated and observed compositional

results are as follows for products prepared as outlined in

the general method of synthesis and the calculated results

are based on the general formula Gdx(attached DTPA

unit)y(average esterified polysaccharide unit)1. GdDTPA-

Inulin, calculated for

Gd0.52(C14H17N309)0.57(C5.5H9.0205.11)2.0H20: Gd, 17.24; C,








31.60; H, 4.82; N, 5.05. Found: Gd, 17.16 +/- 1.43; C,

31.77; H, 4.77; N, 4.90. GdDTPA-Dextran 9400, calculated

for Gdo.36(C14H17N309)0.36 (C6H11.2106.04)'0.3H20: Gd,

15.06; C, 35.28; H, 4.81; N, 4.02. Found: Gd, 15.4 +/- 0.4;

C, 35.16; H, 5.18; N, 3.92. GdDTPA-Dextran 40200,

calculated for Gd0.31(C14H17N309)0.36

(C6H11.0605.85)'0.4H20: Gd, 13.30; C, 36.19; H, 4.95; N,

4.13. Found: Gd, 13.7 +/- 0.6; C, 36.52; H, 5.44; N, 4.03.

GdDTPA-Dextran 70800, calculated for Gd.36(C14H17N309)0.36

(C6H10.8305.75)*0.8H20: Gd, 14.91; C, 34.92; H, 4.92; N,

3.98. Found: Gd, 14.6 +/- 0.5; C, 35.11; H, 5.43; N, 4.00.

GdDTPA-Dextran 487000, calculated for Gd0.04(C14H17N309)0.06

(C6H11.2306.05)'0.1H20: Gd, 2.99; C, 39.02; H, 5.96; N,

1.20. Found: Gd, 2.6 +/- 0.3; C, 39.02; H, 6.17; N, 1.02.

For the product in which the highly loaded DTPA-Dextran

487000 was used, the calculated for Gd0.78(C14H17N309)0.79

(C6H10.5006.05)*1.5H20: C, 32.92; H, 4.36; N, 5.33. Found:

C, 32.40; H, 4.59; N, 5.34. The product could not be

evaluated by ICP analysis as it was too insoluble in

deionized water for solution preparation.



Characterization of Compounds

Elemental analysis. All elemental analysis were

conducted by the Department of Chemistry's Spectroscopic

Services. Samples of each of the compounds were submitted








for evaluation immediately following lyophilization of the

products.

Infrared analysis. The carbonyl stretching

frequencies of the aminopolycarboxlyate esters of the

polysaccharides, the unmodified acids, and their gadolinium

complexes were evaluated by Fourier Transform Infrared

spectroscopy (Nicolet 5DXB FTIR). Several of the prepared

cyclic anhydrides of DTPA were also evaluated by infrared

analysis of the carbonyl stretching region. All spectra

were obtained as Nujol mulls of the samples using sodium

chloride plates. The scans ranged from 2500 to 1000

wavenumbers. No solvent subtraction technique was employed

as characteristic Nujol stretching frequencies did not

interfere with those of the compounds or complexes in the

spectral region evaluated.

Inductively coupled plasma analysis. Weighed samples

of each of the complexes (Mettler AE163 balance) were

dissolved in deionized water and evaluated for mass percent

gadolinium by ICP (Perkin Elmer Plasma II Emission

Spectrometer). A calibration curve of emission counts

versus gadolinium concentration was obtained at the start of

each session following ICP initialization. Dilutions of a

standard solution of GdC13 in 1% HNO3 (Aldrich Chemical Co.)

were used to obtain the calibration curves.

Each sample solution was evaluated five times with 30

seconds read delay between successive evaluations. The








average of the trials and their standard deviations (in

parts per million) was taken as the accepted solution

concentration after conversion to units of molarity. Three

solutions of each complex were subjected to ICP analysis,

the results averaged, and mass percent gadolinium

calculated. In addition to verification of mass percent

gadolinium, the ICP was used in identical fashion to

evaluate gadolinium concentrations for all solutions for

subsequent use in in vitro relaxivity studies and in vivo

imaging studies (Chapter 4).

Nuclear magnetic resonance analysis. Spectra of some

of the DTPA esterified polysaccharides were obtained in

deuterium oxide. A Varian XL-VXR 300 nuclear magnetic

resonance instrument was used.

Potentiometric titrations. All potentiometric

titrations were conducted at 25.0C (0.050C precision, I2R

Thermowatch Model L6 1000 SS) using a standard pH meter

(Orion Research Digital pH/Millivolt Meter 611) and a

semimicro combined pH electrode (Orion Research Gel Filled).

All solutions were purged with argon for 1/2 hour prior to

use. Solutions containing from 0.12 to 0.14 mmole DTPA or

esterified DTPA were prepared in 100 mL of argon purged 0.1

M KNO3 solutions. Metallation was accomplished by titration

of the esters with a solution of 18.8 mM Gd3+ in 0.0100M

HNO3. The mole ratio of Gd3+ added per DTPA moiety was one

to one. Following metallation, the added HNO3 was









neutralized with KOH prior to potentiometric titration.

Titrations were conducted using 0.100 M KOH and constant

ionic strength was maintained with KNO3. Approximate log Ka

values for the ligands were obtained using the computer

program PKAS,62 and refinement of these values and the

approximated thermodynamic stability constants for the metal

complexes was achieved with the computer program BEST.62

Ester hydrolysis studies. Solutions containing 15 mM

DTPA moiety of the esterified polysaccharides or 15 mM

gadolinium in the esterified metal chelates were prepared in

pH = 7.4 phosphate buffer of 0.15 M ionic strength. The

solutions were placed in a water bath maintained at 37.00C

for 4 days. Portions of each solution were removed at

regular intervals and subjected to HPLC analysis using the

same phosphate buffer of 0.15 M ionic strength to elute the

samples. Detection was at 235 nm and a Waters Protein Pac

125 column was used. The relative areas under the curves in

each elution profile for the intact elutionn peak for DTPA

esterified polysaccharide) and hydrolysed ester elutionn

peak for DTPA) were used to calculate the percent

decomposition (hydrolysed ester peak area / intact ester

peak area) for the esterified polysaccharides. The relative

areas under the curves in each elution profile for the

intact elutionn peak for GdDTPA-polysaccharide) and

hydrolysed esters elutionn peak for GdDTPA) were used to

calculate the percent decomposition (GdDTPA peak area /








GdDTPA-polysaccharide peak area) for the gadolinium

complexes of the esterified polysaccharides.

