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BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. 19, 248-252 (1990)
Characterization of Glutathione Conjugates of
Chlorambucil by Fast Atom Bombardment and
Thermospray Liquid Chromatography/Mass
Spectrometry
Deanne M. Dulik,? 0. Michael Colvint and Catherine Fenselaug
Department of Pharmacology and Molecular Sciences and Oncology Center, Johns Hopkins School of Medicine, Baltimore,
Maryland 21205, USA
Chlorambucil @-(di-2chloroethyl)amino-y-phenylbutyric acid) is a bifunctional alkylating agent which exhibits
acquired drug resistance upon repeated dosing in humans. This compound reacts with glutathione both nonenzymatically and enzymatically in the presence of immobilized microsomal glutathione-S-transferases to produce
several glutathione conjugates. These conjugates result from displacement of one or both chlorines by the nucleophilic cysteine sul!l~ydrylmoiety of glutathione. The mono- and diglutathionyl conjugates of chlorambucil were
purified by reversed-phase high-performance liquid chromatography and characterized by positive ion fast atom
bombardment mass spectrometry. In addition, the mono- and dihydroxy hydrolysis products of chlorambucil were
characterized by positive ion thermospray liquid chromatography/mass spectrometry (LC/MS). The glutathione
conjugates of chlorambucil did not produce molecular ion species in thermospray LC/MS mode, but gave characteristic ions at m/z 147 corresponding to fragmentation of the glutathione moiety. The formation of glutathione
conjugates of this class of alkylating agents may play a role in the development of acquired drug resistance.
Glutathione (y-glutamylcysteinylglycine, GSH) is the
most abundant intracellular nucleophile in mammalian
cells, present at concentrations of approximately 5 mM.
Chlorambucil (p-(di-2-chloroethyl)amino-y-phenylbuty- Several studies have suggested a relationship between
GSH and the transferases involved in the enzymatic forric acid, I) is a bifunctional nitrogen mustard alkylating
mation of glutathione conjugates (GST) and the develagent which has been used extensively in the treatment
opment of ADR. Among these observations are: (a) in
of ovarian and head/neck carcinomas. This compound,
the presence of alkylating agents such as melphalan,
along with several others in its therapeutic class, have
intracellular levels of GSH are rapidly depleted;* (b)
been associated with the development of acquired drug
cells which are made resistant to alkylating agents by
resistance (ADR), in which repeated dosing of the agent
repeated exposure show higher initial levels of both
results in a lack of target cell cytoxicity and therefore
GSH and GST;3 and (c) depletion of intracellular GSH
reduced clinical effectiveness. The development of ADR
by GSH synthetase inhibitors, such as buthionine sulhas been associated with several possible factors, includfoximine, causes a concomitant increase in the cytoing (a) decreased drug uptake into target cells, (b)
toxicity of administered alkylating agent^.^ These
increased DNA repair mechanisms, and (c) increased
results suggest a plausible relationship between the
reaction of the alkylating agent with intracellular
reaction of glutathione and alkylating agents and the
nucleophiles with resulting loss of cytotoxic potential.'
development of acquired drug resistance.
CI--H C--H c \ N ~ C H 2 - C - H 2 - C H 2 - C c l o n
The objective of this study was to synthesize and
characterize the reaction products between chlorambuCI--H c-n c'
2
2
cil and glutathione. Conjugates of chlorambucil were
prepared under non-enzymatic conditions and enzymatically using immobilized microsomal glutathione-SI
pansferases
from cynomolgus monkey liver. The use of
t Present address: Department of Drug Metabolism, Smith Kline &
'immobilized
GST for synthesis of glutathione conjuFrench Research Laboratories, L-711, P O Box 1539, King of Prussia,
gates of a variety of electrophilic substrates has been
Pennsylvania 19406-0939, USA. Author to whom correspondence
should be addressed.
described previously by our l a b ~ r a t o r yA
. ~combination
3 Present address: Department of Pharmacology, Oncology Center,
of fast atom bombardment (FAB) mass spectrometry
Johns Hopkins School of Medicine, 600 N. Wolfe Street, Baltimore,
and thermospray liquid chromatography/mass specMaryland 21205, USA.
trometry (LC/MS) were employed for structural charac8 Department of Chemistry, University of Maryland Baltimore
County, 5401 Wilkens Avenue, Baltimore, Maryland 21228, USA.
terization of the resulting conjugates.
