430 Chen. Res. Toricol. 1991,4, 430-436

Formation, Stability, and Rearrangements of the Glutathione Conjugates of Butadiene Monoxide: Evidence for the Formation . -

of Stable Suifurane Intermediates

Jane E. Sharer, Renee J. Duescher, and Adnan A. Elfarra* Department of Comparative Biosciences and Environmental Toxicology Center, University of

Wisconsin, Madison, Wisconsin 53706

Received November 13, 1990

Butadiene monoxide, a toxic metabolite of 1,3-butadiene, is a substrate for the human placentai glutathione (GSH) S-transferase. The products have been identified as S42-hydroxy-3-bu- ten-l-y1)glutathione (I) and S-(l-hydroxy-3-buten-2-yl)glutathione (11). S-(Chydroxy-2-bu- ten-l-y1)glutathione (111), which was formed chemically, was not detected. 'H NMR analysis of I1 was consistent with its structure, but spectra of I indicated a 1:l equilibrium between I and the sulfurane tautomer (IVB) formed by intramolecular displacement of the hydroxyl group by the sulfur atom. The ratio of I to IVB did not change whether the spectrum was obtained a t pH 3,7, or 9 in the presence or absence of LiC10,. Incubations of I a t pH 7 or 9 for 5 days a t 25 "C or for 7 h a t 50 "C, in the presence or absence of nucleophiles plus LiC104, did not affect the HPLC profile of I. Storage of I at -20 "C for 30 weeks, reflux at pH 7.8 for 5 h in the presence of GSH, or incubations a t pH 2 for 5 h at 55 "C in the presence of GSH or 2-mercaptoethanol, however, resulted in the conversion of I to I11 (10-30%). Treatment of I with HzOz resulted in formation of the corresponding sulfoxide (V) and sulfone (VI), which blocked the formation of 111. NMR and chemical reactivity studies of I11 indicated an initial 1:l equilibrium between I11 and the five-membered ring sulfurane (VIIB) formed by intramolecular displacement of the hydroxyl group by the sulfur atom. This equilibrium was shifted slowly to 100% I11 by an apparent cis-trans isomerization of 111. Enzymatic formation of I and I1 was dependent on incubation time and protein and butadiene monoxide concentrations. The V,, (I and I1 combined) was nearly 500 nmol/(mg of proteinamin), with nearly equal amounta of each conjugate being formed. These results suggest that GSH conjugation occurs a t high rates in vivo to produce I and 11. Whereas I1 is chemically stable, I is a novel GSH conjugate because it tautomerizes to a stable sulfurane which slowly rearranges to yield 111.

Introductlon 1,3-Butadiene, a toxicant and carcinogen, is used ex-

tensively in the industrial production of synthetic rubber and plastics ( I ) . Recent studies have identified butadiene monoxide (BM,' 3,4epoxy-l-butene), a direct-acting mu- tagen, as the major metabolite of l,&butadiene both in vivo and in reactions catalyzed by mouse and rat liver micro- somes (2-4). When 1,3-butadiene was incubated with rat liver microsomes and NADPH, both enantiomers of BM were detected (5). Incubations of racemic BM with DNA or 2'-deoxyguanosine resulted in the formation of the re- gioisomers 7- (2-hydroxy-3-buten- l-y1)guanine and 7- (1- hydroxy-3-buten-2-yl)guanine (6). These studies suggest that metabolism of 1,3-butadiene to yield BM and sub- sequent reactions of BM with critical macromolecules may be involved in the mechanism of l,&butadiene-induced toxicity.

Several epoxides have been shown to be conjugated with GSH, a major cellular nucleophile which is present in various mammalian cells (7,8). Although these findings suggest that conjugation via GSH S-transferases may play a significant role in BM metabolism and toxicity, the re- action of BM with GSH was not previously investigated. BM is known to undergo both 1,a-direct addition and 1,4conjugate addition, depending on the nucleophile and the reaction conditions used (9). It is possible that, de-

