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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 evWy 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. 








Modified Proteins As Therapeutic Agents 1 

Conjugates of Amatoxins with Proteins 6 

Toward Optimal Conjugation of Small Molecular Weight 

Cytotoxic Agents to Proteins 10 

Rationale for the Current Work 13 


Introduction 16 

Materials and Methods 17 

Results 22 

Discussion 29 



Introduction 38 

Materials and Methods 40 

Results and Discussion 45 



Introduction 57 

Materials and Methods 58 

Results 69 

Discussion 72 



Introduction 79 

Materi al s and Methods 80 

Results 84 

Discussion 98 





ABGG a-amanitinyl -7' -azobenzoylglycyl glycine 

ABGG-GLU a-amanitinyl -7' -azobenzoylglycylglycyl glucosamine 

ABH a-amanitinyl -7' -azobenzoyl -1 ,6-di aminohexane 

AMA a-amanitin 

BSA bovine serum albumin 

CD circular dichroism 

cinn-HCl trans -cinnamaldehyde-HCl fumes; TLC spray reagent 

CT RNAP II calf thymus RNA polymerase II 

dg-DMSO perdeuterated dimethylsulfoxide 

EDC 1 -ethyl -3- (3-di methyl ami no) propyl car bod i imide 

EDTA ethylenediaminetetraacetic acid 

Fab univalent antigen-binding antibody fragment 

(Fab 1 ),, divalent antigen-binding antibody fragment 

FAB-MS fast atom bombardment mass spectroscopy 

HEPES N^-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 

HPLC high-performance liguid chromatography 

IT immunotoxin 

OMA 6' -0-methyl -a-amanitin 

OMAA 6' -0-methyl al do-a-amani t i n 

OMA-X (X = CN, COOH, gly, NHp, 

and pro) amanitin derivatives defined in the text 

OMDA 6' -0-methyl dehydroxymethyl -a-amanitin 

OML 6' -0-methyl amanull in 


PABGG N^4-aminobenzoylglycylglycine 

PABGG-GLU N_-4-aminobenzoylglycylglycyl glucosamine 

PIPES piperazine-N,N_^-bis(2-ethanesulfonic acid) 

PMR proton magnetic resonance 

PNBGG N^-4-nitrobenzoylglycylglycine 

PNBGG-GAL N^-4-nitrobenzoylglycylglycylgal actosamine 

PNBGG-GLU N^4-nitrobenzoylglycylglycylglucosamine 

Rp 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 

TEA triethyl amine 

TFA trifluoroacetic acid 

TLC thin-layer chromatography 

TMS tetramethyl s i 1 ane 

TSP sodium 3-trimethylsilyl -d.-propanoate 

UTP uridine triphosphate 

UV ultraviolet 

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), 
a-amanitin, was chemically modified to generate aldehydic derivatives 
which were suitable for reductive coupling to proteins. 

Periodate oxidation of 6 1 -0-methyl -a-amanitin (OMA) at neutral pH 
generated a mixture of two amanitin aldehydes which underwent ready 
interconversion. These two forms of 6' -O-methylaldo-a-amanitin (OMAA) 
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" 5 M, respec- 
tively. Conversion of the aldehyde moiety of OMAA to carboxyl and 
nitrile groups yielded derivatives with K.'s of 1 X 10 M and 
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 a-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 K.'s of approximately 10 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; Olsnes and Pihl, 1982; 
Thorpe et al., 1982), especially since the hybridoma method (Kbhler 
and Mil stein, 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 conjugates 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- 
ments 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'K 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 enzymatical ly (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 conjugates. 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. 

Immunotoxins 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 lysosomes. 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 Fiume, 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 
(Fig. 1-1). These peptides exert their toxic action by inhibiting the 








\ / 


CH 2 


H 3 C R? 
° \ / <L 

i i i c 

CH 2 H CO 

(A^V NH CH 3 
0=S^ N N-C V .C-R, CH-CH 
H £ 



CH 2 

CH 2 -CH 3 

Fig. 1-1. Structures of some pertinent amanitins. 



B- amanitin 










CH0HCH 2 0H 


CH0HCH 2 0H 





0CH 3 


0CH 3 

CH 2 0H 

0CH 3 

CH0HCH 2 0H 











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, S-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 o-amanitin (Faulstich and Trischmann, 1973; Preston et 
al., 1981) which have been referred to as ABH and ABGG (short for 
a-amanitinyl-7 1 -azobenzoyl -1,6-diaminohexane and a-amanitinyl -7 1 -azo- 
benzoylglycylglycine; 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 theortetical 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 carbamyl and amidino groups 
are similar in size and shape; they differ principally in that the 
amidino group bears a positive charge, while the carbamyl 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. 1-2 shows the sites at which o-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 B) is subject to 