T, relaxivity studies. Standard solutions of each of

the GdDTPA-polysaccharide complexes and Magnevist were

prepared in pH = 7.4 phosphate buffer. The ionic strength

of the buffer was 0.15 M (isotonic with blood) and was

deoxygenated by one hour of argon bubbling. Gadolinium

concentrations of each of the solutions was evaluated by

inductively coupled plasma emission spectroscopy. Dilutions

of each of the standard solutions with the buffer were then

made such that one mL total volumes of solutions containing

1, 2, 4, and 5 mM Gd3+ were obtained. The solutions were

sonicated to thoroughly mix for 15 minutes.

Approximately 0.5 mL of each of the solutions was

placed in 5 mm NMR tubes which had been previously cleaned

with 0.1 M EDTA solution to remove trace metal ions, rinsed,

and oven dried. A sealed internal coaxial reference tube

(prepared from 1.5 1.8 mm Kimax capillary tubing) contain-

ing deuterium oxide and methanol was used to provide the NMR

lock signal and spectrum reference. The NMR tubes were

sealed with rubber septa and stored at 40C.

The NMR measurements were obtained using a JOEL FX100

Fourier Transform NMR spectrometer operating at 99.55 MHz

resonance frequency with a probe temperature of 25C. The

method for solution T1 evaluation was similar to that out-

lined by Brown and Johnson63. An inversion recovery pulse






55

sequence was used and consisted of a 1800 pulse followed by

time Tau and a 900 pulse. A total of 4096 data points were

collected per scan over a spectral width of 500 Hz. Each

scan required 4.09 seconds acquisition time and four free

induction decay curves were obtained per T value. The

average of the 4 scans was recorded for each of the T

values. A pulse delay of 2.5 seconds was used for all

gadolinium containing solutions. A pulse delay of 10 sec-

onds was used for T1 evaluation of the buffer solution.

Using the display monitor only, the approximate T1 of

each solution was obtained by varying values of T manually

such that the water proton signal intensity approached zero.

These approximate values ranged from 5 to 150 msec and were

dependent on gadolinium concentration. The Tinfinity data

for all gadolinium containing samples was collected at T =

one second to assure accuracy in the measurement. A total

of 9 to 14 T values, excluding Tinfinity, were used for each

of the solutions and ranged within 50 msec of the manually

approximated T1 of the solution.

A stacking program (software supplied with IIMR) was

used to obtain spectral plots of signal intensity versus

frequency as a function of T. A printout of signal inten-

sity and T values was obtained after phasing and estab-

lishment of a baseline for the Tinfinity spectrum for each

solution. All intensity data for other r values were








derived relative to the established Tinfinity spectral

baseline.

All plots were obtained using Grapher version 1.75

(Golden Software, Inc.). Plots of signal intensity versus T

were obtained as a preliminary determination for accept-

ability of the data. The signal intensity values at each T

and at Tinfinity were then used in the following function:

ln (Iinfinity Ir / 2Iinfinity) = -(T/T1) + K

Iinfinity = Signal intensity at Tinfinity
I = Signal intensity at T

T1 = Solution spin-lattice relaxation rate

K = Solvent contribution to total 1/T1

Plots of -ln (Iinfinity IT/2Iinfinity) versus T were

obtained for each sample and a linear least squares fit was

used to obtain 1/T1 (the slope of the line) for the sample.

The process was repeated for the solvent and each concentra-

tion of each of the compounds. Relaxivity plots (1/T1

versus gadolinium concentration) were obtained.

Viscosity and Carrier-Complex Interaction Evaluation.

All studies under this heading involved the collection of T1

data under identical conditions to those given for the T1

relaxivity studies. Values for Tinfinity of 2.5 seconds for

gadolinium containing solutions and 10 seconds for all other

solutions were used. All solutions were prepared in pH =

7.4 phosphate buffer of 0.15 M ionic strength.









To ascertain possible T1 effects due to solution vis-

cosity, buffer solutions containing 5, 22, 43, 104, 380,

572, 936, 1300, 1716, and 2652 mM glucopyranose (in the form

of dextran 9400) were prepared. T1 evaluation was conducted

at 250C. A plot of relaxivity versus glucopyranose

concentration was obtained.

To evaluate possible T1 effects due to interaction

between the metal chelate and the free (unmodified) portions

of polysaccharide, several solutions were prepared. Buffer

solutions containing 1, 2, 4, and 5 mM gadolinium in GdEDTA,

GdDTPA, and GdDTPA-dextran 40200 were prepared and T1

evaluated. Identical solutions were then prepared and to

the 1, 2, 4, and 5 mM gadolinium solutions 2.63, 5.26,

10.52, and 13.15 mM glucopyranose in the form of dextran

40200 was added, respectively, and evaluated by T1 measure-

ment.

To evaluate possible suppression of T1 due to polysac-

charide metal chelate hydrogen bonding and / or steric

restriction of exchanging water molecules, the results of

the previous study were used along with T1 results for a set

of solutions prepared with isopropanol present. Buffer

solutions containing 1, 2, 4, and 5 mM gadolinium in GdEDTA

and GdDTPA-dextran 40200 and 2.63, 5.26, 10.52, and 13.15 mM

glucopyranose, respectively, were prepared with 800 mM

isopropanol (Fisher Scientific Co.) in each solution and T1








evaluated. Plots of relaxivity versus gadolinium

concentration were obtained.



