[CONTRIBUTION FROM THE
LABORATORIES O F THE ROCKEFELLERINSTITUTE MEDICALRESEARCH]
FOR
CHEMICAL REACTIONS OF MUSTARD GAS AND RELATED C0MPOUNDS.I 111. THE REACTION O F MUSTARD GAS WITH METHIONINE
WILLIAM H. STEIN
AND
STANFORD MOORE
Received March 99,1946
In the first paper of this series (l), the reaction of mustard gas (H) with the thioether sulfur of thiodiglycol was described. Since methionine is a protein constituent, and in addition plays an important r81e in biological transmethylation reactions, it seemed of interest to determine whether H was capable of reacting with the thioether sulfur of this amino acid. When H is shaken at room temperature with an aqueous solution of methionine at pH 3, only 10% of the theoretically possible acid is liberated, indicating that 90% of the H has reacted with methionine to form a sulfonium salt. When tfhe same reaction is carried out a t pH 8.5, 6OY0 of the H combines with methionine to yield a sulfonium salt. Under similar conditions 40% of the H reacts with the carboxyl group of sodium hippurate, and 8 to 15% combines with the €-amino group of benzoyllysineamide [see (2)l. These data indicate that H reacts with methionine-sulfur more readily than it does with either the amino or carboxyl groups of the aforementioned substances. From the reaction mixture resulting when H is shaken with methionine in acid solution the sulfonium base I has been isolated as a crystaliine azobenzene sulfonate.2 It will be noted that I is derived from one molecule of H and 2 molecules of methionine, and is analogous in structure to the sulfonium salt formed when H is allowed to react with thiodiglycol(1). Because of the unfavorable properties of I, the pure azobenzene sulfonate was only isolated in low yield. CHs
2"
1 This work was done in whole under Contract No. OEMsr-313 between The Rockefeller Institute for Medical Research and the Office of Scientific Research and Development, which assumes no responsibility for the accuracy of the statements contained herein. The experiments were performed in the period January 1942-August 1944. 2 The analytical data presented in the experimental section indicate that the salt is composed of one mole of the sulfonium base and four moles of azobenzene sulfonic acid, two of which neutralize the two sulfonium groups, and two of which neutralize the two a-amino groups. 681
682
WILLIAM H. STEIN AND STANFORD MOORE
The fact, however, that during the reaction of H with methionine very little acid is liberated, is strong indication that I is the major product of the reactionThis supposition is further supported by the earlier studies on the analogous re. action of H with thiodiglycol, in which it was shown that the sulfonium salt derived from one molecule of H and 2 molecules of thiodiglycol was formed in excellent yield (1). In the discussion to follow, therefore, it is assumed that I is the principal product formed as a result of the reaction of H with methionine. When the aqueous solution resulting from the reaction of 13 g. of H with 20 g. of methionine is heated a t 100" for 2 hours, a heavy, water-insoluble oil (3.2 g.) CH2 CH2Sf. CH2 CHzCH COOH
/
/CH2CH2S*
+ 2H+ + 2 CH2CHsCHCOOH
S
\
CHZCH2S. CHs
I
OH
I
NHz
FIQ.1
separates, which has an unpleasant, persistent odor. It has been identified as a trisulfide, bis(methylthioethyl)sulfide, of the following structure : CH3SCH2CH2SCH2CH2SCH3
(11)
Upon oxidation with nitric acid, the trisulfide was transformed into the trisulfoxide. CHaSCHzCH2SCHzCHzSCHa The trisulfide (11)probably arises from the decomposition of the sulfonium salt (I) in the manner given in Figure 1. According to Figure 1, yhydroxy-a-aminobutyric acid (IV) also should be formed during the decomposition of the sulfonium salt. Additional evidence for the validity of the above reaction scheme was provided by the isolation of I V aa a salt of p-hydroxyazobenzene-p'-sulfonic acid, from which the free amino acid IV could be obtained.
REACTIONS OF MUSTARD GAS.
