436
Chem. Res. Toxicol. 1991, 4, 436-444
(20) Owsley, D. C.,Helmkamp, G. K., and Rettig, M. F. (1969) Episulfonium salts. Detection of an unusual intermediate in the reaction of a stable episulfonium salt with chloride ion. J . Am. 91,5239-5242. Chem. SOC. (21)Adzima, L.J., Duesler, E. N., and Martin, J. C. (1977)Reactions and crystal and molecular structure of an unsymmetrical spirosulfurane. Manifestations of hypervalent bond polarization in a sulfurane. J . Org. Chem. 42, 4001-4005. (22) Roberts, J. J., and Warwick, G. P. (1961)The mode of action of alkylating agents. Biochem. Pharmacol. 6, 205-216. (23) Peterson, L.A., Harris, T. M., and Guengerich, F. P. (1988)
Evidence for an episulfonium ion intermediate in the formation of S-[2-(lvl-guanyl)ethyl]glutathionein DNA. J. Am. Chem. SOC. 110,3284-3291. (24) Foureman, G. L., and Reed, D. J. (1987)Formation of S42(M-guanyl)ethyl]adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionylconjugates of 1,2-dichloroethane. Biochemistry 26, 2028-2033. (25) Pearson, P. G., Soderlund, E. J., Dybing, E., and Nelson, S. D. (1990)Metabolic activation of 1,2-dibromo-3-chloropropane:Evidence for the formation of reactive episulfonium ion intermediates. Biochemistry 29, 4971-4981.
Carbamoylation of Peptides and Proteins in Vitro By S- (N-Met hylcarbamoy1)glutathione and S-(N-Methylcarbamoyl)cysteine, Two Electrophilic S-Linked Conjugates of Methyl Isocyanatet Paul G. Pearson,* J. Greg Slatter,t Mohamed S. Rashed,g Deog-Hwa Han," and Thomas A. Baillie* Department of Medicinnl Chemistry, School of Pharmacy, BC-20, University of Washington, Seattle, Washington 98195 Received January 25, 1991
The reactivity toward peptides and proteins of S-(N-methylcarbamoy1)glutathione (SMG), the glutathione conjugate of methyl isocyanate, and the corresponding cysteine adduct, 5'-(Nmethylcarbamoy1)cysteine (SMC), was investigated with the aid of in vitro model systems. Incubation of SMC or a trideuteriomethyl analogue of SMC with either the reduced or oxidized forms of oxytocin afforded similar mixtures of mono-, bis- and tris-N-methylcarbamoylated peptides. Structure elucidation of the mono and bis adducts by fast atom bombardment tandem mass spectrometry indicated that carbamoylation of oxytocin occurred preferentially a t Cys-6 and that Cys-1 and/or Tyr-2 were secondary sites of modification. Upon incubation of S[N-([14C]methyl)carbamoyl]glutathione(14C-SMG)with native bovine serum albumin (BSA), radioactivity became bound covalently to the protein in a time- and concentration-dependent fashion. "Blocking" of the lone Cys-34 thiol group of BSA in the form of a disulfide prior to exposure of the protein to 14C-SMGfailed to decrease significantly the extent or time course of this covalent binding. It is concluded that carbamate thioester conjugates of MIC are reactive, carbamoylating entities which can donate the elements of MIC to nucleophilic functionalities on peptides and proteins. Free thiols appear to be preferred sites for such carbamoylation processes, a phenomenon that may have important toxicological consequences in the pathology of tissue lesions induced by MIC and related isocyanates.
Introduction Following the catastrophic release of MIC' (Figure 1) into the atmosphere in Bhopal, India, on December 2-3, 1984, which resulted in the deaths of more than 3000 inhabitants (I, 21, considerable attention has focused upon the adverse effeds of this highly toxic agent. In individuals exposed acutely to MIC, the most prevalent clinical symptoms were severe eye and respiratory tract irritation (3), and it has been estimated that some 10% of the exposed population in Bhopal suffered pathological changes in the lung associated with emphysema (4). Studies of the +A preliminary account of these studies was presented at the Second International Sympoeium on Maee Spectrometry in the Life Sciences, San Francisco, CA, Sept 1989. Present address: The Upjohn Laboratories, Kalamazoo, MI 49001. Present address: Lederle Laboratories, Pearl River, NY 10965. Present address: Medicinal Toxicology Research Center, Inha University, Inchon, Korea.
