Preparation and application of bifunctional chelates for biological electron transfer studies and preparation of monoclo...

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Preparation and application of bifunctional chelates for biological electron transfer studies and preparation of monoclonal antibodies
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Emad, Mehrzad, 1961-
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Chelates   ( lcsh )
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Chemistry thesis Ph. D
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Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 140-147)
Statement of Responsibility:
by Mehrzad Emad.
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Typescript.
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Vita.

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PREPARATION AND APPLICATION OF BIFUNCTIONAL CHELATES
FOR BIOLOGICAL ELECTRON TRANSFER STUDIES AND PREPARATION OF
MONOCLONAL ANTIBODIES
















By

MEHRZAD EMAD


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


1991















ACKNOWLEDGMENTS

The partial support of the Division of Sponsored

Research for the work involved in the preparation of EDTA

derivative bifunctional chelate is gratefully acknowledged.

The support for the preparation of antimetalloporphyrin

antibodies by the National Institute of Health is

appreciated. I thank Dr. D. E. Richardson, chairman of the

graduating committee, for his guidance and support during

the past few years. I thank the members of the graduating

committee for their help. Special thanks go to Dr. Paul

Kline and Linda Green at the College of Medicine Hybridoma

Lab at the University of Florida for their help in the

immunology portion of the antibody project. I generously

thank Richardson's research group for their support and

comments during the years. I would especially like to thank

Francis Armitage, Jay Bongers, Matt Ryan and Fran Sunders

for their friendship. I would like to thank my two brothers

Mehdi and Behzad. I would also give special thanks to my

mother Azar Habib that I have dedicated this dissertation

to. Without her support and efforts during the years I

would not be where I am. Special thanks go to my uncle

Samad Habib for his personal comments and advice over the

years. I gratefully thank MetaGene Corporation for their

help and support for the past two years as my employer, and

special thanks go to Randy Fischer for his understanding and

ii









patience during this time. Thanks go to William Randolph

(Randy) Martin, a special friend. I have enjoyed his

company and sense of humor for the past two years. Best

wishes and many thank to Christ Leopold for his help in

educating me about computers and his help with the word

processing. My special thanks and best wishes go to

Elizabeth Ann Stevens for her friendship and help during the

write up of this dissertation. At the end many thanks go to

my dear friend and falconry partner Mr. Stan M. Warden for

his friendship, instructions and support in the past few

years.


iii















TABLE OF CONTENTS


page

ACKNOWLEDGMENTS . . ii

TABLE OF CONTENTS . . iv

ABSTRACT . . ix

CHAPTERS

1 INTRODUCTION . . 1

Bifunctional Chelates . 1
Porphyrins as Bifunctional Chelates 4
Potential of Antimetalloporphyrin
Antibodies as Catalytic Antibodies 6
Electrochemistry of the Co(III)tetra-
sulfonatotetraphenylporphyrin 8
Positively Charged Water Soluble Porphyrin
and its Cobalt Complex as Mediators in
Glucose/Glucose Oxidase System 8

2 PREPARATION OF A BIFUNCTIONAL CHELATE FOR
BIOLOGICAL ELECTRON TRANSFER STUDIES 12

Introduction . 12
Advances in Biological Electron Transfer 13
Model Systems for Biological Electron
Transfer . 13
Properties of the Metal Complex in the New
Model Met by the RuEDTA Bifunctional
Chelate . .. 17
Choice of Secondary Functional Group on
the BC . . 18
Advantages of the New Model System 19
Model Reactions for Preparation of la Using
Carboxylic Acid Activating Agents 19
Activation of Carboxylic Acids with Carbodi-
imide and Chloroformate Activating
Agents . . 20
Experimental . 22
Synthesis of Phenylglycinonitrile (12) 22
Synthesis of N,N'-Diacetyl-l-
phenylethylenediamine (13) 23









Synthesis of N,N'-Diacetyl-l-(p-nitro-
phenyl)ethylenediamine (14) 24
Synthesis of 1-(p-Nitrophenyl)ethylene-
diamine Dihydrochloride (15) 25
1-(p-Nitrophenyl)ethylenediaminetetraacetic
acid (16) . 25
p-Aminophenylethylenediaminetetraacetic
acid (17=la) . .. 26
Synthesis of 1-(p-Haloacetamidophenyl)-
ethylenediaminetetraacetic
acid (18) . 27
Synthesis of p-Methylacetanilide 28
Isobutyl chloroformate in toluene 28
Isobutyl chlorofromate in
Dimethylformamide . 28
Isobutyl chloroformate in
acetonitrile . 29
Synthesis of 4-Acetamidopheynl-
acetic Acid . 29
Synthesis of 4-Iodoacetamidophenyl
Acetic Acid (19) . 29
Synthesis of N,4-Anilinedi-
acetic Acid (20) . 30
NMR Studies of the Reactions of Haloacetyl
Compounds with Ethylmethylsulfide 31
Results and Discussion . 31
Model Compounds and Model Reactions 34
Results of the NMR Studies of the Reaction
Between Ethylmethylsulfide and Halo-
acetyl and Haloalkyl Compounds 36
Modification of the Method of the Prepara-
tion of Iodoacetyl compounds 38
Conclusion . . 39

3 PREPARATION OF MONOCLONAL ANTIBODIES AGAINST A
WATER SOLUBLE METALLOPORPHYRIN 40

Introduction . 41
Background Information . 43
Immunology . 43
Haptens . 44
Antibody Structure . 45
Structure of the Binding Site of the
Antibody . 48
Catalytic Antibodies . 50
Hybridoma Techniques . 53
Enzyme Linked Immunosorbent Assay
(ELISA) . 55
Competition ELISA . 55
Choice of Hapten . 57
Experimental . . 58
Synthesis of Tetraphenylporphyrin (TPP) 59









Synthesis of Tetrasodium meso-tetra(p-
sulfophenyl)porphyrin (TPPS4) 59
Synthesis of 5,10,15,20-Tetrakis(p-
sulfophenyl)porphinate-cobalt(III)
(CoTPPS4) . 60
Synthesis of 5-(4-carboxyl)-10,15,20-
tetraphenylporphine (TPPC) .. .62
Synthesis of 5-(4-carboxyphenyl)-10,15,20-
trisulfophenyl-porphine(TPPS3C) 63
Synthesis of 5-(4-carboxyphenyl)-10,15,20-
trisulfophenyl-porphinate-cobalt(III)
(CoTPPS3C) . 63
Synthesis of 5-(4-carboxyphenyl)-10,15,20-
trisulfophenyl-porphinatezinc(II)
(ZnTPPS3C) .'. 64
Preparation of CoTPPS3C-Bovine Serum
Albumin . 65
Preparation of CoTPPS3C-Key Hole Limpet
Hemocyanin (KLH) . 66
Preparation of p-Nitrobenzoic acid-KLH 66
Preparation of Hybridoma Cultures 66
Primary and Secondary Screening of MC for
Specific Antibodies . 67
Primary screening . 67
Secondary screening . 67
Screening of the MC Supernatant Against
Hapten KLH, PNB-KLH and KLH 68
Determination of the Relative Binding
Affinity of Test Antibodies by Inhibition
ELISA . . 68
Results and Discussion . 68
ICP Elemental Analysis for CoTPPS3CNa4 68
Presence of inorganic sulfate salts 69
Incomplete metallation of the ligand 69
HSO4-Counter ion . 70
ICP Elemental Analysis for ZnTPPS3C 70
Preparation of Conjugates . 71
Preparation of BSA-Hapten Conjugate for
Immunization . 71
Qualitative determination of CoTPPS3C-BSA
conjugation . 71
Quantitative determination of CoTPPS3C-BSA
conjugation . 72
Preparation and Characterization of
Conjugates of KLH with CoTPPS3C
and PNB . .. 74
Preparation and Isolation of Specific
Antibodies .. 76
Screening of the MC supernatant against
Hapten-KLH, PNB-KLH and KLH by Inhibition
ELISA . . 77
Conclusion . . 85









4 ELECTROCHEMISTRY OF A NEGATIVELY CHARGED
PORPHYRIN AND ITS Co(III) COMPLEX 87

Introduction . . 87
Background Information . 88
Site of Electron Transfer on CoTPPS4 89
Modifiers and Promoters (Mediators) 90
Choice of Modifiers and Promoters 92
Electrochemistry Studies of TPPS4 AND
CoTPPS4 in DMF . 93
Materials and Methods . 93
Use of Modifiers and Mediators 95
Electrochemistry of CoTPPS4 and TPPS4 in
Dimethylformamide (DMF) 96
Results and Discussion . 96
Determination of the True Surface Area of
the Gold Working Electrode by Using
Cyclic Voltammetry . 97
Cyclic Voltammetry of CoTPPS4 in Aqueous
Solution . 98
Cyclic Voltammetry of CoTPPS4 in Aqueous
Solutions by Using Modifiers and
Promoters . 99
Cyclic Voltammetry of TPPS4 and CoTPPS4 in
0.2 M TEAP/DMF . 101
i) Diffusion coefficient of CoTPPS4 and
TPPS4 in 0.2 M NaClO4/DMF 105
ii) Determination of the heterogeneous
rate constant of CoTPPS4 and
TPPS4 . 106
Electrochemistry of CoTPPS4 in H20/DMF Mixed
Solvent . 109
Conclusion . . 113

5 meso-TETRAKIS(N-METHYL-4-PYRIDYL)PORPHINE AND
ITS COBALT(III) COMPLEX AS MEDIATORS IN
GLUCOSE/GLUCOSE OXIDASE SYSTEM 116

Introduction . 116
Porphyrins as Mediators . 121
Background Information . 122
Materials and Methods . 123
Mediation of Electron Transfer in GLU/GOD
System . 124
[Co(III)TMPY]4'/5+ as a Mediator in
GLU/GOD System . 125
Results and Discussion . 126
CpCpCOOOHFe as a Mediator in the GLU/GOD
System . 126
Electrochemistry of CoTMPy in Phosphate
Buffer (pH=7.5, U=0.2) 127


vii









Co(III)TMPY as a Mediator in the GLU/GOD
System . 128
Comparison of the CpCpCOOHFe mediation with
Co(III)TMPy . 128
Problems Associated With the Use of
Co(III)TMPy and TMPy as Mediators in
GLU/GOD System . 131
Conclusion . 132

6 SUMMARY AND CONCLUSIONS . 134

REFERENCES . . 140

BIOGRAPHICAL SKETCH . 148


viii















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


PREPARATION AND APPLICATION OF BIFUNCTIONAL CHELATES FOR
BIOLOGICAL ELECTRON TRANSFER STUDIES AND PREPARATION
OF MONOCLONAL ANTIBODIES

by

Mehrzad Emad

December 1991



Chairman:Dr. D. E. Richardson
Major Departments:Chemistry

The synthesis of a bifunctional chelate derivative of

EDTA with an iodoacetyl side functional group was

investigated. The synthesis of this compound based on the

published methods produces two products Model reactions

were conducted to find optimal conditions for production of

the acetylated product.

A bifunctional chelate derivative of water-soluble

tetra[p-sulfonatophenyl]porphyrin, TPPS4, was used to

generate and isolate antibodies against the metalloporphyrin

tetra[p-sulfonatophenyl]porphinatocobalt(III), CoTPPS4.

Covalent binding of a metalloporphyrin to bovine serum

albumin and keyhole limpet hemocyanin was accomplished by

conjugation with 5-(4-carboxyphenyl)-10,15,20-

trisulfophenylporphinatocobalt(III), CoTPPS3C, where one of

ix









the sulfonate functional groups on the CoTPPS4 is replaced

with a carboxylic acid functionality. The preparation of

this metalloporphyrin is described. Several mass cultures

were isolated and screened by ELISA. Mass cultures 2B5, 2B9

and 2C3 were specific for the antigen. The 2B9 mass culture

was cloned, and a monoclonal antibody was isolated that

proved to be highly specific against CoTPPS3C.

To provide basic data on the oxidation-reduction

chemistry of the porphyrins used in antibody development,

the electrochemistry of TPPS4 and CoTPPS4 was investigated

in aqueous solution in a pH range of 1-14 by using various

electrodes. The electrochemistry of the metalloporphyrin

was also investigated by using several modifiers and

promoters.

The electrochemistry of CoTPPS4 and TPPS4 in DMF/TEAP

was investigated. The diffusion coefficient and

heterogeneous rate constants of CoTPPS4 and TPPS4 were

calculated by using a planar gold working electrode. The

electrochemistry of CoTPPS4 in DMF/H20 mixed solvent was

investigated as a potential medium for electrochemical study

of the interaction between an antibody and CoTPPS4.

The role of tetramethylpyridiniumporphyrin (TMPy) and

its Co3+ (Co(III)TMPy) as mediators in the electrochemical

glucose/glucose oxidase system was investigated in the next

section. The catalytic role of Co(III)TMPy in this system

was investigated.















CHAPTER 1
PREPARATION AND APPLICATION OF BIFUNCTIONAL CHELATES
FOR BIOLOGICAL ELECTRON TRANSFER STUDIES AND PREPARATION OF
MONOCLONAL ANTIBODIES


Introduction


Two classes of bifunctional chelates' are investigated

in this study. In the first section of the introduction

bifunctional chelates and their potential application in

preparation of a new model system for electron transfer

studies of cytochrome c (cytc) is described. In the

following section preparation of porphyrin-binding

antibodies and their potential as catalytic antibodies are

described.

Electrochemical studies of tetrasulfonatotetra-

phenylporphyrin (TPPS4) and its Co(III) complex (CoTPPS4)

are then introduced. Finally, the role of tetrakis(4-N-

methylpyridyl)porphine (TMPy) and its Co(III) derivative

(CoTMPy) as mediators in an electrochemical glucose-glucose

oxidase system is described.