I11
683
There is evidence, however, that the decomposition of I does not proceed solely according to Figure 1. The yield of trisulfide is far below the theoretical quantity to be expected from this scheme. Moreover, the recovery of considerable quantities of methionine after heat treatment of I indicates that only about one-quarter of it decomposes according to the above scheme, while the remaining three-quarters decomposes to regenerate methionine. In a protein molecule both the amino and the carboxyl groups of methionine are bound in peptide linkage. In order to learn whether the methionine-sulfur in a structure of this type can react with H, the reaction of carbobenzoxymethionineamide with H was investigated. It is noteworthy that H reacts with carbobenzoxymethionineamides to yield a sulfonium salt. When 0.25 cc. of H is allowed to act upon 2.3 g. of carbobenzoxymethionineamide in 100 cc. of 50% alcohol (alcohol is necessary in order to keep the carbobenzoxymethionineamide in solution), about half of the H hydrolyzes, and the other half reacts to form a sulfonium salt. Upon heating a t 100” in water, this sulfonium salt decomposes to yield HCI, and considerable amounts of carbobenzoxymethionineamide. The characteristic odor of the trisulfide which is formed on decomposition of I can also be detected here, although no water-insoluble oil separates. The yield of sulfonium salt is lower in the case of carbobenzoxymethionineamide than it is in the case of free methionine. This may be due, in part, to the fact that 50% alcohol was employed as solvent in the reaction. In this connection, it was shown earlier (1) that the reaction of H with thiodiglycol to yield a sulfonium salt was inhibited by the presence of alcohol. In the case of a protein, the reaction of H with methionine-sulfur may be more complicated than in the model experiments discussed above. It seems unlikely that two methionine-sulfurs would be oriented in space in a manner permitting both chlorines of one H molecule to react with two methionines. It is much more probable that one chlorine of an H moleculewould react with a methionine-sulfur, and that the other would react either with some other favorably situated reactive group in the protein molecule, or with water. It should be pointed out that the over-all sequence of reactions starting with H and methionine, and ending with the trisulfide 11, involves the transfer of the thiomethyl groups from 2 methionine molecules to a second substance, in this case a diethylsulfide residue. In other words, a “transthiomethylation” has occurred, the thiomethyl group of methionine having been labilized by sulfonium salt formation. It will be recalled that the methylsulfonium salt of methionine has been prepared by Toennies (3). As will be shown in a subsequent paper of this series (4), the methylsulfonium salt of methionine, in contrast to the sulfonium salt I, is quite stable. The lability of the methyl or thiomethyl group in a methionine sulfonium salt thus depends upon the structure of the other group attached to the sulfonium sulfur. Whether or not these findings have a bearing on the mechanism of biological transmethylation reactions remains for further investigations to determine.
684
WILLIAM H. STEIN AND STANFORD MOORE
EXPERIMENTAL
Extent of reaction of H with methionine. H (0.5cc.) was shaken a t room temperature for 18 hours with a solution of 2.4 g. of methionine in 26 cc. of 0.31 N HCl (pH about 3). An aliquot of the resulting clear solution was titrated in 90% alcohol with thymolphthalein as the indicator. The main body of the reaction mixture was neutralized, heated at 100' for 2 hours, and a n aliquot again titrated as before. The increase in acid produced on heating was taken as a measure of the sulfonium salt decomposed. The results indicated that 90% of the H had reacted t o yield the sulfonium salt. On heating, the aqueous solution became cloudy and acquired a strong, unpleasant odor due t o the formation of the trisulfide (11) (see below). At alkaline pH values (about 8.5) the reaction of H with methionine is not as complete as at pH 3. H (0.5 cc.) was shaken with 2.4 g. of methionine and 700 mg. of NaHCOs in 25 cc. of 0.5 N NaOH. The extent of the reaction was determined as described above, except t h a t the reaction mixture was acidified and COZremoved in vacuo prior t o the titrations. The results indicated that abobt 60% of the H reacted t o give the sulfonium salt. Isolation of the Azobenzenesulfonate o j I . H (0.5 cc.) was shaken for 24 hours at room temperature with 2.5 g. of methionine in 25 cc. of 0.65 N HC1. T o the resulting clear solution 5 cc. of N HCl was added, the solution concentrated t o a sirup in vacuo, and absolute ethanol added, which caused the precipitation of an oil. The ethanol was decanted, the oil dissolved in 2 to 3 cc. of water, and again precipitated with ethanol. The alcohol precipitation was repeated once more in order to ensure the removal of the hydrochloride of any unreacted methionine. The oil was dissolved in water and a solution of 5 g. of azobenzene-p-sulfonic acid in water added. The mixture set to a crystal mass almost immediately. After standing a t 0" the crystals were filtered off and washed with water. For recrystallization the salt was suspended in 50 cc. of acetone and 40 cc. of water was added. Any undissolved material was removed and the filtrate was set in an open beaker t o allow the salt t o crystallize slowly as the acetone evaporated; yield 700 mg. The salt was recrystallized i n the same manner for analysis, and dried i n a vacuum desiccator over Drierite; yield 300 mg. of the tetraazobenzenesulfonate of I. Anal. Calc'd for C&~~NloO1&.3 HzO: C, 50.0; H, 5.0; N, 9.4; "2-N, 1.9; S, 15.1; HeO, 3.6. Found: C, 49.6; H, 5.2; N, 9.3; NHz-N, 2.2; S, 15.4; HzO,3.8. The decomposition of I . H (10 cc.) was shaken for 24 hours with a n aqueous solution of 20 g. of methionine. The resulting clear solution was heated at 100' for 2 hours, cooled, and extracted three times with ether. The ethereal extract was dried over NazS04,and after the ether had been removed in vacuo, 3.2 g. of a n oil remained. After two distillations i n vacuo, 1.2 g. of an oil, distilling at 93-94' under 0.2 mm. pressure, was obtained. Anal. Calc'd for CsH&: C, 39.5; H , 7.7; S, 52.8. Found: C, 39.3; H, 7.8; S, 53.0. The sulfur content of such sulfides, sulfoxides (see below), and of H derivatives is frequently found t o be considerably too low when determined by the Pregl method. Hence, the Carius method was used in this case. For the preparation of the sulfoxide, 350 mg. of the sulfide was added t o a few CC. of concentrated HNOS and the mixture allowed t o stand for 5 minutes. Two volumes of water was added, the solution concentrated t o dryness under reduced pressure, alcohol added, and the solution concentrated again. The residue was taken up i n alcohol, 10 volumes of ether added, and the solution stored at 0". The crystalline product was removed and recrystallized from ethanol; yield 200 mg.; m.p. 155-160" with decomposition. After two additional crystallizations from alcohol the decomposition range was raised t o 165-169". Anal. Calc'd for C6H&Ss: C, 31.3; H, 6.1; S, 41.7. Found: C, 31.1; H, 6.1; S, 41.7. The sulfur was determined by the Parr bomb method.