CH 2 OH 



Fig. 1-2. 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 alkyl 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' -O-methyl-a-amanitin (OMA) with sodium 
periodate to yield 6' -O-methylaldo-a-amanitin (OMAA) was first 
reported in 1970 (Wieland and Fahrmeir). Like amanullin (Fig. 1-1), 
OMAA was demonstrated to be nontoxic when injected into mice. However, 
when the aldehyde function of OMAA was reduced with sodium borohydride 
to a hydroxyl group, the resultant derivative, 6' -0-methyl dehydroxy- 
methyl-o-amanitin (OMDA), proved to be toxic to mice. These and other 
data prompted the hypothesis that the r-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 chromatographical ly 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-HCl) 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-HCl 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. j)-Tolylsulphonylmethylnitrosamide and 
tetramethylsilane (TMS) were obtained from Aldrich Chemical; sodium 
periodate, sodium borohydride, D-glucose, and d fi -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 (Murr.) 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 recrystal lization from methanol. 

Estimates of the concentration of aqueous amanitin solutions were 
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% 
methanol ic trans -cinnamaldehyde and subsequent exposure to HC1 fumes 
(cinn-HCl). 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 0DS column (0.94 X 25 cm, Du Pont) 
which was protected by a 0.5 \xm prefilter (Rainin) and a C5 guard 
cartridge (Bio-Rad). Samples for HPLC were filtered through 0.22 urn 


Mil lex 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 [ H]UTP was employed for labeling the 

product. The assay was performed as follows: The reaction tube with 
10 ul 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 
ul. 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 -55-11 calculator. 

Synthesis of Amanitin Derivatives 

Synthesis of 6' -0-methyl-g-amanitin . To 30 umol 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 £-tolylsulphonyl- 
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 ml. The yield was 14.6 
umol (49%); 14.4 umol (48%) of the AMA was recovered unchanged. TLC: 
Rp, 1-0.47; color-reaction, violet. 

Synthesis of 6'-0-methylaldo-a-amanitin under neutral conditions . 
To 20.3 umol of OMA in 1.0 ml of water was added 0.5 ml of a solution 
containing 22.0 umol of sodium periodate. After 5 min of reaction in 
the dark at room temperature residual sodium periodate was quenched by 
the addition of 10 ul 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 umol (92%). TLC: R p , 1-0.43; 
color-reaction, rust-red. 

Synthesis of 6' -0-methyldehydroxymethy1-a-amanitin . OMA (2.0 
Mmol) 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 0MAA-IA and 
0MAA-IB eluted between 57 and 67 ml and between 85 and 95 ml, respec- 
tively. To 0.20 umol each of 0MAA-IA and 0MAA-IB was added 1.0 umol of 


sodium borohydride in 80 ul of water. After gentle mixing, this was 
permitted to react for an hour in the dark at 23°C. Residual sodium 
borohydride was quenched by reaction with 10 ul 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: R p , 1-0.51; color-reaction, 

Synthesis of 6' -0-methylaldo-a-amanitin under acidic conditions . 
To 0MA (8 umol) in 0.75 ml of 50 mM sodium phosphate, pH 3.0, was 
added sodium periodate (8.8 umol) in 30 ul of water. After reaction 
for 5 min at 23°C 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 umol, 88%) 
eluted between 30 and 35 ml. TLC: Rr, 1-0.61; 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 d g -DMS0. 

Synthesis of 6' -0-methyl-q-amanitin 

The synthesis of 0MA 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 0MA using diazomethane (Little, 1984) have demonstrated 


that the choice of reaction conditions is quite critical. Both reac- 
tant stoichiometrics 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 stoichiometrics 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 
l 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' -O-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-l). 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 (eluting at 
















10 20 30 40 



Fig. 1 1 -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 acetonitri le: 0.05% TFA (22:78) as mobile phase. The 
lower panel shows the purification of OMAA-I with 22% acetonitri le as 
the mobile phase. Fractions contained 1.0 ml. 


0.006 - 


E 0.004 



10 0.003 


O 0.002 



o o.ooi 



0.001 - 


12 18 24 6 12 


Fig. 1 1-2 . HPLC analysis of components of OMAA-I. Samples (30ul) 
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 d fi -DMS0 showed only a yery 
small peak in the region expected to contain the signal of an 
aldehydic proton (6=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. 1 1 -3 . CD spectra of OMA (dashed line) and OMAArl (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-HCl, 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-g-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. 1 1 - 1 ) . 
This form of OMAA will be referred to as OMAA-II. 

Spectral and TLC properties of the periodate oxidation product . 
The material prepared and purified under acidic conditons 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-HCl; 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 
dg-DMSO showed the general features expected of a methyl amanitin 
(Wieland et al., 1983) and also a peak of appropriate size at 6=9.55 
as expected for an aldehyde (Fig. 1 1-4) . 