Bifunctional Chelates

Bifunctional chelates are a class of compounds

introduced by Meares and coworkers in the mid 1970s.1 These

molecules are able to complex a metal ion while covalently

binding to other molecules via a secondary functional group.

1









2
Since the first preparation of such compounds, they have

been used in a variety of areas such as therapeutic

medicine, magnetic resonance imaging, and immunology.1-6

The initial compounds prepared by Meares and coworkers

are analogues of the hexadentate ligand

ethylenediaminetetraacetic acid EDTA, in which the

ethylenediamine portion has been modified to contain an

aniline, (la) or benzylamine group, (lb).1



HO H2C
HO' -H2 N H N OH



CH
R= H (0

NH NH
2 2
EDTA la lb


The amines of the R group in (la) and (lb) are converted

into reactive functionalities for reaction with other

molecules such as proteins.6

In an earlier biological electron transfer study, a

[Ru(NH 3)5]2 ion was attached to a specific site on cytc

molecule. The resulting cytc-[Ru(NH3),5]2 was used to

investigate the electrochemical communication between a

redox center at the surface of the protein and the Fe3/2

center in the metalloprotein.7 It was therefore of interest









3
to devise methods for covalently linking EDTA type metal

complexes to cytc at a specific amino acid side chain.

Protein modification studies indicate that iodo- and bromo-

acetyl functional groups have reactivity with sulfur

nucleophiles such as cysteine, 2, and methionine, 3.8-11




NH3 NH3
H-C-COO H-C-COO
I I
CH2-SH CH2-CH,-S-CH3




2 3

Published work indicates that methionine 65 residue of cytc

can be specifically modified by using small haloacetyl

compounds (e.g. iodoacetamide and iodoacetic acid).8 This

methionine is located near the binding site of cytc with its

biological redox partners.12"' It was decided to attempt the

use of a haloacetyl derivative of an EDTA bifunctional

chelate in order to specifically attach a redox active metal

ion near the methionine 65 residue in cytc. The first part

of this dissertation describes the work conducted to

investigate the preparation of an EDTA type bifunctional

chelate with a haloacetyl secondary functional group. Model

compounds have been investigated to explore potential

synthetic routes to such bifunctional chelates. The








4
iodoacetyl derivative of p-aminophenylacetic acid, 4, was

prepared as a model for the bifunctional chelate.




0

2 CI C-"OH



4


Reaction of the model and a number of haloalkyl compounds

with ethylmethylsulfide, 5, has been followed by NMR and

compared to the reaction of methionine with alkyl halides.


CHCH 2-S-CH3

5

Porphyrins as Bifunctional Chelates

Another class of bifunctional chelates studied in this

work are derivatives of porphyrin, 6.









5

The most abundant naturally occurring water soluble

porphyrin, protoporphyrin IX, 7,



H2CH H
CH CH CH2
/N HN

NH N

CH3 / H3
HO-j-CH2CH2 CH2C-OH
o 2o



7



aggregates in aqueous solutions.14-17 This problem hindered

and complicated many potential applications of this

porphyrin or its derivatives in the present research. As a

result, a number of non-aggregating synthetic porphyrins and

metalloporphyrins have been used in this work. The

aggregation in synthetic porphyrins can be diminished by

introducing bulky side chains to the structure of these

macrocycles, and the water solubility of synthetic

porphyrins can be increased by addition of polar or ionic

side chains to the porphyrin or bulky side chains.

For example, in 5,10,15,20 tetraphenylporphyrin (TPP) 8a,

the phenyl ring is perpendicular to the heme plane, which

reduces the intermolecular pi overlap responsible for

aggregation in porphyrins. When a polar or ionic side chain









6

is added, electrostatic repulsion further reduces

aggregation and favors monomeric porphyrins and

metalloporphyrins in solution. Some of the synthetic

porphyrins used in this work are TPP and its derivatives,

meso-tetrakis(4-sulfonatophenyl)porphyrin, TPPS4, 8b, and

meso-tetrakis(4-carobxyphenyl)porphyrin, TPPC4, 8c.













R R


R H, TPP
S03H ,TPPS4
COOH, TPPC4
8



Potential of Antimetalloporphyrin Antibodies as Catalytic
Antibodies

An enzyme is evolved to accommodate and recognize a

substrate or the transition state of a chemical reaction,

and an antibody is produced by the immune system to

recognize and specifically bind an antigen. Some enzymes

require a cofactor in order to catalyze chemical reactions.

The cofactor may be a metal complex which often by itself

has some catalytic activity that is enhanced once coupled

with the protein to form the holoenzyme.20 This









7

investigation included a study of metalloporphyrin binding

antibodies that in many respect mimic enzyme/cofactor

assemblies.

Several synthetic and natural metalloporphyrins have

catalytic properties. The combination of a metallo-

porphyrin binding antibody and a metalloporphyrin could

result in a complex similar to a holometalloenzyme. The

metalloporphyrin-Mab structure might resemble that of a

number of hemoproteins such as cytc and cyt P450. Any of

the functions associated with these metalloproteins could

potentially be achieved by the metalloporphyrin-Mab complex.

At the time this work was initiated, there was little

precedence for metal-complex binding antibodies. Chapter 3

of this dissertation describes the steps leading to

production, isolation, and characterization of a Mab

developed against the negatively charged cobalt

tetrasulfonatotetraphenylporphyrin (CoTPPS4), 9. A

negatively charged metalloporphyrin was chosen since metal

derivative of positively charged porphyrins such as

tetramethylpyridiniumporphyrin tend to be light sensitive.

Also, negatively charged porphyrins better resemble the

negatively charged naturally occurring protoporphyrin IX, 7.

We chose the Co34 complex of sulfonated tetraphenyl-

porphyrin, CoTPPS4, since this metalloporphyrin is monomeric

and stable under physiological conditions and the metal can

bind other ligands in solution.












H H






H 0-g 0 sOH





9



Electrochemistry of the Co(III)-tetrasulfonatotetraphenyl-
porphyrin

Electrochemical techniques are a potentially powerful

tool in studying an electrochemically active antigen in its

binding to an antibody. Efforts to obtain a cyclic

voltammogram of CoTPPS4 using different electrodes in the pH

range 1-14 as well as the use of a number of electrode

modifiers and electrochemical mediators are presented in

chapter 4. Included is the study of the electrochemistry of

TPPS4 and CoTPPS4 in dimethylformamide (DMF) with

tetraethylammonium-perchlorate (TEAP) as electrolyte.

Titration of a solution of CoTPPS4 in DMF with water has

been followed by cyclic voltammetry and is discussed.


Positively Charged Water Soluble Porphyrin and its Cobalt
Complex as Mediators in a Glucose/Glucose Oxidase
System

In recent years there has been considerable interest in

methods to couple the glucose (GLU)/glucose oxidase (GOD)









9

redox reaction to an electrode surface to be applied to

glucose sensors and fuel cells.21C- In the absence of a

mediator glucose oxidase oxidizes glucose to gluconolactone.

The reduced glucose oxidase (GOD(red)) regenerates upon

reduction of oxygen to form hydrogen peroxide and the

oxidized glucose oxidase GOD(ox), Figure (1).


GLU + GOD(ox) > gluconolactone + GOD (red(I)

GOD(red) + 02 > GOD(ox) + H202 (II)


Fig. (1-1). Redox reactions in the GLU/GOD system in the
presence of 02


In the more common method of determining GLU

concentration in solution, the H20 concentration in

solution is measured electrochemically and used to measure

the glucose concentration by using H202 concentra-

tion/glucose concentration profiles.21"i One of the problems

associated with this method is the effect of uncertain

oxygen concentration in the solution on the H02 level.

Therefore, other oxidants have been considered for this

purpose, including a number of organic dyes and some small

inorganic metal complexes. H. A. 0. Hill21c" has used

derivatives of ferrocene (Cp2Fe), 10a,

10a R=H

b =COOH

c =H2NCH2CH2 Fe









10

as mediators to couple the reaction between GLU and glucose

oxidase, Figure (2).


GLU + GOD(ox) > gluconolactone + GOD(red) (III)

GOD(red) + CP2Fe(III) > GOD(ox) + Cp2Fe(II) (IV)

Cp2Fe(II) < Cp2Fe(III) (V)

Fig. 1-2. Electrochemical reactions in the GLU/GOD system
in the presence of ferrocene derivatives.


In an electrochemical cell, the reduced ferrocene is

oxidized by keeping the solution at a fixed oxidizing

potential to that of ferrocene, resulting in the generation

of a catalytic current proportional to the GLU concentration

in the solution, Figure (1-3).

Chapter 5 describes the results of the investigation of

positively charged tetramethylpyridiniumporphyrin (TMPy),

11, and its cobalt complex (CoTMPy), 12, as mediators in the

GLU/GOD system. The diaqua complex of CoTMPy was shown to

be a suitable mediator for this system under the conditions

studied.





.H



H3CN NCH













C6
/N

H CN /NCH3




12


S1pA


I I_(a)
l I I i


0.0 0.1


0.2 0.3
E/V vs. SCE


0.4 0.5


Fig. 1-3. D.C. cyclic voltammogram of ferrocene
monocarboxylic acid (a) in the presence of D-glucose and (b)
(a) plus glucose oxidase in phosphate buffer (pH=7.5,
U=0.2).21c















CHAPTER 2
SYNTHESIS OF A SITE SPECIFIC BIFUNCTIONAL CHELATE FOR
BIOLOGICAL ELECTRON TRANSFER STUDIES


Introduction


Bifunctional chelates were synthesized to covalently

bind metal ions to proteins.' These compounds were designed

to chelate a metal ion and bind a protein molecule via a

secondary functional group and have found wide spread

application in a variety of areas such as protein structure

determination, immunology and spectroscopy since they were

first prepared in 1970's.2-6 The application of these

chelates in the past did not require any selectivity in

their reaction with the various amino acid side chains on

the protein surface. The purpose of this chapter is to

study and outline the synthesis of a site-specific

bifunctional chelate (BC) with a haloacetyl secondary

functional group in light of their potential application for

biological electron transfer (ET) studies. Haloacetyl

compounds have widespread application in labeling sulfur

containing amino acids such as methionine and cystine in

protein chemis-try.7-11 A site specific BC is of potential

value in areas such as protein structure determination,









13

enzyme binding site determination, and macromolecule

spectroscopy techniques such as ESR and NMR.9


Advances in Biological Electron Transfer

Electrochemistry of proteins and biological electron

transfer (ET) studies have received increasing attention in

recent years.13a Electron-transfer reactions of cytc have

been studied extensively since this metalloprotein is well

characterized.13c Examples of these studies include the

interaction of cytc with cytochrome c oxidase and cytochrome

c reductase, various electrodes, small molecules, and

inorganic complexes.12,13,21-24

One of the areas under investigation in the biological

ET studies is the distance dependence of the rate of the

redox reactions.6,25'26 Of interest in these studies are the

distances, during the approach of the electroactive

molecules, at which the transfer of the electron begins to

take place and the mechanism of the actual transfer of

electrons in proteins. These studies are complicated where

the redox site is located deep within the complex structure

of the protein. To simplify these investigations several

model systems are developed and studied.


Model Systems for Biological Electron Transfer

In a model system developed by Isied and Vasilian26 two

metal centers were linked by a series of amino acid chains

at fixed distances. Intramolecular ET studies in this model









14

system revealed that it is possible to transfer electrons

across saturated bonds over long distances, 10-14 A, Figure

(2-1).25-3 In the next model system, designed and prepared

by H. B. Gray7 and coworkers, and S. Isied and coworkers

separately31, a [Ru(NH3) 5]2 complex was attached to cytc via

a coordinate-covalent bond at a fixed Fe-Ru distance of 11.5

A, and the electron transfer between Fe(III) and Ru(II) in

this system was studied. Protein degradation, HPLC and NMR

revealed that Ru was coordinated to histidine residue 33 on

the surface of cytc, Fig (2-2).7'31

In the latter model system the Ru center is bound to

the cytc surface, which is remote from the binding site of

the protein, and the potential difference between the Ru and

Fe centers is fixed in this system. It is of interest to

develop a similar model system where the metal center on the

surface of cytc is bound close to the binding site of the

protein and the potential difference between the metal

centers can be altered.

To change the potential difference between the two

metal centers, other ruthenium complexes of the type

Ru(NH )4(X)L where X is not NH3, have been used to prepare

new Ru-cytc adducts. An alternate method is to change the

metal center in cytc or the ligands on the Ru center in the

adduct. The [Ru(NH3)5s]2 complex, however, is inert under

the conditions tolerated by cytc.

















complex bridge k(25 'C). s'


CH3 CH3





0


9 0 0
J NAI
I .C. 6 AR 0 1 1


GlyPhe-iso






GlyLcu-iso






GlyGly-iso







PhcPhc-iso


8.6 X 10-O






15 X 10'6






9.9 X 10-'







11.6X 10-'


Fig. 2-1. Intramolecular electron transfer rates across
dipeptides.26















































Fig. 2-2. Heme packing diagram of ruthenium-modified
cytochrome c (His-33) derivative.31









17

A model system is proposed where the Ru center is

attached to a site close to the binding site region of

cytc.12'13'21 In this system EDTA bifunctional chelate is used

to covalently bind Ru to cytc. The reactive functionality

on the bifunctional chelate is an iodoacetyl group. 1-(p-

Bromoacetamidophenyl)ethylenediaminetetraacetic acid, 22b,

bifunctional chelates was prepared by Meares from the

precursor p-aminophenyl-EDTA.' p-Aminophenyl-EDTA is used

to prepare a number of bifunctional chelates by

modifications of the aniline functional group, Fig (2-3).40




SCN pCH2meA CH3(CH26 N CH2EDTA








Fg (23 ESCNtof aCHlEb
syth s, Pd fro p-a-iN2ob yET ()TA
TN BiCH2EDTA Bi 2 un EDTA t a 2-
la, 17 ep



-EDTA = BrCH N ED
HNCH2OO 22 H
00 22b


Fig. (2-3) Examples of various bifunctional chelates
synthesized from p-aminobenzyl-EDTA (lb).1


Properties of the Metal Complex in the New Model Met by the
RuEDTA Bifunctional Chelate

Electrochemistry of the EDTA complex of Ru(III) was

studied by Matsubara and Cruetz.21" Their experiments ,in









18

accordance with the work done in our laboratory, indicated

the presence of a substitutable site occupied by water on

this metal complex. In the same work the effect of ligand

substitution on the reduction potential (E1/2) of this

complex was studied which is represented in table (2-1).