INTRODUCTION
0887-6 134/90/04O248-O5 $05.00
0 1990 by John Wiley & Sons, Ltd.
Received 24 July 1989
Accepted I November 1989
GLUTATHIONE CONJUGATES OF CHLORAMBUCIL
MATERIALS AND METHODS
Chemicals
Chlorambucil was kindly provided by Dr John Hilton,
Oncology Center, Johns Hopkins School of Medicine.
Reduced glutathione was obtained from Sigma Chemical Company (St Louis, Missouri). All other chemicals
were reagent grade or better and were used without
further purification. Hydrolysis products of chlorambucil were prepared by reaction of chlorambucil in 0.1 M
NaOH for 1 h at room temperature.
Immobilized enzyme synthesis of glutathione conjugates
Cynomolgus monkey liver microsomal g1utathione-Stransferases were immobilized onto cyanogen bromideactivated Sepharose 4B by a published method.' Incubation mixtures contained chlorambucil (1.0 mM),
reduced glutathione (3.0 mM) and packed Sepharose
beads carrying immobilized glutathione-S-transferases
(15 ml packed beads) in aqueous phosphate buffer (0.1
M, pH 7.4). Total reaction volume was 20 ml. Reactions
were run at 37°C for 1 h. A control non-enzymatic
reaction was run in the absence of immobilized enzyme.
After 1 h, the immobilized protein was removed by filtration through a coarse fritted funnel. The filtrate was
concentrated using solid-phase extraction methods
(Sep-Pak,
C 8
cartridge,
Waters,
Milford,
Massachusetts) and the organic eluent (methanol) was
evaporated to dryness before thin-layer chromatography (TLC) and mass spectrometric analysis.
Thin-layer chromatography
TLC was done using aluminum-backed silica-gel plates
(5 x 10 cm, E. Merck, Darmstadt) for crude reaction
mixtures after solid-phase extraction. The mobile phase
was ammonium hydroxide-absolute ethanol (70 : 30).
After air drying, components of the incubation mixtures
were visualized with ninhydrin spray reagent (0.2% in
ethanol) and heated to 100°C on a hotplate for 5-10
min to produce a blue-purple color.
High-performance liquid chromatography (HPLC)
Incubation mixtures were purified using a Beckman
Model 421 HPLC system with Model llOA pumps.
Conditions were: c,, column (Brownlee, 100 x 4.6 mm,
5 pm); mobile phase, solvent A, 0.1 M ammonium
acetate, pH 6.8, solvent B, methanol; linear gradient,
10-100% solvent B in 25 min; flow rate 1.0 ml min-'.
Ultraviolet (UV) detection was done at 254 nm (Kratos
Spectroflow Model 783 variable UV detector). Reaction
products were collected into 50 ml round-bottom flasks
and frozen on dry ice immediately. HPLC eluents were
evaporated under vacuum and the residues stored at
- 80 "C before analysis by mass spectrometry. Peak
areas were integrated on an Isaac 41A Interface Module
(Cyborg, Corp., Boston, Massachusetts) and Apple IIe
computer.
249
Mass spectrometry
Positive ion FAB mass spectra were obtained using a
Kratos MS50 double-focusing magnetic sector mass
spectrometer with a Kratos FAB source and DS-90
data system (accelerating voltage 8 kV, resolution 3000).
Cesium iodide-glycerol was used to calibrate over the
mass range 92-1005 u. Samples were dissolved in 25-50
pl methanol; thioglycerol was the liquid matrix.
Samples were bombarded with a xenon atom beam at
7-8 kV kinetic energy. Spectra were plotted as averages
of several scans at a scan rate of 10 s/decade. Positive
ion thermospray LC mass spectra were acquired on a
Finnigan TSQ-45 triple-quadrupole mass spectrometer
in the Department of Drug Metabolism, Smith Kline &
French Research Laboratories. A Finnigan thermospray ion source was used. No electron filament or
discharge electrode was employed. Ion source conditions were : block temperature 240 "C; vaporizer temperature 120°C; repeller voltage +25 eV. Spectra were
recorded in 1.95 s scans over the mass range 120-650 u.