Chemical reactivity of the periodate oxidation product . When both 
sodium chlorite and sodium periodate were added to a solution of OMA 
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 yery 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- 
















— CM 


— CD 





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 CD 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" and 1350 cm" 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. 1 1 -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 ambient 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 wery 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-HCl, and reduction to 
form OMDA (Wieland and Fahrmeir, 1970; Faulstich et al., 1973). The 


CH 2 0H 

H3C? H0H TA . H 3 C N CH 

CH > o CH 

n I H n I H 


H » I H n I 

H,C \ / 


' 3 \ 

CH N — CH- 

•C-N — CH-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. An 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 d g -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 OMAA-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-I I, 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-I I 
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 (OMAA-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 joer se_ does not cause the alteration in the 
toxin's conformation. This indicates that the hydrogen bond hypothesis 
for the altered conformation (Faulstich et a!., 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 possiblity of two chromatograph- 
ically separable diastereomeric forms. There have been reports of the 
ready formation of cyclic alkanolamides from the periodate oxidation 
of an appropriate diol (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 


OMAA-II at mildly acidic pH's. The interconversion between alkanol- 
amides and their constituent aldehyde and amide are known to proceed 
yery 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 alkanol amide 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, crystal lo- 
graphic (Kostansek et al., 1978; Wieland et al., 1983) and NMR spec- 
troscopic (Wieland et al., 1983) studies indicate that the a-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 
conformational^ 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. 1-1, Rp). These variants correspond to three levels of 
hydroxylation: (1) no hydroxy! ation, (2) a hydroxy! group on the 
r-carbon, or (3) hydroxyl groups on both the y- and 6-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 additon, the semisynthetic 
derivative OMDA has been prepared which contains a -y-hydroxyv aline 
residue. This hydroxyvaline residue is a close homolog of the isoleu- 
cine residue of amanullin. Thus, it is not surprising that OMDA and 
6 1 -O-methylamanullin (OML) have similar inhibitory activity toward CT 
RNAP II (Cochet-Meilhac and Chambon, 1974). A totally synthetic ana- 
toxin analog which has norvaline (an amino acid without a B-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-l). 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 
recrystal lized (Jentoft and Dearborn, 1979) and kept desiccated over 
PpOg. Hydroxyl amines-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. 


CH 2 OH 




X S H 


CHj CH 2 







och 3 

OMA- amine 

OMA- nh 2 

OMA- pro 

-NH 2 

• CH 2 


CH 2 
\ / 


Fig. III-l. 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. TIC 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, NO 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 a!., 

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 (BShlen et al., 1980) with the aid of Sep Pak C18 cartridges 
(Waters Associates). The C18 cartridges were prerinsed 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 0MA-NH 2 
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 methanol ic 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 umol of lyophilized 
0MAA. The reaction was allowed to proceed in the dark at 23°C for 18 


days. The solution was concentrated to dryness at 30°C 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 30°C 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 30°C in vacuo to remove the acetonitrile, 
supplemented with 10 ml of water, and lyophilized. The final yield was 
0.55 umol (28%). TLC: R p 1-0.60, 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 mrnol ) and sodium 
cyanoborohydride (31.5 mg, 0.5 mrnol ) were dissolved in sufficient 10% 
methanol to make 5.0 ml of solution. Lyophilized 0MAA (2.0 umol) was 
dissolved in 0.10 ml of this solution. After 96 h in the dark at 23°C 
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 umol (84%). TLC: R F , 
1-0.46, II -0 .46; color-reaction, violet. 

Synthesis of OMA-pro . A methanol ic solution (0.10 ml) containing 
0.5 M L-proline and 0.05 M sodium cyanoborohydride was added to 2.0 
umol 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 30°C, redissolved in 15% acetonitrile, and rechromatographed 
by HPLC using this solvent at 0.5 ml/min. The product (1.45 umol, 73%) 
eluted between 59 and 77 ml. TLC: Rp, 1-0.37, II-0.36; color-reaction, 

Synthesis of 0MA-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 umol of 0MA. To this was immediately 
added 10 ul 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 23°C 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 30°C to remove acetonitrile the toxin solution was 
desalted again to yield 0.85 umol (47%). TLC: R p 1-0.49, 1 1 - : . 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 
mmol ) 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 umol of lyophilized 0MAA. After 72 h in the dark at 23°C 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 umol, 58%) eluted between 36 and 45 ml. TLC: R p , 1-0.63, 
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 
0MA-NH 2 (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 0MA-NH ? . 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 0MA-NH ? , which contains the related 2,4-diamino- 
3-methylbutanoic acid residue, might display comparable lability. 
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 




H 3 C-CH H 3 C-CH 

1 H , h 

-C. C „N-CH- _ c N _ CH _ 

H O H o 

IV +CN" \v /NaCNBH, III 

v / H 

H OH x c 

* / I 

H3C-C-C-H * H 3 C-CH 

/ \ \ 3 1 H 

-C^ r „N-CH- -C^~^N-CH- 

1 x I I ^ ■ 

HO Ho 


Rv ,r' ■;: . t^/ 

+ RR'NH 

+ H + // R R' 

V / 

N© - H 2° / N 



H 3 C-CH H 3 C-CH 

1 H 1 H 

-C^ r ,N-CH- NaCNBH, ~C. C ^N-CH 

1 * I ► I * ' 

H Ho 


+ CN 

R R 1 

\ / R 

R R' 

\ / 






3 c 


-c - 


-C. C .N 






H 3 C -CH 

w CN 



-C C ,N-CH- 

H *o 



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. III-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 0MA-NH ? 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-NHL has 


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

Reductive coupling of OMAA 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. OMA-gly and OMA-pro can now be 
obtained in approximately 75% yield. 