Table (2-1). Formal Reduction Potential of RuIII(EDTA)L-
/RuI(EDTA)L2-21b


L E,, V(vs NHE) AE ,
Va

water -0.01 + 0.01 0.070
thiocyanate +0.07 + 0.01 0.065
pyridine +0.10 + 0.01 0.060
imidazole +0.10 + 0.01 0.060
isonicotinamide +0.16 + 0.01 0.060
pyrazine +0.24 + 0.01 0.060
acetonitrile +0.26 + 0.01 0.060



Choice of Secondary Functional Group on the BC

lodoalkyl and bromoalkyl groups are known for their

specific reactivity toward thiol or thioether functional

groups on proteins.32abc Modification of cytc with

iodoacetic acid and iodoacetamide for NMR studies by Moore

et al.33'34 indicates that these molecules bind specifically

to methionine-65 residue on the surface of the cytc which is

located close to the exposed edge of the heme. This region

is known to be the active site region of the protein in its

reactions with its biological redox partners, cytochrome c

oxidase and reductase.12,13,21-25,35 Thus, the haloacetyl









19

derivative of p-aminophenyl-EDTA, la, was chosen as the

bifunctional chelate.



Advantages of the New Model System

A model system using the EDTA based BC has two

advantages over the system previously developed by Sutin and

Gray:

1) In this system one can change the potential difference

between the Fe in cytc and the metal-EDTA by ligand

displacement on the metal center.

2) The haloacetyl secondary functional group of the BC

specifically targets cystine or methionine residues.

Because of the nature of cytc, only one residue

(methionine 65), which is closer to the active site of

the protein than histidine 33, will be modified.


Model Reactions for Preparation of 1-(p-Iodoacetamido-
phenyl)EDTA, 22c, Using Carboxylic Acid Activating
Agents

p-Aminophenylethylenediaminetetraacetic acid, la, is

the precursor in the synthesis of l-(p-Iodoacetamido-

phenyl)EDTA, 22c.


y=l, X=I
y=0, X=Cl
y=0, X=Br
y=0, X=I


EDTA-(CH) NH CO-CH -X
2 Y-/ 2


21
22a
22b
22c, 18










20

Figure 2-4 presents the multistep synthesis in the

preparation of la.




V AcHN. NHAc ACHN% NMc
3 a* -/Ac2O HNOS HOMC/HCI

CII I NH3I EDTA EDTA

HOOC-0lar 105 P/C, N&OH
NO NO NH

." iA la, 17

EDTA


Fig. 2-4. Scheme for preparation of la.


The acylation reaction of la with several haloacetyl

acylating agents was investigated for the preparation of 22c

by using model compounds, since preparation of la is lengthy

and tedious. The haloacetyl acylating agents that were

allowed to react with the model compounds were prepared by

activating the corresponding haloacetic acid precursor with

carbodiimide38a,b,39 and chloroformate36a,b,c,37 carboxylic acid

activating agents.


Activation of Carboxylic Acids With Carbodiimide and
Chloroformate Activating Agents

Chloroformates and carbodiimides are often used to

activate carboxylic acids under such mild conditions

necessary for peptide synthesis, nucleotide synthesis and

protein modification. 36-39 The carboxylic acid or its salt is









21

allowed to react with the carbodiimide reagent to form an

acylisourea intermediate. This inermediate is unstable and

highly susceptible to nucleophilic attack at the carboxylic

carbon. Reaction between a nucleophile and the intermediate

results in the formation of urea and the carboxylic acid

derivative, Figure 2-5.



0 o R
R-N-C-N-R + HO-C-R' -- R-C-O-C-NH
NR
4R
9 R H* 0 9
R'-C-O-C-NH + Nu:----R'-C-Nu + R-N-C-N-R
NR H H

Fig. 2-5. Activation of carboxylic acids by carbodiimide
reagents.


Reaction of a chloroformate agent with a carboxylic

acid results in the formation of an unstable mixed anhydride

intermediate and an equivalent of HCl(g). Addition of a

nucleophile to the intermediate will result in formation of

a molecule of alcohol, the carboxyl acid derivative and

CO2(g), Figure 2-6.

In the chloroformate reactions the nucleophile can

react with either of the carbonyl groups of the mixed

anhydride intermediate. However the carbonyl from the

starting carboxylic acid is more reactive and leads to the

generation of the desired product. Formation of the kinetic

product leads to the desired product which also leads to

formation of CO2(g) as a side product that leaves the









22

solution. Chloroformate and carbodiimide agents were used

in order to activate a-haloacetic acids, and allowed to

react with aniline functional group containing model

compounds; however, the chloroformates gave better results

and are described in the experimental section.



9 9 9 9
RO-C-CI + HO C -R' R'-C-O-C-OR + HCI


9 9 H+ 9
R'-C-O-G-OR + Nu:-- R'C- Nu + C02 ROH


Fig. 2-6. Activation of carboxylic acids by chloroformate
reagents.




Experimental

p-Aminophenylethylenediaminetetraacetic acid was

prepared following literature methods.40,41 Specific

procedures used in this work are described below. All

reagents were purchased from Aldrich Chemical Company. A

Nicolet FT/IR and Varian 300 Mhz NMR were used for analysis

in this work.

Phenylglycinonitrile (12)

The starting material, (12), was synthesized as the

hydrochloride salt by Steiger's method.42 In a well

ventilated hood 25.0 g (0.51 mol) of NaCN and 30.0 g (0.57

mol) of NH4Cl were added to 100 mL of deionized water and

stirred at room temperature. A solution of 53.0 g (0.50









23

mol) benzaldehyde in 100 mL of MeOH was added to the

aqueous solution after all the solid had dissolved. The

solution turns cloudy immediately, turns orange in color

within a half hour and increases in color intensity with

time. After 2 h resulting dark red solution was diluted

with 250 mL of water and extracted with 3x80 mL of benzene.

The combined benzene extracts were acidified with HCl(g) and

the precipitate hydrochloride product salt was collected by

filtration and washed with 100 mL of cold acetone. A

sample of the free base was obtained by dissolving the salt

in a saturated Na2CO3 aqueous solution and extracting with

diethylether. The organic layer was dried over MgSO4 and

solvent was removed under reduced pressure. mp (HC1 salt)

172-173 C, (lit. 170-171 C)42; IR(free base), 2229 nm

(C-N), mp (free base) 53-54.


N,N'-Diacetvl-l-phenvlethylenediamine (13)

Reduction of (12) was accomplished by catalytic

hydrogenation using acetic anhydride treated Raney-Ni

catalyst.43 Two-and-a-half grams of Ra-Ni catalyst were

washed with 2x20 mL portions of absolute EtOH followed by

2x20 mL of acetic anhydride (Note: The catalyst should be

kept wet with solvent at all times since the dry material

will ignite up on standing in air). Treated catalyst, 16.5

g (0.1 mol) of (12) and 12.5 g (0.15 mol) of anhydrous

sodium acetate, were added to 125 mL of acetic anhydride in

a 500 mL Parr pressure bottle. The bottle was placed in a









24

parr hydrogenation apparatus and agitated at 50C while

maintaining the hydrogen gas pressure at 45 psi. After 3 h

an additional 1.0 g of treated Ra-Ni catalyst was added to

the heterogeneous mixture and the reduction was continued

for another 5 h when the rate of hydrogen loss was minimal

(0.1 psi/h). The mixture was filtered to remove the

catalyst and the insoluble material and the filtrate was

evaporated under reduced pressure. The green paste obtained

at this point was extracted with several portions of hot

ethylacetate, concentrated and cooled to obtain crystals of

(13). The mother liquid can be concentrated to obtain a

second crop of crystals. The 67% yield indicated by in the

literature was never obtained in our reductions and the

maximum yield achieved was 34%. IR indicates complete loss

of 2229 nm (CN) peak. Anal. Calcd for C1612 N202; C, 64.88; H,

7.56; N, 12.44; Found: C, 65.43; H, 7.32; N, 12.72; mp 157-

1580 C, (lit.40155-1560 C).



N,N'-Diacetvl-1-(p-nitrophenvl)ethylenediamine (14)

Compound (13) (5.0 g, 22.7 mmol) was added slowly to 16

mL of 90% fuming nitric acid at -40C over a half hour while

stirring. After 5 h the solution was carefully poured over

100 g of ice, and approximately 20 g of Na2CO3 was added

until the excess acid was neutralized. The aqueous solution

was extracted with 100 mL of ethylacetate, and the organic

layer was dried with MgSO4 and filtered. The solvent was









25

removed under reduced pressure, and the crude product was

crystallized from acetone/n-hexane to give 1.30 g (4.9 mmol)

of product (yield=21%). The NMR spectra of the product

indicates that the solid obtained is exclusively the para

isomer of (14).40 mp 174-1760 C, (lit.40 172-1740 C). H

NMR(65 MHz, CD3OD): 8.35, 8.17 (d, 2 Ar H's), 7.68, 7.50 (d,

2 Ar H's), 5.15 (t, 1H), 3.50 (d, 2H), 2.00 (s, 3H), 1.83

(S, 3H).


1-(p-Nitrophenvl)ethylenediamine Dihydrochloride (15)

Compound (15) was obtained in 95% yield by refluxing

1.1 g (4.1 mmol) of (14) in a mixture of 7 mL concentrated

HCl and 5 mL glacial acetic acid for 24 h. The product

precipitated as the reaction vessel cooled to room

temperature and was collected by filtration. Anal. Calcd

for C8H 3N32Cl2, C, 37.19; H, 5.20; N, 15.57, Found: C,

38.81; H, 5.16; N, 16.53, Dcmp. 235-2400 C, NMR(65 MHz,

D20): 3.65 (d, 2H), 4.80 (q, 1H), 7.65 (d, 2H), 8.35 (d,

2H).40


1-(p-Nitrophenvl)ethylenediaminetetraacetic Acid (16)

This step of the synthesis was a combination of the two

methods in references 29 and 36. Six hundred milligrams (2.4

mmol) of (15) was dissolved in minimum amount of water and

the pH was adjusted to 9 with 7M KOH. A pH 7 solution

containing approximately 5 equivalent of bromoacetic acid

was prepared and added slowly over a 5 h period to a stirred









26
solution of (15) at 500 C, while maintaining the pH at 9

after each addition. The pH was maintained at 9 for another

9 h, at 10 for the next 10 h and 13.5 for the following 24

h. The reaction mixture was finally acidified to pH 1 with

concentrated HC1 and left at 4 C for 4 days. The yellow

mixture obtained at this point was filtered and the solid

was dissolved in 5 M NaOH and applied to an anion

exchange(Dowex 1x80 20-50 mesh) column in the format

form."4 The column was eluted with a 0-5 M gradient of

formic acid and the fractions were monitored at 280 nm. The

fractions containing the product were stored at 40 C until

the clear, needle shaped crystals of pure (16) were formed.

Anal. Calcd for C16 N010-0.5 HCOOH: C, 44.45; H, 4.61; N,

9.15, Found: C, 44.55; H, 4.91; N, 9.74, H NMR (300 MHz,

D20): 8.24 (d, 2H, J=7.5), 7.44 (d, 2H, J=7.5), 3.94

(broad, 1H), 3.28 (m, 7H), 2.97 (d, 2H), 2.63 (d, 2H), 2.46(

d, 1H).


p-Aminophenylethylenediaminetetraacetic Acid (17=la)

This compound was obtained by catalytic reduction of

the nitro-phenyl group of (16) in alkali solution.40 The

pH of a solution of 100 mg (0.2 mmol) of (16) in 50 mL of

water was adjusted to 11.5 using dilute NaOH solution at

0C. After bubbling the solution with N2(g) for a half

hour, 30 mg of 10% Pd/C was added and hydrogen was bubbled

though the solution instead. The reaction was monitored for

5 h at this time which the NMR of the solution indicated









27

quantitative conversion of the nitro substituted phenyl

hydrogens to a more shielded type at higher field. The

heterogeneous mixture was filtered, lyophilized and the

product solid was stored at 0C in the dark. NMR and

fluorescamine reagent,i.e. used to show the presence of 1-

amine functional groups2, were used for analysis. H NMR

(300 MHz, D20): 7.03 (d, 2H, J=7.0), 6.81 (d, 2H, J=7.0),

3.88 (d, 1H), 3.26 (m, 5H), 3.10 (m,2H), 2.90 (d, 2H), 2.48

(d, 1H).