Results and Discussion



Evaluation of Synthetic Methods

The method of synthesis that was developed in this work

for highly loaded GdDTPA-polysaccharides required three

steps: preparation of a suitably reactive anhydride of DTPA;

reaction of the anhydride with the polysaccharide in a

suitable anhydrous mixed solvent system under conditions

that did not result in polysaccharide degradation; and

reaction of the DTPA esterified polysaccharide with

gadolinium ion. There was no disclosed precedence for the

polyaminopolycarboxylic acid esterification of

polysaccharides at the inception of the project.

The first esterified product, a model compound, was

obtained for the reaction of dextran 9400 with methyl-red

(p-dimethylamine azo benzene-o-carboxylic acid, Fisher).

The heterogeneous reaction took place in toluene with a

stoichiometric excess, relative to the number of moles of

carboxylic acid function present from methyl-red, of

trifluoroacetic anhydride.

HPLC was used following all esterification reactions to

evaluate the reaction success, evaluate the product purity

following dialysis, and evaluate the integrity of the






59

polysaccharide following reaction. The reaction success was

determined from the relative areas of the HPLC elution

profiles for the esterified product relative to the

unreacted acid (methyl-red in the model case and DTPA or

TTHA in subsequent esterifications). The elution profile

for the methyl-red esterification of dextran, shown in

Figure 3.1, indicated that approximately 16% of the methyl-

red initially present in the heterogeneous mixture had

reacted.

Following extraction into aqueous solution and the

removal of the unreacted methyl-red by dialysis, the UV-

visible spectrum of the compound was obtained in pH = 7.0

phosphate buffer and compared with the spectra of unmodified

methyl-red obtained in buffers of differing pH (Figure 3.2).

The normal indicator behavior64 of methyl-red was absent in

the esterified product and suggests that the carboxyl group

protonation, required for indicator function, was blocked by

ester bond participation. Shown in equation 3.1, the

reaction of methyl-red with the polysaccharide required the

formation of an intermediate species, a trifluoroacetate

anhydride of methyl-red, which activates the methyl-red

carbonyl function to esterification.


R-C(O)OH + [CF3C(0)]20 -- R-C(0)-O-C(O)CF3

R-C(O)-O-C(0)CF3 + HO-R' -- R-C(0)-0-R' + HO-C(0)CF3

R = Remaining functionality of methyl red.
R' = Remaining functionality of polysaccharide. (3.1)
















0.8-




0.6


41 < 1
o.4 A B




0.2
I



0.0- .-------,,
5 9 13 17
Elution Volume (mL)



Figure 3.1. HPLC elution profile for product mixture
obtained from reaction of methyl-red with dextran 9400
(solid line, B) and HPLC elution profile for unmodified
dextran 9400 as obtained from sugar assay of HPLC
fractions (dashed line, A).




















/ S
I



I/ C
I \
I ---- 1qm


0.0 I
250


350 450
Wavelength (nm)


Figure 3.2. UV-Visible spectra of methyl-red
esterified dextran in pH = 7.0 phosphate buffer (solid
line, A), unmodified methyl-red in pH = 7.0 phosphate
buffer (dashed line, B), and unmodified methyl-red in
pH = 5.6 acetate buffer (dashed line, C).


0.8
*



0.6
-



s 0.4 -
0
0


0.2 -






62

The same esterification reaction conditions which were

successful when methyl-red was used resulted in only 1%

reaction when GdTTHA was used. Solid GdTTHA has a free

(noncoordinated) carboxyl group65 and, based on the observed

low reactivity toward esterification, the result suggests

that either the free carboxyl group in the solid phase is

coordinated or fluxional when GdTTHA is in solution, or, the

extreme low solubility of the complex in toluene is not

conducive for the esterification under these conditions.

The reaction success of ethyl- and isobutyl-mixed

anhydrides of DTPA with polysaccharides under various

reaction conditions is shown in Table 3.1. The results

strongly suggest that homogeneous reaction conditions are

required for high reactivity of the anhydrides with the

polysaccharides. The following additional observations were

made: 1) solvents that provided homogeneous reaction

conditions for reactions involving inulin did not

necessarily provide the same for dextran preparations; 2)

the activation of the polysaccharides with n-butyl lithium

did not provide reproducible results; 3) inulin, with a much

greater number of primary alcohol functions available for

esterification relative to glucopyranose based

polysaccharides, reacted to a significantly greater extent

than dextran 9400 under the same reaction conditions.














Table 3.1


Mixed anhydride reactions with polysaccharides,
the reaction conditions, and percent anhydride
reacted for a series of attempted preparations.


Reactant Polysac.a Solventb Temp./ Catalyst %Reactedc
Anhydride Time Added


i-butyl D pyr/DMSO Reflux/2h none 0

i-butyl I pyr/DMSO "/" 7

ethyl I pyr Reflux/lh 7

i-butyl D+ DMSO 50C/15min 5-25

ethyl D benzene RT/12h 0

ethyl I1* "/," 1-5

ethyl D l:lpyr/form RT/60h <1

ethyl D "f/" 600C/3h Zn2+ 24

i-butyl D "/" 600C/17h Zn2+ 22



aD = dextran 9400, I = inulin, polysaccharidee activated by
n-butyl lithium, = heterogeneous reaction conditions, all
other reactions were homogeneous.

bpyr = pyridine; DMSO = dimethylsulfoxide; form = formamide,
unless a ratio is given, all solvent mixtures were prepared
such that the more polar solvent was added to the mixture
of polysaccharide in the less polar solvent until the
polysaccharide dissolved.

CObtained from HPLC elution profiles, all values 2.








Equation 3.2 represents the reaction to prepare the

mixed anhydride and the subsequent reaction of the anhydride

with the alcohol function of polysaccharides to produce the

DTPA esters.