pending on the regioisomer formed (Figure l), BM con- jugation with GSH may represent either a detoxication or activation reaction. The BM conjugates may be less toxic than BM since they are expected to be more readily ex- creted into bile or urine. Conjugate I, however, may form an episulfonium ion by intramolecular displacement of the allylic hydroxyl group by the sulfur atom. The role of such an episulfonium ion in BM-induced toxicity would depend on ita rate of formation, stability, and chemical reactivity at physiological conditions. Thus, to investigate the role of GSH S-transferases in the metabolism and toxicity of BM, studies to characterize the chemical and enzymatic reactions of racemic BM with GSH were initiated in our laboratory. The present investigation demonstrates a high capability of GSH 5'-transferases to metabolize BM to yield the GSH conjugates I and 11. Furthermore, this paper reports the first evidence that GSH conjugates can form stable sulfurane intermediates.

Experlmental Procedures Materials. Racemic BM, 2,4-dinitrofluorobenzene, tri-

fluoroacetic acid, tetraethylammonium chloride, and LiClO, were obtained from Aldrich Chemical Co. (Milwaukee, WI). GSH, human placental GSH S-transferase, catalase, and 2-mercapto- ethanol were purcheeed from S i Chemical Co. (St. Louie, MO). HPLC-grade acetonitrile was obtained from EM Science (Gib-

* To whom correspondence should be addressed at the Depart- ment of Comparative Biosciences, University of Wisconsin School of Veterinary Medicine, 2015 Linden Dr. W., Madison, WI 53706.

Abbreviationr: BM, butadiene monoxide; GSH, glutathione; I, S- (2-hydroxy-3-butan-l-yl)glutathione; 11, S-( l-h~xy-3-butan-2-yl)glu- tathione; III, S-(~hydro.y-2-butan-l-yl)gluta~one; FAB-MS, fast atom bombardment mass spectrometry.

@ 1991 American Chemical Society

Glutathione Conjugates of Butadiene Monoxide Chem. Res. Toxicol., Vol. 4, No. 4, 1991 431

spectrometer (Manchester, United Kingdom) equipped with a saddle field fast atom bombardment gun. Approximately 1 mg each of conjugates 1-111, the sulfoxide, and the sulfone of I dis- solved in H20 was analyzed with a spectrum ranging from 200 to 600 m/z , using a 3-nitrobenzyl alcohol matrix. NMR Spectrometry. Proton NMR spectra of components

1-111, the sulfoxide or the sulfone of I dissolved in D20 was obtained on a Bruker spectrometer (Karlsruhe, Germany) at 500 MHz with chemical shifts reported in ppm from sodium 3-(tri- methylsily1)tetradeuteriopropionate. Proton NMR spectra of I and 111, in D20 with the pH adjusted to 3, 7, or 9 with DCl or KOD, in the presence or absence of LiClO, (5 mM), were also obtained.

Electronic Absorption Spectra and Derivatization. Electronic absorption spectra of 1-111 were recorded between 200 and 250 nm using the analytical HPLC system described above. Derivatization of I and 11 was carried out by mixing samples (0.5 mL of 1 mg/mL solution) of I or 11 with 0.1 mL of 2.0 M KOH/2.4 M KHC03 buffer, and 0.5 mL of 1.5% 2,4-dinitrofluorobenzene in ethanol (IO). Reactions were allowed to proceed for 24 h, after which the samples were filtered by using 0.2-pm LC 13 acrodisc membrane fiiters (Gelman Sciences, Ann Arbor, MI) and analyzed by HPLC using a Waters Nova-PAK (3.9 X 75 mm) reverse-phaw C-18 column (Milford, MA) and acetonitrile/water/trifluoroacetic acid (pH 4.5) as the mobile phase. The gradient used began at 15% acetonitrile for 6 min, then increased to 45% over 10 min, and remained linear for 1 min before falling back to 15% over 3 min. Absorbance was measured at 365 nm during the separation of the 2,4-dinitrophenyl derivatives of I and 11. Scans were re- corded between 220 and 400 nm.

