Title: Chemical modification of alpha-amanitin to yield derivatives suitable for conjugation to proteins via reductive alkylation
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Permanent Link: http://ufdc.ufl.edu/UF00102851/00001
 Material Information
Title: Chemical modification of alpha-amanitin to yield derivatives suitable for conjugation to proteins via reductive alkylation
Physical Description: Book
Language: English
Creator: Mullersman, Jerald Eric, 1956-
Copyright Date: 1986
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Bibliographic ID: UF00102851
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: ltuf - AEK6956
oclc - 15624652

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Words cannot fully express the gratitude that I wish to offer to

the many people who have encouraged and assisted me during the execu-

tion of this work. They have provided me with an environment in which

I have greatly enjoyed working and learning.

Many thanks go to Dr. James F. Preston who has shared with me his

delight in the mysteries of nature for many years. He has provided me

with numerous opportunities to test and develop my scientific abili-

ties and has given me patient and insightful counsel on innumerable


I would also like to thank the other members of my committee,

Drs. Hoffmann, Boyle, Small, and Wakeland. Each has earnestly sought

to enhance my education at e ery stage of this work.

In addition, I would like to gratefully recognize the assistance

of many other faculty members. Dr. Lonnie Ingram has helped at every

turn, sharing freely of equipment, advice, and his good nature. Dr.

John Gander provided advice on several matters and instruction in the

use of the CD spectrometer and digital polarimeter. Sandra Bonetti and

Roy King were instrumental in the acquisition of NMR spectra and ele-

mental analysis data, respectively.

I want to thank fellow graduate students, Mike Little, Tony

Romeo, and Dave Dusek, for their friendship and receptive ears.



ACKNOWLEDGEMENTS. ...................... .... ... ... ..

LIST OF ABBREVIATIONS.... ..... ..............

ABSTRACT.. .. . . .. .. . . . . . . .

Modified Proteins As Therapeutic Agents...........
Conjugates of Amatoxins with Proteins.............
Toward Optimal Conjugation of Small Molecular Weight
Cytotoxic Agents to Proteins.................
Rationale for the Current Work. ........ ......

Introduction .. . . . .. .. . . . . .
Materials and Methods...... .... ... .......
Results.......... . .. .. . . . . . . .
Discussion... . .. . .. . . .. . . . .

Introduction.. ... . . . . . . . . . .
Materials and Methods.... ..... .... .... ...
Results and Discussion.....................

Introduction............ ... ... ...... .....
Materials and Methods.......... ... .......
Results.... . . . . . . . , ,
Discussion... .. . . .. .. .. . . .. .

CYAN080ROHYDRIDE... ........... ......
Introduction...... . .. . . . .. . . . ..
Materials and Methods.....................
Results........... . . . . . . . . . . .
Discussion...... . . . . . . . . . . .

CHAPTER VI. CONCLUSIONS........ ... ...........

BIBLIOGRAPHY... . .. . . . . . . . . .. ..

BIOGRAPHICAL SKETCH.......................................











BSA.................bovine serum albumin

CD..................circular dichroism

cinn-HC1........,...trans-cinnamaldehyde-C fumes; TLC spray reagent

CT RNAP II..........calf thymus RNA polymerase II

d6-DMSO.............perdeuterated dimethylsulfoxide


EDTA................ethylenediaminetetractc acid

Fab.................univalent antigen-binding antibody fragment

(Fab')2..........divalent antigen-binding antibody fragment

FAB-MS..............fast atom bombardment mass spectroscopy

HEPES...............N-2-hydroxyethylpipeaznN-2thesloc acid

HPLC................high-performance liguid chromatography




OMA-X (X =CN, C00H, gly, NH
and pro).......amanitin2dlerivatives defined in the text





PIPES...............piperazine-N,N'-bis(2ehnsloi acid)

PMR................,proton magnetic resonance




RF...............index of TLC mobility; quotient of analyte and
and solvent front migration distances

RNA.................ribonucleic acid

RNAP................RNA polymerase

SMWCA...............small molecular weight cytotoxic agent

TCA.................trichloroacetic acid


TFA.................trifluoroacetic acid

TLC.................thin-layer chromatography


TSP.................sodium 3-trimethylsily1-d4-propanoate

UTP.................uridine triphosphate


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



December, 1986

Chairman: James F. Preston, III
Co-chairman: Edward M. Hoffmann
Major Department: Microbiology and Cell Science

A toxic peptide which inhibits RNA polymerase II (RNAP II),

e-amanitin, was chemically modified to generate aldehydic derivatives

which were suitable for reductive coupling to proteins.

Periodate oxidation of 6'-0-methyl -a-amanitin (0MA) at neutral pH

generated a mixture of two amanitin aldehydes which underwent ready

interconversion. These two forms of 6'-0-methylaldo-a-amanitin (0MAA)

were reduced to the corresponding alcohol with sodium borohydride, but

were inert to treatment with sodium chlorite at pH 2.0 or 3.0. A

chemically distinct form of OMAA was produced by periodate oxidation

at pH 2.0. Spectral data, in combination with the finding that this

compound is easily oxidized to the corresponding carboxyl derivative

by sodium chlorite at pH 2.0, indicated that this latter form of OMAA

contains a free aldehyde group.

The reductive amination of OMAA with several different amines

proved to be very slow, even when high reactant concentrations were

used. The products of reaction with ammonium acetate, glycine, and

L-proline were all relatively poor inhibitors of calf thymus RNAP II

with K 's of 1.7 X 10-7 M, 2.5 X 10-7 M, and 7 X 10-6 M, respec-
tively. Conversion of the aldehyde moiety of OMAA to carboxyl and

nitrile groups yielded derivatives with K.'s of 1 X 10-7Man
1 n
3 X 10- M, respectively. These data, in conjunction with those

previously published, suggest that introduction of ionizable groups
into this part of the amanitin molecule substantially diminishes its

inhibitory potential.

An aromatic amine attached to an amino sugar via a dipeptide

linker was synthesized and coupled to the 7-position of the hydroxy-

tryptophan residue of (r-amanitin to yield a novel azo amanitin

(ABGG-GLU). Marked effects of borate, temperature, cyanoborohydride

concentration, nature of the sugar residue, and pH upon the rate of

conjugation of a model compound to bovine serum albumin (BSA) were

delineated. Conjugation of ABGG-GLU itself to BSA was complicated by

side reactions. Several combinations of reaction conditions were

examined to identify ways to lower the rate of loss of ABGG-GLU to

these side reactions. Raising the concentrations of BSA and ABGG-GLU

provided the most effective means of achieving this goal. ABGG-GLU-BSA

conjugates demonstrated Ki's of approximately 10-7 M, which are
comparable to those obtained by others for amanitin-BSA conjugates.

The potential utility of this conjugation method is discussed.



Modified Proteins as Therapeutic Agents

Study of the chemical modification of proteins began largely as

an approach to understanding the role that particular chemical moie-

ties play in the structural integrity and function of proteins. Selec-

tive modification is still applied in this way and is an area of in-

vestigation in which significant strides continue to be made. In addi-

tion to its continued use in research activities, protein modification

is currently used to produce a large variety of products. For example,

proteins labeled with various enzymes and dyes are being used as very

important scientific and diagnostic tools.

A relatively new area which has been under development is the

application of modified proteins as therapeutic agents. The approaches

to using modified proteins as therapeutic agents are legion (Sezaki

and Hashida, 1984; Zaharko et al., 1979). Some simply aim at utilizing

a protein as a convenient polymeric support to which a drug can be

attached with the hope of a favorable alteration of the drug's pharma-

cokinetic properties (Hurwitz et al., 1980), but considerable atten-

tion has been focused more recently on the use of proteins which bind

to specific structures in order to help a therapeutic agent to "home

in" on the cell or tissue which is the desired target.

This latter avenue has been most extensively studied with the

hope of developing better ways to treat neoplastic conditions, since

current therapies for many cancers are associated with high levels of

morbidity and mortality and often have relatively low efficacy. Anti-

bodies that recognize tumor-associated antigens (Zalcberg and

McKenzie, 1985) have come to be the preferred target-specific carriers

of therapeutic agents (Ghose et al., 1983; 01snes and Pih1, 1982;

Thorpe et al., 1982), especially since the hybridoma method (Kijhler

and Milstein, 1975) has made antibodies of a single specificity avail-

able in almost unlimited quantities.

Conjugates of Antibodies with Protein Toxins

The development of modified antibodies as cancer therapeutic

agents has proceeded primarily along two lines, conjugates made with

enzymes and those made with a variety of small molecular weight cyto-

toxic agents (SMWCAs). The enzymatic conjugates have been prepared

mainly with toxins which inactivate ribosomes and are called immuno-

toxins (ITs) (Blythman et al., 1981). The ribosome-inactivating pro-

teins which have been employed to prepare ITs (reviewed in Stirpe and

Barbieri, 1986) include ricin (Jansen et al., 1982; Raso, 1982;

Vitetta et al., 1982), diphtheria toxin (Moolten et al., 1982),

gelonin, mordeccin, abrin, pokeweed antiviral protein, and saporin
(Thorpe et al., 1985a).

Although many ITS have shown potent and specific toxicity toward

cells in vitro, application of these conju ates to the treatment of

tumor-bearing animals has generally yielded discouraging results. As a

consequence, clinical application of ITs has evolved most rapidly only

in the few circumstances where they can be used in vitro. This has

mainly been limited to freeing bone marrow of unwanted populations of

cells before it is transplanted (e.g., Filipovich et al., 1985).

In part because of the great promise of monoclonal antibodies and

their conjugates in many therapeutic and diagnostic areas, several

studies have been undertaken during the last few years in order to

identify those factors which influence the localization of antibodies

and their conjugates in whole animals. The important parameters which

have been identified by these studies include the kinetics of conju-

gate access to the target cells and the kinetics of conjugate clear-

ance. [These factors and many other issues related to the in vivo

performance of antibody conjugates have been critically reviewed

(Poznansky and Juliano, 1984).] From the available data, it appears

that the accessibility of tumor cells to conjugates is most strongly

correlated with the molecular weight of the conjugate. Smaller frag-

nents of antibodies have more rapid access to the cells which lie in

extravascular tissue spaces (Herlyn et al., 1983; Houston et al.,

1980). The localization of (Fab')2 fragments to target cells is not

only more rapid but more extensive than that of whole antibody. How-

ever, the utility of very small antibody fragments, such as Fab frag-

ments, may be limited by their loss from the circulation due to glo-

merular filtration (Arend and Silverblatt, 1975; Covell et al., 1986).

An even more important consideration is that the conjugate must not be

quickly swept from the bloodstream. Three groups of workers have re-

cently reported their studies on the mechanism of rapid clearance of

ricin and its conjugates from the circulation (Bourrie et al., 1986;

Skilleter and Foxwell, 1986; Worrell et al., 1986a; Worrell et al.,

1986b). They have found that these proteins were cleared from the

bloodstream in minutes by hepatic nonparenchymal cells which recognize

the mannose-containing oligosaccharides borne by ricin. This has

prompted efforts to modify ricin either enzymatically (Foxwell et al.,

1985) or chemically (Skilleter et al., 1985; Thorpe et al., 1985b) in

order to abrogate this unwanted problem. Pokeweed antiviral protein is

not a glycoprotein (Stirpe and Barbieri, 1986) and so would not be

expected to be cleared from the circulation by the same sort of mech-

anism as ricin. In fact, Ramakrishnan and Houston (1985) have reported

circulatory half-lives for pokeweed antiviral protein-antibody conju-

gates of approximately 24 hours.

Conjugates of Antibodies with Small Molecular Weight Cytotoxic Agents

The other major approach to the production of novel cancer thera-

peutic agents by means of modification of antibodies has utilized

SMWCAs. In most instances, investigators have employed chemotherapeu-

tic drugs which are already being applied to the treatment of cancer.

Thus, the antibody moiety is intended to provide additional specifi-

city of action to a drug which already has some specific antitumor

activity. Drugs which have been commonly employed include methotrexate

(Garnett et al., 1983; Kanellos et al., 1985; Kulkarni et al., 1981),

daunorubicin (Arnon and Sela, 1982; Shen and Ryser, 1981), vindesine

(Ford et al., 1983), radioisotopes (Badger et al., 1985; Meares et

al., 1984), and various alkylating agents (de Weger et al., 1982).

These conjugates have typically had much lower potency in vitro than

those prepared with the protein toxins. Despite this, experiments with

animals and small clinical trials with cancer patients have, in some

cases, been quite encouraging.

Some of the same factors outlined above (e.g., size of the conju-

gate) which modulate the efficacy of the protein toxin conjugates in

vivo, may also influence the efficacy of the SMWCA conju ates. In

addition, there may be other mechanisms by which SMWCA conjugates are

cleared rapidly from the circulation by the reticuloendothelial sys-

tem. This will be discussed in greater detail below.