1-(p-Haloacetamidophenyl)ethylenediaminetetraacetic Acid
(18)

Iodoacetylchloride, iodoacetic anhydride,

bromoacetylbromide and bromoacetic anhydride were used for

the synthesis of haloacetyl-EDTA compounds from (la) by

Meares' method.45 In all cases 230 umol of the halogenated

compound was dissolved in a minimum amount of water and the

pH was neutralized with NaBF4. In the iodo compounds

solubility was enhanced by addition of acetone to the

aqueous solution. Two hundred and fifteen pmol of (la) was

dissolved in 0.5 mL of water and titrated with the solution

of halogen compound. The reaction was tested with

fluorescamine for the complete consumption of the amino

compound. Excess halogenated reagent was removed by

extracting the solution with several 1.5 mL portions of Et2 0

and testing the organic layer with 4-(p-nitrobenzyl)-

pyridine. The pH of the solution was adjusted to 2.3 with 6









28

M HC1, and the solution was left over night at 00 C during

which a precipitate formed. The precipitate was filtered

and washed with dilute cold HC1 solution and dried over P205

in a vacuum.


Synthesis of p-Methylacetanilide

Isobutyl chloroformate in toluene. Acetic acid (3.0 g,

0.05 mol) and 5.1 g (0.05 mol) of triethylamine were added

to 20 mL of toluene at room temperature and stirred. Once

all the acid dissolved the solution was cooled to 0C and

6.83 g (0.05 mol) of isobutyl chloroformate in 10 mL of

toluene was added dropwise. After a half hour

triethylammonium chloride precipitate formed and 5.36 g

(0.05 mol) of p-toluidine in 30 mL of toluene was added

slowly to the reaction mixture. The ice bath was removed

and the reaction was stirred at room temperature for 6 h

during which gas evolution was observed. The solvent was

removed and the product was recrystallized from water. IR

and melting point of the product are identical to the

product obtained from the reaction of acetic anhydride and

p-toluidine in water. mp 147-1480 C, (lit.46148.50 C),

yield= 31.1%.

Isobutyl chloroformate in dimethylformamide. This

reaction was tried in DMF as solvent. However, in the time

span of this procedure only small amount of product was

detected by NMR.









29

Isobutyl chloroformate in acetonitrile. The reaction in

acetonitrile was followed exactly as that in toluene. The

white NH4Cl precipitate formed initially dissolved after a

few minutes. At the end of the 6th hour the solvent was

removed and the product was triturated with toluene to leave

behind the ammonium chloride salt. The triturate was

evaporated under reduced pressure and the obtained solid was

recrystallized from water. mp 144-1460, (lit.41 148.5),

yield=42% .


Synthesis of 4-Acetamidophenylacetic Acid

Acetic acid (1.5 g, 0.025 mol) and triethylamine (2.54

g, 0.025 mol) were added to 70 mL of toluene with stirring

at 0C. Isobutyl chloroformate (3.51 g, 0.025 mol) was

added slowly to this solution and stirred for a half hour.

The ice bath was removed and the heterogeneous mixture was

filtered to remove the triethylammonium chloride

precipitate. The supernatant mixed anhydride solution was

added slowly to a solution of 3.79 g (0.025 mol) of PAP in

25 mL of IM KOH and stirred vigorously for 6 h at room

temperature. The final solution was acidified with

concentrated HC1 to pH=1.5 and filtered to obtain the

product which was further dried over CaSO4 under vacuum. mp

164-167 C, (lit.48 1670 C), MI+=193, yield=66% .


Synthesis of 4-Iodoacetamidophenylacetic Acid (19)

lodoacetic acid(2.36 g,0.0125 mol) and triethylamine









30

(1.26 g 0.0125 mol) were added to 50 mL of toluene and

cooled to 0 C. Isobutyl chloroformate (1.73 g,0.0125 mol)

was added to this solution with stirring over half hour.

The precipitated triethylammonium chloride which formed was

removed by filtration. PAP (1.51 g,0.01 mol) was dissolved

in 10 mL of 1 M KOH and added to the supernatant mixed

anhydride solution drop by drop over a 2 h period. The

mixture was stirred vigorously for an additional 5 h at 00 C

and acidified with Concentrated HC1 solution. The aqueous

solution was extracted with EtAc (3x75 mL) and washed with

dilute HC1 solution. The combined organic layers was dried

over MgSO filtered and evaporated under reduced pressure

to obtain a pink solid. A sample of the solid was further

chromatographed on an anion exchange column in the format

form and eluted with a gradient of 0-5 M formic acid The

first peak to elute was used for NMR analysis. mp 163-

164.50 C, NMR: 3.47 (s, 2H), 3.75 (s, 2h), 7.14 (d, J=8.6,

2H), 7.40 (d, J=8.6, 2H).

NOTE: Preparation of the mixed anhydride can be carried out

in acetonitrile as solvent to increase the miscibility of

the two solvents,i.e. water and acetonitrile.


Preparation of N,4-Anilinodiacetic Acid (20)

Bromoacetic acid (1.50 g,ll mmol) was added to a

solution of 1.5 g (10 mmol) of p-aminophenylacetic acid in

50 mL of IN NaOH solution. The reaction was stirred in









31

water bath at 50 c and tested at intervals with

fluorescamine for the presence of primary amine. Once all

the amine was utilized(5 h) the solution was extracted with

Et 0 to remove excess bromoacetic acid. The pH of the

aqueous solution was adjusted to 1.5 with concentrated HC1

and extracted with Etac (3x100 mL). The combined Etac

extracts was dried over MgSO4, filtered and evaporated to

give a white precipitate. The crude product was

crystallized from Etac/hexane and used for proton NMR.

H NMR (DMSO-ds): 3.60 (s, 2H), 4.42 (s, 2H), 7.28 (m, 4H),

8.60 (s, 2H)


NMR Studies of the Reactions of Haloacetyl Compounds with
Ethylmethylsulfide

Reaction of iodomethane, iodoacetamide, iodoacetic

acid, bromo acetophenone, and (19) with ethylmethylsulfide

were followed by NMR. In every case a slight excess of

ethylmethylsulfide in DMSO-d6 was added to the halogenated

compound and observed with a 300 Mhz NMR instrument within

0.5-3h.

Results and Discussion

Bifunctional chelates were designed to bind and label

macromolecules like proteins non-selectively via a covalent

bond.4 It was of interest in this investigation to obtain a

bifunctional chelate which binds sulfur containing amino

acid residues in proteins exclusively. Compound (18) is a

bifunctional chelate with the potential of binding sulfur









32

nucleophiles. The compounds of interest in this work were p-

(halo-acetamide)benzyl-EDTA, 21, and p-(haloacetamido)-

phenyl-EDTA, 22.



21 y=l, X=I
22a y=0, X=Cl EDTA-(CH ) NH CO-CH -X
22b y=0, X=Br 2 Y 2
22c, 18 y=0, X=I


which can be synthesized from the appropriate aldehyde or

amino acid as the starting material.1'2 The synthetic steps

represented in scheme (3) for the synthesis of (la) was

initially developed by Diammanti et al.4 and were further

modified by 0. A. Gansow39.

Preparation of 22b from the aniline precursor and

bromoacetylbromide was introduced by Meares et al.1'45 Both

Meares and Gansow have cited the synthesis of 22b; however,

Meares made use of elemental analysis to confirm the

synthesis of the final bromoacetyl derivatives47, while

Gansow suggests that the procedure of Meares results in a

mixture of products.39 The multistep synthetic steps

represented in scheme (3) were conducted to obtain p-

aminophenylethylenediaminetetraacetic acid, la. In the next

step bromoacetylbromide, iodoacetylchloride, iodoacetic

anhydride, bromoacetylbromide and bromoacetic anhydride were

allowed to react with la under conditions described by

Mears.45 However, in all cases the NMR of the phenyl region

indicated the presence of a product mixture, Figure (2-7).



























Fig. 2-7. NMR of the phenyl region from the product of the
reaction of la and iodoacetic anhydride.

This mixture is attributed to the double functionality of

the acylating agents, which can lead to at least two

products, i.e. an alkylation product and an acylation

product, Figure 2-8.



NH-CH2-CO-L'

/
EDMA- CH Q NH + L-CH2-CO-L'


L-CH2-CO- NH -





Fig. 2-8. Production of acylation and alkylation products in
the synthesis of 18.









34

To investigate the final step for preparation of (18)

and because of the lengthy and tedious workup of (la) model

compounds and carboxyl group activating agents were used to

prepare the acetylated product exclusively.


Model Compounds and Model Reactions

A number of carboxylic acids were activated with

carbodiimide and/or chloroformate agents and reacted with

the compounds selected to model the synthsis of l-(p-

Haloacetamidophenyl)ethylenediaminetetraacetic Acid, 18.

After several attempts using both activating agents

chloroformates agents were chosen for the modeling studies

therefore only these reactions are described.

The acetylation step of the model compounds under a

variety of experimental conditions was investigated. The

product mixture obtained in a number of cases is difficult

to separate by conventional chromatography and discern by

proton NMR; however, with controlling the temperature and

solvent conditions a method was developed to produce the

product of the desired reaction (i.e., acylation).

Toluene, dimethylformamide (DMF), ethyl acetate (Etac)

and acetonitrile were used as solvents to find a suitable

medium for the preparation of the mixed anhydride

intermediate in the model reactions. Toluene proved to be

the easiest solvent to work with while acetonitrile gave the

best yield.









35

In the first model reaction the amide derivative of p-

toluidine and acetic acid was synthesized. The methyl group

on p-toluidine models the simplest alkyl side chain attached

to an aniline molecule at the para position. This reaction

was performed under various experimental conditions to

obtain the best yield and most suitable conditions with

respect to the synthesis of 16a-c. In the next set of model

reactions p-aminophenylacetic acid (PAP), 4, was used as a

model for (la) instead of toluidine and further reacted with

the mixed anhydride of acetic acid. Since the target

molecule, (18), is water soluble, the mixed anhydride of

acetic acid was prepared in toluene and added to a solution

of PAP in IM KOH. This solvent system was applied to the

final model reaction between PAP and iodoacetic acid. This

reaction however, did not turn out to be as straightforward

as the previous model reactions. In several attempts for

the preparation of the iodoacetyl derivative of PAP a solid

product was obtained which had the correct NMR chemical

shifts for the expected product but the wrong integration

values. Compound (19) was obtained when unlike the

previous model reactions a solution of PAP was added very

slowly to a solution of freshly prepared iodoacetic acid

mixed anhydride. The reaction of PAP with iodoacetic acid

mixed anhydride has the potential to generate two products.

The structure of the products were determined by following








36
the reaction of several haloalkyl and haloacetly reagents

with ethylmethylsulfide.
0
--CH-CC H
1-CH N C OH


19
Results of the NMR Studies of the Reaction Between
Ethylmethylsulfide and Haloacetyl and Haloalkyl
Compounds

Ethylmethylsulfide was selected as a model for

methionine relative to methionine-65 in cytc. Ethylmethyl-

sulfide was allowed to react with bromoacetophenone,

iodoacetamide, iodoacetic acid and iodomethane and the

reactions were followed with NMR, Figure 2-9.



0 ,CH2CH3 ., / CH2CH3
R-C-CH2-X R-C-CH2-8: X
CH3 CH3
(X-CI, Br, I)


Fig. 2-9. Formation of the sulfonium ion in the reaction of
EMS with alkylhalides.


The result of these experiments and the NMR chemical shifts

of N,4-diacetic acid aniline, 24,



o0 C-HN 0
C- CH -HN CI-L-C.
HCY 2 Nj-jf -OH









37

were used to confirm the structure and the chemical shifts

of (19). Figure (2-10) represents a typical spectrum

obtained from the reaction of methyliodide with EMS.



in CD30D







I I TV i



-4- -r-,- .- .- .. .. J




Fig. 2-10. NMR spectrum of the reaction mixture of EMS with
methyliodide.


The chemical shift values of (19) were assigned from

the comparison of the chemical shift of (24) to (19) and the

reaction of EMS with the four haloacetyl compounds. Table

(2-2) is a summary of the results obtained from these

reactions.

The shift in the NMR peaks of EMS in reaction with (19)

are 0.23, 0.94, and 0.97 for the corresponding a,b,and c

peaks. These values are well within the range of the values

of the reaction of the model alkyl halide compounds with EMS

presented in table (2-2).













Table 2-2. The effect of formation of the sulfonium ion on
the NMR peak shifts of the ethylmethylsulfide in reaction
with bromoacetophenone, iodoacetamide, iodoacetic acid and
iodomethane.


EMS(ppm) EMS(ppm) APPM

*CH3CH2 1.21(a) 1.47+0.03(a') 0.26+0.03

*CH-S 2.04(b) 2.95+0.04(b') 0.92+0.05

CH33-CH 2.47(c) 3.44+0.07(c') 0.96+0.0


Modification of the Method for the Preparation of Iodoacetyl
Compounds

In the reaction of acetic acid with PAP, a solution of

activated acid was added to PAP. This reaction resulted in

yields as high as 75%. However, when iodoacetic acid was

used in the order just described, i.e. adding the mixed

anhydride solution to PAP, a mixture of products was

obtained which proved to have the correct relative NMR peak

positions but the wrong integration values. The 3.47 P and

the phenyl peaks had almost twice the integration values as

that expected. Finally the synthesis was repeated, however,

the solution of the 4-aminophenylacetic acid was added

slowly to the solution of iodoacetic acid mixed anhydride

and allowed to react. This reaction produced the desired

product. The best results were obtained when a solution of

freshly prepared mixed anhydride of the acid in toluene or









39

acetonitrile was added slowly to a solution of PAP in 1M

NaOH at 0C.

