Agric. Biol. Chem., 43 (10), 2017-2020, 1979 2017

ƒÀ-Elimination Reaction of Antimicrobial L-5-Alk(en)yl-

thiomethylhydantoin-S-oxides

Satoshi TAHARA, Hiroyuki OKAMURA, YUZO MIURA and Junya MIZUTANI

Department of Agricultural Chemistry , Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan

Received July 19, 1978

The antimicrobial L-5-alk(en)ylthiomethylhydantoin-(•})-S-oxides (RHSO) decompose

spontaneously via ƒÀ-elimination under physiological conditions to give 5-methylenehydantoin and alk(en)yl thiosulfinates as antimicrobial principles. Attempts have been made to elucidate

the mechanism of ƒÀ-elimination reaction of RHSO in a buffer solution. The ƒÀ-elimination of

propyl derivative (I) at specified conditions of temperature and pH followed first-order kinetics. A linear relationship between the first-order rate constant and the concentration of hydroxide

ion was also observed. Therefore, it has been shown that the ƒÀ-elimination of I obeys the

second-order kinetics. The second-order rate constant from L-5-propylthiomethylhydantoin-

(+)-S-oxide [(+)-I] in water at 37•Ž was 4.20•}0.31•~103/sec. M.

As shown in our previous papers,1,2) the L- fore, the ƒÀ-elimination reactions of two dia-

5-alk(en)ylthiomethylhydantoin- (•})-S- oxides stereoisomeric substrates, (+)-I and (-)-I

(RHSO) decomposed via a ƒÀ-elimination re were carried out in a buffer solution at 37•Ž. action under physiological conditions to give As shown in Table II, nearly constant values

5-methylenehydantoin and highly antimicro were obtained by the calculation using the bial alk(en)yl thiosulfinates. In this paper, experimental results according to the equation

we deal with L-5-propylthiomethylhydantoin- for first-order kinetics (k=l/t In 100/(100-ƒÔ)).

(+)-S-oxide [(+)-I], (-)-S-oxide [(-)-I] and The results indicate that the fl-elimination re-

(+)-S-oxide [(+)-I] as model compounds to actions of (+)-I and (-)-I obey the pseudo- investigate the degradation mechanism of first-order kinetics, i.e., first-order in the sul RHSO. The degradation reactions of these foxide and the first-order rate constants for

sulfoxides were followed by determining the (+)-I and (-)-I at 37•Ž, pH 6.51 are 3.74 resulting propyl propanethiosulfinate. In •} 0.34•~10-4/sec and 4.11•}0.18•~10-4/sec, practice, the thiosulfinate was quantitatively respectively. Although the degradation of converted into the corresponding disulfide (-)-I probably takes place a little easier than which was analyzed by gas chromatography. that of (+)-I, the stereochemical effect of the At first the effect of temperature on the de sulfur atom does not seem to be so great any gradation of (•})-I was examined. The re how. action was carried out in a buffer solution pH Since the degradation of the hydantoin sul 6.51 at 17•`57•Ž for 30 min. The results are foxides seemed to depend on hydroxide ion, shown in Table I. The preliminary experi the pseudo-first-order rate constants (ƒÈ1) ments revealed that (•})-I decomposed more were calculated from the degradation per rapidly in alkaline solutions. These facts centages of (+)-I in the reaction mixtures of suggested that the degradation depended on various pH (Table III) and the ƒÈl was plotted the temperature and the concentration of as a function of the concentration of hydro hydroxide ion in the reaction mixture. There xide ion ([OH_??_]). As in Fig. 1, experimental Hydantoin Derivatives of S-Alk(en)yl-L-cysteine-S- data points are approximately on a straight oxides. Part IV. Part III, see ref. 1. line which passes the origin (•›). The result 2018 S. TAMARA, H. OKAMIJRA, Y. MIURA and J. MIZUTANI