*
effects of MIC in animal models revealed a similar pattern of pulmonary irritation, mediated by a direct action on the lining epithelium of the nasal cavity and major airways (5, 6). Peripheral emphysema and severe ocular irritation were also evident in animal studies (7). Somewhat surprisingly, in view of the high chemical reactivity of MIC (8) and its short half-life in aqueous media [estimated to Abbreviations: MIC, methyl isocyanate; SMG, S-(N-methylcarbamoy1)glutathione; 'C-SMG, S-[N-([lrC]methyl)carbamoyl]glutathione; SMC, S-(N-methylcarbamoy1)cysteine;CD8-SMC, S-[N-(trideuteriomethyl)carboyl]cysteine; SEC, S-(N-ethylcarboyl)cynteine; NMF, N-methylformamide;GSH, glutathione; BSA, bovine serum albumiq MMTS, methyl methanethiobulfonate;DTE,dithioqthritol; D'IT, dithiothreitol; MCPBA, m-chloroperoxybenzoicacid; TEAF,triethylammonium formate, HPLC, high-performanceliquid chromatography; LC-MS, thermospray liquid chromatography-mass spectrometry;FABMS, fast atom bombardment mass spectrometry;MS MS, tandem maas spectrometry; CID, collision-induced dimciation; PLC, faat protein liquid chromatography. Signal multiplicities in nuclear magnetic resonance (NMR) spectra are designated as follows: a, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet.
B
0 1991 American Chemical Society
Reactive S-Linked Conjugates of Methyl Isocyanate ,NHz
R1\ CbN-C-O MIC
SH
I
Rd
NCO-SCH2CH, C W
SMC
Ri-C&&-H:
-
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CD,-SMC
Rl-Rz-CI+
SDMC
:
SH
I
H2N-CYS-TYR-ILEOLN-ASNCYS-PRO-LEU-GLY-NH2 Oxytocin (Reducad)
S
I
S
I
H2N-CYS-TYR-ILE-GLN-ASN-CYS-PRO-LEU-GLY-NHz
Oxytocin (Oxidized)
Figure 1. Structures of compounds referred to in the text. be approximately 2 min (9)], systemic effects involving organs remote from the primary site of exposure have been reported in survivors of the Bhopal disaster (10,11).These long-term effects, some of which have also been noted in animal studies, have included infertility (12),myelotoxicity (13,14), and disorders of the cardiovascular, gastrointestinal, musculoskeletal,and immunological systems (4).In this context, it is of interest to note that, in recent studies in rodents exposed to 14C-MICvapor, radioactivity from inhaled MIC entered the systemic circulation rapidly and was distributed widely to extrapulmonary organs (6),while in a separate investigation, significant levels of radioactivity were found to be bound covalently to hepatocellular proteins with 30 min of administration of MIC to rodents (15). The chemical nature of these 14C-labeledmaterials (or material) distributed in blood and tissues following exposure to W-MIC remains unknown, but it is possible that a portion of the inhaled MIC undergoes biotransformation in the lung to chemically less reactive products which cross the alveolar-blood barrier and enter the systemic circulation. Therefore, the possibility arises that metabolites of MIC may mediate the extrapulmonary toxicities of the parent isocyanate. Although very little information is available on the metabolic fate of MIC in mammalian systems, the presence of high concentrations of glutathione (GSH) in lung endothelial fluid (16)suggests that inhaled MIC may react chemically with this thiol to form the corresponding glutathione conjugate, SMG (Figure 1). Support for this hypothesis derives from the observation that MIC reacts readily with GSH in aqueous media to form SMG (17),and in preliminary in vivo studies, we have shown that when rats are given MIC by intraperitoneal injection, SMG is excreted a metabolite in bile (18)and the corresponding mercapturic acid derivative is present in urine (19). Moreover, SMG has proven to be a toxic species in its own right in that concentrations in the low millimolar range were cytotoxic toward rat hepatocytes in vitro, while levels as low as 10 r M inhibited the growth of murine TLX5 lymphoma cells in culture (20).Although the biochemical basis for these cytotoxic properties remains unknown, it is possible that SMG reverts to free MIC under physiological conditions in a fashion similar to that reported for the structurally related GSH conjugates of allyl and benzyl