When the mixed anhydride of the iodoacetic acid was

added to a solution of PAP some of the product, 19, is

initially formed. This product contains an iodoalkyl

functional group which on standing in alkali aqueous

solution will react with some of the remaining PAP. The

product of this reaction is (25) which by inspection will

have the same or very similar NMR as (19) with different

integration.





HO .-CH -@NH-CH- gHN- C



25



Conclusion

The synthesis of a bifunctional chelate derivative of

EDTA with haloacetyl reactive functional group was

investigated in light of preparation of a new model for

biological ET studies. Reaction of bromoacetyl bromide,

bromoacetic anhydride, iodoacetyl chloride and iodoacetic

anhydride with Ib, in attempts to prepare such bifunctional

chelate resulted in a mixture of products under the

conditions described in the literature. Model reactions

were conducted and investigated to develop a method for









40

preparation of the haloacetyl derivatives of lb. These

reactions make use of chloroformate carboxyl group

activating agents to exclusively prepare the N-

haloacetylated product of the model compounds. Based on the

similarity between the structure of the model compounds

studied and lb this method has potential application in

preparation of bifunctional chelates with haloacetyl

reactive functional group.















CHAPTER 3
PREPARATION OF MONOCLONAL ANTIBODIES AGAINST A
WATER SOLUBLE METALLOPORPHYRIN


Introduction


In the recent years, great progress has been made in

the development of antibodies that not only bind but also

chemically transform antigenic substrate molecules. The

initial production of catalytic antibodies derived form

Pauling's ideas on the mechanism of catalysis by enzymes.49-51

Pauling had proposed that a catalyst or an enzyme can

increase the rate of a chemical reaction by stabilizing the

high energy transition state of the reaction relative to

reactants hence lowering the activation energy barrier.

In 1986 an antibody (Ab) generated against a phosphate

ester molecule was shown to catalyze the hydrolysis of

analogous carboxylic esters.50'51 A phosphate ester was

chosen as the antigen to simulate the proposed structure for

the transition state of the carboxylic ester hydrolysis

reaction. Since that time antibodies generated against

other reaction transition state analogues have been shown to

have catalytic activity in more challenging chemical

transformations such as amide bond hydrolysis19"47 and









42

formation19'52'53, Claisen rearrangement19,54a-c and Diels-Alder

reactions.19,55

It has been of interest to develop antibodies that

incorporate cofactors in their catalytic function.56 At the

time this work was initiated only one Ab against a metal

complex had been reported.57 These monoclonal antibodies

were derived against metal complexes of

ethylenediaminetetraacetic acid. At that time however,

antibodies against metalloporphyrins, a commonly encountered

metal containing prosthetic group in biological systems, had

not been reported. The purpose of this investigation was to

generate and characterize metalloporphyrin- binding

antibodies.

This chapter presents the work conducted in

preparation, isolation and characterization of a monoclonal

antibody (Mab) against the metal complex CoTPPS4 9.

Preparation of 5-(p-carboxy),10,15,20-trisulfonato-

tetraphenylporphyrin (TPPS3C) 27, is described. The

cobalt derivative of this chelate (CoTPPS3C) was prepared

and conjugated to BSA and used to immunize mice. KLH-

CoTPPS3C and p-nitrobenzoic acid-KLH conjugates were

prepared and used in ELISA to screen and isolate antibodies.

A Mab was isolated, and the specifity of the clone and

several mass cultures was examined by a series of inhibition

studies.

















/N,




H O-C i-C1
0

27




Background Information


Immunology

The immune system provides one of the body's defense

mechanisms against invasion by foreign agents such as

bacteria or viruses.18 Immunology is defined as the study

of the mechanisms of that portion of the immune system

involved in the specific immunity. Specific immunity is the

defense mechanisms that is involved or stimulated by

exposure to foreign substances. These responses increase in

magnitude with each successive exposure to the same foreign

agentss.

The B and T white cells of the immune system are

responsible for recognition and ultimately elimination of

foreign agents, and they reside in the secondary lymphoid

tissue, i.e. lymph nodes and spleen. It is the B cells that

produce antibodies after they are triggered by the invading









44

agent. Antibodies function in detection and labeling of

foreign agenst and are globular glycoproteins that bind to

an invading agent and mark the agent for destruction and

removal by phagocytes. In this manner antibodies have

double recognition capability. They recognize the invading

agents that as a result is recognized by phagocytes."

A molecule that induces an immune response and

formation of antibodies is referred to as antigen.18 An

antigen often induces the production of more than one Ab.

A property of antibodies is their specifity for binding to

only one type of invading agent or antigen.19 Specific

molecular components of the agent or antigen are bound by a

specific Ab and are collectively referred to as antigenic

determinants or epitopes.

With the recent developments in hybridoma technology

and screening techniques, antibodies specific to individual

antigens can be obtained in high purity against proteins and

an array of smaller species and molecules. The cells

responsible for secretion of the specific antibodies are

isolated, cultured and cloned to obtain large quantities of

pure antibodies. An Ab isolated from a pure cloned cell

line is referred to as a monoclonal antibody (Mab) which

binds specifically to an epitope on the antigen.18


Haptens

Small molecules often can not elicit an immune response









45

by themselves since there seems to be size and charge

restriction for a foreign molecule to be recognized as an

antigen by the immune system. When small molecules are

conjugated to macromolecules such as proteins or polylysine

they are recognized as an antigenic determinant of the

macromolecule by the immune system. Small molecules which

elicit an immune response only after conjugation to

macromolecules are know as haptens.


Antibody Structure

The simplest type of Ab known as the G type

immunoglobulin (Ig) or IgG is a monomeric immunoglobulin

composed of two identical halves, Figure (3-1). Each half

contains a light (L) chain and a heavy (H) chain, named

after their relative molecular weights, and the general

formula for an IgG molecule is H2L2. Each light chain is

bound covalently to a heavy chain via a disulfide bridge at

the carboxy end of the light peptide chain. The light chain

is approximately 200 amino acids long with an average weight

of 25,000 D where the heavy chains are more versatile in

their size and have molecular weights in the range of

50,000-77,000 D.18

Various Ab types are basically oligomers of the IgG

type. These groups vary in the number and orientation of

monomers present in the structure, Figure (3-2), and are

bound together mainly via intermolecular disulfide bonds.18









46

Treatment of IgG antibodies with the enzymes pepsin and

papain leads to different Ab fragments, Figure (3-3).

Papain cleaves the heavy chain just at the point where the

carboxy end of the light chain ends and two identical

fragments are resulted. Each fragment is composed of one

light and a part of the heavy chain and is referred to as

the antigen binding fragment (Fab). Other cleavage sites by

papin results in fragments totally composed of the heavy

chain (Fc fragments). Fragment Fab, results from treatment

of the Ab with pepsin and contains the two F b which are

attached together at the hinge region of the heavy chains.

The other fractions resulted from pepsin treatment are

small polypeptide chains.18

Amino acid sequencing and immunochemistry studies of

IgG antibodies reveals that the light chain of most

vertebrate antibodies are of two immunochemically distinct



r antigen binding site

N

light chain e. C.

^ C carbohydrate
heavy chain r A;- IfNA. Vn


Fig. 3-1. Structure of an IgG antibody.




















































(b)


Fig. 3-2. Structure of (a) human secretary IgAl antibody and
(b) human IgM antibody.









48












Fig. 3-3. Fragments from enzyme digestion of an IgG
antibody.


forms called Lambda (A) and Kappa (K). Starting from the

carboxy end, these studies also indicate a large degree of

homology in the first 100 amino acid residues. This part of

the primary structure is called the constant region (CL).

The last 100 residues are referred to as the variable region

(VL) has a large degree of variation in the amino acid

sequence. Similar to the light chain, heavy chains are

partitioned to variable and constant regions based on their

amino acid sequence. Since the amino acids in the variable

region of a light and a heavy chain of each IgG molecule

together construct the Ab binding site, each IgG unit is

able to bind two identical antigens.


Structure of the Antigen Binding Site of the Antibody

The ability of an organism to generate antibodies

against a large number of antigens is the direct result of

its genetic diversity which is reflected in the amino acid

sequence of the antigenic binding site of the antibodies.

The genes that code the light and heavy chains of the









49

antibodies can encode a minimum of 108 combinations of

proteins. This variability in the Ab production and

composition gives the immune system the flexibility to

produce antibodies against almost any antigenic determinant

with a high degree of specifity.49

The three dimensional structure of any protein is

completely determined by its amino acid sequence. The

extensive sequence diversity of the antigen binding site of

the heavy and light variable regions posses structural

problems because certain amino acid sequences are incapable

of folding into soluble and/or stable proteins. In an Ab

molecule this problem is resolved by confining the diversity

of the amino acid sequence to three short stretches of amino

acid with in the variable region of the light and the heavy

chains. These highly divergent regions are referred to as

the hypervariable regions and are held in place by the more

conserved regions of the amino acid sequence referred to as

the framework regions.

In the Ab binding site the three hypervariable regions

of the light and the heavy chains are brought together in

space to form the Ab binding site. The three dimensional

structure of the Ab binding site complements the three

dimensional surface structure of an antigen. Therefore the

hypervariable regions are called the complementarity-deter-

mining regions, CDR.











Catalytic Antibodies

Antibodies can be prepared to elicit catalytic

properties following three strategies, as follows

a) Stabilization of the transition state (enthalpic

effect). The molecular interaction between a Mab and an

antigen or hapten is very similar to an enzyme-substrate

interaction. Like antibodies, enzymes are proteins.

Enzymes catalyze a variety of chemical reactions with a high

degree of specifity for substrate structure and few side

reactions.

In the recent years antibodies generated against

analogues of transition states of chemical reactions have

been shown to have catalytic activity.19'49'51'58 Transition

state analogues were used because a transition state is

short lived and therefore cannot be recognized by the immune

system to generate antibodies against it. Molecules which

best resemble the high energy transition state complex of

various chemical transformations have been prepared and used

as haptens to generate antibodies. For example an Ab

generated against the phosphate ester (28) was designed to

model the transition state in the rate determining step of

hydrolysis of (29), Figure (3-4)

In the rate determining step of ester hydrolysis

reactions, figure (3-4), the trigonal planar structure of


















R'.*4 NH



-0 NH R


29 \
R (C CO/wcOCy, l R



Fig. 3-4. Ester hydrolysis diagram, the phosphonate ester
was used to generate an antibody that increased the rate of
this reaction.



the carbonyl carbon will transform to a tetrahedral in the

high energy transition state. During this process the C=0

bond length will increase and the charge distribution on the

atoms directly involved in the reaction changes. A

phosphate ester (28) was used as a hapten to model the TS in

the hydrolysis reaction of (29) and elicit antibodies.

Monoclonal antibodies were isolated against this transition

state analogue and some of these antibodies were able to

catalyze the hydrolysis of esters and increase the rate of

the reaction by a factor of ca. 103.51


b) Lowering of the activation free energy by an

entropic effect. An enzyme or catalyst can lower the

activation barrier of a chemical transformation









52

entropically. For example, the enzymes may function in

bringing the necessary components together in the correct

orientation to affect the entropy of activation in the rate

determining step. Jencks and Page have discussed that

entropic effects can account for effective molar

concentrations as high as 108 M in enzyme catalyzed-

reactions.59 For a system to increase the rate of a

reaction it does not necessary have to bind preferentially

with the activated complex. The Ab can function as an

"entropic trap" to bring together the necessary components

for the formation of the TS hence lower the entropy of the

activation of the reaction.19 This strategy has been used

to design antibodies that entropically catalyze

lactonization60, Calisen rearrangement54a-c, Diels-Alder55,

and transacylation reactions.52


c) Haptens as cofactors to antibodies. Holoenzymes are

a group of enzymes composed of a protein and a non protein

portion called a cofactor without which the enzyme can not

function. A cofactor is typically either an organic

molecule, a metal ion or a metal-containing prosthetic

group.20

In metalloenzymes the metal complex often has catalytic

activity that is enhanced by the protein environment. For

example, the catalytic property of the iron porphyrins in

the conversion of hydrogen peroxide to water and oxygen is

enhanced in the enzyme catalase.20









53

Removal of the cofactor from the structure of a

metalloenzyme or a metalloprotein results in a protein with

a cavity for binding to a metal or a prosthetic group. The

spatial relationship between the protein and the cofactor

resembles the structural relationship between an Ab and an

antigen. This structure/function relationship has been the

motivation in this work for development and generation of

antibodies against metal complexes that might function as

metalloprotein or metalloenzyme mimics.

Metalloporphyrins are among the most vesratile and

interesting metallocofactors. At the time this work

started, no antibodies against porphyrins or metallo-

porphyrins were reported in the literature. This chapter

describes the preparation of monoclonal antibodies against a

metalloporphyrin. The Mab-porphyrin complex might have any

of the properties associated with the hemoproteins such as

peroxidase activity, electron transfer and oxygen

transfer. 61a,b


Hybridoma Techniques

The steps involved in the preparation of a Mab are

presented in Fig (3-5). Carbodiimide, N-hydroxysuccinimide

(NHS) or chloroformates are used to link the hapten to a

carrier macromolecule, which is usually a protein like BSA,

KLH, or polylysine.








54

Typically a mouse is immunized with the conjugate or

antigen. The animals with good Ab response as determined by


Conjugate

Hapten Carrier
\__Fused Cells (Hybridomas)



spleen Cells i Myeloma Cells



+-[ Screening
+- Steps

Clone

Fig. 3-5. Steps in the generation of hybridomas and
isolation of monoclonal antibodies.