LC conditions were: RP-300 HPLC column (Brownlee,
100 x 4.6 mm, 5 pm); solvent A, 0.1 M ammonium
acetate adjusted to pH 5.3 with glacial acetic acid,
solvent B, methanol; linear gradient, 20% solvent B to
70% solvent B in 20 min; flow rate 1.1 ml min-'. A UV
detector (Kratos Spectraflow Model 783) was placed in
line before the mass spectrometer and the eluent was
monitored at 254 nm.
RESULTS AND DISCUSSION
The HPLC chromatogram for the enzymatic reaction
mixture between chlorambucil and reduced glutathione
is shown in Fig. 1. Seven HPLC peaks were observed,
designated as A-G. HPLC product G (retention time
19.3 min) coeluted with an authentic sample of chlorambucil and gave an identical FAB mass spectrum. HPLC
products A and B coeluted with products obtained by
reaction of chlorambucil with 0.1 M NaOH, suggesting
that they are hydrolysis products formed by displacement of one or both of the chlorines in the
chloroethyl side chains by water. FAB mass spectral
analysis of purified peaks A and B gave no useful structural information when analyzed in several liquid
matrices and in the presence of 0.1% HCl. The mono
and dihydroxy hydrolysis products of a structural
analog, melphalan, were also resistant to FAB analysk6
Thermospray LC/MS analysis of product A (retention
time 4.0 min) produced a protonated molecular ion at
m/z 268, corresponding to the dihydroxy analog of chlorambucil (Fig. 2). Thermospray LC/MS analysis of
product B (retention time 5.3 min) produced a molecular ion M" at mlz 250, corresponding to the cyclic aziridinium ion form of the monohydroxy analog of
chlorambucil (Fig. 3).
Four ninhydrin-positive products were identified
from enzymatic reaction mixtures containing chlorambucil and reduced glutathione (rf = 0.64, 0.58, 0.50 and
0.32). These products corresponded to HPLC fractions
C, D, E and F, suggesting that four glutathione conjugates were present. The FAB mass spectrum of HPLC
s-
D. M. DULIK, 0. M. COLVIN AND C. FENSELAU
250
HO-H2C-H2C,N
0
\ /
CH2-CH
HO-H C-H C'
2
2
2
-CH-COOH
2
E
mlz
0
5
15
10
25
20
Figure 2. Positive ion thermospray LC/MS spectrum of HPLC
product A, dihydroxy chlorambucil.
TIME (rnin)
Figure 1. HPLC chromatogram of the reaction mixture of chlorambucil and reduced glutathione in the presence of immobilized
microsomal glutathione-S-transferases (UV detection at 254 nm).
Designated products are: A, dihydroxy chlorambucil; B, monohydroxy monochloro chlorambucil; C, diglutathionyl chlorambucil;
D, monohydroxy monoglutathionyl chlorambucil; E, 4(glutathiony1)phenylbutyricacid; F, monochloro monoglutathionyl
chlorambucil; G, chlorambucil.
product C (retention time 8.0 min) shows a protonated
molecular ion at mass 846, corresponding to the diglutathionyl conjugate of chlorambucil (Fig. 4). The FAB
mass spectrum of HPLC product D (retention time 8.8
min) contains a protonated molecular ion peak at m/z
557 and a sodium adduct peak at m/z 579, corresponding to the monohydroxy monoglutathionyl conjugate
(Fig. 5). This glutathione adduct of a hydrolysis product
of chlorambucil is of special interest, since the analogous conjugate was not a major metabolite of the structurally similar alkylating agent, melphalan.6 The FAB
mass spectrum of a minor HPLC product E (retention
time 11.2 min) gave a weak signal at m/z 470 using 2nitrophenyl octyl ether as the liquid matrix. The mass
H C
M+
2 I
100
%I
50
M+N~)+
272
F,3TTT7 I
m/z
Figure 3. Positive ion thermospray LC/MS spectrum of HPLC
product B, monohydroxy chlorambucil (cyclic aziridinium ion
form).
%I
I
x 20
50
200
300
400
800
900
Figure 4. Positive ion FAB mass spectrum of HPLC product C, diglutathionyl chlorambucil. SG = glutathionyl.