TABLE 1. EFFECT OF REACTION TEMPERATURE TABLE III. PSEUDO-FIRST-ORDERRATE CONSTANTS ON THE DEGRADATION OF L-5-PROPYLTHIO- DETERMINEDFROM THE DEGRADATIONPERCENTAGES METHYLHYDANTOIN-(•})-S-OXIDE [(•})-I] OF L-5-PROPYLTHIOMETHYLHYDANTOIN-(+)-S-

The reaction conditions are shown in the text. OXIDE[(+)-I] IN THE REACTIONMIXTURES

Results of duplicate experiments were averaged. OF VARIOUSpH The degradation reactions were carried out in M/10

phosphate buffer solutions at 37•Ž for 30 min. The degradation percentages were averaged values of

duplicates.

a In the buffer solution pH 6.51, for 30 min.

TABLE II. ƒÀ-ELIMINATION REACTIONS OF

L-5-PROP Y LTHIOMETHYLHYDANTOIN-(+)-S- OXIDE [(+)-I] AND (-)-S-OXIDE [(-)-I]

IN THE BUFFER SOLUTION (pH 6.51) AT 37•Ž a Calculated from the ionic product of water a t 37•Ž, [H_??_] [OH_??_=2.40/1014.b

ƒÈ1=1/t In 100/100-ƒÔ, t=1800 sec.

a Calculated from the equation, ƒÈ=1/t In 100/100-ƒÔ

The averaged values of k, i.e., the apparent first

order rate constants (37•Ž, pH 6.51): k1 from (+)-1, (3.74•}034)•~10-4/sec

k1 from (-)-I, (4.11•}0.18)•~10-4/sec * Asterisked values were excluded when the averages

were calculated. FIG. 1. Relationship between the Pseudo-first-

order Rate Constant (ƒÈ1) and the Concentration of

Hydroxide Ion ([OH_??_]) in the Reaction Mixture . indicates that ƒÈ1 is directly proportional to The ratios of ƒÈ1 to [OH_??_] at each experimental point [OH_??_], namely ƒÈ1=ƒÈ2[OH_??_] in equation. are as follows: 2.82, 3.73, 4.05, 4.35, 4.44 and 4 .44 The ƒÀ-elimination of (+)-I is consequently •~101/see•EM. The mean except for the first value was

shown to be E2 in nature, i.e., first-order in calculated to be 4.20•}0.31•~101/sec•EM. the sulfoxide and first-order in hydroxide ion, respectively. The second-order rate constant The degradation pathway is summarized in

(ƒÈ2) for (+)-I at 37•Ž, 4.20•}0.31•~103/sec. M, Fig. 2. The substrate molecule and hydroxide was obtained as a man value of the experimen ion associate to form a transient intermediate, tal data of ƒÈ1/[OH_??_]. which gives 5-methylenehydantoin and an From the facts described above, we may alkane(or alkene)sulfenic acid . The latter suggest the degradation of L-5-alk(en)ylthio- undergoes dehydrating bimolecular con methylhydantoin-(•})-S-oxides (RHSO) to densation immediately to form an alk(en)yl be the nature of base-catalyzed E2 reaction. thiosulfinate. ƒÀ-Elimination of L-5-Alk(en)ylthiomethylhydantoin-S-oxides 2019

refluxing periods of 18-24 hr in 1 N hydro-

chloric acid, 3 hr in 1 N sodium hydroxide and

24-48 hr in a neutral solution were necessary for complete disappearance of the alliin homo

log.

In recent years, ƒÀ-elimination reactions of

sulfoxide10) and selenoxide derivatives11) have

frequently been utilized in the field of organic synthesis to introduce an olefinic bond into

the synthetic intermediate.

In any event, we have not found so far

such a labile sulfoxide as RHSO which undergo ƒÀ-elimination with E, mechanism in a neutral FIG. 2. Second-order ƒÀ-Elimination Reaction of L-

5-Alk(en)ylthiomethylhydantion-(•})-S-oxides aqueous solution at ambient temperatures.