Chem. Res. Toxicol., Vol. 4,No. 4,1991 437 isothiocyanate (21). Indeed, the results of recent pilot studies with synthetic SMG support this hypothesis insofar as incubation of SMG with cysteine in vitro resulted in the concomitant formation of free GSH and the corresponding cysteine conjugate, SMC (Figure 1) (18). In light of the above considerations and the growing evidence that GSH conjugation of certain types of reactive intermediate occurs in a reversible fashion (22),we have speculated that SMG, formed as pulmonary metabolite of inhaled MIC, may serve as a vehicle by which the highly reactive isocyanate can be transported via the systemic circulation to distant organs. Liberation of free MIC at cell surfaces (23)or, possibly, a t intracellular sites would be expected to result in extensive carbamoylation of biological macromolecules, leading, in turn, to extrapulmonary toxicities of inhaled MIC. The objectives of the present investigation were to examine the reactivity of SMG and SMC toward amino acid residues on peptides and proteins in order to establish whether these carbamate thioester conjugates of MIC indeed function as carbamoylating agents toward biological nucleophiles. Oxytocin, which has been employed successfully as a trapping agent for electrophilic xenobiotic metabolites (24), was selected as a convenient model peptide for this work, while BSA was adopted for studies of protein carbamoylation in view of the known immunogenicity of several isocyanates (25)and the demonstrated utility of this soluble macromolecule as a target for covalent modification by chemically reactive drug metabolites (26,27). The presence of accessible cysteine residues in these model compounds (two in oxytocin and one in BSA) was considered to be an advantage since it provided an opportunity to assess the role of the nucleophilic sulfhydryl group as a target for carbamoylation reactions mediated by SMG and SMC.
Materials and Methods Materials. BSA (99%) and oxytocin (99%)were purchased from Calbiochem (La Jolla, CA). GSH (reduced and oxidized forms),cysteine, and methyl methanethiolsulfonate (MMTS)were obtained from the Sigma Chemical Co. (St. Louis, MO). Dimethylcarbamoylchloride, dithioerythritol (DTE),dithiothreitol (DTT),5,5’-dithiobis(2-nitrobenzoicacid) (DTNB),ethanethiol, MIC, ethyl isocyanate, sodium dihydrogen phosphate, sodium monohydrogen phosphate, and m-chloroperoxybenzoic acid (MCPBA,8045%) were purchased from the Aldrich Chemical Co. (Milwaukee, WI). H2180(97-98 atom % excess leg)was purchased from Cambridge Isotope Laboratories (Woburn, MA). Samples of SMG, S-[N-([14C]methyl)carbamoyl]glutathione (14C-SMG;specific activity = 0.265 mCi mmol-’), SMC, S-[N(trideuteriomethyl)carbamoyl]cysteine(CD,-SMC; 97.8 atom % excess 2Hg,2.2 atom % excess 2H2), and SEC were prepared as described previously (17,201.The N,N-dimethyl homologues of SMG and SMC (SDMG and SDMC, respectively)were obtained by synthesis, as described below. General Methods. Protein concentrations were determined by the method of Lowry et al. (28) on a Gilford Stasar I1 spectrometer using BSA as a standard. Protein thiol content was assayed according to Ellman (29). Analytical thin-layer chromatography (TLC) was performed on Analtech silica gel TLC plates (0.25-mm thickness) with 1-propanol/acetic acid/water (101:5 v/v) as mobile phase, and the spots were visualized by exposure to iodine vapor. Synthetic Procedures. Samples of SDMG and SDMC were prepared by reacting GSH or cysteine, respectively, with ethyl N,N-dimethylthiocarbamatesulfone. Details of these procedures follow: (A)S-(NJVN-Dimethylcarbamoy1)glutathione(SDMG). Ethanethiol (6.2 g, 100 mmol) was added to a solution of dimethylcarbamoyl chloride (10.8 g, 100 mmol) in pyridine (79.1 g, 1.0 mol) (30). The resulting mixture was heated under reflux for 2 h, and the product was distilled at atmospheric pressure to afford ethyl N,N-dimethylthiocarbamate(4 g, 30 mmol; yield =
Pearson et ai.