Immun~otoxins Versus Conjugates Made with Small Molecular Weight
Cytotoxic Agents

As of yet, a clear choice between ITs and SMWCA conjugates as the

superior alternative cannot be made. Each type of conjugate has its

own distinct advantages and disadvantages. Because of the extremely

high potency of the enzymatic toxins (Yamaizumi et al., 1978), ITs

need only contain a single molecule of toxin per antibody molecule.

Consequently, any deleterious effect on the antigen-binding activity

of the antibody molecule may be small. On the other hand, the low

molecular potency of the SMWCAs makes it desirable to prepare conju-

gates with as many SMWCA molecules linked to each antibody molecule as

can be tolerated. Unfortunately, difficulties with low solubility of

the conjugates and loss of antigen-binding activity often intervene at

relatively low levels of conjugation. For example, antibody conjugates

prepared with methotrexate have been reported to lose solubility and

antigen-binding activity with greater than ten (Kulkarni et al., 1981)

or twelve (Kanellos et al., 1985) molecules of methotrexate per anti-

body molecule. Ford et al. (1983) indicated that antibody conjugates

with more than eleven molecules of vindesine became poorly soluble.

Some workers have sought to circumvent this problem by linking the

SMWCA first to a highly soluble carrier and then coupling the loaded

carrier to an antibody. For example, Garnett et al. (1983) conjugated

an average of 32 molecules of methotrexate to each molecule of human

serum albumin. This drug-albumin conjugate was then linked to a mono-

clonal antibody. Another group of workers has used poly-L-glutamic

acid as an intermediate carrier for daunorubicin (Kato et al., 1984;

Tsukada et al., 1984). An advantage of the SMWCA conjugates is the

relative insensitivity of the SMWCAs to lysosomal hydrolases, while

the protein toxins are apparently very susceptible to proteolytic

digestion in 1ysosomes. Ammonium chloride has been found to greatly

increase the potency of ITs, presumably by suppressing the function of

lysosomes in the target cells (e.g., Jansen et al., 1982). Also in

their favor, the conjugated SMWCAs need not greatly increase the mo-

lecular weight of the antibody, while the protein toxins generally

have significant molecular weights (greater than 30,000 daltons). As

already discussed above, the higher molecular weight may have a sig-

nificant negative impact upon the rate at which the conjugate can

reach the tumor from the intravascular space.

Conjugates of Amatoxins with Proteins

Conjugation of amatoxins to proteins, which is the subject of

this work, has been studied in several laboratories (reviewed in

Faulstich and Finme, 1985). Amatoxins are the constituents of certain

mushrooms which cause the vast majority of fatal mushroom poisonings.

Work by Wieland and his colleagues has revealed the amatoxins to be

cyclic octapeptides which have several unusual amino acid residues

(Fi. I1).These peptides exert their toxic action by inhibiting the

H3C\ R2

C t O CH2 H CO
/ 'CtH ,`C/C"C"H NH
,N O= S '-', R



Fig. I-1. Structures of
amanitin R
a-amanitin OH
B-amanitin OH
v-amanitin OH
amanull in OH


some pertinent amanitins.
-2 -3


DNA-dependent RNA polymerases which are responsible for transcription

of nuclear genes. The inhibition of the class II enzyme, which gener-

ates messenger RNA, is particularly potent with a K of approximately
10-' M (reviewed in Wieland, 1983).

The first reports concerning amatoxin-protein conjugates arose

from efforts to prepare amatoxin-specific antisera. An amatoxin with a

free carboxyl group, R-amanitin, was linked to bovine serum albumin

(BSA) with the aid of a water-soluble carbodiimide. When administered

to rabbits, these conjugates proved to be much more toxic than the

free toxin (Bonetti et al., 1976; Cessi and Fiume, 1969). Subsequent

pathological studies showed that this increased toxicity was due to

uptake of the conjugate by Kupffer cells (Derenzini et al., 1973).

Thus, this represented accidental targeting of the toxin into the

reticuloendothelial system by virtue of its conjugation to BSA. An

increase in toxicity of this conjugate relative to free toxin was also

demonstrated for macrophages in culture (Barbanti-Brodano and Fiume,

1973; Fiume and Barbanti-Brodano, 1974).

A number of conjugates have also been prepared with azo

derivatives of a-amanitin (Faulstich and Trischmann, 1973; Preston et

al., 1981) which have been referred to as ABH and ABGG (short for

a-amanitiny1-7'-azobenzoy1-1,6-diaminohene and a-amanitiny1-7'-azo-

benzoylglycylglyci ne; previously called ADH and ADGG). Both of these

amatoxin derivatives have been coupled to proteins with the aid of a

water-soluble carbodiimide. When the toxicity of conjugates made from

BSA and ABH was tested on several cell lines, a positive correlation

between toxicity and phagocytic rate was found (Hencin and Preston,

1979). Conjugates prepared with the lectin concanavalin A proved to be

potent cytotoxins, the toxicity of which could be greatly diminished

by the addition of certain sugars which are bound by the lectin and

thus inhibit binding to the cell surface (Hencin, 1979). Linkage of

ABGG to a monoclonal antibody which recognizes Thy 1.2, an antigenic

marker of mouse T cells, yielded a conjugate with potent, specific

toxicity in vitro toward cells which bear the Thy 1.2 antigen (Davis

and Preston, 1981).

Because of certain characteristics of amatoxins, the amatoxin-

antibody conjugates fall within a class which is distinct from either

ITs or SMWCA-antibody conjugates. Amatoxins are like the ribosome-

inactivating enzymes used in ITs in that they are nonspecific cyto-

toxins, but they are like the SMWCAs in that they have a relatively

small molecular weight and resistance to hydrolases. Someday the

amatoxin-antibody conjugates may prove to be useful as therapeutic

agents and, as members of a distinct class, they may be uniquely

suited to some applications.

The amatoxins share with the SMWCAs a relatively low molecular

potency compared to the ribosome-inactivating toxins. Thus, one can

anticipate the need for relatively high levels of conjugation to anti-

body, especially in systems where there are few target antigenic sites

per cell. However, for amatoxin-antibody conjugates the importance of

retaining antigen-binding activity and favorable pharmacokinetic be-

havior (i.e., long circulatory half-life and rapid penetration to tar-

get cells) is more critical than for the conjugates made with the

chemotherapeutic drugs. This is true because the drugs possess consid-

erable selective toxicity toward many types of neoplastic cells with-

out being conjugated to an antibody, while amatoxins do not. Amatoxin-

protein conjugates can potentially kill any cell which takes them up.

These considerations have prompted a search for a mode of conjugation

of SMWCAs (as exemplified by amanitin) to antibodies which would re-

present a theoretical optimum.

Toward Optimal Conjugation of Small Molecular
Weight Cytotoxic Agents to Proteins

Because of the wide utility of chemically modified antibodies, a

number of investigators have compared the effect of different kinds of

modifications on the ability of the antibodies to retain their anti-

gen-binding activity. Most of the currently employed conjugation meth-

ods rely upon reaction with the amino groups of the antibody. This is

because many of the amino groups are disposed toward the surface of

the protein, they are reactive as nucleophiles at neutral to mildly

alkaline pH's, and they can be modified with high selectivity with

several classes of reagent.

A reasonable premise might be that for the conjugation of any

given chemical entity (e.g., SMWCA) to an antibody, a method which

preserves the native charges of the antibody should produce the least

possible alteration in its structure. In fact, modifications which

preserve the positive charge of the antibody amino groups, including

reductive alkylation and amidination, generally have a lesser effect

on antigen-binding activity than those modifications which abolish the

charge of the antibody amino groups (see below). Both amidination and

reductive alkylation are reactions which are highly specific for amino

groups of proteins. The utility of amidination is somewhat limited by

the harsh conditions required for generating the imidate esters which

are the amidinating reagents. On the other hand, reductive alkylation

with sodium cyanoborohydride (Borch et al., 1971) and an aldehyde has

received increasing attention as an excellent method for specifically

and gently modifying proteins (Hutchins and Natale, 1979).

Cohen and Becker (1968) have shown that the precipitin activity

of several different anti-hapten antibodies was greatly inhibited by

low levels of carbamylation, while it was preserved at high levels of

amidination with ethyl acetimidate. The carbamy1 and amidino groups

are similar in size and shape; they differ principally in that the

amidino group bears a positive charge, while the carbamy1 group is

uncharged. Thus, this study demonstrated especially well the impor-

tance of preserving the native charges of the antibody. Studies of

this kind have prompted the introduction of amidinating reagents for

the haptenation of antibodies to be used in hapten-sandwich tech-

niques. Wofsy and his colleagues (1978) have synthesized azo haptens

which can be coupled to antibodies at levels greater than thirty hap-

ten moieties per antibody molecule with good preservation of the anti-

gen-binding activity. A similar reagent for introducing the dinitro-

phenyl group has recently been reported which can be used to prepare

conjugates with up to thirteen dinitrophenyl groups per antibody mole-

cule without loss of antigen-binding activity (Hewlins et al., 1984).

Radiolabeling of antibodies by reductive methylation with tri-

tium-labeled sodium borohydride and formaldehyde has gained in popu-

larity because of the high degree of preservation of antigen-binding

activity which is achieved with this method (Tack and Wilder, 1981).

Reductive alkylation has, however, not been frequently used to attach

larger chemical moieties to antibodies despite the good results which
have been obtained with reductive methylation.

In addition to deleterious effects on antigen-binding activity,

chemical modifications of antibodies which remove positive charges may

also depress their circulatory half-life. Several kinds of chemical

modification have been found to promote clearance of proteins from the

circulation of mammals. These modifications include acylation, reac-

tion with formaldehyde, and dinitrophenylation. Much of the work on

acylated proteins has focused on lipoproteins (Brown et al., 1980).

Cells of the reticuloendothelial system bear specific, high-affinity

receptors for certain acylated proteins (Mahley et al., 1980;

Nagelkerke et al., 1983; Pitas et al., 1985). Formaldehyde-modified

albumin is taken up by the same cells, but apparently by a separate

receptor (Blomhoff et al., 1984; Horiuchi et al., 1985; Horiuchi et

al., 1986). Dinitrophenylated albumin has also been found to be

cleared by the reticuloendothelial system, primarily in the liver

(Kitteringham et al., 1985; Rhodes and Aasted, 1973; Skogh, 1982;

Skogh et al., 1983). These three classes of chemical modification each

reduce the number of native positive charges on the proteins and thus

make them more anionic. In the case of the receptor for acylated lipo-

proteins it has been specifically shown that certain polyanions can

compete for binding to the receptor (Mahley et al., 1980). This sug-

gests that the anionic character of the modified protein contributes

to its affinity for this receptor. Two groups of workers have observed

a correlation between increasing localization to the liver of anti-

bodies coupled with diethylenetriaminepentaacetic acid, a metal che-

lator, and increasing levels of conjugation (Anderson and Strand,

1985; Sakahara et al., 1985). In these studies, the chelator was

linked to the antibodies by an acylation reaction. Winkelhake (1977)

has also noted substantial decreases in the circulatory half-life of

an acylated antibody, while reductively methylated antibody was

cleared much more slowly.

Unfortunately, there is relatively little information concerning

the fate of SMWCA-antibody conjugates in vivo. The information ob-

tained in other systems, as described above, suggests that the most

frequently used method of preparing SMWCA-antibody conjugates (i.e.,

acylation) may not be optimal. The available data also suggest that a

conjugation method which preserves the native charges of the antibody,

such as amidination or reductive alkylation, may provide better reten-

tion of antigen-binding activity and longer circulatory half-life.

Rationale for the Current Work

The studies described below have been directed toward the devel-

opment of a practical method by which a suitable amatoxin derivative

can be conjugated to proteins by means of reductive alkylation. Devel-

opment of a method of this kind would provide a future opportunity to

examine its usefulness relative to existent methods for conjugation of

amatoxins to antibodies, especially with regard to any effects on

antigen-binding activity and circulatory half-life in animals.

Preparation of an amatoxin useful for reductive alkylation

requires the introduction of an aldehyde group into the molecule.

Fig. I-2 shows the sites at which a-amanitin (AMA), the most readily

available amatoxin, can be easily and selectively modified. The

dihydroxyisoleucine residue (site A) is susceptible to oxidation by

periodate to yield an aldehyde (Wieland and Fahrmeir, 1970). The

7-position of the hydroxytryptophan residue (site 8) is subject to

C H,





Fig. I-2. Sites for selective chemical modification of a-amanitin.

electrophilic substitution (Faulstich and Trischmann, 1973; Morris and

Venton, 1983). For example, relatively complex aromatic amines can be

diazotized and reacted with AMA to yield azo derivatives like ABGG.

Also, the hydroxyl group of the hydroxytryptophan moiety (site C) has

been alkylated with alky1 halides to prepare useful derivatives

(Faulstich et al., 1981).

Two of these three possible sites were chosen for evaluation.

Since periodate oxidation of the dihydroxyisoleucine residue yields an

aldehyde function in high yield, the feasibility of using this ap-

proach was examined. Also, since the azo derivative ABGG had already

been utilized to synthesize several potent conjugates, a derivative of

ABGG which contained an aldehyde function was prepared and studied.