ELISA screens (see below) are sacrificed and the spleens (or

lymph nodes) are removed. A cell suspension of the spleen

or lymph nodes is prepared, and the cells are fused with

myeloma cell line. Only a small portion of these cells fuse
successfully. The fused cells (hybridomas) have the

immortality of the myeloma cells and the metabolic
capability of spleen cells. Some of these hybridomas will
also have the Ab generating ability of the spleen cells.

The fused cells are plated and tested for Ab generation by
using ELISA or radioimmunoassay (RIA) in culture wells. The

wells with positive result will further be screened and









55

cloned. Cloned cell lines are made of one cell line and

produce monoclonal antibodies.49


Enzyme Linked Immunosorbent Assay (ELISA)

Two versions of the ELISA method were used in this

work. In the first method, a hapten-portein conjugate in

buffer solution is incubated on a plastic culture plate

where small quantities will absorb onto the plastic surface,

Figure (3-6). The plate is washed and a solution of test Ab

is added. The plate is washed subsequently to remove

unbound proteins. At this point an anti-mouse

immunoglobulin Ab is added to the plate, and the plate

subsequently washed to remove the free anti-immunoglobulin.

The anti-immunoglobulin which binds to the CE or CL region

of the sample antibodies is covalently attached to an enzyme

where the end product of the enzyme

reaction is colored. The substrate is added to the plate,

and the amount of the test Ab is measured by assessing

optical density (O.D.) scanning of the plate.


Competition ELISA

Competition assays measure the relative affinity of the

antigen for the binding site of the Ab molecule and can be

used to test the specifity of the Ab for the hapten. A

conjugate of the hapten and a protein other than the one

used for immunization of the mouse, is prepared and used to




























Enzyme-Linked Immunoabsorbent Assay (ELISA).


L12J
I j,' ^ '


Competition or Inhibition ELISA.


Fig. 3-6.


Fig. 3-7.









57

coat a culture plate. A solution of the competitor or

inhibitor compound of known concentration is incubated with

the test Ab, and the solution is subsequently added to the

culture plate. The steps following are as in standard

ELISA.

If the Ab has a large affinity for the competitor the

amount of free Ab available to bind with the conjugate is

reduced, resulting in a lower O.D. reading. An Ab with a

low affinity for the conjugate will result in a high O.D.

reading, indicatesing no specifity of the Ab for the hapten.

If the competitor used in this assay is the original hapten,

the result will indicate whether the Ab is specific for the

hapten and not any portion of the hapten rather than a

portion of the protein in the conjugate used in

immunization.


Choice of Hapten

Naturally occurring porphyrins, such as protoporphyrins

IX, were not used as haptens for this work since they

polymerize in buffered aqueous solutions.14 The cobalt3

derivative of the synthetic water soluble 5,10,15,20-

tetrasulfonatotetraphenylporphyrin (TPPS4) was selected as

the basic hapten because this metalloporphyrin is monomeric

under physiological conditions at mM concentrations.15'16

The Fe3*TPPS4 complex which better resembles the more

abundant naturally occurring iron protoporphyrins, was not

chosen since at neutral or slightly basic pH it dimerizes









58

via an oxo bridge.17'62 The Co3 complex of this porphyrin

(CoTPPS4) is monomeric under physiological pH and was chosen

as the hapten. To attach CoTPPS4 to a carrier molecule,

CoTPPS3C, 30, which has a conjugatable carboxylate

functionality, was prepared and conjugated to carrier

molecule. This complex resembles CoTPPS4 except one

sulfonate group is converted to a carboxyl group to allow

covalent binding of this complex to the carrier.











N/ --CO -N \




0


30



Experimental

C, H, and N analysis were conducted at in the

department of chemistry at the University of Florida. NMR

spectra were obtained by using a 300 Mhz Varian instrument.

ICP-ES analysis was conducted by using a Beckman ICP

instrument. In these analyses a calibration plot of the

element under study was obtained by using commercially











available standard solutions. This plot was used to measure

the concentration of that element in the analyte solutions.

The Fe3 complex of TPPS4, FeTPPS4, the positively

charge tetramethylpyridiniumporphyrin salt, TMPY, and its

cobalt derivative, CoTMPY, were purchased from Mid Century

Chemical company. Tetracarboxytetraphenylporphyrin (TPPC4)

was prepared following published methods.63b The cobalt

complex of this water soluble porphyrin (CoTPPC4) was a gift

from Dr.R. Drago's group in the inorganic division of the

University of Florida chemistry department.


Synthesis of Tetraphenylporphyrin (TPP)

5,10,15,20-Tetraphenylporphyrin(TPP) was synthesized by

method of Alder et al.63' Freshly distilled pyrrole (11.04

g, 0.24 mole) and 29.28 g (0.24 mole) of benzaldehyde were

added to 1 L of refluxing propionic acid. After 0.5 h the

mixture was cooled to room temperature. The heterogeneous

mixture was filtered by using a sintered-glass vacuum

funnel. The solid left behind was thoroughly washed with

hot methanol followed by a hot water wash.

Anal. Calcd for C 44H30N 40: C, 85.90; H, 4.92; N, 9.12,

Found: C, 82.47; H, 4.75; N, 8.80, Glass impurity in sample.

H NMR in CDC13: 7.78 (m, 3H), 8.26 (dd, 2H), 8.89 (s, 2H).


Synthesis of Tetrasodium meso-tetra(p-sulfophenvl)porphyrin
(TPPS4)

The method of synthesis was an adaptation of the method









60

introduced by Srivastava and Tsutsui." Five hundred mg of

TPP was dissolved in 20 mL of concentrated H2SO4 The

green solution was heated on a steam bath overnight. Once

the mixture was cooled to room temperature, 300 g of ice was

added. Excess acid was neutralized by addition of a sludge

of lime to the mixture. Neutralization is apparent by the

permanent purple color of the mixture. The precipitate

CaSO4 was removed by filtration and was washed with minimum

amount of water which was added to the filtrate. The

solvent was evaporated under reduced pressure and the purple

solid was dissolved in minimum amount of methanol. Reagent

grade acetone was added to the methanol solution until the

porphyrin precipitated out of solution. This step was

repeated 6-7 times. At this point the solid was removed

from the cold solution by filtration on a fine sinter glass

vacuum funnel and was dried over P20s in vacuum. The NMR

indicated exclusive para substitution on the phenyl rings.

NMR in DMSO-d6: 8.09 (d, 1, J=8.06 hz), 8.22 (d, 1,

J=8.06), 8.88 (pyrrole)(s, 1), UV-Vis: (solvent H20), 630,

577, 548, 512, 410, 289.


Synthesis of 5,10,15,20-Tetrakis(p-sulfophenyl)porphinate-
cobalt(III) (CoTPPS4)

The metallation of TPPS4 was accomplished by the

general method for metallation of porphyrins developed by

Alder.65 One hundred milligram of the sodium salt of TPPS4

was added to 35 mL of refluxing dimethylformamide. After









61

all the solid had dissolved 5% molar excess CoCl2 was added.

A spot of solution was used to periodically check the

presence of phosphorescence on Wattman paper under UV light.

Formation of the cobalt complex of porphyrins quenches the

phosphorescence of porphyrins observed in UV light. At the

point where no phosphorescence was observed another 5%

excess of CoCd2 was added and the solution was refluxed for

an additional hour. The solvent was removed and an aqueous

solution of the obtained solid was chromatographed on

Amberlite CG-120 cation exchange in the Na form in order to

remove excess cobalt. Water was removed under reduced

pressure and sample was characterized by UV-Vis

spectroscopy.

UV-Vis:(in phosphate buffer pH=8, U=0.2), 424, 538, (in

80% ethylene glycol/phosphate buffer), 420, 544, 250-350

(Sh).








/ N-qo-N



H0-9 OH









62

Synthesis of 5-(4-carboxyl)-10,15,20-tetraphenylporphine
(TPPC)

Synthesis of the hapten was an adaptation of the method

introduced by Lavallee.66

Benzaldehyde (20 mL, 0.2 mol) and 4-carboxybenzaldehyde

(10.5 g, 0.07 mol) were added to 1 1 of propionic acid and

heated in a 2000 mL 3 neck round bottom flask to reflux

temperature. Freshly distilled pyrrole (19 mL, 0.27 mol)

was added slowly to this hot solution and allowed to reflux

for 1 hr. The mixture was transferred to a 3000 mL

Erlenmeyer flask and 1 1 of ethylene glycol was added.

After the solution cooled to room temperature it was

stoppered and left in the freezer overnight and filtered by

using a medium size fritted glass funnel the following

morning. A precipitate was collected which was

chromatographed on an 18 x 9 cm silica gel (200-400 mesh)

column and eluted with CH2Cl2. The only compound to elute

with this solvent corresponds to tetraphenylporphyrin.

After separation of this compound the solvent was switched

to 2.5% ETOH in CH2Cl2 which eluted the next band

corresponding to the product.

NMR in CDCl1: 7.77 (s, 3), 7.74 (s, 6), 8.21 (dd, 6H,

J=1.6, 2.01), 8.34 (d, 2H, J=8.07), 8.50 (d, 2H, J=8.43),

8.85 (m, 8H).









63

Synthesis of 5-(4-carboxyphenyl)-10,15,20-trisulfophenyl-
porphine(TPPS3C)

TPPC (500 mg) was dissolved in 30 mL of concentrated

sulfuric acid and heated on a steam bath for 12 hrs. The

product was precipitated by addition of 3-5 fold excess

acetone. The solid was filtered and recrystallized by

repeated dissolving in hot methanol and precipitation by

acetone. After several precipitations the acidic solid was

dissolved in methanol and neutralized by addition of 1,10

phenanthroline. The solution was then concentrated and

passed through an Amberlite CG (100-200 mesh) cation

exchange column in the sodium form. Water was removed under

reduced pressure and the purple color tetrasodium salt of

TPPS3C was obtained.

Uv-VIS:(in 80% ethylene glycol/phosphate buffer), 644,

589, 550, 514, 418. (in DMF), 649, 590, 548, 424, 512, 418.

NMR in CD3OD: 8.28 (d, 1H, J=8.1), 8.34 (m, 6H), 8.44 (d,

1H, J=8.04), 8.95 (Broad).


Synthesis of 5-(4-carboxyphenyl)-10,15,20-trisulfophenyl-
porphinate-cobalt(III) (CoTPPS3C)

The cobalt complex of TPPS3C was prepared following the

method developed by Alder.65 One hundred mg of TPPS3C and

10% molar excess of CoCl 2.6H20 were added to 30 mL of

refluxing DMF. The reaction was followed by using the

phosphorescence of the free ligand, which is quenched after

metallation of the porphyrin as described for preparation of

CoTPPS4. After the reaction was complete the solution was









64

cooled to room temperature and the solvent was removed under

reduced pressure. The excess metal ions were removed by

cation exchange chromatography of the obtained solid by

using amberlite CG-120 resin in the Na+ form. Water was

removed under reduced pressure and the sample was analyzed

by ICP-ES and UV spectroscopy.

UV:(in pyridine), 550, 433, 263. (in H20), 537, 423,

320. ICP-ES:(Co/S), Calc. (14/23), Exp. (1.4+0.03/

3.0+0.11).


Synthesis of 5-(4-carboxyphenyl)-10,15,20-trisulfophenyl-
porphinatezinc(II) (ZnTPPS3C)

Tetrasodium salt of TPPS3C (140 mg) and 150 mg of

Zn(OAC)2.2H20 were dissolved in 25 mL of refluxing DMF.

Qualitative monitoring of the phosphorescence of this

solution using UV light does not indicate the extend of

metallation since zinc porphyrins are also phosphorescent.

However the growth of two UV peaks at 598 nm and 558 nm

indicates the formation of the zinc porphyrin complex.

After 2 hr of reflux 100 mg excess of the zinc salt was

added to the reaction mixture and the heating was continued

for 8 hr when the absorbance at the two peaks was no longer

changing. The solvent was removed under reduced pressure

and the excess metal was removed by cation exchange

chromatography as described for the CoTPPS3C preparation.

Uv-VIS:(in DMF), 598, 558, 425. ICP-ES: (Zn/S), Calc.

(21.11/31.02), Exp. (15.9+0.30/23.9+0.50).









65

Preparation of CoTPPS3C-Bovine Serum Albumin

The conjugation of the hapten to BSA was an adaptation

of a literature method.67 CoTPPS3C (36 mg of 10%

purity,3.22 umol) was dissolved in 7 mL of DMF at room

temperature. One hundred microliters each of 32.2 uM

solutions of N-hydroxy-succinimide (NHS) and 1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide methiodide (DMECI)

in phosphate buffer (pH=6.0, U=0.0125) were added to the

porphyrin solution with stirring. After 2 h 4.3 mg of BSA

in 27 mL of phosphate buffer (pH=8.0, U=0.2) was added to

the mixture and allowed to react for 2.5 h at room

temperature. The solution was then dialyzed against several

changes of fresh phosphate buffer (pH=8.0, U=0.2), while

periodically checking the dialysate buffer solutions for the

soret band of the porphyrin. After several buffer exchange

the protein solution was vacuum concentrated by using a

Micro-prodicon concentrator (Model MPDC-15) fitted with a

15,000 MW cut off membrane. Concentrating the conjugate

under pressure by using an AMICON concentrator is a faster

method; however, our previous attempts using this method had

resulted in denaturation of the protein. The concentrated

protein was further loaded on a 1.5 cm x 60 cm Sephadex G-50

gel column and eluted with phosphate buffer (pH=8.0, U=0.2)

and monitored at 228 nm wavelength. The fractions in the

single peak observed were concentrated by using the Micro-

prodicon concentrator. The concentrate was then









66

chromatographed by using a 1.5 cm x 40 cm Sephadex G-200 gel

column. A single peak eluted at the proximity of the peak

observed for unmodified BSA. The fractions were combined,

concentrated and used for further analysis.