438 Chem. Res. Toxicol., Vol. 4, No. 4, 1991 30%). This thioester (665 mg, 5 mmol) was dissolved in CHC13 (25 mL) and added to a solution of m-chloroperoxybenzoicacid (MCPBA; 4.33 g, 25 mmol) in CHC13 (25 mL). After 2 h at ambient temperature, the m-chlorobenzic acid and residual MCPBA were removed by cooling and filtering the solution. The filtrate was washed with 5 % aqueous sodium carbonate solution and dried over anhydrous MgSO,, and the solvent was removed in vacuo to afford ethyl NJ-dimethylthiocarbamate sulfone (3.22 g, 20 m o l ; yield = 60%)as a yellow oil. A portion of this material (272 mg, 1.6 mmol) was added to a solution of GSH (254 mg, 0.8 mmol) in methanol (30 mL) and triethylamine (10 mL), and the resulting mixture was stirred at ambient temperature for 1 day. The solvent was removed under reduced pressure, the residue was dissolved in water (40 mL), and the pH was adjusted to 2.0 by the addition of HCl(1 M). This solution was washed twice with ether, and the aqueous phase was removed and lyophilized. Extraction of the dry residue with methanol gave the desired product, SDMG (232 mg, 0.62 mmol; yield = 77%). TLC: R = 0.44, 'H NMR (D20): 6 4.63 (1 H, dd, J = 7.5 and 5 Hz; kys a-CH); 3.98 (2 H, s; Gly CH,); 3.82 (1 H, t, J = 5 Hz; Glu a-CHI; 3.45 (1 H, dd, J = 12.5 and 5 Hz) and 3.21 (1 H, dd, J = 12.5 and 7.5 Hz; Cys 0-CH,); 3.00 and 3.02 [6 H; N(CH3),]; 2.52 (2 H, t, J = 5 Hz; Glu y-CH,); and 2.16 (2 H, m, Glu @-CHz).13C NMR (D2O): 6 29.1 (Glu @),34.3 and 34.4 (Cy5 and Glu y), 39.7 and 39.8 [N(CHJZ], 44.6 (Gly CHZ), 56.7 (Glu a),56.8 (Cy5 @),172.2 (SCO), 175.1 (Glu C02H), 176.3 and 176.4 (Gly COPH and Glu CONH), and 177.6 (Cys CO). (B) S- (NJVN-Dimethylcarbamoy1)cysteine (SDMC) Ethyl NJV-dimethylthiocarbamatesulfone (272 mg, 1.6 mmol), prepared as outlined above, was added to a stirred solution of cysteine (100 mg, 0.8 mmol) in methanol (15 mL) and triethylamine (6 mL) under a nitrogen atmosphere. The reaction mixture was stirred at ambient temperature for 3 days, following which the solvent was removed in vacuo. Workup of the product, as described above for SDMG, afforded SDMC (100 mg, 0.52 mmol; yield = 65%). TLC; R, 0.51. 'H NMR (D20): 6 4.02 (1 H, dd, J = 7.5 and 5 Hz; Cys a-CH); 3.56 (1 H, dd, J = 12.5 and 5 Hz) and 3.34 (1 H dd, J = 12.5 and 7.5 Hz; Cys B-CH,); and 3.00 and 3.02 [6 H; N(CH3),]. 13C NMR (D,O): 6 33.1 (Cys a),39.3 and 39.5 [N(CH,),], 57.3 (Cys P), 171.5 (SCO), and 174.5 (COZH). Instrumentation. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian VXR 300 spectrometer operating at 300 MHz for 'H or at 75 MHz for 'BC nuclei, respectively. Samples were dissolved in D20, and chemical shifts are expressed in parts per million (6) downfield from sodium 3-(trimethylsilyl)-lpropanesulfonate as internal standard. LC-MS was performed on a Vestec Model 201 instrument, equipped with a Hewlett-Packard 59979C ChemStation data system and LKB 2150 HPLC pump. Separation of conjugates was performed on an Altex Ultrasphere C18reversed-phase HPLC column (15 cm X 4.6 mm i.d.; 5 pm) with a mobile phase of CH3CN/Hz0 (2.5:97.5 v/v) containing 50 mM NHIOAc (pH 4.5), delivered at a flow rate of 0.75 mL min-'. In order to achieve optimal thermospray ionization of the analytes, CH3CN/H20(41 v/v) was added postcolumn to the effluent at a flow rate of 1.0 mL min-' to generate a final mobile-phase composition of 45% CH3CN at the TSP interface. Under these conditions, the thermospray interface parameters for optimal sensitivity were found to be T,= 133 "C, Tz= 211 "C, T3 = 232 "C, and block = 274 "C. Fast atom bombardment (FAB) mass spectra were recorded at an accelerating voltage of 8 kV on a VG 70-SEQ hybrid tandem instrument of EBQQ geometry, equipped