Stability Studies. Stabilities of 1-111 were studied by HPLC analysis after incubations for different time intervals, at various pHs and temperatures. Sample solutions (2 mg/mL) were made with 100 mM phosphate buffer at pH 2 and 7 or 100 mM borate buffer at pH 9 and incubated for up to 5 days at room temperature or heated up to 7 h in a 50 "C water bath. In some experiments, stabilities of I and III were studied in the presence of 5 mM GSH, 2-mercaptoethanol, tetraethylammonium chloride, or GSH plus LiCIO1. Moreover, the stabilities of I and its sulfoxide or sulfone form were studied by HPLC analysis before and after reflux at pH 7.8 for 5 h in the presence or absence of 5 mM GSH.

Enzymatic Incubations of GSH S-Transferase and BM. Purified human placental GSH S-transferase was dissolved in a buffer (pH 7.4) containing 0.1 M KH2P04, 0.15 M KCl, and 1.5 mM ethylenediaminetetraacetic acid. This enzyme, a 7r class glutathione S-transferase, was used due to its commercial availability in the purified form. Incubations of BM and GSH (10 mM) in the presence or absence of the enzyme were carried out at 37 OC in a Dubnoff metabolic incubator (Chicago, IL) with continuous shaking; total reaction volume was 0.5 mL. Solutions were incubated for 4 min before addition of BM to start the reaction. At the appropriate times, reactions were stopped and protein was precipitated with a 3.3% reaction volume of 50% trichloroacetic acid (final pH = 2). Samples were filtered and analyzed by HPLC as described above for the characterization of the synthetic standards. Enzymatic activity was corrected for activity obtained without the enzyme. Electronic absorption spectra of biosynthetic conjugates were performed before and after derivatization with 2,4-dinitrofluorobenzene, as described above.

wo + CSB

9Q 08

t H0-@-3G

08 bo

I I 1 I 1 1 Figure 1. Structures of the three possible regioisomen formed by the reaction of BM with GSH.

stown, NJ), and NMR supplies were obtained from Wilmad Glans Co. (Buena, NJ). All other chemicals were of the highest grade commercially available.

Syntheeis of B M 4 S H Conjugates. GSH (0.215 g; 0.7 "01) was dissolved in 10 mL of acetone and water (1:l v/v) and the pH adjusted to 7.8 with triethylamine. A molar excess of BM (0.1 mL; 1 mmol) was added, and the solution was held at reflux for 5 h. Excess BM was removed by extraction three times with ethyl ether in a 1:2 volume ratio and the aqueous solution rotary evaporated to near dryness. Distilled deionized water (10 mL) was added and the pH adjusted to 3.5 with acetic acid. The solution was then passed through a Bio-Rad Chelex 100 (Na+) 0.8 X 4 cm column (Richmond, CA) to remove metal cations and a Bio-Rad AG 50W-X8 (H+) 0.8 X 4 cm column to remove the subsequent contaminating Na+ ions. These ion-exchange treatments were necessary to obtain a solid crude product upon rotary evaporation and lyophilization of the solution. Crude yield of product was 237 mg, or 90% of theoretical yield.

HPLC Purification of BM-GSH Conjugates. HPLC sep- aration of crude material was performed by using a Gilson gra- dient-controlled HPLC system (Model 302 pumps; Middleton, WI) equipped with a Bio-Rad AS-100 HRLC automatic sampling system, a 3-cm Brownlee ODS guard column (San Jose, CA), and a Beckman Model 167 scanning absorbance detector (Irvine, CA) on a Beckman Ultrasphere 5-pm ODS reverse-phase analytical column (4.6 X 250 mm) (San Ramon, CA) with UV detection at 220 nm. By use of a mobile phase of 4% acetonitrile and 0.1% trifluoroacetic acid in water, pH 2.0, a t a flow rate of 1 mL/min, three major peaks were recovered at retention times 14.3, 15.7, and 17.0 min which were assigned labels I, 11, and 111, respectively. The limit of detection attained by the standard curve using this system is approximately 0.5 nmol I, 11, or I11 per 20-pL injection volume. Bulk separation of crude material was accomplished with a l-mL injection volume onto a Beckman Ultrasphere bpm ODS reverse-phase semipreparative column (10 X 250 mm) with a mobile phase of 2.7% acetonitrile and 0.1% trifluoroacetic acid in water, pH 2.0, a t a flow rate of 3 mL/min. Peaks I, 11, and 111, which had retention times of 25.1, 28.7, and 32.5 min, re- spectively, were collected by using a Gilson 203 microfraction collector. Peak solutions were each rotary evaporated, passed through the ion-exchange columns, and lyophilized as previously described. HPLC recovery of the combined I, 11, and I11 com- ponenta was nearly 85%; ratios of I, 11, and I11 by weight in the crude mixture were nearly 95:1, respectively. Purities of the conjugates, as determined by HPLC, were 98.5%, >99%, and 92% for I, 11, and 111, respectively.