The oxidation of 6'-0-methyl-a-amanitin (0MA) with sodium

periodate to yield 6'-0-methylaldo-a-amanitin (0MAA) was first

reported in 1970 (Wieland and Fahrmeir). Like amanullin (Fig. I-1),

OMAA was demonstrated to be nontoxic when injected into mice. However,

when the aldehyde function of OMcAA was reduced with sodium borohydride

to a hydroxyl group, the resultant derivative, 6'-0-methyldehydroxy-

methyl-a-amanitin (0MDA), proved to be toxic to mice. These and other

data prompted the hypothesis that the y-hydroxyl of the dihydroxy-

isoleucine (residue 3) sidechain was critical for the toxic action of

amatoxins (Wieland and Fahrmeir, 1970).

After it was learned that the amatoxins express their toxicity by

means of potent inhibition of RNA polymerase II (RNAP II), studies

showed that OMAA was a very poor inhibitor of RNAP II (Buku et al.,

1971), while amanullin was found to be a potent inhibitor (Cochet-

Meilhac and Chambon, 1974). The low toxicity of amanullin in mice has

been rationalized in terms of unusual pharmacokinetic properties. A

possible explanation for the low inhibitory activity of OMAA toward

RNAP II was found when its circular dichroism (CD) spectrum was

discovered to be greatly different from that of other amanitins. This

alteration in the CD spectrum and attendant loss of toxicity were

interpreted as being due to a change in the conformation of the

peptide backbone. It was proposed that the conformational change was

induced by the formation of an intramolecular hydrogen bond to the

oxygen of the aldehyde (Faulstich et al., 1973). Further support for

an altered conformation of OMAA has come from radioimmunoassay

studies. Faulstich and Wieland (1975) found that more than 120 times

as much OMAA as compared to AMA was required to displace half of the

radiolabeled amatoxin from amatoxin-specific antibodies.

Unpublished work by Dr. James F. Preston has suggested that there

are actually at least two products which result from the periodate

oxidation of OMA and which can be chromatographically separated on a

column of Sephadex LH-20 eluted with water. These oxidation products

demonstrated the same mobility on TLC and reacted with trans-

cinnamaldehyde and HC1 (cinn-HC1) to yield the rust color which is

distinctive of OMAA (Wieland and Fahrmeir, 1970). Each one also was

reduced by sodium borohydride to a compound with the characteristics

of OMDA, including violet color reaction with cinn-HC1 and potent

inhibition of calf thymus RNA polymerase II (CT RNAP II).

The nature of these two periodate oxidation products was reeval-

uated here in order to determine whether one or both components should

be used in reductive alkylation studies and to obtain insights into

their chemistry which might be pertinent to the intended conjugation

to protein amino groups.

Materials and Methods

Reagents. All chemicals were analytical reagent grade unless

otherwise specified. e-Tolylsulphonylmethylnitrosamide and

tetramethylsilane (TMS) were obtained from Aldrich Chemical; sodium

periodate, sodium borohydride, 0-glucose, and d6-dimethylsulfoxide

(dg-DMSO) from Sigma Chemical; and sodium chlorite and amidosulfonic
acid from Alfa Chemical. Solvents and trifluoroacetic acid (TFA) were

obtained from Fisher Scientific. Water used in the experiments

described in this and subsequent chapters was deionized and then

distilled from glass.

Toxin. The amatoxin AMA was purified from carpophores of Amanita

suballiacea (Murt.) Murr. collected in the Gainesville, Florida, area

by a modification of methods previously described (Little and Preston,

1984). Briefly, the toxin was obtained as follows: Coarsely chopped

carpophores (800 g) were extracted with 1.2 1 of methanol for

approximately 24 h. The filtered and concentrated crude toxin solution

was extracted three times with three volumes of diethyl ether. The

defatted aqueous phase was then mixed with nine volumes of methanol

and refrigerated overnight. This was then filtered to remove the

precipitated polar compounds (primarily carbohydrates and salts).

After concentration the toxin was partially purified by column chroma-

tography on Sephadex LH-20 eluting with 50% methanol, on Bio-gel P-2

eluting with water, and on Sephadex LH-20 eluting with water. The

remaining impurities were removed by high performance liquid chromato-

graphy (HPLC) on a Zorbax ODS column (0.94 X 25 cm, Du Pont) eluting

with 15% acetonitrile and/or by recrystallization from methanol.

Estimates of the concentration of aqueous amanitin solutions were
-1 -1
based upon an extinction coefficient at 304 nm of 15,400 M cm (cf.

Cochet-Meilhac and Chambon, 1974). Because of the pH-dependence of its

absorbance spectrum, measurements of absorbance at 304 nm of AMA were

made in 5 mM sodium phosphate buffer, pH 7.0. Yields of amanitin

derivatives were calculated from these estimates of concentration and

the volume of the solutions.

Analytical Procedures

Spectroscopy. Absorbance measurements were made with the aid of a

Gilford 2400 spectrophotometer. Absorbance spectra in both the ultra-

violet (UV) and visible ranges were obtained on either a Beckman 25

spectrophotometer or a Hewlett-Packard 8451A diode array spectrophoto-

meter. Circular dichroism (CD) spectra were recorded on a Jasco J-500C

spectropolarimeter with the aid of Jasco IF-500 and Okidata IF-800

data processing equipment. Proton magnetic resonance (PMR) spectra

were obtained on a Nicolet NT-300 spectrometer operating at 300 MHz in

the Fourier transform mode.

Chromatography. Thin-layer chromatography (TLC) was performed on

0.25 mm layers of silica gel 60 F254 (Merck). TLC solvent system I

contained 1-butanol: acetic acid: water (4:1:1). Amatoxins were

detected on chromatograms by means of their quenching of the layer's

fluorescence and by their color reaction after being sprayed with 2%

methanolic trans-cinnamaldehyde and subsequent exposure to HC1 fumes

(cinn-HC1). HPLC was performed with a Waters model 6000A pump which

was equipped with a U6K injector. Elution of compounds was detected by

a Gilson Holochrome variable wavelength absorbance monitor. Samples

were chromatographed on a Zorbax ODS column (0.94 X 25 cm, Du Pont)

which was protected by a 0.5 pm prefilter (Rainin) and a C5 guard

cartridge (Bio-Rad). Samples for HPLC were filtered through 0.22 pm

Miller GV or SR filters (Millipore).

RNA polymerase assay. Calf thymus RNA polymerase II (CT RNAP II)

was prepared and stored as previously described (Preston et al.,

1975). Components of the reaction mixture were those of Cochet-Meilhac

and Chambon (1974) except that C"H]UTP was employed for labeling the

product. The assay was performed as follows: The reaction tube with

10 pl of amanitin solution was incubated at 37*C for 10 min. The

enzyme solution was added to this and allowed to incubate for 10 min

more at 37*C. The reaction was started by adding a solution containing

the nucleoside triphosphates to yield a final reaction volume of 100

l.After 10 min at 37"C the reaction was stopped and processed for

scintillation counting as previously described (Preston et al., 1975).

Counting was performed on Beckman LS-133 and LS-8000 liquid scintil-

lation counters. Inhibition data were analyzed according to Dixon

(1953). The best fit of lines to data points was estimated by the

method of least squares using the statistical functions of a Texas

Instruments TI-55-II calculator.

Synthesis of Amanitin Derivatives

Synthesis of 6'-0-methy1-a-amanitin. To 30 plmol of AMA in 10.5 ml
of ice-cold methanol were added 4.5 ml of an ice-cold ethereal-

ethanolic solution (de Boer and Backer, 1954) of diazomethane which

was generated by the action potassium hydroxide on e-tolylsulphony1-

methylnitrosamide and adjusted to 0.10 M with ice-cold ether. The

diazomethane solution was titrated according to Arndt (1943) prior to

use. (Note: Diazomethane is a toxic and potentially explosive gas. It

should only be prepared and used in a well-ventilated hood, preferably

behind a safety shield, in glassware which lacks ground or abraded

surfaces.) The reaction mixture was permitted to rise to room tempera-

ture in a water bath in the dark. After 30 min the remaining diazo-
methane and the bulk of the ether were evaporated under a stream of

nitrogen. The resultant solution was concentrated to dryness in a
rotary evaporator at 30*C, redissolved in water, filtered, adjusted to
22% acetonitrile, and subjected to HPLC with the same solvent mixture.

The desired product eluted between 24 and 32 mi. The yield was 14.6

plmol (49%); 14.4 pmol (48%) of the AMA was recovered unchanged. TLC:

RF, I-0.47; color-reaction, violet.
Synthesis of 6'-0-methylaldo-a-amanitin under neutral conditions.
To 20.3 pmol of OMA in 1.0 ml of water was added 0.5 ml of a solution

containing 22.0 pmol of sodium periodate. After 5 min of reaction in

the dark at room temperature residual sodium periodate was quenched by
the addition of 10 pll of ethylene glycol. The solution was filtered,

adjusted to 22% with respect to acetonitrile, and subjected to HPLC
eluting with 22% acetonitrile. The desired products eluted between 28

and 45 ml. The yield was 18.8 pmol (92%). TLC: RF, I-0.43;
color -react ion ru st -red .

Synthesis of 6'-0-methyl dehydroxymethy1 -a- amani tin. 0MA (2.0
pmol) was oxidized with periodate as described above. The components
of the reaction mixture were separated by HPLC eluting with 18%

acetonitrile in order to achieve complete separation of the products.
The resultant fractions were placed on ice as they were collected.

Under these chromatographic conditions the products OMAA-IA and

DMAA-IB eluted between 57 and 67 ml and between 85 and 95 ml, respec-
tively. To 0.20 pmol each of OMAA-IA and OMAA-IB was added 1.0 pmol of

sodium borohydride in 80 pl of water. After gentle mixing, this was

permitted to react for an hour in the dark at 230C. Residual sodium

borohydride was quenched by reaction with 10 pl of 1.0 M D-glucose for

a further hour under the same conditions. The reaction mixture was

rinsed from the vial with 1.0 ml of 22% acetonitrile and then chroma-

tographed by HPLC using the same solvent. A single product eluted

between 29 and 34 ml in 75-90% yield. TLC: RF, I-0.51; color-reaction,

Synthesis of 6'-0-methylaldo-a-amanitin under acidic conditions.
To OMA (8 pimol) in 0.75 ml of 50 mM sodium phosphate, pH 3.0, was

added sodium periodate (8.8 pmol) in 30 pl of water. After reaction

for 5 min at 230C in the dark, the solution was adjusted to 22% with

respect to acetonitrile and applied to HPLC eluting at 1 ml/min with
acetonitrile: 0.05% TFA (22:78). The desired product (7.0 Clmol, 88%)

eluted between 30 and 35 ml. TLC: RF, I-0.61; color-reaction, violet.
One-half of this material was prepared for PMR studies. After the

acetonitrile was removed at 30*C in vacuo, the sample was lyophilized

and redissolved in 1.0 ml of d6-DMSO.


Synthesis of 6'-0-methy1-a-amanitin
The synthesis of OMA has been patterned after that of Wieland and

Fahrmeir (1970). The major disadvantage of this synthetic route is the

use of the dangerous diazomethane; alternatively, AMA can be methyl-

ated with methyl iodide (Faulstich et al., 1981). Studies on the

synthesis of OMA using diazomethane (Little, 1984) have demonstrated

that the choice of reaction conditions is quite critical. Both reac-

tant stoichiometries and concentrations are important. At stoichio-

metries and/or concentrations less than the optimum the yield of OMA

falls rapidly, but virtually quantitative recovery of input toxin is

achieved as the sum of AMA and OMA. At stoichiometries and/or concen-

trations greater than the optimum the yield of OMA falls because of

the production of byproducts, one of which has been identified as

1',6'-N,0-dimethyl-a-amanitin (Little, 1984). The conditions described

here are similar to those of Little (1984) and provide for recovery of

virtually all of the input toxin as the sum of OMA and recovered AMA.

OMA shares many characteristics in common with AMA, including

potent inhibition of CT RNAP II, UV absorbance spectrum, and violet

color reaction with trans-cinnamaldehyde. However, it can be readily

distinguished from AMA on the basis of a distinct CD spectrum and the

resistance of its UV absorbance spectrum to bathochromic shift upon

alkalinization (Faulstich et al., 1973; Faulstich et al., 1981).

Products of Periodate Oxidation of 6'-0-methyl-a-amanitin under
conditions of Neutral pH

HPLC purification of the periodate oxidation products. Separation

of the products of periodate oxidation of OMA by reverse-phase HPLC

yielded two components, just as was seen previously when the sepa-

ration was performed by conventional chromatography. These two

entities will be referred to as OMAA-IA for the material eluting first

and OMAA-IB for the material which eluted later (Fig. II-1). When the

HPLC fractions were held at room temperature, reanalysis at intervals

of the purified components by HPLC demonstrated a slow intercon-

version of the two forms (Fig. II-2). Within 2 h OMAA-IA elutingg at







IO 20 30 40 50


Fig. II-1. Preparative HPLC purification of various forms of OMAA
on a reverse-phase C18 column. The upper panel shows the purification
of OMAA-II with acetonitrile: 0.05% TFA (22:78) as mobile phase. The
lower panel shows the purification of OMAA-I with 22% acetonitrile as
the mobile phase. Fractions contained 1.0 ml.