with an Ion-Tech fast atom gun and a VG11/250 data system. Samples were dissolved in methanol/l M HCl(1:l; 50 pL), and aliquots (1 pL) were added to a glycerol matrix containing 0.1 M HC1. Ionization was achieved by bombardment with a primary beam of xenon atoms (8-keV energy),and conventional FAB spectra were recorded via the data system. Collision-induced dissociation (CID) was performed in the f i t (rf-only) quadrupole region employing argon as a collision gas (3.5 x 10" Torr) and a collision energy of 35 eV (laboratory frame of reference). The desired parent ion was transmitted into the collision quadrupole by adjusting the field strength of the magnetic analyzer, and the quadrupole mass filter was scanned between 50 and 1400 Da over 10 s. Daughter ion spectra were recorded via the data system, and 5-10 scans were summed in
.
the multi-channel analysis mode. Mass assignments were made following centroid calculations, and the spectra are presented in continuum format. Low-pressure liquid chromatography was performed by using a Pharmacia FPLC system (Pharmacia, Piscataway, NJ), consisting of an LCC-500 controller interfaced to two P-500 pump% and an MV-7 injector. The column effluent was monitored at 280 nm (2 AUFS) with a Pharmacia UV-1 single-path monitor equipped with a 10-mm path length flow cell; the output of the detector was recorded with an LKB 2220 integrator. Column fractions (8 mL) were collected in a Cygnet fraction collector a t 4 min intervals. Radioactivity measurements were carried out by liquid scintillation counting in a Packard Tricarb 200CA instrument. Quench corrections were performed by reference to an external standard. Exchange Reactions with Carbamate Thioester Conjugates. In an extension of the study on the exchange phenomenon between SMG and cysteine (I@, these two species were allowed to react at 37 "C in an aqueous buffer (pH 7.4) made up in H2'80. This buffer was prepared by dissolving sufficient anhydrous sodium dihydrogen phosphate and sodium monohydrogen phoephate in H2180(1 mL) to yield a final pH of 7.4. Aliquots (50 pL) of the reaction mixture were withdrawn over intervals up to 2 h, mixed with an equal volume of a solution of the internal standard (SEC; 500 pM in the mobile phase for HPLC), and analyzed directly by LC-MS. The incorporation of label into SMC was determined from the relative abundances of the '80 and '80 species of the [M NH4].+adduct ions (at m/z 196 and 198, respectively). In separate expenments, SDMG (1 mM) and SDMC (1 mM) were allowed to react with cysteine (5 mM) and GSH (5 mM), respectively, in phosphate buffer (pH 7.4,100 mM) at 37 "C. After a period of 120 min, aliquots (50 pL) of the reaction mixtures were removed and processed for LC-MS analysis as described above. In this case, disappearance of the substrate and formation of the corresponding product were quantified on the basis of the abundances of the ions at m/z 193 (MH+ for SDMC) and m/z 147 (glutamic acid fragment ion from SDMG; 30). Under the HPLC conditions used for LC-MS analyses, the conjugates had retention times of 5.7 min (SDMC) and approximately 15 min (SDMG). Carbamoylation of Oxytocin by Carbamate Thioesters. Oxytocin was first desalted by using a modification of the method described by Moon and Kelly (32).A C18 SepPak solid-phase extraction cartridge (Waters Associates, Milford, MA) was prewashed with methanol (2 X 3 mL) and water (2 X 3 mL). oxytocin (1 mg, 1 pmol) was dissolved in water (400 pL) and applied to the cartridge, which was then washed with water (2 X 3 mL). The oxytocin was eluted with 70% methanol in water (3 mL), the methanol was removed under a stream of nitrogen, and the residual aqueous solution was