Synthesis of t he Sulfoxide and Sulfone of Conjugate I. Conjugate I was oxidized by dissolving it in 15% H202 and 50 mM K2HP04 at pH 7. The reaction was allowed to proceed for 6 h at 4 "C, after which the reaction was stopped by removing excess H202 with 150 units of catalase/mL. Samples were sep- arated by HPLC on the analytical system described previously with a mobile phase of 1% acetonitrile and 0.1% trifluoroacetic acid, pH 2. Two new distinct peaks with retention times of 8.3 and 8.8 min were observed. These peaks were then collected after separation on the semipreparative HPLC system described above (retention timea of 28.6 and 30.3 min) with a mobile phase of 100% H20 and 0.1% trifluoroacetic acid, pH 2.

Mass Spectrometry. Positive ion FAB-MS analysis was performed by using a Kratoe M S - m ultrahigh-resolution mass

Results Identification, Stabil i ty, and Rearrangement of the

Synthetic GSH Conjugates of BM. Reaction of racemic BM with GSH gives three regioisomers (Figure l), which were separated by HPLC. Ratios of I, 11, and 111 by weight in the crude mixture were nearly 9:5:1, respectively. Identification of the isomers was achieved by examination of their FAB-MS spectra (Figure 2) and their proton NMR spectra (Figure 3). Mass spectra for each of 1-111 yielded a pseudomolecular ion of mlz 378, consistent with the expected molecular weight of a GSH conjugate of BM. Peaks at m/z 358,274, and 232 correspond to lois of water, cleavage at the carbon-sulfur linkage of cysteine, and loss of glutamic acid, respectively. Proton NMR spectra of

Chem. Res. Toxicol., Vol. 4, No. 4, 1991 Sharer et al. 432

h U .I

a 3

Y

I4

G c(

.I U I a

Figure 2. Positive ion FAB-MS spectra of conjugates I, 11, and I11 (panels A, B, and C, respectively).

1-111 show the presence of the GSH and BM residues. The assignment of structures of 1-111 was achieved by com- paring the chemical shifts, multiplicities, and integration ratios of the various protons in the spectra of 1-111 with those of published spectra of allyl alcohol, allyl mercaptan, and other GSH conjugates (11-13). These assignments were also based on the use of homonuclear decoupling and the expected greater downfield chemical shift of hydrogens attached to an sp2 carbon or a carbon with hydroxyl sub- stitution relative to hydrogens attached to an sp3 carbon or a carbon with thioether substitution, respectively.

Whereas the proton NMR spectra of I1 and I11 were consistent with their open-form structure, the spectrum of I indicated a 1:l equilibrium between the open form and the sulfurane or episulfonium ion tautomer, which possibly formed by intramolecular displacement of the allylic hy- droxyl group by the sulfur atom (Figure 3A). The splitting of the peaks corresponding to H3 (6 = 5,91,5.76), H4 (6 = 4.32, 3.50), H5H6 (6 = 3.71, 2.94), and the protons on the cysteine @-carbon (6 = 3.10,2.75) is consistent with these structural assignments. Integrations of all of the split signals indicated a 1:l ratio between each tautomer. The insufficient resolution of the multiplets a t 3.10,2.94, and 2.75, which was possibly due to the presence of diaste- reomers, did not allow an accurate estimation of J values, but further evidence for the structural assignments was obtained by using homonuclear decoupling. Saturation of the H3 proton of I a t 5.91 was used to identify the H4 proton of I at 4.32, and saturation of the H3 proton of IV at 5.76 was used to identify the H4 proton of IV. These results and the finding that the glycine and glutamine protons in the NMR of peak I (Figure 3A) exhibited dif- ferent chemical shifts from the glycine and glutamate protons of peak I1 (Figure 3B) provide evidence against the possibility that peak I was a mixture containing a second diastereoisomer from peak 11. Indeed, the observed chemical shift differences between the split signals and the absence of similar splitting5 in the spectra of I1 and I11 indicated that the splitting observed in the spectrum of I was not due to resolution of signals of the diastereomeric pair of I, but rather to formation of an episulfonium ion or a sulfurane tautomer. The finding that the signal at- tributed to the H4 proton of the cyclic tautomer of I had a chemical shift (3.5 ppm) similar to that of the H4 of I1 (Figure 3) is inconsistent with an ion pair formula for the structure of the tautomer, but rather indictea the formation of a sulfurane intermediate.