0.005 -

E 0.004


O 0.002-

O 0.001



0 6 12 18 24 O 6 12 18 24

Fig. II-2. HPLC analysis of components of OMAA-I. Samples (30pl)
of OMAA-IA (upper tracings) and OMAA-IB (lower tracings) were
rechromatographed on a reverse-phase C18 column with 22% acetonitrile
at 2 ml/min at 2 h (panel A) and 26 h (panel B) after their initial
purification by preparative HPLC.

about 14 min) was contaminated with significant amounts of OMAA-IB

which eluted at about 19 min (upper portion of panel A) and after 26 h

further conversion was apparent (upper portion of panel B). An even

more dramatic conversion of OMAA-IB to OMAA-IA was observed (lower

portion of panels A and B).

Spectral and TLC properties of the periodate oxidation products.
The two OMAA-I components have UV absorbance spectra which are

indistinguishable from each other and very similar to that of OMA.

When held on ice, the interchange between the two products described

above was retarded. Indeed, when HPLC fractions which derived from

separation of the periodate oxidation products of OMA were placed on

ice immediately after they were collected, no contamination of one

form with the other could be detected by HPLC analyses repeated over

48 hours. This aided in the acquisition of CD spectra of the two

products in their pure forms. Thus, HPLC fractions were placed on ice

as they were collected and warmed to room temperature just before the

CD spectrum was obtained. HPLC analysis also showed that fractions did

not undergo significant transformation to the other form during the

time at room temperature which was needed for acquisition of the CD

spectrum. The CD spectra of the periodate oxidation products were

indistinguishable from each other and from that previously published

for OMAA (Fig. II-3).

A PMR spectrum of OMAA-I dissolved in d6-DMS0 showed only a very
small peak in the region expected to contain the signal of an

aldehydic proton (a=9.5-10). In addition, the spectrum lacked the

sharp definition of peaks which has been characteristic of the PMR

spectra of other amanitin derivatives (data not shown).





250 300



Fig. II-3. CD spectra of OMA (dashed line) and OMAA5I (solid
line) in water at concentrations of approximately 4 X 10" M.

Chemical reactivity of the periodate oxidation products.

Reduction of OMAA-IA and OMAA-IB with sodium borohydride produced in

75-90% yield a toxin derivative with the properties expected of OMDA.

The reduction products from these forms of OMAA were indistinguishable

in terms of UV absorbance and CD spectra (which were identical to

those of OMA), TLC and HPLC mobility, violet color reaction with

cinn-HC1, and inhibition of CT RNAP II with K. of 3.4 + 0.2 X 10-9 M.

In contrast to their ready reduction with sodium borohydride, neither

OMAA-IA nor OMAA-IB was susceptible to the action of the oxidant

sodium chlorite at pH 2.0 or 3.0 (Launer and Tomimatsu, 1954; Lindgren

and Nilsson, 1973).

Product of Periodate Oxidation of 6'-0-methyl-a-amanitin at pH 3.0

HPLC purification of the periodate oxidation product. In contrast

to the reaction at neutral pH, periodate oxidation of OMA at mildly

acidic pH's yielded only a single product which was distinct from the

periodate oxidation products described above and which could be puri-

fied by reverse-phase HPLC using an acidic mobile phase (Fig. II-1).

This form of OMAA will be referred to as OMA~A-II.

Spectral and TLC properties of the periodate oxidation product.
The material prepared and purified under acidic conditions had many

properties which were strikingly different from those of OMAA-IA and
OMAA-IB. TLC analysis of HPLC fractions (or of the reaction mixture)

showed predominantly a single component with higher mobility which

stained violet with cinn-HC1; there was trace contamination with the

rust-staining OMAA-I forms. TLC analysis of these same HPLC fractions

in solvent system I after they were neutralized with sodium phosphate

buffer and immediately spotted onto a TLC plate showed primarily the

rust-staining OMAA-I and a trace of the violet-staining OMAA-II. The

UV absorbance and CD spectra of OMAA-II were indistinguishable from

those of OMA. However, within thirty minutes of its neutralization

with sodium phosphate buffer the CD spectrum of OMAA-II changed to

that typical of OMAA-IA and OMAA-IB. A PMR spectrum of OMAA-II in

d6-DMS0 showed the general features expected of a methylamanitin
(Wieland et al., 1983) and also a peak of appropriate size at a=9.55

as expected for an a~dehyde (Fig. II-4).

Chemical reactivity of the periodate oxidation product. When both

sodium chlorite and sodium periodate were added to a solution of OMIA

which was buffered at pH 2.0 or 3.0, a novel product was generated.

This product has been identified as the carboxylic acid (0MA-C00H)

which results from the oxidation of the aldehyde function of OMAA (see

chap. III).


The chemical nature of OMAA is of interest for at least two

reasons. First, since OMAA-I has an altered conformation, detailed

information about its nature can provide some insight into the forces

which maintain amatoxins in their toxic conformation. Second, know-

ledge of its chemistry provides a sound base upon which the planning

of semisynthetic modifications to this part of the toxin can rest. It

is perhaps because the chemical nature of OMAA has been misunderstood

that very little in the way of synthetic modification of the aldehyde

group has been reported since the preparation of OMAA was first de-

scribed by Wieland and Fahrmeir in 1970. The only report of a modifi-











E re


o .



*r- rdI
LL. 3





cation of this kind has been the synthesis of a dinitrophenylhydrazone

in modest yield by reaction of a periodate oxidation product of AMA

with dinitrophenylhydrazine (Morris and McSwine, 1983).

Until recently, OMAA has been thought to be a true aldehyde.

Faulstich et al. (1973) proposed that the altered conformation of OMAA

(as evidenced by its unique CO spectrum) was due to an intramolecular

hydrogen bond between the carbonyl function of the aldehyde and the

peptide backbone. Garrity and Brown (1978) reported an infrared spec-
trum of OMAA with bands at 2800 cm-1 and 1350 cm-1 which were thought

to arise from the carbonyl of the aldehyde group. This view has been

challenged by Morris and McSwine (1983) who were unable to detect a

signal characteristic of an aldehydic hydrogen in the PMR spectrum of

a periodate oxidation product of AMA. They have proposed that the

aldehyde of aldoamanitins exists primarily as an intramolecular adduct

with a nucleophile such as an amide nitrogen (e.g., Fig. 11-5). The

data presented in this chapter support this hypothesis, but do not

provide direct proof of it.

An important indication that OMAA-I does not exist primarily as a

free aldehyde is its stability. OMAA-I has been manipulated and stored

for years in the unbient atmosphere in our laboratory without loss of

the material. This is atypical of most true aldehydes, which usually

must be stored under an inert atmosphere to prevent oxidation.

The available data suggest that the two forms of OMAA-I, although

chromatographically distinct, are very similar in other respects. They

both resemble OMAA, as it has been previously described by others, in

terms of CD spectrum, color reaction with cinn-HC1, and reduction to

form 0MDA (Wieland and Fahrmeir, 1970; Faulstich et al., 1973). The


\ I

I O,

Hz, C CH
\ I

11 I
-C-N-C H--

- C -N-CH -C- N -CH -

" I



\ /C*




Fig. II-5. Scheme of periodate oxidation of the dihydroxyiso-
leucine sidechain and hypothetical intramolecular reaction of the
aldehyde group. The asterisk marks the new chiral center formed by the
intramolecular reaction.

studies with the HPLC-purified forms of OMAA-I have shown that they

slowly interconvert in solution at room temperature. n equilibrium

between the two components is reestablished from one of the purified

forms over the course of several hours. This slow rate of intercon-

version is in sharp contrast with the short period of time (no more

than a few minutes) which is required to establish the equilibrium

following the periodate oxidation of OMA in neutral solution. A simple

explanation for this discrepancy might be that neither form of OMAA-I

is the entity first generated by the periodate oxidation (i.e., the

free aldehyde), but rather they derive from it. Thus, the free alde-

hyde, when formed under conditions of near-neutral pH, rapidly and

almost completely converts to the two forms of OMAA-I. The components

of OMAA-I might be interconverting by a slow back-reaction to the free

aldehyde followed by formation of the other component.

The PMR spectrum of OMAA-I in d6-DMS0 showed only a very small

signal with a chemical shift which would be expected for an aldehyde.

Thus, our PMR spectral data confirm those of Morris and McSwine

(1983). The inertness of 0MAA-I to reaction with sodium chlorite has

provided a further piece of evidence that supports the hypothesis that

it is not primarily a free aldehyde.

The discovery that periodate oxidation of OMA under mildly acidic

conditions generates a distinct entity has added greatly to our under-

standing of the nature of OMAA. This novel product, OMAA-II, appears

to be a free aldehyde by spectroscopic (characteristic signal by PMR)

and chemical (oxidation to carboxylic acid by sodium chlorite) crite-

ria. The demonstration by both TLC and CD spectroscopy that OMAA-II

changes to OMAA-I within minutes of neutralization of the solution

lends credence to the hypothesis outlined above that the periodate

oxidation of OMA yields a free aldehyde (0MAA-II) which converts

rapidly to the two forms of OMAA-I in solutions of near-neutral pH.
The finding that the CD spectrum of OMAA-II is indistinguishable

from that of OMA is very interesting. This suggests that the presence

of the aldehyde group per se does not cause the alteration in the

toxin's conformation. This indicates that the hydrogen bond hypothesis

for the altered conformation (Faulstich et al., 1973) is probably

incorrect. The altered conformation of OMAA-I is associated with sev-

eral characteristics, including a PMR spectrum which is not character-

istic of an aldehyde, inertness to oxidation by sodium chlorite, and

the presence of two similar, yet chromatographically distinguishable,
forms. The probable explanation for this behavior of OMAA-I is that it

exists in solution as a mixture of two diastereomers which are gener-

ated by the reaction of the aldehyde moiety with an intramolecular

nucleophile. The a-amino group of the hydroxytryptophan residue is a

reasonable candidate as this nucleophile on several grounds. As shown

in Fig. II-5, reaction of the aldehyde group with this amide nitrogen

to yield an alkanolamide would form a five-membered ring (thermody-
namically favored?) with a new chiral center arising from the r-carbon

of residue 3 (marked with an asterisk). The generation of this addi-

tional chiral center provides for the possibility of two chromat'ograph-

ically separable diastereomeric forms. There have been reports of the

ready formation of cyclic alkanolamides from the periodate oxidation
of an appropriate dio1 (e.g., van Tamalen et al., 1960). This proposal

for the structure of OMAA-I also provides a ready rationale for both

the inertness of OMAA-I to sodium chlorite and the stability of

DMAA-II at mildly acidic pH's. The interconversion between alkanol-

amides and their constituent aldehyde and amide are known to proceed

very slowly at mildly acidic pH's, but at much higher rates with

increasing pH (Hubert et al., 1975; Jencks, 1969, p. 495). Thus, at

the mildly acidic pH's at which the oxidation with sodium chlorite was

attempted OMAA-I was locked in the alkanolamide form and could not be

oxidized. On the other hand, the reduction of OMAA-I with sodium boro-

hydride was conducted at near-neutral pH where the conversion to free

aldehyde would be permitted. When OMA is oxidized in an acidic medium,

the formation of the alkanolamide is inhibited and OMAA-II, the free

aldehyde, can be isolated or oxidized by sodium chlorite. However,

when a solution of OMAA-II is neutralized the thermodynamically more

stable OMAA-I rapidly forms.

Although the proposed structure of OMAA-I is strictly hypothet-

ical, it is worthwhile to consider how the toxin's conformation might

be altered by this sort of intramolecular reaction. First, crystallo-

graphic (Kostansek et al., 1978; Wieland et al., 1983) and NMR spec-

troscopic (Wieland et al., 1983) studies indicate that the al-amino of

the hydroxytryptophan residue is hydrogen bonded to the terminal

carbonyl of the asparagine residue. Formation of the alkanolamide

would necessarily disrupt this hydrogen bond and perhaps destabilize

the native conformation. Also, formation of the alkanolamide would

generate what has been referred to as a "bridged lactam". Methods for

intentionally introducing bridged lactams into peptides have been

recently developed and applied to the synthesis of peptides with

conformationally constrained backbones (Freidinger et al., 1980;

Freidinger et al., 1982).

As will be seen in the next chapter, the chemical nature of

OMAA-I seems to have a very large impact upon the rate and course of

its reductive amination. Additional observations will be presented and

discussed which indirectly support the proposed structure of OMAA-I.