taken to dryness by lyophilization. Preparation of reduced oxytocin was carried out by a modification of the method of Liebler et al. (24).Thus, oxytocin (1 mg,1 pmol) was dissolved in TEAF buffer (20 mM, pH 7.7; 500 pL) containing DTT (10 mg, 13 pmol), and the mixture was allowed to stand at ambient temperature for 2 h. Excess DTl' was then removed by extraction into ethyl acetate (4 X 1 mL), and the aqueous phase containing the reduced oxytocin was used directly for studies of the carbamoylating properties of SMC and CD3-SMC. These experiments were performed as follows. Samples of reduced or oxidized oxytocin (1 mg, 1 pmol) were dissolved in TEAF buffer (20 mM, pH 7.7; 1 mL) and allowed to stand (10 min) at 37 "C in 1.5-mL Eppendorf tubes before being treated with either SMC or CD3-SMC (1.78 mg, 10 pmol) in TEAF buffer (500 pL). After a period of 6 h a t 37 "C, the reaction mixtures were lyophilized and the dried residues were stored a t -20 "C prior to being analyzed by FAB-MS/MS. Covalent Modification of Bovine Serum Albumin by leSMG. In order to assess the ability of SMG to carbamoylate proteins, and more specifically to react with protein sulfhydryl groups, "C-SMG was incubated with either native BSA or with "thiol-blocked" BSA in which the single free thiol (Cys-34) of the protein is protected with an S-methyl functionality. In the case of native BSA, the free thiol content was determined experimentally to be 15 nmol mg-', corresponding to 1 nmol of thiol/nmol of BSA (27). "Thiol-blocked" BSA was prepared by dissolving BSA (1.0 g, 15 mmol) in phosphate buffer (100 mM, pH
+
Reactive S-Linked Conjugates of Methyl Isocyanate 7.4;9 mL) and treating the solution with freshly distilled methyl methanethiolsulfonate (MMTS; 34 pL, 20 equiv), according to the method of Miwa et al. (2s).Separationof "thiol-blocked" BSA from excess MMTS was performed by FPLC. Aliquots (2mL) of the crude reaction mixture were applied to a column of Sephadex G-25(19cm x 1 cm)and eluted with phosphate buffer (100mM, pH 7.4) at a flow rate of 2 mL min-'. Under these conditions,BSA eluted between 8 and 16 min, while MMTS eluted between 20 and 28 min. Fractions containing BSA were pooled and reduced in volume to less than 10 mL with a Diaflow Model 52 ultrafiltration apparatus equipped with PM30 semipermeable membranes (Amicon,Danvers, MA), the ultrafiitrate from which was free of detectable protein. The residual BSA solution was diluted to 10 mL, the pH was readjusted to pH 7.4,and the sample was stored at 4 O C as "thiol-blocked"BSA. Solutions of native BSA (1.5mg, 23 nmol) or 'thiol-blocked" BSA (1.5mg, 23 nmol) were diluted with phosphate buffer (100 mM, pH 7.4)to a final incubation volume of 100 pL, vortexed briefly, and warmed to 37 "C in a water bath. At time zero, 12 tubes containing native and 'thiol-blocked" fractions were treated with either 2-or 10-pL portions of "C-SMG (23nmol pL-' in buffer). Each tube was vortexed briefly and then incubated with gentle shaking at 37 O C . Tubes were removed at 20 min, 45 min, 2 h, and 4 h, frozen in liquid nitrogen, and stored at -20 "C until analyzed. All incubationswere performed in triplicate. Tubes were then thawed, diluted with phosphate buffer (40pL), and vortexed, and aliquota (100 pL) were analyzed by chromatography on a column of Sephadex G-25 (19cm X 1 cm), using Tris-HC1buffer (20mM, pH 7.5)as mobile phase at a flow rate of 1 mL m i d . Fractions correspondingto the modified BSA, which eluted between 12 and 18 min, were collected, and aliquots of each were counted for radioactivity and assayed for protein content. The results are expressed as nanomoles of 14C-SMGequivalents bound per nanomole of protein (mean f SD, N = 3).