A.

P F

Y i ' i ' j ' i ' i ' i

G I P C. I

ppm

Figure 3. 500-MHz proton NMR spectra of conjugates I, 11, and I11 (panels A, B, and C, respectively).

The reactivity and stability of I were further investigated by studying the effects of various nucleophiles, ionic species, and pH on the HPLC profile and NMR spectra of I. The ratio of the two tautomers did not change whether the proton NMR spectrum of I was obtained at pH 3, 7, or 9 in the presence or absence of the strong electrolyte LiC104. These results indicate that the rates of formation and destruction of the cyclic tautomer of I were independent of the pH or polarity of the medium. Incubations of I at pH 7 for 5 h at 50 "C in the presence or absence of 2-mercaptoethanol, tetraethylammonium chloride, GSH, or GSH plus LiC104 or incubations at pH 9 for 5 days at 25 "C in the presence of GSH did not affect the HPLC profile of I. These results indicate high stability and weak electrophilicity of the cyclic tautomer of I, which is consistent with a sulfurane rather than an episulfonium ion structure.

Long-term storage of I for 30 weeks at -20 "C resulted in a 10% conversion of I to 111, whereas extended storage of I1 did not exhibit any decomposition. These results suggest that the mechanism of formation of I11 may not be the lP-conjugate addition of GSH to BM, but rather the intramolecular rearrangement of I or ita sulfurane form under the synthetic conditions to yield 111. Further evi- dence for this hypothesis is indicated by the finding that nearly 10-20% of I was converted to I11 after reflux at pH

Glutathione Conjugates of Butadiene Monoxide

40-

20-

Chem. Res. Toxicol., Vol. 4, No. 4, 1991 433

Mil 394

329

I

J 1 f a s t

m O H S G ( I I I A )

( V I I A ) ( V I I B ) ( I I I B ) Figure 4. Proposed mechanism for the rearrangement of con- jugate I to conjugate 111.

7.8 in presence of GSH, conditions that were similar to those used to synthesize the GSH conjugates of BM. In addition, I was converted to I11 (30%) by incubation at pH 2 for 5 h at 55 "C in the presence of GSH or 2- mercaptoethanol. Incubations of I without GSH or 2- mercaptoethanol, however, did not lead to the formation of 111. Moreover, when subjected to the synthetic condi- tions, the sulfoxide and sulfone forms of conjugate I (see below for characterization of these compounds), which cannot form an episulfonium ion or sulfurane tautomer, indicated no conversion to conjugate I or 111.

The NMR spectrum of I11 obtained shortly after the synthesis and isolation of the GSH conjugates was con- sistent with the presence of a 1:l equilibrium between the open form and the five-membered sulfonium ion or sul- furane intermediate resulting from intramolecular dis- placement of the allylic hydroxyl group by the sulfur atom. Integrations indicated only one proton each where the signals for H3H4 (4.05 ppm) and H6H6 (3.2 ppm) were assigned. Two additional protons were, however, found to integrate in the range (3.7-3.9 ppm) of the glycine and glutamate a-protons (data not shown). The spectrum (Figure 3C) obtained a month after storage of I11 at -20 OC revealed an absence of this equilibrium possibly because of a cis-trans isomerization of IIIA to IIIB (Figure 4). Studies of the stability of the cyclic tautomer of I11 and attempts to trap the presumed sulfonium ion with nu- cleophiles, similar to the experiments described for I, were undertaken: the ratio of IIIA to the cyclic tautomer did not change whether the NMR spectrum of 111 was obtained at pH 3, 7, or 9; incubations of newly isolated I11 at pH 7 or 9 at 50 "C in the presence or absence of GSH did not affect the HPLC profile of 111, but incubations at pH 2 in presence of GSH led to the conversion (15%) of I11 to I (data not shown). These results indicated the stability and weak electrophilicity of IIIA and its tautomer, which is consistent with the sulfurane (VIIB), rather than the sulfonium ion (VIIA), structure for the cyclic tautomer of IIIA.