Oxidative Transformations of OMAA 

Reaction of OMAA-I with hydroxylamine^O-sulfonic acid produced 
OMA-CN in 60% yield after a period of a few days. This reaction relies 
upon the formation of an O-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). 

OMAA-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 CD spectra of all of the new derivatives were 
indistinguishable from that of OMA (Fig. II-3). Also, their color- 
reaction with cinn-HCl was violet compared with the rust color ob- 
tained with OMAA-I. As expected, only OMAA-NH- reacted with fluores- 
camine to yield a fluorescent product. FAB-MS of 0MA-C00H and 0MA-NH 2 
revealed mass ions of the appropriate size for the proposed structures 
(Figs. II 1-3 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 K. of 3 X 10" 9 M. 
OMA-gly, OMA-pro, 0MA-C00H, and 0MA-NH 2 were much weaker inhibitors 
with Kys 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-NHL 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 K. was determined for the contents of at least 
two, and usually three, fractions across a peak. The material in the 


o • 

C_3 (/> 

01 l/> 

•1- o 
U_ Q. 


O <C 



T — i — I — i — I — i — I — r 
O O O O 
GO CD <3- C\J 



x 10- 


> 6- 

-ai8 -0.12 

-0.06 0.06 0.1 

0MA-NH 2 , uM 


Fig. 1 1 1 — 5 . Dixon plot showing the apparently noncompetitive 
inhibition of CT RNAP II by OMA-NH 2 . The concentrations of UTP used 
were 0.75 uM (circles), 1.10 uM (squares), and 1.50 uM (triangles). 


peak was subjected to additional chromatographic steps until it 
appeared homogeneous with respect to inhibition of CT RNAP II. The K, 
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 CD spectra, provides a basis for interpreting their diminished 
affinities in terns of interactions between the enzyme and the newly 
introduced substituents. In this regard, OMA-NHL 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 Y-carbon atom of residue 2 ( see Figs. 1-1 and III-l). While 
amanitins OMDA and OML, which have a hydroxy! 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, 0MA-NH ? 
which bears an amino group is a much poorer inhibitor. At first 
glance, the primary difference between OMA-Nhk 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 
hydroxy! 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 K.'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 Mn in the RNAP assay system by the amino acid 


Since OMA-NH^, which bears a positive charge, was a relatively 
poor inhibitor of CT RNAP II, it was anticipated that 0MA-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 
OMA-Nhk. 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 amanitins 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 anatox- 
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 hydroxy! 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 contain the N^-4-nitrobenzoylglycylglycine moiety 
linked to an amino sugar (D-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-ethyl- 
3-(3-dimethylamino)propylcarbodiimide (EDO, N^-2-hydroxyethyl- 
piperazine-N' -2-ethanesulfonic acid (HEPES), and sodium 3-trimethyl- 




V H 

1 s* 





CH 2 OH 

PROT- n'© 

N c- H 

hcn; r 




CH 2 OH 







CH 2 






CH 2 OH 

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


si lyl -d.-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 NaOH, EDC (0.575 g, 
3.0 mmol), and glucosamine HC1 (0.86 g, 4.0 mmol). After 12 h of 
reaction at 23°C 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: R p , II 1-0.68, IV-0.34. 
For C 17 H 22 N 4 1Q calculated: C 46.16, H 5.01, N 12.66; found: C 45.22, 
H 5.13, N 12.32. [a] 25 =+21°. ^=1.2 X 10 4 M" 1 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, 
aromatic) . 

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: R p , III-0.68, IV-0.34. For 
C 17 H 22 N 4°10 calculated: C 46.16, H 5.01, N, 12.66; found: C 45.10, 
H 5.11, N 12.23. [<x] 25 =+32°. e 2 68 =1,2 X l0 ^ M_1 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 







- CM 




- CD 



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 E 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 urn Mil lex-GV filters (Millipore) and added aseptically to 

autoclaved vials. All reactions contained 1.0 mg/ml (1.51 X 10 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 20°C 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 
u.1 were removed aseptically and placed on ice. The chilled solution 
was adjusted to 10% TCA with 40 ul 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 5°C in order to concentrate 


the conjugate solution to approximately 50 ul . The conjugates were 
diluted and concentrated three more times in a similar fashion. The 
second wash was with 0.1 M di sodium 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 

4 -1 -1 
and 300 nm were determined to be approximately 4.54 X 10 M cm and 

3.0 X 10 M cm , respectively. The comparable values for the PNBG6 

derivatives were found to be 1.0 X 10 4 M" 1 cm" 1 and 3.95 X 10 3 M" 1 
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: A 30Q from PNBGG = 1.21(A 30 q) - 0.0832(A 27g ) . Based upon 
the contribution to A~ no 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 


200 250 300 



Fig. IV-4. UV absopbance spectrum of PNBGG-GLU in water at 
approximately 2.5 X 10" 3 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 Concentration on 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 A,, 7 q or A, Q0 (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 37°C than at 20°C). 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/ml 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 37°C; circle, pH 8.0 at 37°C; inverted triangle, pH 9.0 at 
20°C; square, pH 8.0 at 20°C. 