Results Exchange Reactions with Carbamate Thioester Conjugates. Under the incubation conditions employed in our preliminary studies (18)which led to extensive exchange of the N-methylcarbamoyl moiety between SMG and cysteine, and between SMC and GSH, no exchange was found to occur with the corresponding NJV-dimethylcarbamate thioesters. Thus, incubation of SDMG (1 mM) with cysteine (5 mM) for 3 h failed to generate SDMC at levels greater than 25 pM (the limit of detection of the LC-MS assay), while SDMC similarly failed to react with GSH. Thioesters of tertiary carbamates, therefore, appear not to carbamoylate cysteine or GSH under conditions of physiological pH and temperature. In contrast, the secondary carbamate derivatives SMG and SMC again proved to be effective carbamoylating agents toward cysteine and GSH, although when incubationswere performed in a medium enriched in H;W, neither product was found to have incorporated the isotopic label. This lack of l80 incorporation serves to eliminate from consideration an exchange mechanism that involves hydrolysis of the starting conjugate to N-methylcarbamic acid or equilibration of MIC with N-methylcarbamic acid in aqueous media, prior to carbamoylation of the acceptor thiol. Carbamoylation of Reduced Oxytocin by Carbamate Thioesters. When SMC was allowed to react with reduced oxytocin in aqueous buffer and the products were analyzed directly by FAB-MS, evidence was obtained for the presence of at least three carbamoylated peptides whose protonated molecular species (MH+)were present at m/z 1066 (mono adduct), 1123 (bis adduct), and 1180 (tris adduct) (Figure 2A). Substitution of CD3-SMC for unlabeled SMC in the carbamoylation reaction led to shifta in these m/z values of 3,6, and 9 Da, respectively (Figure 2B), consistent with the formation of mono-, bis-, and tris-N-methylcarbamoyl derivatives of oxytocin (M, of MIC = 57). In addition to these adducted peptides, the FAB
Chem. Res. Toxicol., Vol. 4, No. 4, 1991 439
1123
II
IO66
1000
'
7100
1200
b!s-(CDd.AddUci
'0°4
MH. 1129
\
0
1000
1100
1200
mlz
Figure 2. Partial FAB mass spectra of the mixture of products formed upon reaction of SMC (A) or CDS-SMC(B)with reduced oxytocin. Reactions were carried out in TEAF buffer (20mM pH 7.7)at 37 O C for 6 h, following which samples were lyophilii and analyzed directly by FAB-MS. spectra depicted in Figure 2 revealed the presence of oxidized oxytocin (MH+ at m/z 1007) as well as residual substrate (reduced oxytocin; MH+ at m/z 1009). On the basis of these findings, it is apparent that SMC serves as an efficient carbamoylating agent with respect to reduced oxytocin, although the conventional FAB spectra provide no information on the sites at which the peptide becomes modified in these experiments. In order to address the latter issue, the products of reaction with SMC and CD3-SMCwere analyzed by FAB-MS/MS, when the individual MH+ species were subjected to CID and the resultant spectra of daughter ions recorded. The CID daughter ion spectrum of reduced oxytocin itself is reproduced in Figure 3 and is characterized by a series of structurally informative B, and Y,," fragment ions which reflect the primary amino acid sequence of the native peptide (33,34). Prominent features of this spectrum include the B6 ion at m/z 725, which results from cleavage between Pro-7 and Cys-6 with charge retention on the N-terminal portion of the peptide, and the complementary Yfl ion at m/z 285, which comprises the three C-terminal amino acid residues. Additional diagnostic fragment ions are as noted in Figure 3. Comparison of this daughter ion spectrum with those of the products of reaction of oxytocin with either SMC or CD3-SMC served to indicate the sites of carbamoylation of this peptide, as exemplified below for the mono and bis adducts derived from CDS-SMC (Figures 4 and 5). The daughter ion spectrum of m/z 1069, the MH+ ion of [N-(trideuteriomethyl)carbamoyl]oxytocin(Figure 4), afforded a series of ions indicative of the presence of two regioisomeric peptides. In the case of the major component, the B6 ion appeared at m/z 785, an increment of 60
Pearson et al.