Characterization of Oxidation Products of Conju- gate I. The FAB-MS spectrum of the sample obtained after lyophilization of the HPLC component which had

21 'f

;%\ OH H

B* M-273

7 '7 I :*k OH H

Figure 5. Positive ion FAB-MS spectra of the sulfoxide (A) and sulfone (B) forms of conjugate I.

w

Figure 6. 500-MHz proton NMR spectra of the sulfoxide (A) and sulfone (B) forms of conjugate I.

a 30.3-min retention time revealed a pseudomolecular ion of mlz 394, consistent with the expected molecular weight of the sulfoxide form of I (Figure 5A). FAB-MS of the HPLC component which had a 28.5-min retention time gave two ions of m / z 273 and 136 (Figure 5B). These ions are conistent with the molecular weights of the two frag- ments formed by cleavage of the cysteine carbon-sulfur linkage of the sulfone form of I. Proton NMR analysis of these oxidation products (Figure 6) is also consistent with these assignments. Oxidation of the sulfur atom of I re- sulted in a downfield shift of the hydrogens in the prox-

434 Chem. Res. Toxicol., Vol. 4, No. 4, 1991 Sharer et al.

nmol/ (mg of protein-min) for total conjugate production.

l/[Butadiene monoxide] (mM)-1

Figure 7. Human placental GSH S-transferase catalyzed reaction of BM and GSH: effects of varying enzyme concentration (A), incubation time (B), and BM ConcentratiOnS (C). Valuea represent the results obtained from typical experiments (A and B) or meane f SD of the results obtained from 3 experiments (C).

imity of the sulfur atom. Still evident is the signal splitting of the H6He, H4, and the cysteine @-protons. This, however, may be due to strong electrostatic interactions between the hydroxyl group and the oxygen on the sulfur atom.

Enzymatic Formation and Characterization of Conjugates. HPLC analysis of the human placental GSH S-transferase catalyzed reaction of BM and GSH yielded two peaks, which were not detected in the absence of BM and were considerably larger than those detected in the absence of the enzyme (data not shown). These peaks had retention times and electronic absorption spectra (A, = 214 nm) similar to those of synthetic I and 11; 111 exhibited a 207-nm A, in ita electronic absorption spectrum (data not shown). Further evidence for the identity of the biosynthetic GSH conjugates of BM was obtained by HPLC analysis of their 2,4-dinitrobenzene derivatives. Again, the HPLC retention times (7.0 and 6.3 min for conjugates I and 11, respectively) and electronic absorption spectra (Amm = 362 nm) of the 2,4-dinitrobenzene deriv- atives of biosynthetic metabolites were identical with those of synthetic I and I1 (data not shown). These results demonstrate that conjugation of BM with GSH occurs enzymatically in vitro, producing the two regioisomeric products I and 11. Formation of I and I1 was dependent on enzyme and BM concentrations and incubation time (Figure 7). As indicated above, I was the major synthetic product; however, approximately equal amounts of I and I1 were formed enzymatically. Double-reciprocal plotting of data (Figure 7C) indicated a V,, of nearly 500