(RHSO). Moreover, the degradation products, alk(en)yl

The reaction pathway was studied by using L-5- thiosulfinates, have potent antimicrobial

propylthiomethylhydantoin-S-oxide (1, R=n-C3H7). activity. Therefore, the hydantoin sulfoxide is one of convenient precursors which gradually

release the biologically active principles under DISCUSSION physiological conditions in analogy with ƒÀ- Although great many, ƒÀ-elimination reactions chloroethylphosphonic acid as an ethylene have been intensively studied so far,3,4) the generator for plant tissues,12) base-catalyzed decomposition of aliphatic It is also well known that alk(en)yl thiosul sulfoxides was reported for the first time in finates are characteristic precursors of fresh 1963 by Hofmann et at.5) Kinetic studies6) flavors of Allium plants. We will describe in potassium tert-butoxide-dimethyl sulfoxide the results on non-enzymatic simulation of showed the elimination of aliphatic sulfoxide garlic flavor formation by using L-5-allyl- was E2 in nature, i.e., first-order in base and and L-5-methylthiomethylhydantoin-(•})-S- first-order in sulfoxide. It was also reported oxides in our subsequent paper. by Kingsbury and Cram7) that 1, 2-diphenyl-

1-propyl phenyl sulfoxide underwent decom PROCEDURES position with the five-membered Ei mechanism.

A quantitative study of the hydrolysis of 1. Compounds. L-5-Propylthiomethylhydrantoin-

S-methyl-L-cysteine-S-oxide (an alliin homo (•})-S-oxide[(•})-I], (+)-S-oxide [(+)-I] and (-)-S- log) by boiling in 1 N hydrochloric acid and oxide [(-)-I] were prepared as previously described,13) Optical properties of the used compounds were as also in an alkaline or even in a neutral solu follows: (•})-I, [ƒ¿]12D-45•‹ (c=1, H2O); (+)-I, [ƒ¿]12D tion, was carried out by Ostermayer and +38•‹ (c=1, H2O); (-)-I, [ƒ¿l12D-122•‹ (c=1, H2O). Tarbell.8) They showed that most of the material was converted to pyruvic acid, am 2. GLC and quantitative analyses. Gas chromato monia, dimethyl disulfide and methyl me graphy was carried out as shown in our previous paper.1) thanethiosulfonate. The sulfur-containing Since the volatile disulfides are conveniently analyzed by gas chromatography, the propyl propanethiosulfinate products were estimated to be formed from arising from I was converted to dipropyl disulfide or methyl methanethiosulfinate, which had been ethyl propyl disulfide via reaction (1). Some funda shown to disproportionate to them under the mental ex periments required for such a determination experimental conditions. The reaction pro- R-S(•¨O)-S-R+2 R•L-SH•¨2 R-SS-R•L+H2O (1) ducts (primary ones) are analogous to those of R-SS-R+R•L-SS-R•L_??_2 R-SS-R•L (2) alliin-alliinase system9) and partially to those of RHSO in a neutral buffer solution.1) But R-SS-R+R•L-SH_??_R-SS-R•L+R-SH (3) 2020 S. TAHARA, H. OKAMURA, Y. MIURA and J. MIZUTANI

were carried out, and we confirmed that the recovery 2-3) Effect of pH on the degradation of (+)-L

of disulfide from (•})-I via reaction (1) was 99%. The The degradation of (+)-I in six m/10 phosphate buffer

exchanges of alkyl groups between two disulfides via solutions (pH 5.63, 5.91, 6.24, 6.46, 6.64 and 6,82) was

reaction (2) and the reduction of disulfide by alka determined. (+)-I (10mM) was left at 37•Ž for 30 min nethiol via reaction (3) were negligible under our in each buffer solution. The reaction mixtures were

analytical conditions. treated in the same way as described in 2-2). The

The disulfides were determined quantitatively by standard ratio of peak area of ethyl propyl disulfide to comparing the peak areas with those of isoamyl alcohol that of isoamyl alcohol was obtained from the reaction

as an internal standard, Since I (10-25mM) was com mixture pH 8.11 left at 37•Ž for 60 min.