100 150 




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 
shown) . 

Further Studies on the Effect of Sodium Cyanoborohydride 

Concentration, Borate Concentration, and pH on the Conjugation of 

The inital 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 yery 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 


100 200 




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/ml BSA, 10 mM nickel (II) chloride, 
and 6 mM sodium citrate in 0.2 M sodium borate, pH 8.0, at 37°C. The 
triangles, squares, and circles represent samples at 25, 50, and 75 
hours of reaction, respectively. 





e> 30- 





Fig. IV-7. Effect of borate concentration on the extent of conju- 
gation of PNBG6-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 37°C. Inverted 
triangle, 0.2 M; square, 0.5 M; upright triangle, 1.0 M; and circle, 
2.0 M sodium borate. 







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/ml 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-D-aldohexoses, 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 20°C 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 cyanoborohydri de-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 amino analog of PNB6G-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 
described (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-l): 

In the first method PABGG (0.126 g, 0.50 mrnol ) and glucosamine 
hydrochloride (10.8 g, 50 mrnol ) 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 NaOH with continuous rapid stirring. EDC (0.192 g, 1.0 
mrnol) was added to this solution four times at 30 min intervals; 
during this time the reaction was allowed to proceed at 23°C 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 


H 2 N \ VoNHCH 2 CONHCH 2 COOH + H 2 N< o 

+ .EDC 






2 N^ ^C0NHCH 2 C0NHCH 2 C0NH 


OH >CH 2 0H 







OH >CH 2 OH 

1) NaN0 2 /HCl 

2) AMA 


pOH >-CH 2 0H 



Fig. V-l. 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 30°C , 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 mrnol , 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 mrnol) in 45 ml of water was added 5 ml of 1.0 M sodium 
formate, pH 3.5. The solution was sparged 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 
catalyst was then removed by filtration through Whatman # 40 paper; 
the catalyst was washed with 50% acetonitrile until the A ?7 o of the 
filtrate was negligible. The combined filtrates were concentrated to a 
small volume in vacuo at 50°C and aoplied 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 mrnol, 82%) eluted in fractions 
51 to 58. TLC: R p 1-0.31, III-0.56;. For C 17 H 24 N 4 8 calculated: 
C 49.51, H 5.87, N 13.58; found: C 47.23, H 6.28, N 13.18. [a] 25 =+16°. 
e 278 =1.38 X 10 4 M" 1 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 umol) was dissolved in 0.75 
ml of ice-cold 0.1 M HC1. The PABGG-GLU was diazotized by addition of 
30 pi 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 umol 
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 umol, 55%) eluted between 32 and 40 
ml. After evaporation of the acetonitrile in vacuo at 30°C the final 
product was desalted by the Sep Pak C18 procedure. TLC: 1-0.17, 
II 1-0.44. 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 

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 

4 -1 -1 
of 4.54 X 10 M cm ; 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: 3.24A 3 g 5 /(A2 7 g-0.88A 3 gr) . 

The fate of ABGG-GLU in the reaction mixtures was determined by 

analytical HPLC. Duplicate samples (2-10 ul 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 710B 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-l). When initial efforts to 
reduce PNBGG-GLU failed, a first method was developed by which PABGG 
was coupled to the amino group of D-glucosamine with the aid of EDC. 
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 formate 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 a-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 
aryl amine function of PABGG-GLU was diazotized in dilute HC1 (Fig. 
V-l) in which the potent nitrosating agent nitrosyl 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 10" 9 M, while 
that of ABGG was 3 X 10 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 



h "* Q. 


- o 


- CD 

- CO 

1 T 

200 250 300 


Fig. V-3. UV absorbance spectrum of PABGG-GLU at 1.8 X 10 M in 
5 mM sodium phosphate, pH 7.0, (light line) and 0.1 M HC1 (dark line). 

■* o 

3 S_ l— 
+J 14-T3 

CD T3 


-t-J <-^ 




-C O 


Q- • 


00 .-H 


O i— 1 









^ -a 

•r- c 


T3 «3 










(data not shown). FAB-MS studies of AB66-GLU supported the predicted 
structure, showing mass ions of the expected sizes (Fig. V-5). 