Discussion The results presented in this paper clearly show that the

reaction of BM with GSH at pH 7.8 leads to the formation of three regioisomers, I, 11, and 111, in weight ratias of 951, respectively. The findings that I rearranges to I11 in a time-, pH-, and temperature-dependent manner and that I11 was not detected when human placental GSH S- transferase was incubated with BM and GSH suggest that 111 did not form by a 1,4conjugate addition of GSH to BM, but rather by a rearrangement of I to I11 (Figure 4). Moreover, these resulta indicate that the chemical reaction of BM with GSH occurs by an sN2 mechanism in which GSH, in a 2:l preference, attacks the sterically less hin- dered carbon of the oxirane ring of BM. While I1 may also be formed by an SN1 mechanism which involves the for- mation of the resonance-stabilized allylic carbonium ion, our results indicate that the overall contribution of such a mechanism to product formation is much less than that of the sN2 mechanism. It is of interest to note that the reaction of BM with 2‘-deoxyguanosine in acetic acid gave nearly equal amounts of the regioisomeric products arising through nucleophilic attack by the N-7 of the purine ring at the two oxirane carbons of BM, whereas reaction of BM with organometallic nucleophiles preferentially gave products resulting from 1,Cconjugate additions (6, 9). Taken together, these results provide further evidence that regioselectivity of BM reactions with nucleophiles depends on the nucleophile and the reaction conditions.

Inveetigation of the physical and chemical characteristics of the GSH conjugates 1-111 provided clear evidence that each of these conjugates has its unique physical and chemical properties. NMR spectra of I indicated a 1:l equilibrium between the open form and the sulfurane in- termediate (IVB) as evidenced by the chemical shifts and splitting of signals of the protons in the immediate vicinity of the sulfur atom and the finding that the H4 proton of the tautomeric isomer of I had a chemical shift similar to that of the H, of I1 (Figure 3). Moreover, the lack of similar splittings in the NMR spectra of 11 and 111 and the finding that I can rearrange to I11 by reflux under con- ditions of synthesis or by extended storage at low tem- perature are consistent with the formation of the sulfurme IVB. The finding that reflux of I at pH 7.8 in the absence of GSH, or incubation at 50 O C at pH 2 in the absence of GSH or 2-mercaptoethanol, did not lead to the detection of I11 provides further evidence for the formation of the sulfurane intermediate IVB; in the presence of nucleo- philes, IVB may decompose to the more electrophilic ep- isulfonium ion IVA, which may rearrange to yield III. The sulfoxide and sulfone forms of I, which cannot form an episulfonium ion or a sulfurane intermediate, did not re- arrange to similar structures. In addition, lengthy storage of 11, which is kinetically less likely to form the epi- sulfonium ion, does not lead to the formation of I11 or I. Thus, rearrangement of I to 111 (Figure 4) is likely to occur through the episulfonium ion (IVA) or sulfurane inter- mediate (IVB). The finding that incubations of I at pH 7 or 9 in the presence of nucleophiles and LiC104, .an electrolyte that is commonly used to increase the polarity of the solvent (14), did not affect the HPLC profile of I indicates the weak electrophilicity of the cyclic tautomer of I, which is also consistent with the formation of IVB rather than IVA.

Smit et al. (15) described the chemical reactivity of episulfonium ions as highly dependent on the extent of formation of tight ion pairs and tetravalent sulfur (sulfu-

Glutathione Conjugates of Butadiene Monoxide

rane) intermediates, which would be significantly less electrophilic than the episulfonium ion. Cysteine conjugate episulfonium ions have been recently detected by NMR in the presence of perchloric acid or super acid (16, 17). Moreover, reactive episulfonium ions and sulfurane in- termediates have been implicated in the acid-catalyzed rearrangements of thiol adducts of arene oxides (18,19). NMR observation of a sulfurane intermediate has been reported upon reaction of cyclooctene-S-methylepi- sulfonium ion salt with chloride ion to yield cyclooctene as the final product (20). Furthermore, the X-ray struc- tures of a number of sulfuranes have been reported (21). These include diaryldialkoxyspirosulfuranes, which exist in equilibrium with zwitterionic resonance structures. Thus, ample evidence for the formation of sulfurane and episulfonium ion intermediates has been demonstrated. Formation of IVB rather than IVA is consistent with the NMR of I, the rearrangement of I to 111, and the inability to trap the episulfonium ion with nucleophiles or to detect the rearrangement of I to 11.