R-C(O)OH + C1-C(0)O-X --- R-C(O)OC(0)O-X

R-C(0)OC(0)O-X + HO-R' R-C(0)O-R' + CO2 + XOH

R = Remaining functionality of DTPA.
X = Remaining functionality of alkyl format.
R' = Remaining functionality of polysaccharide. (3.2)


As shown in equation 3.2, the use of mixed anhydrides in

esterification processes results in the simultaneous

formation of primary alcohols upon reaction of the mixed

anhydride with the polysaccharide. Since the

nucleophilicity of primary alcohols may be comparable with

saccharide secondary alcohol functions, it is likely that

the observed low reactivity of the mixed anhydrides towards

dextran was due to the competing reaction of the primary

alcohol with the mixed anhydride to form the alkyl ester of

the polyaminopolycarboxylic acid. Although it is highly

probable that this competitive reaction was occurring, the

moderate heating of either the iso-butyl or ethyl mixed

anhydride with dextran in 1:1 volume ratio of pyridine and

formamide in the presence of Zn2+ resulted in significant

reaction success, shown in Table 3.1. This reaction success

provided a calculated degree of loading which approximated

0.2 mole DTPA per mole repeating unit of the polysaccharide.






65

The use of a cyclic anhydride of the reactant acid, as

shown in equation 3.3, does not involve the simultaneous

production of competitive primary alcohols upon

esterification of the desired polysaccharide alcohol

function. This reactivity of the cyclic anhydrides makes

their application to esterification reactions with secondary

or kinetically inhibited primary alcohol functions

considerably more suitable in terms of expected yield than

when mixed anhydrides are used.



0 0
,-c 7 0-R
R-N 0 + HO-R' --> R-N (3.3)
SC' -C O-H
6 0

R = remaining functionality of aminopolycarboxylic acid
R' = remaining functionality of polysaccharide



The use of a mixture of anhydrous pyridine and

formamide results in homogeneous reaction conditions for the

esterification of dextrans59 and inulin and reduces the

extent of competitive hydrolysis of the cyclic anhydrides

expected in aqueous media. The degree of DTPA conjugation

was significantly effected by the presence of Zn2+ in the

reaction mixture. The presence of trace quantities of zinc

ion, added to the solvated polysaccharide before the

addition of the reactant anhydride, resulted in a 30 fold

increase in the level of DTPA loading relative to when no

zinc ion was present. This result suggests that Zn2' is









catalytic in the esterification reaction involving

polysaccharides and is supported by the reported catalytic

activity of ZnC12 in the acetate esterification of

monosaccharides.66 Additionally, the zinc ion may function

to remove some of the random coiling commonly exhibited by

some polysaccharides when in solution,67 and, in so doing,

may have served to reduce steric crowding near the reactive

alcohol functions.

As shown in Table 3.2, a high percentage of the

anhydride reacted when the commercially available dicyclic

anhydride was used to esterify dextran and inulin in 1:1

volume ratio of pyridine and formamide in the presence of

Zn2+, however, HPLC elution band broadness of the product

was observed. In the work of Gibby et al.52, the use of the

dicyclic anhydride to prepare highly crosslinked products

resulted in considerable elution band broadness when a

Sephacryl 400 column was used. When the dicyclic anhydride

was used in our work, strong chromatographic evidence for

substantial product crosslinking was observed when elution

profiles were obtained from the Protein Pac HPLC column,

shown in Figure 3.3. Product elution occurred near the void

volume of the column (4.6 mL as assessed by Blue Dextran)

and was very broad as was the eluting band at higher elution

volumes. Also shown in Figure 3.3, a broad band near 9 mL,

similar to that observed when the dicyclic anhydride was

used, was observed when a cyclic anhydride, prepared using a







67

1:3.2 mole ratio of DTPA to acetic anhydride, was reacted in

the presence of trace quantities of trifluoroacetic

anhydride (TFAA is generally viewed as an esterification

catalyst for reactions with cellulose68).


Table 3.2


Cyclic anhydride reactions with polysaccharides,
the reaction conditions, and percent anhydride
reacted for a series of attempted preparations.


Reactanta Polysac.b Solvent Temp./ Catalystc %Reactedd
Anhydride Time Added


d-dicy D* toluene reflux/3h TFAA 0

d-dicy D 1:1 pyr/form 600C/24h none 0

d-dicy D "/" 60C/3.5h Zn2+ [88]
RT/2h

d-dicy I "/" Zn2+ [67]

d-1:3.2 D "/" 650C/24h TFAA 16,[8]

d-mocy D "/" 650C/4h Zn2+ 42,[7]
& RT/24h

d-1:1.0 D "/" 600C/3h 23
& RT/12h

d-1:2.0 D "/" 600C/3.5h none 0
& RT/12h

d-1:2.0 D "/" 600C/lh Zn2+ 34
& RT/12h

continued next page












Table 3.2--continued.


Reactanta Polysac.b Solvent Temp./ Catalystc %Reactedd
Anhydride Time Added

d-1:3.2 D 1:1 pyr/form 50uC/4h Zn2" 59
RT/2h

D(402) "/" 60

I /" I" 72

t-l:1.0 I l:lpyr/form 600C/3.5h Zn2+ 78
& RT/12h

t-1:2.0 D "/" 600C/3h 15

t-1:3.0 D "/" "/" 36

t-1:3.2 D "/" "/" 53

t-1:3.8 Not Attempted


ad- = DTPA; t- = TTHA; d-dicy = DTPA dicyclic anhydride
(Aldrich); D-mocy = DTPA monocyclic anhydride prepared as
outlined by Halpern, et al.58; ratios indicate moles DTPA
or TTHA to moles of acetic anhydride used to prepare the
cyclic anhydride. For example, t-l:3.0 is a cyclic
anhydride of TTHA which was prepared by the reaction of a
ratio of 1 mole of TTHA with 3.0 moles of acetic anhydride.
bD = dextran 9400; D(402) = dextran 40200; I = inulin;
* = heterogeneous reaction conditions, all other reactions
homogeneous.

cTFAA = trifluoroacetic anhydride; Zn2+ added as either
solid ZnC12 or a mixture with anhydrous pyridine.

obtained from HPLC elution profiles, bracketed numbers
represent percent reacted anhydride which formed cross-
linked product (if 2 percentages are reported) or the
percent reacted anhydride which formed products with very
broad elution profiles. All values are 2.
