440 Chem. Res. Toxicol., Vol. 4, No. 4, 1991 _ _380 .._ _508 . _ _ 622 _ _ .725 --. 822 ---
267
743
6%
502
388
MH*
_935- -
1009
285 '6
725
0
200
400
600
800
loo
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1200
m/z
Figure 3. Spectrum of daughter ions formed by CID of the MH' parent (m/z 1009)of reduced oxytocin. The structure of the native peptide and the proposed origins of daughter ions are as indicated. Conditions for FAB-MS/MS analyses are given in the text.
q,
'6
705
H+
69
'8
995
A1200 Figure 4. S ectrum of daughter ions formed by CID of the MH+ parent (m/z 1069) of [N-(trideuteriomethyl)carbamoyl]ox~in. The p r o p 2 origins of daughter ions are indicated according to the convention depicted in Figure 3. Asterisks denote daughter ions arising from a minor regeoisomer carbamoylated at Cys-1 or Tyr-2. Conditions for FAB-MS/MS analyses are given under Materials and Methods.
Da (CD,NCO) over the corresponding ion in the spectrum of the native peptide, while the Yfl ion remained unchanged at m l z 285. This information requires that carbamoylation must have occurred at some point between Cys-1 and Cys-6. Further analysis of the spectrum shown in Figure 4 revealed that the m/z values for ions Yi' through Y," shifted by 60 Da relative to the native peptide, an observation that can be accommodated only by carbamoylation at Cys-6. Therefore, the major mono adduct of oxytocin is the product of reaction at the internal Cys residue. Upon closer inspection of this daughter ion spectrum, it is apparent that the 60-Da shift in the Y," through Y," sequence ions ia not complete, in that Y/ ions present in the spectrum of oxytocin itself (at m/z 388,502, 630, and 743) still are present, albeit at low relative abundance. Since these ions all are daughters of the carbamoylated parent ( m l z 10691, the only explanation
for their presence in this spectrum is that the sample contains a second mono adduct which is modified at the N-terminus. However, since CID of oxytocin and ita carbamoylated derivatives does not result in cleavage of the linkage between Cys-1 and T~I-2,it is not possible on the basis of these daughter ion spectra alone to discriminate between adduct formation at the two alternative sites. Therefore, the minor mono adduct of oxytocin is identified as the product of reaction at either Cys-1 and Tyr-2 (or may be a mixture of both carbamoylated forms). On the basis of the abundance ratios of the respective Y," ions (at m/z 448 and 388) for the major and minor mono adducts of oxytocin, it may be estimated that carbamoylation of the peptide at Cys-6 predominates by a factor of approximately 5 over the formation of isomeric structures. The daughter ion spectrum obtained upon collisional activation of mlz 1129, the MH+ species of the [N,h'-
Chem. Res. Toxicol., Vol. 4, No. 4, 1991 441
Reactive S-Linked Conjugates of Methyl Isocyanate
7
MB 29
101
V i
448
%
5(
y3-
285
I
Y7‘
803
_.
1200
mh
Figure 5. Spectrum of daughter ions formed by CID of the MH’ parent (m/z 1129) of [Nfl-bis(trideuteriomethyl)carbamoyl]o~in. The proposed origins of daughter ions are indicated according to the convention depicted in Figure 3. Conditions for FAB-MS/MS analyses are given under Materials and Methods.
bis(trideuteriomethyl)carbamoyl]oxytocin adduct, is reproduced in Figure 5. In this case, the intense B6 ion appears at mlz 845,120 Da higher than the corresponding fragment in the CID spectrum of native oxytocin. Once again,the Y3))ion appears at mlz 285, indicating that both of the modified residues are located within the six Nterminal amino acid residue domain. Since the [ Y fragment is present at m/z 448 (60Da higher than in oxytocin), the location of one of the carbamoylated sites was determined to be Cys-6. Ions Y