Preparation of CoTPPS3C-Key Hole Limpet Hemocyanin(KLH)

Conjugation of KLH with CoTPPS3C was accomplished by

following the procedure for BSA modification except the

reaction buffer solution was Na2CO3 (pH=10 and U=0.2). The

Co03/KLH ratio was used to determine the hapten/KLH ratio by

using the same techniques in determination of hapten/BSA for

CoTPPS3C-BSA conjugate.


Preparation of p-Nitrobenzoic acid-KLH

KLH modification with p-nitrobenzoic acid (PNB) was

carried out in a manner identical to KLH-CoTPPS3C

modification. The concentration of the KLH in the conjugate

was obtained by BCA protein assay as described for BSA.

This value and the extinction coefficient of the protein at

272.8 nm was used to calculate the contribution of KLH

absorbance at this wavelength to the conjugate absorbance.

The absorbance of the solution of the conjugate at 272.8 nm

was measured (corrected for the KLH absorbance) and used to

calculate the PNB concentration in this solution.


Preparation of Hybridoma Cultures

The preparation of the monoclonal antibodies against

CoTPPS4 were conducted in University of Florida ICBR









67
Hybridoma Laboratory at the University of Florida.68 Balb c

mice were injected with CoTPPS3C-BSA conjugate followed by a

booster injection in two weeks. ELISA screens of the sera

of the immunized mice with KLH-CoTPPS3C conjugate were used

to identify Ab producing mice. The mouse(mice) with

positive results was sacrificed and the spleen cells were

used for fusion to produce hybridomas. The hybridomas were

studied in mass culture and screened by ELISA.


Primary and Secondary Screening of MC for Specific
Antibodies

Primary screening. In the primary screening post

fusion supernatant solutions were assessed for the presence

of antibodies by ELISA on plates coated with hapten-KLH

conjugate. Mass cultures with positive results were

expanded, mass cultured and used for secondary ELISA

screening.

Secondary screening. In this step supernatants from

mass culture wells were incubated in plates previously

coated with hapten-BSA, BSA, hapten-KLH, and KLH and used in

ELISA. Antibody(ies) specifically generated against the

hapten will have a high affinity for hapten-BSA and hapten-

KLH conjugates and little if any affinity for either of the

carrier proteins. The affinity of the Ab for an antigen is

directly reflected in the O.D. readings in ELISA.









68

Screening of the MC Supernatant Against Hapten-KLH, PNB-KLH
and KLH

The supernatants from the mass cultures were further

assayed to evaluate the specifity and affinity of the test

antibodies for the hapten, a protein side chain and a polar

aromatic benzene ring. Plates were coated with hapten-KLH

conjugate, PNB-KLH conjugate and KLH and were used in ELISA

as described before.


Determination of the Relative Binding Affinity of Test
Antibodies by Inhibition ELISA

Inhibitors used in these assays were CoTPPC4, CoTPPS3C,

ZnTPPS3C, TPPC4 and TPPS3C. A solution of the MC

supernatant was incubated with various concentrations of the

inhibitor at 4 c over night. One hundred microliter of each

incubated solution was transferred to ELISA plates) which

was previously coated with 100 ul of 5 ug/mL solution of

hapten-KLH conjugate. The plates) was washed and rabbit

antimouse IgG-alkaline phosphatase (Sigma) was added to each

well. After another washing the enzyme substrate, p-

nitrophenylphosphate (Sigma) was added and the O.D. of the

plates was measured at 405 nm for each well.


Results and Discussion

ICP Elemental Analysis for CoTPPS3CNa4

C, H and N elemental analysis of sulfonated

phenylporphyrins often give irregular results.62

Investigators have tried to purify these porphyrins by









69

several chromatographic methods and repeated

recrystallization steps before analysis. Often a residue is

left behind in an elemental analysis instrument which is

speculated to constitute inorganic salts.66 In this work

the S/Co ratio was measured by ICP/ES as a method of

analysis for the hapten, CoTPPS3C. Anhydrous Na2SO4 was

used as the sulfur standard while cobalt standard solution

was obtained commercially and diluted to the desired

concentrations for the construction of calibration plots.

The calculated Co and S concentrations were 14 ppm and 23

ppm respectively for 0.02659 g of pure sample in 25 mL of

aqueous solution. The experimental values were 1.4+ 0.03

for Co and 3.0+ 0.11 for S.

The ICP-ES results indicates a S/Co3 ratio higher than

the calculated value. Possible explanations of this

discrepancy are as follows,

Presence of inorganic sulfate salts: The

neutralization of excess sulfuric acid in the sulfonation

step gives rise to sulfonated salt by products. These salts

could have been carried along with the product resulting in

higher S measurements.

Incomplete metallation of the ligand: The use of

excess cobalt and the high sensitivity of the

phosphorescence of the free ligand however, seem to dispute

this explanation.









70

HSOCounter ion: The Co3 in CoTPPS3C might exist as

the HSO4-salt which leads to unaccounted sulfur.

Because of the discrepancy in the sulfur analysis, Co3+

concentration was used to determine the concentration of the

hapten in solution. The experimental/calculated cobalt

concentration ratio respectively (1.4+0.03/14) for the

CoTPPS3C solution, 0.02659 g in 25 mL of aqueous solution,

indicated that the sample was 10% pure. The impurities were

NaCl and/or Na2SO4 carried over from the cation exchange

chromatography step in purification of the hapten and/or the

neutralization of excess H SO4 in the sulfonation step.

A UV spectrum of a standard solution of the hapten was

obtained and molar absorptivity values for the two wave

lengths with maximum absorbance were calculated. These

values are 2.96 x 105 (M-cm-1) and 1.68 x 104 (M-cm-1) in

deionized water at 426 and 540 nm respectively.


ICP Elemental Analysis for ZnTPPS3C

A 0.01679 g sample of ZnTPPS3C was dissolved in 50 mL

of distilled water and used for ICP analysis as described

for CoTPPS3C. The calculated ratio of S/Zn concentration is

1.48 for this solution. The experimental S/Zn ratio for

this solution was 1.50+0.04 obtained by ICP/ES. These

results indicated that no other sulfur sources than the

metalloporphyrin were present in the sample. The

sulfur/metal ratio for this compound is in better agreement









71

with the calculated value than the CoTPPS3C since Zn+2 does

not need a counter ion in the metalloporphyrin structure.

The ratio of the experimental/calculated for sulfur and

Zn concentrations respectively in this sample solution was

S=23.9+0.50 /31.02 and Zn=15.9+0.30 /21.11. These results

indicate a sample purity of 76%. The molar absorptivities

for this metalloporphyrin in distilled water at three

absorbance maxima are; (3.16 x 103 cm-'M- (594.4 nm), (6.33 x

103 cm-M-1) (555.2) and (1.76 x 104 cm-1M-1) (309 nm).



Preparation of Conjugates

Carbodiimde, N-hydroxysuccinimide or chloroformate

agents are used for covalent binding of small molecules to

proteins and macromolecules.36-39 The water soluble

carbodiimide DMECI and N-hydroxysuccinimde were used for the

preparation of the conjugates in this work.



Preparation of BSA-Hapten Conjugate for Immunization

Qualitative determination of CoTPPS3C-BSA Conjuga-

tion. In the second step of purification of hapten-BSA

conjugate by gel chromatography a single peak eluted from

the sephadex G-200 gel column which corresponded to a

species with a molecular weight similar to BSA which has the

same color and very similar uv-vis spectrum as the hapten.

A UV-Vis spectrum of a solution of the hapten, BSA and the

conjugate CoTPPS3C-BSA are shown in figure (3-7). The









72

spectrum of the conjugate is basically the same as the

spectrum of the hapten with the added absorption peak at 280

nm. This peak is attributed to the absorption of the

tyrosine amino acid residue in BSA.


Quantitative Determination of CoTPPS3C-BSA conjuga-

tion. The extend of modification was determined by the

ratio of the porphyrin concentration to the protein

concentration in the solution of conjugate after the final

chromatographic purification. The porphyrin concentration

was obtained by using the absorbance at the Soret band or

the Co3+ concentration from ICP-ES. For the solution of the

conjugate the porphyrin absorbance at 558 nm was measured

and used to correct the absorbance of the assay solution.

The sulfur to metal ratio from ICP elemental analysis

can be used in order to determine the extent of

modification. However, because of the low concentration of

the protein and inconsistent S readings of our ICP

instrument at the time, an alternate procedure was chosen.

The concentration of the hapten in a solution of the

conjugate was obtained from the UV absorbance of the hapten

at the Soret band and the Co3* concentration from ICP-ES.

The protein concentration in the conjugate was obtained by

bicinchoninic acid (BCA) protein assay.69 A kit for this

assay was purchased from Pierce company. The hapten

concentration obtained by using UV and ICP-ES resulted in a












2.50



2.00


LLJ

<1.50


0
) 1.00
co


0.50


UV-VIS SPECTRA FOR CONJUGATION OF
CoTPPS3C TO Bovine Serum Albumin


0 .00u ,, ,, ,,1 ... ,. .,,,,,,, ,, ,,,, ,
200.00 300.00 400.00 500.00 600.00 700.00
WAVE LENGTH (nm)


Fig. 3-7. Qualitative assessment of BSA-CoTPPS3C
conjugation, (---) BSA, (...) CoTPPS3C, (-) Conjugate.









74

CoTPPS3C/BSA ratio of 22 and 28 respectively with an average

value of 25 haptens/carrier molecule.

The BCA assay makes use of the reduction of Cu2 to Cu^

by the protein which in turn forms a purple complex with BCA

present in the assay solution. This complex has a strong

absorption at 550 nm region of the visible spectra which is

used to construct calibration plots. This assay can measure

protein concentrations as low as 5 ug/mL.69

Trial runs of the assay with solutions of porphyrin and

buffer separately indicated that the porphyrin and the

buffer do not interfere with the assay results. The UV

absorbance in the assay is normally measured at 550 nm.

However the porphyrin had minimal absorbance at this wave

length. In this work the assay solutions were monitored at

558 nm instead. The absorbance of the conjugate in the

assay was corrected for the porphyrin absorbance at this

wavelength.


Preparation and Characterization of Conjugates of KLH with
CoTPPS3C and PNB

For the purpose of screening and isolation of the

various antibodies produced by the immune system against a

single antigen (or hapten) it is necessary to obtain a

conjugate of the same hapten with a different carrier

macromolecule. Since BSA was used as the carrier in the

inoculation of the mouse KLH-hapten and KLH-PNB conjugates

were prepared for screening. KLH is a decameric protein









75

that exists as different oligomers in aqueous solution

depending on pH and ionic strength. The monomer has a

molecular mass of 800,000 Da. At pH 10 this protein

dissociates to monomer and smaller oligomers to a large

extend70d which is more suitable for protein modification.

Conjugates of KLH with the hapten and PNB were prepared in

Na2CO3 buffer solution, pH=10 and U=0.15.

Cobalt ICP-ES analysis and protein assay were used to

asses the hapten/KLH (800,000 Da) ratio. From the results

of these analyses a value of approximately 100 CoTPPS3C

molecules/800,000 Da of KLH was calculated. The analysis of

PNB-KLH conjugate was assessed by an alternate method since

PNB can not be quantitatively analyzed by ICP-ES. PNB has a

molecular weight of 167.12 g/mol and simply composed of

carbon, nitrogen and hydrogen. These elements account for

more than 99% of the KLH protein composition with a monomer

molecular weight of 800,000 Da.

UV spectroscopy was used to obtain a crude value for

PNB/KLH ratio. The molar absorptivity of PNB was calculated

from the absorbance of a 1.1 x 10-3 M solution of PNB

prepared in NaHCO3 buffer (pH=10 and U=0.15) at 272.8 nm.

The molar absorptivity of PNB at 272.8 in this solution is

9.66 x 103 MW-cm-1(57.8 mL/mg.cm).

A 1.11 mg/mL standard solution of KLH in NaHCO3 (pH=10

and U=0.15) was prepared. The molar absorptivity of KLH at

276 nm and 272.8 nm were calculated for this solution. The









76

experimental value of 0.93 mL/mg cm at 276 nm (KLH lambda

max) in the carbonate buffer is in good agreement with the

value of 1.12 mL/mg cm at 278 nm in phosphate buffer (pH=7.4

and U=0.2) reported by Sigma Co. The calculated molar

absorptivity of KLH for the same solution at 272.8 nm

(lambda max of PNB) was 0.91 mL/mg cm.

From the protein assay results an absorbance value for

KLH at 272.8 nm was calculated. This value was subtracted

from the total absorbance of the conjugate solution at 272.8

nm to calculate to absorbance of PNB. Using the

concentration of KLH in the conjugate obtained by the

protein assay and the absorbance of PNB at 272.8 nm a

PNB/KLH ratio of 16.5 for 800,000 Da of KLH was calculated.


Preparation and Isolation of Specific Antibodies

Hybridomas obtained from the fusion of the spleen cells

of a responding mouse and myeloma cells were cultured to

obtain test Ab solutions for the preliminary screening. In

the primary screening, post fusion supernatant solutions

were assessed for the presence of antibodies by ELISA.

Wells with positive results were expanded, mass cultured and

used for a secondary ELISA screening. In this step

supernatant from mass culture wells were incubated in plates

previously coated with hapten-BSA, BSA, hapten-KLH, and KLH

and assayed.