Much of our knowledge about the relationship between structure

and activity of amatoxins which differ at residue 3 (residue 3

corresponds to the dihydroxyisoleucine residue of AMA) has derived

from the existence of three naturally-occurring variants in this

sidechain (Fig. I-1, R2). These variants correspond to three levels of

hydroxylation: (1) no hydroxylation, (2) a hydroxyl group on the

r-carbon, or (3) hydroxyl groups on both the y- and a-carbon atoms

(Wieland, 1983). Increasing extent of hydroxylation has been found to

correlate with a modest increase in inhibitory activity toward CT RNAP

II (Cochet-Meilhac and Chambon, 1974). In addition, the semisynthetic

derivative OMDA has been prepared which contains a r-hydroxyvaline

residue. This hydroxyvaline residue is a close homolog of the isoleu-

cine residue of amanullin. Thus, it is not surprising that OMDA and

6'-0-methyl amanullin (0ML) have similar inhibitory activity toward CT

RNAP II (Cochet-Meilhac and Chambon, 1974). A totally synthetic ama-

toxin analog which has norvaline (an amino acid without a B3-branch) at

this position is a very poor inhibitor of RNAP (Wieland et al., 1981).

However, the significance of this latter finding is unclear, since the

effect of this structural change on the overall conformation of the

peptide has not been examined. One can see that these data relate to

variations in the structure of this sidechain which are both very

conservative and which result in small changes in inhibitory activity.

In particular, no charged groups had been introduced into this residue

at the time that this work was begun.

Although the exact nature of the site on the RNAP molecule to

which amatoxins bind has not been elucidated, some information about

the manner in which amatoxins inhibit RNAP has been gathered. Although

their mode of inhibition is apparently noncompetitive (Cochet-Meilhac

and Chambon, 1974), the amatoxins may inhibit RNAP by binding to a

catalytic subsite. Both genetic (Greenleaf, 1983) and biochemical

(Brodner and Wieland, 1976) studies have implicated one of the large

subunits, which presumably contributes part of the active site, as

being involved in amanitin binding. Also, some recent work has led to

the proposal that amatoxins may inhibit RNAP by binding to a portion

of the catalytic site which is involved in translocation of the

nascent RNA molecule (Vaisius and Wieland, 1982).

Since the amatoxins bear no close resemblance to RNAP's sub-

strates or products, it has been difficult to conceptualize their

mechanism of inhibition and to generate a hypothetical framework upon

which studies of structure-activity relationships can be planned.

Thus, although reductive coupling of OMAA to protein amino groups was

envisioned as a possibly useful route for the conjugation of amatoxins

to proteins, there did not exist a large body of knowledge concerning

the likely effect of this kind of modification on the activity of the

toxin nor was there a theoretical basis upon which to predict such

effects. In addition, the results presented in the previous chapter

suggested that the forms of OMAA which could be useful for conjugation

to protein amino groups, e.g., OMAA-IA and OMAA-IB, do not have a free

aldehyde function and also that the form of OMAA which has a free

aldehyde, OMAA-II, is present in solutions of neutral pH in very small

quantities. It was reasonable to anticipate that this diminished

availability of the aldehyde group would have a negative impact upon

the rate of reductive coupling to amines. However, it was difficult to

predict a prior the magnitude of this effect.

In order to assess the importance of these factors, the reductive

coupling of OMAA-I to some simple amines was examined (Fig. III-1). In

addition, two oxidative transformations of the aldehyde group (to

carboxyl and nitrile functions) have been accomplished. These two

additional alterations of this sidechain provide conservative changes

in the structure and additional important information about the

structural requirements for potent inhibition of RNAP II.

Material and Methods


Reagents. Ammonium acetate, glycine, and L-proline were obtained

from Sigma Chemical. Sodium cyanoborohydride (Sigma Chemical) was

recrystallized (Jentoft and Dearborn, 1979) and kept desiccated over

P205. Hydroxyl amine-0-sulfonic acid was purchased from Alfa Chemical.
Triethylamine (TEA) was obtained from Pierce Chemical. The source of

other reagents has been described in the previous chapter.

Toxin. The syntheses of OMA and OMAA have been described in the

previous chapter. These two compounds served as the starting material

for all of the semisynthetic derivatives described here.








-NHCH2 cooH



OMA gly

OMA- pro

OMA- amine

Fig. III-1. Scheme showing the reductive amination of OMAA.

Analytical Procedures

Spectroscopy and chromatography. Absorbance and CD spectroscopy
and chromatographic procedures were performed as described in chapter
II. TLC sovent system II contained 2-butanol: methanol: 0.5 M sodium

chloride (4:2:1). Fast-atom bombardment mass spectroscopy (FAB-MS) was

performed by Triangle Laboratories (Durham, NC) using a VG 7070E mass

spectrometer. In some cases HPLC solvents contained a buffer instead

of water; this buffer contained 20 mM TFA which was adjusted to pH 3.0

with TEA and will be referred to as TFA-TEA buffer (Cohen et al.,


RNA polymerase assay. The analysis of inhibition of CT RNAP II
was conducted as described in the previous chapter.

Synthesis of Amatoxin Derivatives
Desalting of toxin solutions. Certain toxin solutions were

desalted (Bibhlen et al., 1980) with the aid of Sep Pak C18 cartridges

(Waters Associates). The C18 cartridges were prefinsed with 20 ml of

50% acetonitrile followed by 10 ml of water. Aqueous solutions of

toxin were loaded onto the cartridge at a rate no greater than

1 ml/min. After the cartridge was washed with 15 ml of water, the

toxin was finally eluted with 10 ml of 50% acetonitrile. When OMA-NH2
was desalted, this protocol was modified in that 20 mM ammonium

bicarbonate was substituted for water at each step.

Synthesis of OMA-NH To 10 ml of molar methanolic ammonium

acetate was added 6.3 mg (0.1 mmol) of sodium cyanoborohydride. A

portion of this solution (1.0 ml) was added to 2.0 plmol of lyophilized
OMAA. The reaction was allowed to proceed in the dark at 230C for 18

days. The solution was concentrated to dryness at 300C in vacuo,

redissolved in acetonitrile: TFA-TEA buffer (22:78), and chromato-

graphed by HPLC using the same solvent at 0.5 ml/min. The desired

product eluted as the major product between 21 and 26 ml. This mate-

rial was reconcentrated to dryness at 30*C in vacuo, dissolved in

acetonitrile: TFA-TEA buffer (15:85), and rechromatographed by HPLC in

this solvent at 0.5 ml/min. The toxin derivative eluted from the

column between 45 and 57 ml. This was concentrated at 300C in vacuo to

remove the acetonitrile and then desalted using the appropriate Sep

Pak C18 protocol (see above). The eluate from the Sep Pak C18 car-

tridge was concentrated at 300C in vacuo to remove the acetonitrile,

supplemented with 10 ml of water, and lyophilized. The final yield was

0.55 pmol (28%). TLC: RF, I-0.60, II-0.83; color-reaction, violet.
FAB-MS: [M+H] expected, 902.37; found, 902.33; [M-H] expected,

900.37; found, 900.14.

Synthesis of OMA-gly. Glycine (0.751 g, 10 mmol) and sodium

cyanoborohydride (31.5 mg, 0.5 mmol) were dissolved in sufficient 10%

methanol to make 5.0 ml of solution. Lyophilized OMAA (2.0 pmol) was

dissolved in 0.10 ml of this solution. After 96 h in the dark at 230C

the reaction mixture was taken up in 22% acetonitrile and chromato-

graphed by HPLC using the same solvent at 0.5 ml/min. The desired

product eluted between 16 and 25 ml yielding 1.68 pmol (84%). TLC: RF'
1-0.46, II-0.46; color-reaction, violet.

Synthesis of OMA-pro. A methanolic solution (0.10 ml) containing

0.5 M L-proline and 0.05 M sodium cyanoborohydride was added to 2.0

pmol of OMAA with dissolution of the toxin. After 170 h in the dark at

23*C the reaction mixture was taken up in 22% acetonitrile and applied

to HPLC using the same solvent at 0.5 ml/min. The desired product,

which eluted between 26 and 29 ml, was concentrated to dryness in

vacuo at 300C, redissolved in 15% acetonitrile, and rechromatographed

by HPLC using this solvent at 0.5 ml/min. The product (1.45 plmol, 73%)

eluted between 69 and 77 ml. TLC: RF, I-0.37, II-0.36; color-reaction,

Synthesis of OMA-C00H. A solution (0.4 ml) containing 0.015 M

sodium chlorite, 0.030 M amidosulfonic acid, and 0.10 M sodium phos-

phate, pH 2.0, was added to 2.0 pmol of OMA. To this was immediately

added 10 pl of a solution containing 0.24 M sodium periodate and 0.1 M

sodium phosphate, pH 2.0. After 10 min in the dark at 230C the toxin

solution was desalted using the Sep Pak C18 technique, concentrated to

dryness in vacuo at 40*C, redissolved in acetonitrile: TFA-TEA buffer

(18:82), and chromatographed by HPLC using the same solvent at 1.0

ml/min. The desired product eluted between 47 and 51 ml. After concen-

tration in vacuo at 3000 to remove acetonitrile the toxin solution was

desalted again to yield 0.85 pmol (47%). TLC: RF, I-0.49, II-:0.29;

color-reaction, violet. FAB-MS: [M+H] expected, 917.34; found,

917.27; [M-H] expected, 915.33; found, 915.49.

Synthesis of OMA-CN. Hydroxylamine-0-sulfonic acid (22.6 mg, 0.2

mmiol) was dissolved in sufficient 1.0 M sodium borate, pH 9.0, to make

1.0 ml of solution. A portion of this (0.20 ml) was used to dissolve

2.0 pmol of lyophilized OMAA. After 72 h in the dark at 230C the

reaction mixture was taken up in 22% acetonitrile and chromatographed

by HPLC using the same solvent at 1.0 ml/min. The desired product

(1.16 Ipmol, 58%) eluted between 36 and 45 ml. TLC: RF, I-0.63,
II-0.85; color-reaction, violet.

Results and Discussion

Reductive Amination of OMAA-I

Reaction of OMAA-I with ammonium acetate in the presence of

sodium cyanoborohydride was the first reductive amination reaction

attempted. This reaction would be expected to yield the primary amine

OMA-NH2 (Fig. III-2, structure VI with R=R'=H), the simplest amine

accessible via this route. Several unexpected difficulties were

encountered in the synthesis of OMA-NH2. Even when ammonium acetate
was included at a stoichiometry of 100 to 1 relative to OMAA, the

major products appeared to be dimeric and/or trimeric products (cf.

Borch et al., 1971). That is, they were bound by a cation-exchange

resin (Sephadex SP-50), yet were unreactive with fluorescamine. Their

appearance was suppressed by increasing the molar ratio of ammonium

acetate relative to OMAA from 100 to 500. However, instead of a single

fluorescamine-reactive entity being produced under these new condi-

tions, there were several. The concern was that OMAA and/or the

desired product were undergoing a secondary degradative reaction.

Since it has been well documented that many peptides which contain

residues of 2,4-diaminobutanoic acid are unstable at neutral pH

(Davies et al., 1969; Katrukha et al., 1968; Poduska et al., 1965), it

seemed plausible that OMA-NH2, which contains the related 2,4-diamino-

3-methylbutanoic acid residue, might display comparable liability.

Another area of concern was the tryptathionine residue which is

believed to be susceptible to base-catalyzed destruction (Wieland,

1983). However, when some of these byproducts were isolated by chroma-

tography on Sephadex LH-20, many of them were found to be unstable and








H1 O
aCNBH, Ill


HC C -

C -H

c C'CN CH -
H 'O

- C',N- CH -

-H 2 0


R R'


R\ ,R.






-H+ V


-C '
11 1I

-C N-CH-

, ,N-C~H-





Fig. III-2. Scheme showing the probable reactions and side-
reactions occurring in the reductive amination of OMAA.

to decompose back to the starting material, OMAA (data not shown).

These quasi-stable amines may be a-aminonitriles (Fig. III-2,

structure VII) (Bejaud et al., 1976) which have already been impli-

cated as undesirable, quasi-stable byproducts in the reductive methyl-

ation of proteins (Gidley and Sanders, 1982). One or more of these

amines might also arise from reaction between the intermediate immon-

ium (Fig. III-2, structure V) and an intramolecular nucleophile (such

as an amide nitrogen) to yield an N-Mannich compound (Tramontini,

1973). This intramolecular reaction to form an N-Mannich (Fig. 1II-2,

structure VIII) is comparable to .that proposed in chapter II for the

formation of an intramolecular alkanolamide (Fig. III-2, structure I)

in OMAA. Studies on the stability of N-Mannich compounds of this kind

have been recently reported (Bundgaard, 1985; Loudon et al., 1981).

Their stability increased with decreasing bulk of the substituents on

the amino group (i.e., R and R' in Fig. III-2, structure VIII). The

N-Mannich which might form during the synthesis of OMA-NH2 would bear
the smallest substituents possible (hydrogen) and thus impede the

progress of the reaction on account of its high stability. When the

reaction was permitted to progress for several weeks, most of these

fluorescamine-reactive compounds slowly disappeared and one increased

in amount. As noted above, chromatographic separation on Sephadex

LH-20 of the components of a reaction which still contained multiple

fluorescamine-reactive amatoxin derivatives showed that many of these

entities were unstable and decomposed back to the starting material,

OMAA. This supports the notion that labile compounds like structures

VII and VIII (Fig. III-2) are formed. Probably as a consequence of

these or similar side-reactions, the purified yield of OMA-NH2 has

never exceeded 30% despite numerous efforts to optimize the reaction.