Effects of Alkaline pH and Nickel (II) Ion 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 
37°C 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 

S- <u 
"3 > 

-O -i- 
E +-> 
O rO 

jQ C7> 

E C 
-t-> -a 





- - <3- 

X » 














" O 


r- O 

- ro 


I ' i ' i i i — i i i I o i '| i — r 













I < I 
o o 

<tf- C\J 

- -O 

.-- o 
■ _ in 

•-- o 


' o 

o — 


250 350 450 550 



Fig. V-6. Absorbance spectrum of AB6G-GLU at 8 X 10" M in the 
presence of 10 mM nickel (II) chloride (solid line) and 45 min after 
the addition of EDTA (dashed line). 









Fig. V-7. Effect of incubation of ABGG-GLU at pH 8.0 and 37°C on 
its absorbance spectrum. The ABGG-GLU concentration was initially 
7.2 X 10" M and a sample was diluted 100-fold in order to obtain the 
spectrum. Dashed line, ABGG-GLU at pH 8.0; solid line, ABGG-GLU after 
13 days at pH 8.0 and 37°C. 


pH's as low as 7.0 (see chapter IV). Thus, conjugation of ABGG-GLU to 
BSA at pH 7.0 was compared to pH 8.0. Also, the effect of raising some 
of the reactant concentrations on the rates of conjugation and toxin 
alteration was examined. 

As indicated in Table V-l, increasing the BSA and ABGG-GLU con- 
centrations five-fold produced a three- to five-fold increase in the 
rate of conjugation. As expected, conjugation proceeded more rapidly 
at pH 8.0 than at pH 7.0; the difference in rate was most pronounced 
early in the reaction (after 8 hours). The rate of alteration of the 
toxin did not show a significant dependence on the concentration of 
ABGG-GLU or BSA, but was higher at pH 8.0. 

Under all of these reaction conditions a single major sideproduct 
eluted before ABGG-GLU in the analytical reverse-phase HPLC system. 
ABGG-GLU eluted as two poorly resolved peaks after approximately 10 
min, while the byproduct emerged as a highly symmetrical peak after 
about 8 min. Preparative reverse-phase HPLC of pooled, desalted ultra- 
filtrates from these reactions yielded a quantity of the sideproduct 

which was amenable to analysis. The sideproduct showed a K. (4 X 10 

M) with CT RNAP II and a UV/visible absorbance spectrum which were 

indistinguishable from those of ABGG-GLU. 

Influence of nickel (II) ion, citrate, and sodium cyanoboro- 

hydride on the rate of toxin conjugation and alteration . Additional 

experiments were conducted to determine the importance of the nickel 

(II) ion and sodium cyanoborohydride on the rates of conjugation and 

alteration of the toxin. As noted above, a protective effect of nickel 

(II) ion had been detected in early studies conducted at pH 8.0. In 

those studies citrate was included to permit solution of the nickel 


Table V-l. Effect of pH and reactant concentrations on the 
degradation of ABGG-GLU and on the extent of conjugation of ABGG-GLU 
to BSA. 

of BSA a 


time of 



1 mg/ml 






1 mg/ml 






5 mg/ml 






5 mg/ml 






1 mg/ml 






1 mg/ml 






5 mg/ml 






5 mg/ml 






a All reaction mixtures contained 0.5 M sodium borate, 
0.125 M PIPES, 25 mM nickel (II) chloride, and 25 mM sodium citrate. 
ABGG-GLU was present in a molar concentration which was 240 times that 
of BSA. 

The molar ratio of ABGG-GLU to BSA in the conjugates was 
determined spectrophotometrical ly. 

c The amount of ABGG-GLU which remained in unreacted form in the 
reaction mixtures was determined by analytical HPLC. 


(II) ion in the alkaline solution. The results presented in the sec- 
tion above suggested that the conjugation of ABGG-GLU might be more 
profitably conducted at pH 7.0 rather than pH 8.0. At pH 7.0 nickel 
(II) chloride is quite soluble without the inclusion of a chelator 
like citrate. Therefore, experiments were conducted to examine the 
need for the nickel (II) ion in the reactions at pH 7.0 and the impact 
of citrate upon any effect of the nickel (II) ion (Table V-2). The 
inclusion of citrate in the reaction did not appear to have a pro- 
nounced effect upon either the rate of conjugation or the rate of 
alteration of the toxin, while omission of the nickel (II) ion from 
the reaction resulted in an acceleration of both. When sodium cyano- 
borohydride, nickel (II) chloride, and citrate were all omitted from 
the reaction, loss of the toxin was almost negligible and coupling of 
the toxin to BSA occurred at a ^ery low, but significant, level (cf. 
chapter IV). The conjugate prepared in the presence of nickel (II) 
chloride demonstrated a K. of 1.35 X 10" M (with respect to amanitin 
residues), while that prepared without nickel was slightly less inhib- 
itory with a K. of 6.1 X 10" 7 M. 