1.0



0.8

0,
j 0.6



,o 0.4 '





IPA
0.0
0 2 4 6 8 10 12
Elution Volume (mL)




Figure 3.3. Elution profile for unmodified
polysaccharide (solid line), esterification product
obtained from reaction of dicyclic anhydride (dashed
line, A), and from reaction of mono- and dicyclic
anhydride mixture in presence of trifluoroacetic
anhydride with the polysaccharide (dashed line,B).








Indicated in Table 3.2, the greatest anhydride

reactivity towards the polysaccharides, without

chromatographic evidence for crosslinked products, was

observed for products obtained from the reaction of an

anhydride prepared using a 1:3.2 mole ratio of DTPA or TTHA

to acetic anhydride in 1:1 volume ratio of pyridine and

formamide with Zn2+ present. These reaction conditions were

used subsequently for all large scale preparations of DTPA

esterified polysaccharides. The DTPA cyclic anhydride

prepared in this fashion was a mixture containing both

dicyclic and monocyclic anhydride as supported by infrared

carbonyl stretching frequencies of 1820.7, 1772.1, 1757.7,

and 1639.6 cm-1. The two higher wavenumbers are attributed

to characteristic anhydride carbonyl stretching frequencies

(-C(O)-O-C(O)-) and the lowest wavenumber represents the

central carboxyl group carbonyl stretch (-C(O)OH).69 The

absorbance band at 1757.7 cm-1 represents a terminal

carboxyl group stretch69 (-C(O)OH) that has been shifted in

energy, relative to that of DTPA at 1737.9 cm-1, possibly as

a result of the inductive effect of the neighboring

anhydride function.

Although the TTHA cyclic anhydride prepared by the

reaction of a mole ratio of 1 mole TTHA to 3.2 moles acetic

anhydride was not assessed by infrared spectroscopy, it is

probable that a higher percentage of monocyclic anhydride

was present in the TTHA mixture than in the DTPA mixture









prepared similarly due to the greater number of carboxyl

functions in TTHA. The use of a 1:3.8 mole ratio of TTHA to

acetic anhydride would likely result in a mixture of

monocyclic and dicyclic anhydrides of which the percentage

of each species closely approximated those in the DTPA

cyclic anhydride mixture which was prepared by 1:3.2 DTPA to

acetic anhydride. Improved yield in the polysaccharide

esterification with cyclic anhydrides of TTHA may be

obtained when a mole ratio of 1 mole TTHA to 3.8 moles

acetic anhydride is used to prepare the anhydride mixture.

There are no reports concerning the formation of

ketones on cyclization of aminopolycarboxylic acids upon

heating with acetic anhydride and no evidence was obtained

in this work which supported the formation of ketones.

However, the Blanc rule70 for dicarboxylic acids indicates

that the formation of the anhydride or the ketone will occur

depending on the relative positions of the carboxyl groups:

with 1,4- and 1,5-diacids forming the anhydride and 1,6-

diacids or further removed positions producing the ketone.

If the Blanc rule is applicable to polyaminopolycarboxylic

acids then 80% of DTPA and 87% of TTHA would form ketones

upon reaction with acetic anhydride (based on the carboxyl

positions and possible combinations to form either ketones

or cyclic anhydrides). Based on the observed reactivity of

the compounds formed by reaction of the acids with acetic

anhydride, it is improbable that the Blanc rule applies.








No DTPA chelate crosslinking of the esterified

polysaccharides prepared by this method was detected by HPLC

(Figure 3.4) as evidenced by the lack of eluting band

broadness or distinctly separate peaks at lower elution

volumes relative to the unmodified polysaccharides (refer to

Figure 3.3). The mobilities or elution properties of the

esterified polysaccharides on the Protein-Pak column

material are expected to differ from the unmodified

polysaccharides as a result of the charged DTPA moiety

interacting with the polysaccharide in the esterified

product. The elution bands esterifiedd and unmodified

polysaccharide) differ significantly in terms of band width,

and this observation suggests that the dispersity of the

molecular weights in the polysaccharide starting material

may have been altered (significantly reduced) in the

esterification process. Additionally, the elution bands for

the unmodified polysaccharides of differing average

molecular weight overlapped with one another when the

Protein-Pak column was used. Therefore, the evaluation of

crosslinking by HPLC elution band broadness of the

unmodified polysaccharide and the esterified product could

not be used. Elution band comparison of the purposely

crosslinked esters with the prepared esterified

polysaccharides does, however, allow the conclusion that

significant crosslinking in the prepared compounds was not

detected by the HPLC method.















0.8


I I
0.6



B A'C DI
S0.4


O\ ,



/\ \ ,
\ I
0.0 -





Elution Volume (mL)



Figure 3.4. HPLC elution profile for unmodified dextran
40200 (B, solid), DTPA esterified dextran 40200
following dialysis (A, solid), DTPA esterified dextran
9400 after dialysis of product obtained (C, dashed)
using the developed general reaction conditions, and
unmodified dextran 9400 (D, dashed).









Compound Characterization Results

The DTPA esterified inulin and dextrans of 9400,

40200, and 70800 average molecular weight were found to be

very water soluble. Repetition of the esterification

process resulted in levels of loading averaging 0.38 0.05

mole DTPA per mole of repeating glucopyranose unit of the

dextran compounds and 0.52 0.08 mole DTPA per mole

fructofuranose repeating unit in the inulin compound. The

general esterification method produced a DTPA esterified

dextran 487000 with an average of 0.10 0.02 mole DTPA per

mole of repeating glucopyranose unit, and the products were

readily water solubilized.

A modification in the general method was applied to

subsequent preparations involving dextran 487000, wherein

the heated reactants were cooled to room temperature and

allowed to stir for 30 h prior to product isolation. This

modification resulted in the reproducible production of

substantially higher levels of loading (0.80 0.06 mole

DTPA per mole glucopyranose repeating unit) than the product

produced by the general method. The product (by the

modified general method) was, however, considerably less

water soluble than the product prepared by the general

method.