pletely decomposed in a buffer solution pH 7.4 at 37•Ž for 60 min,1) the amount of disulfide derived from REFERENCES propyl propanethiosulfinate in this reaction mixture was looked upon as that corresponding to 100% de- 1) S. Tahara, Y. Miura and J. Mizutani, Agric. Biol. gradation of I and the degradation percentage of each Chem., 43, 919 (1979). sample was calculated. Little difference was found in 2) S. Tahara, Y. Miura and J. Mizutani, ibid., 41, the recoveries of the disulfide from the reaction mix 221 (1977). tures maintained at 37•Ž for 60 , 120 and 180 min, 3) C. K. Ingold, "Structure and Mechanism in respectively. Organic Chemistry", 2nd Ed., Cornell University 2-1) Effect of temperature on the degradation of (•}) Press, Ithaca, 1969, p. 649. -I The phosphate buffer solutions (pH 6 .51) 4) J. March, "Advanced Organic Chemistry: Reac containing 24.5mM (•})-I were maintained at 17 , 27, tions, Mechanisms, and Structure," McGraw- 37, 47 and 57•Ž for 30 min, respectively. The reaction Hill Book Co., New York, 1968, p. 727. mixtures (0.5ml each) were extracted with 0 .25ml of 5) J. E. Hofmann, T. J. Wallace, P. A. Argabright ether which contained 1ƒÊl of isoamyl alcohol as an and A. Schriesheim, Chem. & Ind. (London), internal standard. Each ether extract (ca, 0.15ml) was 1963, 1243; T. J. Wallace, J. E. Hofmann and transferred onto a mixture of 3ƒÊl of propanethiol and A. Schriesheim, J. Am. Chem. Soc., 85, 2739 0.1ml of 2% sodium carbonate solution and the mix (1963). ture was well shaked. The resulting dipropyl disulfide 6) J. E. Hofmann, T. J. Wallace and A. Schriesheim in the ether layer was analyzed by GLC. The amount J. Am. Chem. Soc., 86, 1561 (1964). of dipropyl disulfide contained in the propanethiol 7) C. A. Kingsbury and D. J. Cram, ibid., 82, 1810 used was determined by the control analysis lacking (1960). (•})-I for correction. 8) F. Ostermayer and D. S. Tarbell, ibid ., 82, 3752 2-2) Determination of apparent rate constants. The (1960). phosphate buffer solution (pH 6.51) containing 10mM 9) A. Stoll and E. Seebeck, Adv. Enzymol., 11, 377 (+)-I or (-)-I was maintained at 37•Ž. The reaction (1951). mixtures (0.5ml each) were pipetted off at each time 10) B. M. Trost, T. N. Salzmann and K. Hiroi, J . Am. and treated in the same way as described in 2-1) except Chem. Soc., 98, 4887 (1976). using ether which contained 0.25ƒÊl of isoamyl alcohol 11) K. B. Sharpless, R. F. Lauer and A . Y. Teranishi, and using ethanethiol in place of propanethiol. The ibid., 95, 6137 (1973); P. A. Grieco, M. Nishizawa, resulting ethyl propyl disulfide was analyzed. The T. Oguri, S. D. Burke and N. Marinovic, ibid., 99, standard ratios of peak area of ethyl propyl disulfide 5773 (1977). to that of isoamyl alcohol corresponding to 100% 12) J. A. Maynard and J. M. Swan , Australian J. degradation of (+)-I and (-)-I were obtained from the Chem., 16, 596 (1963). reaction mixtures adjusted to pH 7.4 and left at 37•Ž 13) J. Mizutani, Y. Miura and S. Tahara, J. Pesticide for 60 min, and then the degradation percentage of Sci., 4, 17 (1979). each reaction mixture was calculated.