Reductive coupling of ORAA to the amino acids glycine and

L-proline proceeded at a much higher rate than the reaction with

ammonium acetate. This higher rate may derive, in part, from the

larger substituents on amino group and a consequent lowering of the

stability of the N-Mannich byproduct. When these reactions were

fractionated by HPLC before the reaction reached completion, novel

compounds which decomposed to OMAA were again noted and in this case

isolated from void-volume fractions. 0MA-gly and OMA-pro can now be

obtained in approximately 75% yield.

Oxidative Transformations of OMAA

Reaction of OMAA-I with hydroxylamine-0-sulfonic acid produced

OMA-CN in 60% yield after a period of a few days. This reaction relies

upon the formation of an 0-sulfonated oxime by reaction of the reagent

with the aldehyde. This then udergoes an elimination reaction to yield

the nitrile and a sulfate ion (Fizet and Streith, 1974).

0MAA-II was generated by the reaction of OMA with sodium perio-

date at pH 2.0 (see previous chapter). By inclusion of sodium chlorite

(Launer and Tomimatsu, 1956; Lindgren and Nilsson, 1973) in this

system, OMAA-II was oxidized quickly to the carboxyl derivative,


Properties of the Derivatives
These derivatives had chromatographic properties expected for

their structures. The charged derivatives generally migrated more

slowly on TLC and had shorter retention times on HPLC than OMAA, while

the relatively nonpolar OMA-CN showed the opposite behavior. In con-

trast to OMAA-I, the CO spectra of all of the new derivatives were

indistinguishable from that of OMA (Fig. II-3). Also, their color-

reaction with cinn-HC1 was violet compared with the rust color ob-

tained with OMAA-I. As expected, only OMAA-NH2 reacted with fluores-

camine to yield a fluorescent product. FAB-MS of OMA-C00H and OMA-NH2
revealed mass ions of the appropriate size for the proposed structures

(Figs. III-3 and III-4).

Instability of the reductive amination products has been detected

after prolonged storage of frozen solutions. This destruction was

associated with loss of the distinctive UV absorbances, e~g., at 304

nm (data not shown). Loss of the compounds was retarded when solutions

were mildly acidic.

None of the new derivatives inhibited CT RNAP II as strongly as

AMA. OMA-CN was the strongest inhibitor with a Ki of 3 X 10-9 M.

OMA-gly, OMA-pro, OMA-C00H, and OMA-NH2 were much weaker inhibitors
with K 's of 2.5 X 10-7 M, 7 X 10-6 M, 1 X 10-7 M, and 1.7 X 10-7 M,

respectively. The inhibition by OMA-gly, OMA-pro, and OMA-NH2 was
studied more thoroughly and showed apparently noncompetitive behavior

(e.g., Fig. III-5). Since the amanitin amines were considerably less

potent inhibitors than other amanitins which might be generated in
small amounts as byproducts of the reaction (e.g., OMDA), steps were

taken to help ensure that a K obtained for the material in a peak was
characteristic of the bulk of the material in the peak and not grossly

biased by the presence of traces of more potent inhibitors. In order

to accomplish this, the Ki was determined for the contents of at least
two, and usually three, fractions across a peak. The material in the










CF) 'r


SI l l 1 j i l l
_ mo co N














- a



+ E

E ,








O m e q ( ( c








O <0

4 Q.

O r-





L O w


- -






































X to
x 0



-0.!8 -0.1 2 -0.0 6 O 0.0 6 0. 1 2 0.1 8


Fig. III-5. Dixon plot showing the apparently noncompetitive
inhibition of CT RNAP II by OMA-NH .The concentrations of UTP used
were 0.75 pM (circles), 1.10 pM (si uares), and 1.50 pM (triangles).

peak was subjected to additional chromatographic steps until it

appeared homogeneous with respect to inhibition of CT RNAP II. The Ki

of the pooled fractions from each final peak was consistent with that

of the constituent fractions (data not shown).

The finding that the inhibition of CT RNAP II by each of the

amanitin amines showed noncompetitive behavior with respect to UTP

provides preliminary evidence that these amanitin derivatives inhibit

this enzyme in a manner analogous to other amanitins, presumably

through localization at an amanitin binding site on the enzyme. This

evidence for inhibition by a mechanism common to other amanitins,

along with the resumption of a "native" conformation as suggested by

the CO spectra, provides a basis for interpreting their diminished

affinities in tems of interactions between the enzyme and the newly

introduced substituents. In this regard, OMA-NH2 is most readily

compared to the previously examined amanitins, OMDA and OML. These

three derivatives differ only with respect to a single group. They

each have a different group of roughly the same size and shape append-

ed to the r-carbon atom of residue 3 (see Figs. I-1 and III-1). While

stanitins OMDA and OML, which have a hydroxyl and a methyl group,

respectively, at this location, bind to CT RNAP II almost as tightly

as the most inhibitory of the naturally-occurring toxins, OMA-NH2

which bears an amino group is a much poorer inhibitor. At first

glance, the primary difference between OMA-NH2 and the other two
amanitins appears to be the positive charge expected for an aliphatic

amino group at near-neutral pH. However, because of its charge the

amino group may also be effectively somewhat larger than the analogous

hydroxyl and methyl groups due to a larger layer of bound water. Thus,

it is difficult to attribute the difference in affinity unequivocally

to the presence of a positive charge, but this seems likely to be the

most important factor. The Ki's of OMA-gly and OMA-pro are much more
difficult to rationalize because of the multiplicity of functional

groups which have been introduced and because of the high likelihood

of chelation of the Mn2+ in the RNAP assay system by the amino acid

mo ieties.

Since OMA-NH2, which bears a positive charge, was a relatively

poor inhibitor of CT RNAP II, it was anticipated that OMA-C00H, which

should bear a negative charge, would be a quite potent inhibitor. It

was surprising to find that it inhibits the enzyme almost as poorly as

DMA-NH2. In contrast, OMA-CN, a semisynthetic derivative which is like

OMDA in that it lacks a group with a charge, is a rather potent inhi-

bitor of CT RNAP II. These data taken together suggest that the por-

tion of the amanitin binding site of CT RNAP II which interacts with

the sidechain of residue 3_ may contain closely spaced positively and

negatively charged moieties. Thus, derivatives with charged sidechains

are strongly repulsed, while derivatives with uncharged sidechains can

bind more strongly. Alternatively, this portion of the binding site

may be somewhat nonpolar so that the addition of highly polar

substituents to this sidechain inhibits binding.

It seems clear that OMAA is not well-suited for reductive

coupling to proteins. Even under forcing conditions with very high

concentrations of amines, the rate of reaction is very slow. However,

some the derivatives described here and future subderivatives may

prove useful in a number of different applications.




The results presented in the previous chapter indicated that

reductive coupling of OMAA to protein amino groups does not represent

a feasible route for the conjugation of amanitin to proteins. Conse-

quently, attention was turned to the preparation of an azo amanitin

derivative which could be employed in this way.

Synthesis of azo ananitins requires diazotization of an aromatic

amine. However, since aldehydes are reactive to diazotizing reagents,

the aldehyde group needed to be introduced in protected or precursor

form. This need for protection is complicated by the fact that amatox-

ins are unstable toward most of the conditions which are used to

deprotect aldehydes or to generate them from a precursor (Wieland,


Many aldoses contain aldehyde groups which are inherently protec-

ted by intramolecular reaction with a hydroxyl group to form a hemi-

acetal. Despite the fact that only a very small proportion of an aldo-

hexose (e.g., glucose) is present in solution in the free aldehyde

form, these sugars have been successfully coupled to proteins by

reductive alkylation with sodium cyanoborohydride (Gray, 1974). Under

the conditions which were initially described, this reaction was quite

slow. However, Roy et al. (1984b) have recently noted that borate

increases the amount of free aldehyde which is in equilibrium with the

cyclic forms of aldoses. This effect was most prominent for glucose

and lactose. They were further able to show that borate greatly in-

creased the rate of reductive coupling of lactose to BSA (Roy et al.,


These observations by Roy and his coworkers suggested a possible

route for preparing an azo amanitin with the desired properties. A

derivative of the already well-studied ABGG was envisioned which would

bear an amino sugar linked to the carboxyl group of the glycylglycine

linker (see Fig. V-I). This chapter describes experiments which were

undertaken to define the feasibility and optimal reaction conditions

for the reductive coupling of N-acylated amino sugars to protein.

Model compounds which contai n the NJ-4-ni troben zoyl glycyl glyci ne moi ety

linked to an amino sugar (0-galactosamine or D-glucosamine) were

prepared and utilized for these studies. The conjugation of these

compounds to protein amino groups is schematically depicted in Fig.

IV-1. The effects of temperature, pH, buffer, and various reactant

concentrations on the reaction rate have been studied.

Materials and Methods


N-4-nitrobenzoylglycylglycine (PNBGG) was obtained from United

States Biochemicals. Nickel (II) chloride, BSA, sodium cyanoboro-

hydride, 2-amino-2-deoxy-D-glucose (glucosamine) hydrochloride,

2-amino-2-deoxy-D-galactose (galactosamine) hydrochloride, 1-ethy1-

3-(3-dimethylamino)propylcarbodiimide (EDC), N-2-hydroxyethyl-

pi perazine-N -2-ethanesulfoni c acid (HEPES), and sodium 3-trimethyl-




H2 O











Fig. IV-1. Scheme illustrating the reductive coupling of
PNBGG-sugar derivatives (or ABGG-GLU) to proteins; "R" represents the
PNBGG (or ABGG) moiety.

sily1-d4-propanoate (TSP) were obtained from Sigma Chemical. Sodium

cyanoborohydride was recrystallized according to Jentoft and Dearborn

(1979). Trichloroacetic acid (TCA) was obtained from Fisher Scien-

tific. Other reagents were obtained as described in the previous


Analytical Procedures

Chromatography. TLC and HPLC were performed as described in the

previous chapters. TLC solvent system III contained 1-butanol: metha-

nol: water (4:2:1); solvent system IV contained 2-butanol: ethyl ace-

tate: water (14:12:5). Column chromatography was also performed with

Sephadex LH-20 and Sephadex SP-50 (Pharmacia Fine Chemicals).

Bio-Beads SM-4 (Bio-Rad) were soxhlet-extracted with methanol for

seven days prior to use.

Spectroscopy. Spectroscopic studies were performed as outlined in

the previous chapters. In addition, optical rotation was determined

with a Jasco DIP-360 digital polarimeter equipped with a 10 cm cell.

Elemental analysis. Elemental analysis was performed by the

instrument facility in the Department of Chemistry, University of


Synthesis of PNBGG Derivatives

Synthesis of PNBGG-GLU. To PNBGG (0.56g, 2.0 mmol) in 4.0 ml of

water was added with stirring 0.18 ml of 10 M Na0H, EDC (0.575 g,

3.0 mmol), and glucosamine HC1 (0.86 g, 4.0 mmol). After 12 h of

reaction at 230C in the dark the resultant gel was dissolved in 100 ml

of water. The solution was added to 125 ml of Bio-Beads SM-4, stirred

for 1 h, and then filtered. The resin was washed for another hour with

100 ml of water and filtered again. Bound material was eluted from the

resin by extracting six times for 10 min with 100 ml of 75% methanol.

The combined methanolic extracts were concentrated in vacuo at 40*C to

a small volume. After filtration through Whatman # 40 paper the

solution was applied to a 2.5 X 15 cm column of Sephadex SP-50 (Na )

and eluted with 100 ml of water. The eluant was concentrated in vacuo

at 50*C to a small volume and applied to a 2.5 X 95 cm column of

Sephadex LH-20 which was eluted with water. Fractions of 7.5 ml were

collected at approximately 0.2 ml/min. The desired product (1.35 mmol,

67%) was located in fractions 52 to 60. TLC: RF, III-0.68, IV-0.34.

For C17H22N4010 calculated: C 46.16, H 5.01, N 12.66; found: C 45.22,

H 5.13, N 12.32. [a]25=+210. e268=1.2 X 104 M1 cm-1 PMR (Fig. IV-2):
3.46-3.99 (m, 6H, sugar); 4.04 (s, 2H, glycine); 4.21 (s, 2H,

glycine); 4.77 (d, 0.4H, J=8.2 Hz, anomeric); 5.20 (d, 0.6H, J=3.3 Hz,

anomeric); 8.04 (d, 2H, J=8.6 Hz, aromatic); 8.37 (d, 2H, J=8.6 Hz,


Synthesis of PNBGG-GAL. The synthesis of PNBGG-GAL was similar to
that of PNBGG-GLU. PNBGG-GAL eluted from the Sephadex LH-20 column in

fractions 59 to 65 (57% yield). TLC: RF, III-0.68, IV-0.34. For

C17H22N4010 calculated: C 46.16, H 5.01, N, 12.66; found: C 45.10,
H 5.11, N 12.23. [n]25=+32o. e268=1.2 X 104 M1 cm-1 PMR (Fig. IV-3):
3.6-4.3 (m, 10H, sugar and glycines); 4.70 (d, 0.5H, J=8.4 Hz,

anomeric); 5.24 (d, 0.5H, J=3.6 Hz, anomeric); 8.03 (d, 2H, J=8.6 Hz,

aromatic); 8.36 (d, 2H, J=8.9 Hz, aromatic).