Based on elemental analysis combined with ICP analysis,

the metallated products contained an average of 0.38 0.06

moles of GdDTPA attached per mole of glucopyranose unit in









dextrans of 9400, 402000, and 708000 average molecular

weight and 0.50 0.03 moles GdDTPA per mole fructofuranose

unit in inulin. As supported by elemental and ICP analysis

results, all the complexes contained an average of 1.00

0.04 mole Gd3+ per mole of DTPA moiety.

The level of loading of GdDTPA on to the

polysaccharides of up to 70800 average molecular weight is

roughly ten times that obtained by Ranney et al.53 in

reacting the bisanhydride of DTPA with Dextran 70,000 in

aqueous media and approximately twice that reported in the

work of Gibby et al.52 which involved the synthesis of a

mixture of crosslinked products from the bisanhydride

(dicyclic anhydride) reaction with dextran 150,000 in

anhydrous media. A recent report by Nycomed researchers71

indicates that their GdDTPA-dextran 75,000 (conjugation bond

type was not disclosed) contained an average of 15 GdDTPA

moieties per polysaccharide carrier. These previous efforts

to conjugate GdDTPA to dextrans resulted in either low

loading of DTPA on the dextran or a continuum of molecular

weight crosslinked products. High loading is a desirable

aspect in macromolecule-bound contrast agents as plasma

expansion is minimized. The dextran conjugates prepared by

the method of synthesis developed in this work resulted in

average molecular weights per gadolinium of only 13% more

than GdDTPA and 11% more than GdDTPA for the inulin

conjugates.









The infrared carbonyl stretching frequencies of the

complexes and the nonmetallated DTPA-polysaccharide esters

in the present work were compared with that of GdDTPA and

DTPA. As shown in Table 3.3, the stretching frequencies of

the DTPA-polysaccharide esters are nearly identical to DTPA.

Similarities of carbonyl stretching frequencies for GdDTPA

and the GdDTPA-polysaccharides support that gadolinium is

chelated by the DTPA esterified polysaccharides.


Table 3.3.


Infrared carbonyl stretching frequencies of
DTPA, DTPA-dextran conjugates, and the
gadolinium complexes.


Compound

DTPA

DTPA-Inulin

DTPA-Dextran 9400

DTPA-Dextran 40200

DTPA-Dextran 70800

DTPA-Dextran 487000


-l a


Wavelength (cm-l)a

1630 1696

1626

1639

1631

1628

1628


H2GdDTPA 1581

GdDTPA-Inulin 1596

GdDTPA-Dextran 9400 1606

GdDTPA-Dextran 40200 1598

GdDTPA-Dextran 70800 1594

GdDTPA-Dextran 487000 1602

a sh = shoulder of lower wavenumber


1737

1734

1737

1737

1736

1738


1704

1700 1738

1696 sh

sh 1737

1702 1723

sh sh

absorption band.








Further evidence to support the lack of significant

crosslinking in the DTPA esterified dextrans as synthesized

by this new method was obtained from the potentiometric

titration studies. As indicated in Figure 3.5, DTPA

exhibits a sharp inflection at 3 moles OH- added per mole

acid function with a less definite inflection between 3 and

5 moles OH- per mole acid function.72 DTPA in solution with

a 1:1 mole ratio of Gd3+ to DTPA exhibits a sharp inflection

at 5 moles OH- per mole acid function3. The potentiometric

behavior of the free acid has been explained based on the

assumed trizwitterionic nature of the ligand in solution as

shown in Formula I.




O 0
II Il
HO-C C--R

H-N+ +N-H +N-H
0-o-- C-o -c-0~o
11 II it
o o 0



R = H in DTPA or esterified alcohol function
in DTPA-Dextran.


Formula I.






































Moles OH- added per mole DTPA


Figure 3.5. Potentiometric titration at 25.0C with
0.1 M KOH of DTPA esterified dextran (A), DTPA (B), 1:1
Gd:DTPA-dextran (C).








Over the mole ratio (mole OH- per mole acid function)

interval from 0 to 3, two carboxyl protons and one ammonium

proton are removed and the remaining ammonium protons are

removed in the interval between 3 and 5.72 In the presence

of a 1:1 mole ratio of Gd3+ to DTPA, the single sharp

inflection at 5 moles OH- per mole acid function represents

the neutralization of 5 moles of hydronium ion displaced by

ligand chelation with one mole of Gd3+.3

Indicated in Figure 3.5, all of the polysaccharide

modified ligands in this work exhibited sharp inflections at

2 moles OH- per mole acid function with ill defined

inflection in the 2 to 4 equivalent range. With Gd3+

present in a 1:1 mole ratio of Gd3+ to conjugated DTPA, the

single step neutralization at 4 equivalents of base added

was observed. Based upon the zwitterion structure in

Formula I and the known potentiometric behaviors of DTPA and

DTPA in the presence of 1:1 molar addition of Gd3, these

observations for the dextran esters of DTPA are consistent

with products in which only one of the carboxyl functions of

DTPA has been modified. For these new ligands, the

remaining carboxyl proton and one ammonium proton are

apparently removed in the 0 to 2 mole OH- per mole acid

function range of the titration with the remaining two

ammonium protons removed in the 2 to 4 equivalents range.

In the presence of 1:1 mole Gd3+ to conjugated DTPA, also

shown in Figure 3.5, 4 moles of hydronium ions are






80

neutralized in the titration and represent the displacement

of 4 moles of hydronium ions per mole of Gd3+ chelated.

These results strongly support the contention that the DTPA

esterified dextrans produced in this work are not

significantly chelate crosslinked as sharp potentiometric

inflections at 1 and 3 equivalents of base would be expected

for DTPA-Dextran and 1:1 Gd3+ with DTPA-Dextran,

respectively, in the crosslinked products.

Following dialysis against 0.1 M EDTA, the mass percent

gadolinium in the GdDTPA-Dextran 9400 and 40200 complexes

was not altered more than 0.4%. The observed absence of

significant metal transfer to EDTA in dialysis with the

prepared polymeric complexes suggests that the level of

loading by DTPA on to the polysaccharides is preventing

significant nonspecific metal binding (characteristic

particularly to dextrans) to the investigated

polysaccharides.73,74 This observation additionally

supports that the thermodynamic stability of the new

complexes exceeds that of GdEDTA (log K = 1017).