The azo coupling reaction has provided a relatively simple means 
of selectively introducing chemically complex moieties into a single 
site on the amanitin molecule. Thus, one can attach a complex linker 
and a conjugable group en bloc . Some refinements of the synthetic 
procedures have been introduced in this work which serve to make this 
route to conjugable derivatives of amanitin more attractive and prac- 
tical. The nitrobenzoyl group has continued to serve as a convenient 


Table V-2. Effect of nickel (II) chloride, citrate, and sodium 
cyanoborohydride on the degradation of ABGG-GLU and on the extent of 
conjugation of ABGG-GLU to BSA. 

components 3 




nickel , citrate, and 



nickel and 






no additions 0.8 96 

All reaction mixtures contained 5 mg/ml BSA, 18 mM ABGG-GLU, 
0.5 M sodium borate, and 0.125 N PIPES. Nickel (II) chloride (25 mM), 
sodium citrate (25 mM), and sodium cyanoborohydride (100 mM) were also 
added as indicated. The reaction mixtures were incubated in the dark 
for 30 hours at 37°C. 

The molar ratio of ABGG-GLU to BSA in the conjugates was 
determined spectrophotometrical ly. 

c The amount of ABGG-GLU which remained in unreacted form in the 
reaction mixtures was determined by analytical HPLC. 


precursor for the necessary aryl amino function (cf. Faulstich and 
Trischmann, 1973; Preston et al., 1981). Catalytic transfer hydrogena- 
tion has proven to be an efficient and selective method for reducing 
the nitro group to an amine without need for a hydrogenation 

The reductive conjugation of aldehydes to proteins has been shown 
to be relatively straightforward by several workers (Jentoft and 
Dearborn, 1979; Schwartz and Gray, 1977; Wong et al., 1985). While the 
alkylation reaction has proven to be very specific for protein amino 
groups, some side reactions have been defined. In reductive methyl a- 
tion of proteins the cyanide which is generated in the course of the 
reaction can react with an intermediate of the reaction to form a 
quasi-stable adduct (Gidley and Sanders, 1982). The inclusion of a 
metal ion, such as the nickel (II) ion, which complexes with cyanide 
has been shown to be effective in overcoming this problem (Jentoft and 
Dearborn, 1980). In the case of reductive methylation some reduction 
of formaldehyde has been observed, especially at lower pH's (Jentoft 
and Dearborn, 1979), but there have been no comprehensive studies of 
the fate of sugars which were being reductively coupled to proteins. 

It was hoped that the recognized side reactions due to cyanide 
and reduction of the aldehyde function could be avoided by including 
the nickel (II) ion in the reaction system and by maintaining a 
slightly alkaline pH. It also seemed prudent to include the nickel 
(II) ion in order to prevent the well -recognized degradation of pro- 
tein by cyanide (Catsimpoolas and Wood, 1964; Catsimpoolas and Wood, 
1966; Wood and Catsimpoolas, 1963). It was noted that the nickel (II) 
chloride produced a profound change in the color and absorbance spec- 


trum of ABGG-GLU. This is likely due to complex at ion of the nickel ion 
by the azo amanitin. The formation of coordination complexes between 
transition metal ions and ^-hydroxyazo compounds has been known for 
many years (Zollinger, 1961). It is conceivable that two ABGG-GLU 
molecules may be able to coordinate with a single nickel (II) ion. 

In initial studies of reductive coupling of an azo amanitin to 
protein a diminution of the color of the reaction was noted after 
prolonged periods of reaction. This color change was most prominent in 
reactions to which no nickel (II) chloride was added. Since the color 
of the azo amanitins derives from the aromatic portion of the mole- 
cule, it was reasoned that this part was being altered. Several possi- 
ble side-reactions were envisioned. One possibility was that the tryp- 
tathionine portion of the molecule was undergoing 0-elimination 
because of the slightly alkaline pH and elevated temperature. Indeed, 
when ABGG-GLU was incubated at 37°C in 0.2 M sodium borate, pH 8.0, 
alteration of the absorbance spectrum was noted over the course of 
several days. It was also conceivable that the azo bridge was being 
reduced; the nickel (II) ion might be protective against the reduction 
by virtue of the formation of the coordination complex. In fact, the 
chemical stability of azo dyes has been noted to be increased by 
complexation with transition metal ions (Zollinger, 1961). 

Experiments were performed in order to determine whether the rate 
of side reactions could be reduced relative to the rate of the conju- 
gation reaction. Increasing the concentrations of ABGG-GLU and BSA has 
been found to have a positive effect in this regard; the rate of con- 
jugation was increased without greatly effecting the rate of loss of 


The major byproduct could be purified and had the same absorbance 
spectrum and K. toward CT RNAP II as ABGG-GLU. It eluted from the C18 
reverse-phase HPLC system before ABGG-GLU, suggesting that it is more 
polar than ABGG-GLU. In contrast to ABGG-GLU which elutes as two poor- 
ly resolved peaks, the byproduct eluted as a single, sharp peak. The 
two components of ABGG-GLU presumably reflect the fact that the sugar 
residue has two anomeric configurations. Altogether, these character- 
istics indicate that this byproduct likely results from reduction of 
the aldehyde group of the sugar to a hydroxyl group. Thus, one might 
expect that factors which make the aldehyde group more available 
(e.g., increased pH) would increase the rate of formation of this 
byproduct. This is, in fact, the pattern which is seen. 