Antibody(ies) specifically generated against the hapten

will have a high affinity for hapten-BSA and hapten-KLH









77

conjugates and little if any affinity for either of the

carrier proteins. The affinity of the ab for an antigen is

directly reflected in the O.D. readings in this type of

ELISA. Table (3-1) presents the O.D. at 405 nm for several

selected mass cultures in this screening. In this assay

mass cultures 2C1, 2B5, 2B9, 2C3 and 3D3 were selected and

used in the next screening steps.


Table 3-1. Mass culture from balb C mice.

PLATE 2A8 2C1 3D1 3D3 2B5 2B9 2E3
COATING *
Hapten/KLH 0.602 1.126 0.576 0.943 NA NA NA
Hapten/BSA 0.949 1.726 0.623 1.462 2.160 1.572 0.702
KLH 0.731 0.716 0.720 0.820 0.362 0.439 0.323
BSA 0.659 0.345 0.688 0.373 0.216 0.383 2.150

Note: (*) are some of the Mass Cultures (MC) selected for
the following screenings.


Screening of the MC supernatant against Hapten-KLH, PNB-KLH
and KLH by Competition ELISA

Figure (3-8(a-e)) presents the results of these assays.

These results indicate that the antibody(ies) generated by

2C1 MC and 3D3 MC are non specific since they show high

affinity for KLH and the two conjugates of this protein.

Therefore they were excluded from the following screening

steps and 2B9 was cloned to produce monoclonal HL211 which

was further proved to be an IgM Ab. HL211 clone, 2B5 MC AND

2C3 MC were used in a final inhibition assay where CoTPPC4,

CoTPPS3C, ZnTPPS3C, TPPC4 and TPPS3C were used as












78







ELISA of 2C1 Mass Culture


1.4-

1.2-

1
E
0.8-
- 0-
^-

0.4

0.2-


0 0.5 1 1.5 2 2.5 3 3.6
-LOG(ReL Conc. of Ab In MC Supernatent)


ELISA of 2B5 Mass Culture


0 0.5 1 1.5 2 2.5 3
-LOG(ReL Conc. of Ab In MC Supernatent)


The result of secondary MC screening.


Fig. 3-8.











79






ELISA of 2B9 Mass Culture


0 0.6 1 1.6 2 2.8 3 8
-LOG(Rel. Cono. of Ab In MC Supernatent)


ELISA of 2C3 Mass Culture


0.o 0.0 0. 1.20 150 181
-LOG(Rel. Cono. of Ab In


Fig. 3-8. (Continued),

















ELISA of 3D3 Mass Culture

Pleme c- d4 wIth
1 Haptan-KILH
-- KLH
*- PNB-KLH
E .


0 o.4

0.2

0 0.3 0. 0.9 120 50 1t81 2.11 2.41 271 83.01 8.31
-LOG(Rel. Conc. of Ab in MC Supernatant)






Fig. 3-8. (Continued).




inhibitors. Because of the nature of these assays a low

O.D. reading corresponds to an Ab with a high affinity for

the hapten. Figure (3-9 a-d) presents the result of these

assays for 2B5 MC and HL211 clone.

During the preparation of antibodies in this work, the

preparation of an antimetalloporphyrin Ab was reported by

Schwabacher et al.71 They had obtained a total of three

monoclonal antibodies against Fe 3 and Co complexes of the

negatively charged tetracarboxyltetraphenylporphyrin

(TPPC4). Monoclonal 13C4 and 13B4 wer against the Fe(III)-

TPPC4 complex and 35F8 was against the Fe(III)-TPPC4

complex. The result of the competition assays of these

monoclonals are presented in table (3-2).

















2B5 MC Inhibition ELISA


-2 0 2 4 6
-LOG Conc.(mM) of Inhibitor


2B5 MC Inhibition ELISA


-2 0 2 4 8 8 10
-LOG Conc.(mM) of Inhibitor


Inhibition ELISA,


Fig. 3-9.











82






HL211 Clone ELISA Inhibition Assay


1 2 3
-LOG Conc.(mM) of Inhibitor


Supernatent diluted 1:0


HL211 Clone ELISA Inhibition Assay


-1 0 1 2 3
-LOG Conc.(mM) of Inhibitor
Supernatent diluted li:


Fig. 3-9.(Continued).









83

The binding constants reported in the literature were

measured by competition ELISA procedure. Table (3-1)

indicates that all three antibodies have the greatest

affinity toward their corresponding antigen and, except for

13C4, they have the least binding affinity toward the free

ligand. 13C4 shows an affinity of at least two order of

magnitude toward Fe(III)-TPPC4 greater than all the other

metalloporphyrins tested in the assay. This Ab has a

binding affinity more than 3 order of magnitude toward the

Fe(III)-TPPC4 than the Co(III)-TPPC4 which seems to indicate

that the metal center is directly involved in the binding

site.


Table (3-2).71 Dissociation constants of (M)TPPC4 obtained
from the work by Schwabacher et al. in the preparation of
IgG antibodies against Co(III)TPPC4 and Fe(III)TPPC4.
(M)TPPC4 Kd, 35F8 against Kd, 13C4 against Kd, 13B4 against
Co(III)TPPPC4 Fe(III)TPPC4 Fe(III)TPPC4

H 2.6 x 10-5 3.3 x 10-6 5.9 x 10-5
Mn3+ 1.3 x 105 1.7 x 10-6 4.7 x 10-6
Fe3+ 3.2 x 10-5 4.1 x 10-' 1.4 x 10-7
Co3+ 6.9 x 10-7 1.4 x 10-5 5.9 x 10-7
Cu2+ 3.6 x 10-5 1.5 x 10-' 3.1 x 10-5
Zn2+ 4.0 x 10-5 1.3 x 10-4 1.4 x 10-6
Sn4+ 1.1 x 10-5 3.0 x 10-5 7.9 x 10-7









84

In the present work 2B5 MC, 2C3 MC and monoclonal HL211

were shown to have the greatest affinity for the antigen

Co(III)-TPPS3C, Figure (3-9). Comparison of the Co(III)-

TPPS3C and TPPS3C assay results indicates that the antigen

is more tightly bound than the free ligand. Comparing the

Co(III)-TPPS3C assay results to Zn(II)-TPPS3C assay results

indicates that the nature of the and/or the charge on the

metal center in the metalloporphyrin is/are of importance in

the binding. The affinity of the antibodies is lower for

the Co(III)-TPPC4 than the antigen. These results indicate

that these antibodies are selective toward both the ligand

and the metal center of the metalloporphyrin used as

antigen. Table 3-3 presents estimated binding constants

obtained from the plots obatined in the competition assays

for 2B5 and HL211 Mab.


Table (3-3). Association constants obtained from the
competition assays of 2B5 MC and HL211 Mab.

Competitor Ka for 2B5 MC Ka for HL211*
CoMCTPPS3 > 107 6.3 x 104
ZnMCTPPS3 6.3 x 105 3.2 x 102
CoTPPC4 2.5 x 105 2.0 x 104
MCTPPS3 4.0 x 105 1.6 x 102
TPPC4 1.6 x 105 1.6 x 102
Supernatent was dilute 1:8 for assay.


The data and results presented in the competition

studies are limited to only one metalloporphyrin with a

divalent metal and no other trivalent metalloporphyrin other









85

than the antigen. However for a better understanding of the

binding site structure and affinity of the antibodies

obtained in this work a more comprehensive competition study

is under investigation. The porphyrins and

metalloporphyrins under investigation are listed below:

Co(III)-TPPS4, Fe(III)-TPPS4 and the free ligand, TPPS4;

Co(III)-TPPS3C, Zn(II)-TPPS3C and the free ligand, TPPS3C;

Co(III)-TPPC4, and the free ligand;

Co(III)-TMPy, Fe(III)-TMPy and the free ligand, TMPy.

The results of these inhibition assays over a large

range of inhibitor concentration will allow a more accurate

calculation of the binding constants. From these results

the nature and specifity of the binding site of the

antibodies will be better understood.


Conclusion

CoTPPS3C was prepared and conjugated to BSA and KLH by

using carbodiimide/N-hydroxysuccinimide reagents. Hapten-

BSA conjugate was used to immunize mice and the KLH

conjugate was prepared and used in the screening and

characterization of the antibodies.

Hybridomas were followed to obtain 2B5 MC, 2C3 MC and

HL211 monoclonal antibodies against the CoTPPS3C antigen.

The results of the inhibition ELISA studies in this work

indicates a high degree of specifity of these antibodies for

the cobalt hapten. Because of the limited number of

inhibitors that were used in the inhibition ELISA assays a









86

more comprehensive assay is under way that involves a larger

number of porphyrins and metalloporphyrins. Further work is

also under progress to obtain other monoclonals against this

antigen by using other mass cultures obtained as a result of

this work to isolate IgG type monoclonals against CoTPPS3C.

IgM antibodies have the disadvantage of precipitating when

bound with the antigen. Preliminary UV antigen binding

studies of a solution of HL211 monoclonal in the presence of

antigen showed a shift in the Soret band of the antigen.

This solution however, formed a precipitate shortly after

addition more metalloporphyrin.















CHAPTER 4
ELECTROCHEMISTRY OF A NEGATIVELY CHARGED PORPHYRIN AND
ITS Co(III) COMPLEX


Introduction


Electrochemistry of naturally occurring

metalloporphyrins has been generally investigated in non-

aqueous solutions.n2ab-" Under physiological conditions

these porphyrins form aggregates, which complicate

electrochemical studies in aqueous solutions.14'15 Synthetic

water soluble non-aggregating porphyrins and

metalloporphyrins have been used as models for naturally

occurring porphyrins. The electrochemical studies of the

synthetic water soluble porphyrins have mainly involved

positively charged tetrapyridylporphyrins, 76a,b,c-80 and

negatively charged synthetic porphyrins have only rarely

been considered.,7581,82

The cobalt(III) derivative of tetrasulfonatotetra-

phenylporphyrin (CoTPPS4) has a reported reduction potential

of -0.65 (vs SCE) in borate aqueous buffer solution.82

Other investigators,83 in accordance with the present

investigation, have tried to reproduce this result; however,

in all cases ill-defined voltammograms have been obtained.









88

The purpose of this study is to find conditions that

allow investigation and characterization of the

electrochemistry of CoTPPS4 and TPPS4 under conditions

applicable to biological systemss. It is finally of

interest to find a method that can be used to study the

interaction of CoTPPS4 with antibodies by electrochemical

means.

In this investigation, cyclic voltammetry of CoTPPS4 in

the pH range of 1-14 was examined by using several

electrodes. The use of promoters and modifiers for the

CoTPPS4 electrochemical studies in aqueous solution is

described. The reduction electrochemistry of TPPS4 and

CoTPPS4 were investigated in DMF/TEAP by using a gold

electrode, and the heterogeneous rate constant (k,) and

diffusion coefficient (D) of these two compounds in DMF

were obtained from these experiments. Finally, the cyclic

voltammetry of CoTPPS4 was investigated in DMF-H20 mixed

solvent in light of the potential application of this system

in metalloporphyrin complex/protein interaction studies.


Background Information

Porphyrins and metalloporphyrins show varied

electrochemical behavior in aqueous solution. Some are not

stable under physiological conditions and tend to adsorb to

electrode surfaces, and, in a number of cases, irreversible

redox behavior is observed. In some instances the

metalloporphyrin will eject the metal ion upon









89

reduction.74'83-8sab Because of such diversity the

electrochemical studies of porphyrins and their metal

derivatives are potentially complicated.


Site of Electron Transfer on CoTPPS4

An investigation of electron transfer of CoTPPS4 with

Cr 3 suggests that the transfer of electrons between the two

species takes place via the negatively charged sulfonate

ionic side chain on the phenyl rings.85a Other

investigations suggest, however, that the axial ligand on

the metal center in the metalloporphyrins is important in

exchange of electron(s) with other electroactive species.78-8

The specific site of electron transfer on metalloporphyrins

is not well determined.

In heterogenous electron transfer, the electroactive

species diffuses toward the electrode surface and efficient

exchange of electrons results when the species can closely

approach the electrode surface. To observe well defined

heterogenous electron transfer of CoTPPS4 in aqueous

solution, knowledge of the site of electron transfer of the

metalloporphyrin is important. If the complication in the

electrochemical behavior is caused by the CoTPPS4/electrode

interaction, the electrode surface can in principle be

modified accordingly to give well behaved electrochemistry

of this metal complex.









90

Modifiers and Promoters (Mediators)

Electrode surface structure in protein heterogeneous

electron transfer studies is of great importance and have

resulted in the development of modifiers and promoters.87-90

These molecules are able to exchange electrons with both the

protein and the electrode surface. Modifiers are molecules

which contain at least two functional groups. One

functional group binds with the electrode surface and the

other with the protein. The interaction of the second

functional group with the protein surface brings the

electrode closer to the electrode surface and results in

facile electron transfer. A promoter (mediator) molecule

functions as a modifier however, is free in solution and

does not bind to the electrode surface strongly.

The protein interacting functional group of a modifier

typically has a charge or can hydrogen bond with the pro-

tein.88-90 Table (4-1) shows some of the modifiers used for

electrochemical studies of proteins.

From the modifiers listed bis(4-pyridyl)bisulfide,

lysine, and X-(Pyridylmethylene)hydrazinecarbothioamide (X-

PMHC) are effective with proteins with positively charged

domain and 2-aminoethanethiol, 2,2'-Dithiobisethanamine and

methylviologen for negatively charged protein domain. The

three isomers of X-PMHC have the property of mediating both

domain type.90 Figure (4-1) shows how the differe




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