The thermodynamic stability constant for GdDTPA-dextran

40200, as obtained from the potentiometric titrations and

assuming independent metal binding by the chelating groups,

was 18.02 0.13 (log K for Gd3+ + L4- GdL1-). Neither

Gibby nor Ranney reported stability constants, however,

recent work by Sherry et al.75 involving the assessment of

thermodynamic stabilities for mono- and di-propyl esters of









DTPA resulted in log K values of 18.91 and 16.30,

respectively. The DTPA esters of dextran in the present

work exhibited acid dissociation constants, shown in Table

3.4, comparable with those obtained Sherry et al.75 for the

mono-propyl ester of DTPA. The stability of the prepared

gadolinium complexes when compared with the evaluated model

compounds in the work of Sherry et al.75 provides further

evidence in support of non chelate-crosslinked products.


Table 3.4.


Acid Dissociation Constants of DTPA-Dextrans
and Propyl Esters of DTPA.


DTPA-dextrana DTPA-PEb DTPA-PE2



pK1 10.0 9.8 9.6

pK2 6.7 6.6 4.8

pK3 4.8 3.8 3.6

pK4 1.7 1.8 low

pK5 low low




aObtained by potentiometric titration (0.1 M KNO3,
25.0C) in this work. All values are 0.2.

obtained by A. D. Sherry et al.75 Values are 0.1,
determined potentiometrically (0.1 M NaCl, 250C).
Mono-propyl and di-propyl esters of DTPA are DTPA-PE1
and DTPA-PE2 respectively.








Hydrolysis Kinetics. An important issue in the

development of macromolecule-bound contrast agents is the

nature of the bond between the metal chelate and carrier.

The covalent attachment of GdDTPA to proteins such as bovine

serum albumin (BSA) has been via the formation of stable

peptide bonds between the carrier and metal chelate.76 The

polysaccharides of the conjugates reported here are

covalently linked with ester bonds to the metal chelate.

Ester hydrolysis has been extensively studied77 and the

effect of different lanthanide ions, including Gd3+, has

been evaluated for amino acid esters of diacetic acids.55

The assumed mechanism for these metal promoted hydrolysis

reactions involves OH- attack at the ester carbonyl group,

and carbonyl activation occurs by metal binding to the ester

carbonyl oxygen.

The ester bonded GdDTPA-polysaccharide conjugates may

exhibit different in vivo excretory pathways than the

peptide bonded conjugates as a result of the Gd3+ promoted

ester hydrolysis. The half-lives for the ester bonds in the

conjugates were obtained from slopes of logarithmic

decomposition plots (first order in conjugate

concentration). The half-life of GdDTPA-dextran 70800 (21

hours) was significantly shorter than the nonmetallated

DTPA-dextran 70800 ester (85 hours) while for the GdDTPA-

dextran 9400 complex and the nonmetallated compound the

half-lives were within experimental error of each other (68






83

and 71 hours, respectively). The experimental error was

4 hours based on the repetitive determination of the HPLC

elution peak areas. The presence of free GdDTPA in

solutions containing the GdDTPA-dextran complexes did not

decrease the half-lives of the complexes but was observed to

increase the half-lives of both GdDTPA-dextran 9400 and

70800 (170 and 89 hours, respectively) to greater than that

observed for their respective nonmetallated conjugate. The

result suggests that gadolinium chelated by the ester bonded

DTPA moiety is necessary for metal assisted ester hydrolysis

to occur and that free GdDTPA (or a contaminant present in

the mixture) adversely affects the rate of hydrolysis.

In working with propyl ester models of DTPA, Geraldes

et al.78 presented nuclear magnetic resonance dispersion

(NMRD) evidence that supports the coordination of the ester

carbonyl oxygen in gadolinium complexes. The results also

tend to reinforce the suggested mechanism for metal promoted

ester hydrolysis. No evidence for ester carbonyl

coordination in the present work was obtained as an expected

stretching mode shift to lower frequencies of the completed

ester carbonyl relative to the free ester carbonyl69 could

not be ascertained from the broad infrared peaks.

One possible explanation for the observed lack of

consistent rates of ester hydrolysis among the gadolinium

conjugates involves the lack of homogeneity in the

























-ppm


-ppm (to TMS)


Figure 3.6. Proton NMR spectra in D20 of (A) DTPA and
(B) DTPA esterified dextran 9400.







85

polysaccharide bound DTPA. In Figure 3.6(a), the proton NMR

spectrum of nonesterified DTPA is shown. The esterification

would result in an additional peak in the spectrum

corresponding to the DTPA methylene protons adjacent to the

ester linkage while the other proton shifts would remain

relatively unchanged. Unmodified dextran has no proton

shifts in the region from 3.0 to 3.6 ppm. As shown in

Figure 3.6(b), the 3.0 to 3.6 ppm region of the spectrum for

the DTPA-dextran 9400 ester contained a large number of

chemical shifts for the DTPA moiety. This result was also

observed for the DTPA-dextran 40200 compound and the DTPA-

inulin compound (unmodified inulin also has no proton shifts

in the 3.0 to 3.6 ppm region). The result suggests that

several different environments exist for the resonating

protons of the DTPA moiety in the ester. Additionally, the

sharp alcohol proton shift(s) of dextrans and inulin (at

approximately 5 to 5.5 ppm) was significantly broadened by

the esterification and suggests that significant

intramolecular hydrogen bonding was taking place in the

ester product. Future studies to investigate ester

heterogeneity should include NMR of the esters in the

presence of a lanthanide shift reagent, such as europium

ion, in an effort to resolve the 3.0 to 3.8 ppm spectral

region.