REACTIONS OF MUSTARD GAS.
III
685
The aqueous phase remaining after removal of the sulfide was freed of chloride ion with Ag,CO* and of Ag+ with Has. The resulting solution was concentrated in uucuo to 300 cc. and stored at 0". Methionine (9.3 g.) was filtered off. The filtrate was concentrated to 50 cc. and another 1.1 g. of methionine recovered. The filtrate was concentrated t o 25 cc. and 75 cc. of alcohol added. After standing a t Oo, a further 4.1 g. of methionine was recovered, making the total yield of methionine 14.5 g. The filtrate from which the bulk of the methionine had been removed was concentrated t o dryness i n vacuo to remove alcohol, and the residue dissolved in 30 cc. of water. An aqueous solution of 2 g. of p-hydroxyazobenzene-p'-sulfonicacid was added, and the solution placed a t 0". The crystals which deposited were recrystallized from hot water; yield 1.1 g. Anal. Calc'd for CIH,NOs.C12H10N204S.HzO: C, 46.4;H, 5.1;N, 10.1;S, 7.7;HzO,4.3. Found: C,46.1;H,5.1;ru',10.2;S,7.8;Hz0,5.7. For the isolation of 7-hydroxy-a-aminobutyricacid, 416 mg. of the hydroxyazobenzene sulfonic acid salt was dissolved in 5 cc. of hot water and 273 mg. of barium acetate monohydrate in 2 cc. of water added. The barium sulfonate precipitated immediately, and after cooling the mixture, was removed. The filtrate was freed exactly of barium and sulfate ions, decolorized with charcoal, and concentrated i n vacuo to a sirup. The free aminoacid crystallized upon addition of alcohol, and was recrystallized from water by t h e addition of alcohol; yield 55 mg.; m.p. 186-187" (dec.). Fischer and Blumenthal (5) reported m.p. 187" (dec.) ,lor this compound. Anal. Calc'd for CdHgNOs: C, 40.3;H, 7.6;N, 11.8. Found: C, 40.2; H, 7.5; N, 11.8. The nitrogen was determined by the micro-Dumas method. The reaction of H with carbobenzoxymethionineamide. H (0.25cc.) was shaken for 24 hours with a solution of 2.3 g. of carbobenzoxymethionineamide in 100 cc. of 50% alcohol. The reaction mixture was concentrated i n vucuo to a small volume to remove the alcohol, and 1.3 g. of unreacted carbobenzoxymethionineamide filtered off. The filtrate was titrated t o phenolphthalein with 0.1 N NaOH. The titer indicated that 55% of the H employed had hydrolyzed. The neutral solution was heated a t 100" for 2 hours, and the acid liberated again titrated. The results indicated that 45% of the H had reacted to form a sulfonium salt which was decomposed by heat with the liberation of acid. The characteristic odor of the trisulfide formed on the decomposition of the sulfonium salt (I)could also be detected. No water-insoluble oil separated, however. On cooling, 0.5 g. of carbobenzoxymethionineamide crystallized and was filtered off. At least this muchcarbobenzoxymethionineamide must, therefore, have been regenerated by the decomposition of the sulfonium salt.
The authors wish to acknowledge their indebtedness t o the late Dr. Max Bergrnann for the constant advice and encouragement which he gave in the course of this research. Thanks are due also t o Mr. Stephen M. Nagy, who performed the microanalyses reported in this paper. NEWYORE,N. Y. REFERENCES
(1) STEIN, MOORE,AND BERGMANN, J. Org. Chem. (paper I this series). (2) MOORE,STEIN,AND FRUTON, J. Org. Chem. (paper I1this series). (3) TOENNIES, J. Biol. Chem., 132,455 (1940). (4) STAHMANN, FRUTON, AND BERQMANN, J. Org. Chem. (paper VI this series) (6) FISCHER AND BLUMENTEAL, Ber., 40,106 (1907).