- Co

Cr) -






- cO,


Conjuation Protocols

Conjugation of PNBGG-GLU and PNBGG-GAL to BSA. BSA stocks were

dialyzed against water or the appropriate buffer before use. Molar
concentrations of BSA were estimated on the basis of E1% of 6.67 at

279 nm (Janatova et al., 1968) and a molecular weight of 66,300 (Reed

et al., 1980). Reaction components were sterilized by filtration

through 0.22 pm Millex-GV filters (Millipore) and added aseptically to

autoclaved vials. All reactions contained 1.0 mg/m1 (1.51 X 10-5 M)

BSA and 3.62 X 10-3 M of a carbonyl compound (PNBGG-GLU or PNBGG-GAL).

Other components were incorporated as indicated below. Reactions were

allowed to proceed in the dark at either 200C or 37*C.

Purification and analysis of conjugates. The BSA conjugates were

freed of unconjugated PNBGG derivatives by one of two methods. In

experiments where the buffer concentration was 0.2 M, samples of 200

pl were removed aseptically and placed on ice. The chilled solution
was adjusted to 10% TCA with 40 pl of ice-cold 60% TCA. After 15 min

the precipitate was pelleted by centrifugation for 5 min in a table-

top centrifuge (Fisher Scientific). The supernatant fluid was decanted

and the pellet dissolved in 0.1 M sodium phosphate, pH 7.0. This

precipitation with TCA was repeated and the final pellet was dissolved

in 0.5 ml of the sodium phosphate buffer.

In experiments where buffer concentrations exceeded 0.2 M the

conjugate was purified with Centricon CM-30 ultrafiltration devices

(Amicon). The sample (0.2 ml) of the reaction mixture was added to the

device and diluted to 2.0 ml with 0.2 M sodium borate, pH 8.0, with

mixing. This was then centrifuged at 5,500 rpm for 2 h in either a

Beckman JA-40 or Sorvall type 30 rotor at SoC in order to concentrate

the conjugate solution to approximately 50 pl. The conjugates were

diluted and concentrated three more times in a similar fashion. The

second wash was with 0.1 M disodium ethylenediaminetetraacetic acid

(EDTA), while the third and fourth washes were with 0.1 M sodium

phosphate, pH 7.0. The final concentrate was diluted with 0.5 ml of

0.1 M sodium phosphate, pH 7.0

The extent of coupling was estimated by comparing the absorbances

at 279 nm and 300 nm. Molar extinction coefficients for BSA at 279 nm

and 300 nm were determined to be approximately 4.54 X 104 -1 cm- and
3 -1 -1
3.0 X 10 M cm respectively. The comparable values for the PNBGG
4 -1 -1 3 -1
derivatives were found to be 1.0 X 10 M cm and 3.95 X 10 M
cm .These extinction coefficients reflect a significant difference

in the absorbance spectra of BSA and the PNBGG derivatives. The

absorbance of the PNBGG derivatives falls slowly in the near UV (Fig.

IV-4), while that of BSA falls rapidly. It is this difference which

has been exploited to provide a method for estimating the extent of

conjugation. Simultaneous equations which incorporated these extinc-

tion coefficients were solved to yield an algorithm (cf. Mishell and

Shiigi, 1980, pp. 345-7) by which the absorbance at 300 nm could be

partitioned into the contributions from BSA and the conjugated PNBGG

derivative: A300 from PNBGG = 1.21(A300) 0.0832(A279). Based upon

the contribution to A300 from each component and their respective
extinction coefficients, the molar ratio of PNBGG derivative to BSA in

the conjugate was easily calculated. Absorbance measurements at 279 nm

and 300 nm were corrected for light scattering by extrapolation from

390 nm, a wavelength at which neither BSA nor the PNBGG derivatives

absorb significantly. Light scattering was assumed to obey Rayleigh's


250 300



Fig. IV-4. UV absogbance spectrum of PNBGG-GLU in water at
approximately 2.5 X 10' M.

theorem ideally and thus to vary with the fourth power of wavelength

(cf. Leach and Scheraga, 1960).


Initial Studies ~on the Effects of pH, Temperature, Nature of Sugar
Residue, Borate, and Sodium Cyanoborohydride Cncefr+rntrationo the
Conjugation of PNBGG-Sugars to BSA

The results of initial studies to examine the influence of sever-

al reaction parameters that were thought likely to be important are

summarized in Fig. IV-5. Conjugation of PNBGG-GLU and PNBGG-GAL to BSA

-was found to be strongly dependent upon sodium cyanoborohydride. In

the absence of sodium cyanoborohydride there was a slow time-dependent

increase in the extent of conjugation, but the degree of conjugation

after 225 hours was less than three PNBGG-sugar molecules per BSA

molecule for all of the conditions examined. There was no significant

change in the A279 or A300 (i.e., the spectral parameters used to
follow the course of the reaction) of the BSA in sham reactions which

lacked PNBGG-GLU or PNBGG-GAL or in reactions where lactose was conju-

gated to the BSA (data not shown). The most important factors affect-

ing the rate of conjugation were the use of borate instead of other

buffer salts (phosphate or HEPES) and the temperature (reactions pro-

ceeded much more rapidly at 370C than at 200C). In accord with the

results of Roy et al. (1984b), the effect of borate on the reaction

rate was more marked for the glucosamine than for the galactosamine

derivative. In fact, in the presence of borate PNBGG-GLU coupled at a

significantly higher rate than PNBGG-GAL, while there was little

difference in their rates of reaction in the absence of borate. The

influence of pH (pH 8.0 versus pH 9.0) was found to be less signifi-

Fig. IV-5. Effect of pH, temperature, and nature of sugar residue
on the extent of conjugation of PNBGG-GAL (upper panel) and PNBGG-GLU
(lower panel) to BSA over time. All reaction mixtures contained 1.0
mg/m1 BSA, 3.62 mM PNBGG derivative, 7.24 mM sodium cyanoborohydride,
10 mM nickel (II) chloride, and 6 mM sodium citrate in a 0.2 M buffer.
Closed symbols represent borate buffer-containing reactions, while the
open upright triangle contained phosphate buffer. Upright triangle,
pH 9.0 at 370C; circle, pH 8.0 at 37*C; inverted triangle, pH 9.0 at
200C; square, pH 8.0 at 20*C.






cant, but reactions did proceed more rapidly at pH 9.0. The inclusion

of nickel (II) chloride as a cyanide-absorbing reaction component

(Jentoft and Dearborn, 1980) had no demonstrable negative effect upon

either the reaction rate or the properties of the conjugate (data not


Further Studies on the Effect of Sodium Cyanoborohydride
Concentration, Borate Concentration, and pH on the Conjugation of

The initial studies described above showed that the glucosamine

residue permitted much higher reaction rates than the galactosamine

residue. Thus, further work was restricted to PNBGG-GLU. The depen-

dence of rate on the sodium cyanoborohydride concentration is present-

ed in Fig. IV-6. Under the conditions utilized the rate increased ra-

pidly with increasing sodium cyanoborohydride concentration and level-

ed off at approximately 0.10 M. Furthermore, under conditions of opti-

mized sodium cyanoborohydride concentration there was no significant

effect of varying borate concentration between 0.2 M and 2.0 M on the

rate of coupling (Fig. IV-7). Variation of the pH between 6.0 and 9.0

had a profound effect on the rate of conjugation (Fig. IV-8). While

there was a large increase in rate between pH 7.0 and 9.0, at pH's

less than 7.0 the rate was very low, almost negligible.


The coupling of sugars to proteins by reductive alkylation with

sodium cyanoborohydride was first demonstrated by Gray (1974). Even

with concentrations of the reactants at near saturation the rate of




OQ 10 20


Fig. IV-6. Effect of sodium cyanoborohydride concentration on the
extent of conjugation of PNBGG-GLU to BSA after various times. All
reactions were conducted with 1 mg/m1 BSA, 10 mM nickel (II) chloride,
and 6 mM sodium citrate in 0.2 M sodium borate, pH 8.0, at 370C. The
triangles, squares, and circles represent samples at 25, 50, and 75
hours of reaction, respectively y.




0 30


O 50 10 0


Fig. IV-7. Effect of borate concentration on the extent of conju-
gation of PNBGG-GLU to BSA at various times. All reactions were con-
ducted with 1 mg/ml BSA, 10 mM nickel (II) chloride, 6 mM sodium
citrate, and 72.5 mM sodium cyanoborohydride in borate buffers of
varying concentration with pH 8.0 and temperature 370C. Inverted
triangle, 0.2 M; square, 0.5 M; upright triangle, 1.0 M; and circle,
2.0 M sodium borate.


.J 32 -


6.0 7.0 8.0 9.0


Fig. IV-8. Effect of pH on the extent of conjugation of PNBGG-GLU
to BSA at various times. All reaction mixtures contained 1 mg/m1 BSA,
0.1 M sodium cyanoborohydride, 10 mM nickel (II) chloride, and 6 mM
sodium citrate in a buffer consisting of 0.2 M sodium borate and 0.05
M PIPES. All reactions were kept at 37*C at the appropriate pH. The
triangles, squares, and circles represent samples analyzed after 25,
50, and 75 hours of reaction time, respectively.

conjugation was very slow. Despite this sluggishness, the linkage of

sugars to proteins by this method has served as an important route for

the synthesis of neoglycoproteins. The demonstration by Roy and his

coworkers (1984a, 1984b) that borate increases the proportion of

acyclic sugar in solution and thus greatly accelerates the rate of

reductive coupling of lactose to BSA has served to enhance the attrac-

tiveness of this approach for conjugation of sugars to proteins. If

this phenomenon of borate-enhanced reductive coupling might also apply

to N-acylated 2-amino-2-deoxy-0-aldobexoses, then its utility could

conceivably be extended to the conjugation of a wide variety of com-

pounds to proteins.

The PNBGG adducts of glucosamine and galactosamine were prepared

as synthetic intermediates of the corresponding ABGG derivatives.

These proved to have characteristics which made them useful for pre-

liminary studies on the coupling of this class of compounds to pro-

tein. In particular, they were quite soluble in water and their UV

absorbance spectra were sufficiently different from that of BSA to

permit estimation of the extent of conjugation based upon spectral


The reductive coupling of both PNBGG-GLU and PNBGG-GAL was

markedly stimulated by 0.2 M borate. As might be expected from the

studies of Roy et al. (1984b), PNBGG-GLU reacted substantially more

rapidly than PNBGG-GAL in the presence of borate, while their rate of

reaction was similar in solutions of other buffers. -Increasing the

temperature from 200C to 37*C was also found to greatly enhance the

rate of coupling, while increasing the pH from 8.0 to 9.0 had a much

smaller effect. The reductive coupling of the PNBGG derivatives to BSA

was strongly cyanoborohydride-dependent, but very slow, time-dependent

conjugation of the PNBGG derivatives to BSA was detected in the

absence of sodium cyanoborohydride. This reaction may correspond to

that previously seen in solutions of glucose with BSA (Baynes et al.,


Optimization of the conjugation reaction was examined further

with the more active PNBGG-GLU. The reaction rate was seen to increase

markedly as the sodium cyanoborohydride concentration was increased to

approximately 0.1 M, beyond which the rate plateaued. This finding is

in sharp contrast to reductive methylation with formaldehyde and

sodium cyanoborohydride where the rate varies little with the cyano-

borohydride concentration (Jentoft and Dearborn, 1979). However, there

have been indications of a similar strong dependence on cyanoborohy-

dride concentration for the rate of reductive coupling of raffinalde-

hyde to proteins (van Zile et al., 1979). Under conditions of opti-

mized sodium cyanoborohydride concentration at pH 8.0, increasing the

borate concentration from 0.2 to 2.0 M had no significant effect upon

the rate.

A more extensive examination of the effect of pH was undertaken.

This showed a large increase in the rate of coupling between pH 7.0

and 8.0. Previous studies on the reductive coupling of sugars to

proteins in buffers other than borate have noted a significant in-

crease in the rate with increased pH (Marsh et al., 1977; Schwartz and

Gray, 1977). This presumably resulted from the fact that sugars have a

higher proportion of their acyclic form in solution at higher pH's

(Roy et al., 1984b). The pH-dependence of conjugation noted here may

derive in part from this effect, but it is likely to be much more

strongly related to the pH-dependence of the interaction between

borate and the sugar.