Relaxivity Results

The manual approximation of T1 for each of the

solutions evaluated by the inversion recovery technique

resulted in raw data plots of signal intensity versus proton

shift as a function of T. The raw data was used to obtain

plots of signal intensity versus 7, an example of which is

shown in Figure 3.7. When converted to logarithmic

functions of intensity versus T plots, linearity was

observed, as shown in Figure 3.8. It was observed that

significant deviation from linearity only occurred at T

values greater than 40 msec from the actual solution T1 and,

for this reason, the manual approximation of T1 and small

increments between T values are required in order to obtain

reliable data. The slope of each of these logarithmic plots

was equal to the T1 of the solution and this information was

used along with the gadolinium concentration in each of the

solutions to construct a relaxivity plot of 1/T1 versus

gadolinium concentration, as illustrated in Figure 3.9.

As shown in Table 3.5, the T1 relaxivities of the

GdDTPA-polysaccharides ranged from 1.5 to 2.3 times that of

GdDTPA at 100 MHz (25C). The increase in T1 relaxivities

for the prepared conjugates relative to that of GdDTPA are

consistent with relaxivities reported for other covalently

attached GdDTPA-macromolecules47,79-81 relative to GdDTPA.

















8000-

6000-


40004 a


I



00 3
N.3
S-2000


-4000V


-6000-

-Rnn-


0 200 400 600 800 1000 1200
Tau (ms)


Figure 3.7. Plot of signal intensity versus r for a
solution containing GdDTPA-dextran 9400, pH = 7.4
phosphate buffer of 0.15 M ionic strength, 100 MHz,
25C.


I I _ _


uvvvv















2.5

a

2.0
I /'

1.5 -



j01* /
w I

0.5 -



0 20 40 60 8
Tau (ms)






Figure 3.8. Natural log plot of signal intensity
versus T for a solution containing GdDTPA-dextran 9400,
pH = 7.4 phosphate buffer of 0.15 M ionic strength, 100
MHz, 250C.
















50-



40-



30-


Gadolinium Concentration, mM
Gadolinium Concentration, mM


Figure 3.9. Relaxivity plot (1/T1 versus gadolinium
concentration) for GdDTPA-dextran 9400, pH = 7.4
phosphate buffer of 0.15 M ionic strength, 100 MHz,
25C.


V.
I


.z20-
*)

91)



10-



0-
0










Table 3.5. T1 Relaxivities at 100 MHz, 1 to 5 mM Gd3+



Compound Relaxivitya Correlation
(mM Gd sec)-1 Coefficientb


GdDTPA 3.8 0.1 0.9993

GdDTPA-inulin 8.3 1.0 0.9206

GdDTPA-dextran 9400 8.7 0.6 0.9970

GdDTPA-dextran 40200 8.1 0.3 0.9991

GdDTPA-dextran 70800 7.1 0.7 0.9907

GdDTPA-dextran 487000 5.8 0.5 0.9969


precision is expressed as population standard deviation and
is equal to [Z(yi-yav )2/n]12, where y = 1/T1 for the
solution evaluated and n = number of sample solutions
tested.

bCorrelation coefficient is equal to [n(Zxy)-(Zx)(Zy)]/
[4(Zx2)-(Zx)2]1/2[4(Zy2)_-(y)2]1/2, where y = 1/T1,
x = gadolinium concentration of solution, and n = number of
samples tested.



The observed increased relaxivities, R1 (Table 3.5) of

the new conjugates, relative to GdDTPA, may be due to

several factors. The paramagnetically induced solvent T1

relaxation contains inner-sphere and outer-sphere relaxation

components.82 The factors affecting the outer-sphere

component are not well defined and an adequate model is not

presently available. Factors that affect the rate of

diffusion of water molecules in and through the outer

sphere, such as hydrogen bonding, are included in the outer-









sphere component. Although hydrogen bonding may, for some

species, contribute greatly to solvent relaxation, the

outer-sphere component is generally considerably smaller

than the inner-sphere term for paramagnetic species with at

least one inner-sphere coordinated water molecule. The

following equations summarize the theoretical treatment of

inner-sphere relaxation parameters.82

R1[Gd3+] = 1/T1 observed

1/T1 observed = 1/T1 paramag + 1/T1 diamag

1/T1 paramag = 1/T1 inner + 1/T1 outer

1/T1 inner = Pq/T1m + Tm

P = mole fraction of metal ion
q = # of water molecules bound to metal
Tim = bound water proton relaxation time
Tm = residence life of bound water

Tlm a 1/Tle + i/Tm + 1/TR
T1 = longitudinal relaxation time of
metal electrons in the complex
TR = metal-chelate-water rotational
tumbling time

R1 a q(Tle + TR)/Tm (for Gd3+ systems, 10 to

100 Mhz)

Based on the above relationships, the higher relaxivities

observed for the conjugates relative to GdDTPA may be due to

one or a combination of the following: (i) an increase in

rotational correlation time (possibly due to

microviscosity)48 by virtue of the rigid attachment of the

metal chelate to the polysaccharide; (ii) an increase in

the number of inner sphere coordinated water molecules







92

and/or their rate of exchange with bulk water molecules82 by

reduction of the denticity of the chelate upon

esterification; (iii) an increase in the number of outer

sphere coordinated water molecules and/or their rate of

exchange83 via entrapment by the polysaccharide, or a

combination of all these effects. Additionally, the

mechanism of inner sphere water ligand exchange with bulk

water molecules may be significantly altered relative to

GdDTPA by the presence of hydrogen bonding between the

coordinated water molecules and the macromolecular

carrier.84 The somewhat lower relaxivities of these

polysaccharide conjugates as compared to protein conjugates

is likely due to the lack of globular tertiary structure in

the polysaccharide complexes that results in lower

rotational correlation times.

Indicated in Figure 3.10, an apparent trend in the

observed relaxivities suggests a decrease in relaxivity with

increasing dextran carrier molecular weight. It was

observed that the polysaccharide alone made no significant

contribution to the solvent relaxation over the

concentration range (less than 200 mM glucopyranose) used in

the T1 studies (Figure 3.11) and increased solvent

relaxation significantly only at high concentrations. At

glucopyranose concentrations comparable with that in the

GdDTPA-polysaccharide solutions, the solvent relaxation was

observed to be within experimental error of the pure solvent