The NMR spectrum of I11 indicated an initial 1:l equilibrium between IIIA and the five-membered ring sulfonium ion, VIIA (Figure 4). Studies of the stability of I11 and attempts to trap the sulfonium ion with nu- cleophiles at pH 7 or 9 indicated the stability and weak electrophilicity of I11 and ita tautomer, which suggests the formation of the sulfurane intermediate VIIB. Addition- ally, the failure to trap the cyclic sulfonium ion with GSH at pH 3 and the observed rearrangement of I11 to I is consistent with the sulfurane rather than the sulfonium ion structure for the cyclic tautomer of IIIA. The for- mation of five-membered ring sulfonium ions has been reported previously in the reaction of 1,Cdihalobutanes with cysteine or GSH (13, 22). These sulfonium ions, however, reacted rapidly with nucleophiles at pH 8-9 and decomposed significantly in the presence of aqueous alkali (pH 9) to yield tetrahydrothiophene. Thus, the stability and reactivity of the five-membered ring sulfonium ions formed from 1 ,Cdihalobutanes differ significantly from that of the sulfurane formed by the rearrangement of IIIA. The ring-closure process of IIIA eventually stopped by an apparent slow cis-trans isomerization of IIIA to IIIB.

The in vitro conjugation of BM with GSH by human placental GSH S-transferase yields approximately equal amounts of I and 11, which suggests that the enzymatic reaction proceeds mostly by a mechanism different than that of the chemical reaction. “Borderline” SN2 mecha- nism, or a mechanism with a significant SN1 component, may explain the results obtained with the enzymatic re- action. In addition, the high rate of the enzymatic reaction suggests that GSH conjugation with BM occurs at high rates in vivo to yield I and 11.

In conclusion, the present data demonstrate a high ca- pability of GSH S-transferase to metabolize BM to yield I and 11. Whereas I1 is a very stable GSH conjugate, I slowly rearranges to yield the regioisomer 111. A mecha- nism, which involves the formation of a stable sulfurane tautomer (IVB), is proposed. The novelty of this mecha- nism lies in the fact that the proposed allylic sulfurane appears to be much more stable than the episulfonium ions formed by the GSH S-transferase catalyzed reactions of GSH with 1,2-dihaloethanes (12, 23-25). This low re- activity of I and 11 indicates that GSH conjugation with BM may prevent modification of DNA and other critical cellular targets and may protect against BM toxicity.

Acknowledgment. This research was supported by NIH Grant GM 40375. J.E.S. was supported by National Institute of Environmental Health Sciences Institutional

Chem. Res. Toxicol., Vol. 4, No. 4, 1991 435

Grant T32 ES07015. NMR spectra were determined at the National Magnetic Resonance Facility a t Madison, which is supported in part by NIH Grant RR02301.

Registry NO. I, 133872-48-7; 11,133872-49-8; 111,133872-50-1; IVB, 133872-51-2; VIIB, 133872-53-4; BM, 22910-58-3; GSH,

CHz=CHCH(OH)CHzS(O)-G, 133886-93-8; CHZ=CHCH(OH)- 70-18-8; GSH S-transferase, 50812-37-8; LiClO,, 7791-03-9;

CH2S(02)-G, 133872-52-3; 2-mercaptoethanol, 60-24-2; tetra- ethylammonium chloride, 56-34-8.

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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'-(N- methylcarbamoy1)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-SMG failed 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 in- habitants (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 ex- posed 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 sur- prisingly, 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-methyl- carbamoy1)glutathione; 'C-SMG, S-[N-([lrC]methyl)carbamoyl]gluta- thione; SMC, S-(N-methylcarbamoy1)cysteine; CD8-SMC, S-[N-(tri- deuteriomethyl)carboyl]cysteine; SEC, S-(N-ethylcarboyl)cynteine; NMF, N-methylformamide; GSH, glutathione; BSA, bovine serum albu- miq MMTS, methyl methanethiobulfonate; DTE, dithioqthritol; D'IT, dithiothreitol; MCPBA, m-chloroperoxybenzoic acid; TEAF, triethyl- ammonium formate, HPLC, high-performance liquid chromatography; LC-MS, thermospray liquid chromatography-mass spectrometry; FAB- MS, fast atom bombardment mass spectrometry; MS MS, tandem maas

liquid chromatography. Signal multiplicities in nuclear magnetic reso- nance (NMR) spectra are designated as follows: a, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet.

0 1991 American Chemical Society

spectrometry; CID, collision-induced dimciation; B PLC, faat protein