Additional experiments were undertaken to evaluate the effect of 
nickel and cyanoborohydride on the course of the reaction at pH 7.0. 
In the absence of nickel the rates of both conjugation and loss of 
ABGG-GLU were substantially higher. The presence of citrate had no 
significant effect upon the course of the reactions which contained 
nickel. In the absence of cyanoborohydride there was very little con- 
jugation of ABGG-GLU to BSA and there was no significant loss of the 
azo amanitin after thirty hours of reaction. This shows that both the 
conjugation and the side reaction(s) are strongly cyanoborohydride- 
dependent under these conditions. The lower conjugation and side- 
reaction rates observed in the presence of the nickel (II) ion probab- 
ly have a complex origin, but may be largely a consequence of complex- 
ation of ABGG-GLU to the nickel. If these complexes form predominantly 
in a stoichiometry of two ABGG-GLU per nickel ion, then the reactivity 


of ABGG-GLU may be diminished on account of its lower effective con- 

The conjugates of ABGG-GLU and BSA prepared here proved to be 
relatively poor inhibitors of CT RNAP II. However, this low level of 
inhibitory activity of amanitin-BSA conjugates has been consistently 
observed by other workers. The significance of a high or low K, for 
the interaction between RNAP II and an amanitin-protein conjugate is 
not at all clear. As of yet, the molecular determinants of these K.'s 
are not known. Despite their high K.'s, amanitin-BSA conjugates have 
in the past proven to be highly cytotoxic to cells in culture 
(Faulstich et al., 1975; Hencin and Preston, 1979; Preston et al . , 

In summary, an efficient method of preparing an azo derivative of 
AMA has been described. Reaction conditions for the coupling of this 
azo derivative to BSA have been optimized so that substantial amounts 
of the toxin can be linked to the protein within a reasonable time. 
The conjugates thus produced have inhibi toy properties toward CT RNAP 
II which are similar to those reported by others. The task of deter- 
mining whether this new method of conjugation can be used to prepare 
amanitin-antibody (or SMWCA-antibody) conjugates which are as good as 
or better than those being generated by current methods lies ahead. 


Two routes for the preparation of aldehyde-containing amanitins 
have been explored. Study of the periodate oxidation products of OMA 
has provided new insights into the nature of OMAA. Under certain 
circumstances, a true aldehyde can be generated by periodate oxidation 
of OMA, but the form of OMAA which has been prepared by others appears 
to exist almost exclusively in a non-aldehydic form. Apparently as a 
consequence of this, the reductive amination of OMAA is difficult. 
Despite these difficulties, a number of new derivatives of amanitin 
have been prepared which bear conservative changes in the sidechain of 
residue 3. A large range of inhibitory potency has been discovered for 
derivatives with relatively small differences in this sidechain. A 
preliminary correlation between the presence of a charged group in 
residue 3 and markedly diminished inhibitory potential has been noted. 

An efficient method for preparing an azo amanitin which bears a 
residue of glucosamine has been described. The glucosamine residue has 
been found to share with lactose a strong tendency to expose its 
aldehyde function in solutions of borate, as evidenced by increased 
reactivity in reductive alkylation reactions in this medium. This 
property has been utilized for the preparation of an azo amanitin 
which bears an aldehyde which is protected within the sugar; it can be 
selectively deprotected in solutions of borate to permit efficient 
reductive coupling of the amanitin to proteins. 



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Jerald Mullersman was born at Fort Huachuca, Arizona, on June 13, 
1956, to Ferd and Wanda Mullersman. He spent his formative years in 
Gainesville, Florida. He subsequently completed undergraduate and 
medical training at the University of Florida. He now resides in St. 
Louis, Missouri, with his wife Bette and two daughters Sarah and 
Emily. Currently, he is serving as a resident in laboratory medicine 
at the Washington University medical center. 


I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 

'James F. Preston, III, Chairman 
Professor of Microbiology and 
Cell Science 

I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 

2 Jm& 

Edward M. Hoffman 
Professor of Micro 
Cell Science 

I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 


Michael D. P. Boyle 
Professor of Immunology and 
Medical Microbiology 

I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 

r Parker A. Small, Jr 

Professor of Immunology and 
Medical Microbiology 

I certify that I have read this study and that in my opinion it 
conforms to acceptable standards of scholarly presentation and is 
fully adequate, in scope and quality, as a dissertation for the degree 
of Doctor of Philosophy. 

Edward K. Wakeland 
Associate Professor of 

This dissertation was submitted to the Graduate Faculty of the College 
of Agriculture and the Graduate School and was accepted as partial 
fulfillment of the requirements for the degree of Doctor of 
Phi losophy. 

December, 1986 

dzk Z'CJA 


Dean^/College of Agriculture 

Dean, Graduate School 


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