GLUTATHIONE CONJUGATES OF CHLORAMBUCIL
(M+H)+
300
400
350
450
500
251
(M+Na)+
550
600
m/z
Figure 5. Positive ion FAB mass spectrum of HPLC product D, monohydroxy monoglutathionyl chlorambucil. SG
spectrum is believed to correspond to 4-(glutathionyl)
phenylbutyric acid. This unusual conjugate is analogous
to that previously reported for melphalan, in which
nucleophilic displacement of the mustard moiety occurs
by glutathione, perhaps through the cyclic aziridinium
ion inte~mediate.~
HPLC product F (retention time 13.0
min) was confirmed by FAB mass spectrometry as the
monochloro monoglutathionyl conjugate of chlorambucil; a protonated molecular ion of mass 575 was
observed as well as a potassium adduct of mass 613
(Fig. 6). Thermospray LC/MS analysis of products C-F
gave no protonated molecular ions under the conditions
employed; a common fragment ion of mass 147 was
observed for each product, resulting from loss of glutamic acid from the intact glutathione conjugate. This
characteristic loss of 147 u in the thermospray LC mass
spectra of other glutathione conjugates has been
and is most likely a result of the thermal
lability of the tripeptide.
Integration of the HPLC/UV peak areas for peaks
A-G demonstrated that the enzyme-catalyzed reaction
of chlorambucil with glutathione produced a modest
two- to fivefold increase in the relative amounts of glu-
C I - H2C-H2C
C-H c'
2
2
GS-H
0
1
\
CH-CH
2
2
-CHZ-COOH
254
'O01
%I
rt
I
I
I
I
j
5
(M+H)+(M+K)+
0
200
250
300
350
400
450
500
550
600
0
650
mlz
Figure 6. Positive ion FAB mass spectrum of HPLC product F,
monochloro monoglutathionyl chlorambucil. SG = glutathionyl.
= glutathionyl
tathione conjugates formed as compared with the nonenzymatic reaction. Formation of product E was
negligible in the non-enzymatic reaction. A marked
decrease in the amounts of peaks A and B were
observed in the enzymatic reaction. In general, the presence of immobilized microsomal glutathione-s-transferases may serve to increase the relative nucleophilicity
of the cysteine sulfhydryl moiety of glutathione and
therefore provide increased competition for nonenzymatic hydrolysis of the active alkylating agent.
These observations suggest that chlorambucil, as well as
other alkylating agents in this class, are substrates for
the microsomal glutathione-S-transferases. No studies
have been done with the cytosolic forms of the enzyme
in our laboratory to date.
In summary, chlorambucil reacts with glutathione to
produce several conjugates which are formed by nucleophilic displacement of one or both chlorines from the
nitrogen mustard side-chain. The products formed are
analogous to those produced by reaction of glutathione
with the alkylating agent melphalan. The glutathione
conjugates are amenable to characterization by positive
ion FAB mass spectrometry and produce fragment ions
in thermospray LC/MS which are characteristic of the
glutathione moiety. The hydrolysis products of chlorambucil are resistant to analysis by FAB mass spectrometry, but produce molecular ions in thermospray
LC/MS mode. This may in part be due to the relative
polarity of the hydrolysis products in the liquid matrix
in FAB which impedes desorption, or perhaps analysis
is hindered due to the presence of interfering buffer
salts. The formation of glutathione conjugates of chlorambucil may play a role in the development of
acquired drug resistance in tumor cells. Further studies
will address the presence of chlorambucil-glutathione
conjugates in sensitive and resistant cells and the possible biochemical mechanisms responsible for the development of ADR.
Acknowledgements
The authors gratefully acknowledge the financial support of US
Public Health Service Grants GM-21248 and CA-16783-11 from the
252
D. M. DULIK, 0. M. COLVIN AND C. FENSELAU
National Institutes of Health and the US Department of Agriculture
Specific Cooperative Agreement 58-579-4-14. We thank Connie
Murphy, Mid Atlantic Mass Spectrometry Laboratory, a National
Science Foundation Regional Instrumentation Facility, for providing
FAB mass spectral data. We thank Dr. John Hilton, Johns Hopkins
School of Medicine, for helpful discussions.
REFERENCES
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(1987).
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35,3405 (1986).
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(1987).
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