This study, in contrast with others, has focused upon the reduc-

tive coupling of complex compounds to proteins using relatively low

reactant concentrations. Most previous workers have utilized concen-

trations which could only be achieved with sugars and highly soluble

proteins. The current studies have defined a system in which compounds

can be coupled to proteins at a practical rate using reactant concen-

trations which may be readily achieved with drugs (or their deriva-

tives) and immunoglobulins. The optimized rate of conjugation attained

here with PNBGG-GLU and BSA appears to approach the rate obtained by

Lee and Lee (1980) for reductive coupling of some free aldehydes to


From the work of Hall (1956) on the pKa of various amines it can

be anticipated that the pKa of protein amino groups which have been

linked to one of the PNBGG derivatives will drop to about 9.0. This

should provide considerable preservation of charge at physiologic pH.

In the next chapter the application of this method to the conju-

gation of amanitin to BSA is described.




The results obtained with the model compounds in the preceding

chapter suggested that preparation of a derivative of AMA containing

D-glucosamine would provide a reasonable route to an amatoxin which

could be conjugated to proteins by means of reductive alkylation.

Preparation of the anino analog of PNBGG-GLU (PABGG-GLU) has permitted

access to a simple synthetic route to an azo amanitin derivative,

ABGG-GLU, which bears the D-glucosamine residue. This new compound is

closely related chemically to the azo amanitin ABGG which has already

been successfully utilized to prepare several conjugates that have

demonstrated potent and specific cytotoxicity (see chapter I).

The conjugation of ABGG-GLU to BSA was studied in order to permit

direct comparisons with the data obtained with the model compounds.

The behavior of ABGG-GLU in this system appeared more complex than

that of PNBGG-GLU in some important respects. Therefore, further

studies were undertaken to identify and optimize parameters which were

felt likely to be particularly relevant to the conjugation of


Materials and Methods

N-4-aminobenzoylglycylglycine (PABGG) was obtained from Dr. James

F. Preston and was prepared by hydrogenation of PNBGG as previously

descri bed (Preston et al., 1981). Piperazine-N,N_-bis (2-ethanesulfonic

acid) (PIPES) was obtained from Sigma Chemical. Other reagents were

obtained or prepared as indicated in previous chapters.

Analytical Procedures

Chromatography, spectroscopy, and elemental analysis. These

methods are fully described in previous chapters.

RNA polymerase assay and source of toxin. The protocols for
examining the inhibition of RNA polymerase activity and for purifi-

cation of AMA are outlined in chapter II.

Synthesis of ABGG-GLU

Synthesis of PABGG-GLU. Two methods for the synthesis of
PABGG-GLU have been developed (Fig. V-1):

In the first method PABGG (0.126 g, 0.50 mmol) and glucosamine

hydrochloride (10.8 g, 50 mmol) were added to 43.5 ml of water in a

flask. The PABGG was brought into solution by dropwise addition of

0.55 ml of 10 M Na0H with continuous rapid stirring. EDC (0.192 g, 1.0

mmol) was added to this solution four times at 30 min intervals;

during this time the reaction was allowed to proceed at 230C in the

dark. After a total reaction time of 2 h the solution was concentrated

in vacuo at 30*C to near dryness. Remaining water was removed as an

azeotrope with ethanol by twice suspending the residue in 25 ml of






02N -~ -s~oOH H20H



H2N O2 2@


1) NaNO2/HC1

2) AMA

~sHO 0


Fig. V-1. Scheme showing the two routes for synthesis of
PABGG-GLU and the synthesis of ABGG-GLU from AMA and PABGG-GLU.

absolute ethanol and evaporating in vacuo at 30*C. The dried residue

was suspended in 50 ml of methanol and filtered through Whatman # 40

paper. The filter cake was washed twice with 25 ml of methanol at room

temperature. The pooled filtrates were concentrated to dryness in

vacuo at 300C dissolved in 10 ml of water, and applied to a 4 X 20

cm column of Sephadex SP-50 (Na ) which was eluted with 500 ml of

water. This aqueous Sephadex SP-50 eluate was concentrated in vacuo at

40*C to a small volume, applied to a 2.5 X 95 cm column of Sephadex

LH-20, and eluted with water at 0.2 ml/min to collect 7.5 ml frac-

tions. The desired product (0.19 mmol, 38%) eluted between fractions

53 and 60.

A second method of preparing PABGG-GLU involved catalytic trans-

fer hydrogenation (cf. Anwer and Spatola, 1980) of PNBGG-GLU. To

PNBGG-GLU (0.50 mmol) in 45 ml of water was added 5 ml of 1.0 M sodium

format, pH 3.5. The solution was sroarged with nitrogen for 15 min and

then 0.375 g of 10% Pd on charcoal (Kodak) was added. This mixture was

stirred for 12 h in the dark at 23*C under a nitrogen atmosphere. The

cata-lyst was then removed by filtration through Whatman # 40 paper;

the catalyst was washed with 50%; acetonitrile until the A278 of the
filtrate was negligible. The combined filtrates were concentrated to a

small volume in vacuo at 500C and applied to a 2.5 X 95 cm column of

Sephadex LH-20. Fractions of 7.5 ml were collected at approximately

0.2 ml/min. The desired product (0.41 mmol, 82%) eluted in fractions

51 to 58. TLC: RF, I-0.31, III-0.56;. For C17H24N408 calculated:
C 49.51, H 5.87, N 13.58; found: C 47.23, H 6.28, N 13.18. [n]25=+16*.

'278=1.38 X 104 M1 cm-1 PMR: 3.4-4.0 (m, 6H, sugar); 4.015 (s, 2H,
glycine); 4.12 (s, 2H, glycine); 4.75 (d, 0.4H, J=8.0 Hz, anomeric);

5.20 (d, 0.6H, J=3.3 Hz; anomeric); 6.87 (d, 2H, J=8.4 Hz, aromatic);

7.70 (d, 2H, J=8.6 Hz, aromatic).

Synthesis of ABGG-GLU. PABGG-GLU (30 pimol) was dissolved in 0.75

ml of ice-cold 0.1 M HC1. The PABGG-GLU was diazotized by addition of

30 pl of 1 M sodium nitrite followed by incubation at 23*C in the dark

for 30 min. A portion of this solution (0.65 ml) was added to 25 pmol

of AMA which was dissolved in 1.20 ml of 0.5 M sodium phosphate, pH

8.0. There was immediate development of a deep purple color. After 5

min the crude product was desalted using the Sep Pak C18 protocol (see

chapter III). The desalted material was dried in vacuo at 50"C,

redissolved in acetonitrile: TFA-TEA buffer (18:82), and purified by

HPLC. The desired product (13.8 plmol, 55%) eluted between 32 and 40

ml. After evaporation of the acetonitrile in vacuo at 300C the final

product was desalted by the Sep Pak C18 procedure. TLC: I-0.17,

III-0.44. FAB-MS: [M+H] expected, 1342.50; found, 1342.54; [M-H] -

expected, 1340.485; found, 1340.50.

Conjugation of ABGG-GLU to BSA
Reaction mixtures were assembled as described in the previous

chapter except that ABGG-GLU was substituted for the PNBGG deriva-

tives. Excess ABGG-GLU was removed from the conjugates by the ultra-

filtration method which was detailed in chapter IV. The only modifi-

cation made in this protocol was that the conjugates were incubated in

the presence of EDTA for a full hour before the second centrifugation.

The extent of conjugation was estimated spectrophotometrically.

The molar extinction coefficient of ABGG-GLU at 395 nm was taken to be
4 -1 -1
1.4 X 10 M cm (Faulstich and Trischmann, 1973); the extinction at

279 nm in 0.1 M sodium phosphate, pH 7.0, was determined to be
4 -1 -1 1%
1.23 X 10 M cm .The E at 279 nm and molecular weight of BSA

(see chapter IV) were used to calculate a molar extinction at 279 nm

of 4.54 X 104 M1 cm-1; BSA does not absorb at 395 nm. Using these

parameters the molar ratio of ABGG-GLU to BSA in the conjugates was

estimated by the formula: 3.24A395/(A279-0.88A395 '
The fate of ABGG-GLU in the reaction mixtures was determined by

analytical HPLC. Duplicate samples (2-10 pl each) were diluted in

0.1 M sodium phosphate, pH 3.0, to yield a final concentration of

approximately 0.2 mM ABGG-GLU. The diluted samples were applied to

HPLC by an automated sample injector (Waters Model 7108 WISP) and

chromatographed at 1 ml/min on a C18 Z-module (Waters) using aceto-

nitrile: TFA-TEA buffer (18:82) as the eluant. The absorbance at 304

nm was monitored and peaks were integrated by a Waters Model 720 Data



Synthesis and Properties of ABGG-GLU

Preparation of PABGG-GLU. Two routes for the synthesis of
PABGG-GLU have been developed (Fig. V-1). When initial efforts to

reduce PNBGG-GLU failed, a first method was developed by which PABGG

was coupled to the anino group of D-glucosamine with the aid of EOC.

This method required a large excess of D-glucosamine in order to pre-

vent polymerization of the PABGG. In the second method PNBGG-GLU was

selectively reduced to PABGG-GLU by catalytic transfer hydrogenation

with palladium on charcoal as the catalyst and sodium format as the

reductant (cf. Brieger and Nestrick, 1974; Anwer and Spatola, 1980).

The products of the two syntheses were found to be identical by seve-

ral criteria, including PMR spectrum (Fig. V-2), UV absorbance spec-

trum (Fig. V-3), TLC mobility, elemental analysis, and optical rota-

Azo coupling of diazotized PABGG-GLU to o-amanitin. A synthesis

of ABGG-GLU was devised which provided significantly increased yield

over previously reported procedures (Falck-Pederson et al., 1983;

Preston et al., 1981). First, because of its low nucleophilicity, the

ary1 amine function of PABGG-GLU was diazotized in dilute HC1 (Fig.

V-1) in which the potent nitrosating agent nitrosy1 chloride is gener-

ated (Challis and Butler, 1968). Second, the azo coupling reaction was

conducted in a sodium phosphate buffer at pH 8.0. At this pH the

phenolic hydroxyl of AMA, which has a pKa of approximately 10 (Falck-

Pederson, 1981), can be expected to be slightly ionized and the ten-

dency of the diazotized PABGG-GLU to be converted to an unreactive

diazotate is slight (Zollinger, 1961). The yield of ABGG-GLU obtained

using these conditions is reliably 50-60% and most of the remainder of
the AMA is recovered in unreacted form.

Properties of ABGG-GLU. ABGG-GLU is in many ways very similar to

its predecessor ABGG. Their UV/visible absorbance spectra (see Fig.

V-4) are indistinguishable. They demonstrated very similar potency of

inhibition of CT RNAP II; ABGG-GLU showed a K. of 4 X 109 M, while
that of ABGG was 3 X 10-9 M. Examination of ABGG-GLU by reverse phase

HPLC under analytical conditions showed two poorly separated peaks.

These apparently represent the two diastereomers which exist in solu-

tion as a consequence of the anomeric nature of the sugar residue. A

similar phenomenon has been observed for PNBGG-GLU and PNBGG-GAL










- 0



O 1.2-



200 250 300

Fig. V-3. UV absorbance spectrum of PABGG-GLU at 1.8 X 10-5 M in
5 mM sodium phosphate, pH 7.0, (light line) and 0.1 M HC1 (dark line).

1 0







L 0 *-I

I "O
C "


.* -O

,~ ..
I .**

7 1.
'*.; I

-~ *..


to CV O

(data not shown). FAB-MS studies of ABGG-GLU supported the predicted

structure, showing mass ions of the expected sizes (Fig. V-5).

Effects of Alkaline pH and Nickel (II) lon on ABGG-GLU

Preliminary trials of conjugation of ABGG-GLU to proteins re-

vealed some unexpected problems. The most readily noticed of these was

that the nickel (II) ion, which was added to absorb the cyanide gener-

ated in the course of the reaction, reacted with ABGG-GLU to form a

complex as evidenced by an altered absorbance spectrum (Fig. V-6).

ABGG-GLU could be recovered intact from the complex by adding EDTA to

the solution, but at least 45 min was required for the action of the

EDTA to be completed.

Also, when conjugation was undertaken at pH 8.0, decolorization

of the solution was noted over a period of days. This was faster in

reactions where the nickel (II) chloride was omitted. The loss of

color appeared to correlate with the appearance of a new toxin-related

entity which had very low mobility on TLC. When ABGG-GLU was placed at

370C in 0.2 M sodium borate buffer, pH 8.0, a slow alteration of the

UV/visible absorbance spectrum was noted (Fig. V-7).

Conjugation of ABGG-GLU to BSA

Influence of pH and reactant concentrations on the rates of toxin

conjugation and alteration. Since difficulties with degradation of the

toxin were noted under the conditions which were developed for cou-

pling the PNBGG derivatives to BSA, means of circumventing this pro-

blem by altering the reaction conditions were sought. It had been

noted in work with PNBGG-GLU that significant coupling occurred at













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