THE FORMATION OF SULFONIUM SALTS FROM ALCOHOLS AND METHIONINE IN SULFURIC ACID*

BY THEODORE F. LAVINE, NORMAN F. FLOYD, AND MARY S. CAMMAROTI

(From The Innstitute for Cancer Research and the Lankenau Hospital Research Institute, Philadelphia, Pennsylvania)

(Received for publication, July 17, 1953)

The activity of “8-adenosylmethionine” as a biological methylating agent (l), the influence of methionine on the production of thiomethyl- adenosine by yeast (a), and the isolation of the methylsulfonium derivative of methionine from plant extracts (3) attest to the biological importance of methionine sulfonium compounds. The present paper deals with the chemistry of methionine sulfonium derivatives.

We have shown (4) that in hot 50 per cent HzS04 methionine and methyl- sulfuric acid form methionine methylsulfonium hydrogen sulfate at a rapid rate. Because hot HzS04 favors the formation of sulfuric acid esters from alcohols, it appeared worth while to investigate the formation of methi- onine sulfonium salts directly from hydroxy compounds, particularly those for which the sulfate (or halide) derivative is relatively inaccessible. A few instances of this approach are recorded in the literature (5, 6).

The reaction of methionine with hydroxy compounds according to Re- action 1 was followed by determining the rate of disappearance of meth- ionine.

RSMe + R’OH + HzSOI - (R) (R’) (Me)S+SO& + Hz0 (1)

A competitive and limiting factor to the above reaction is the self- decomposition of methionine in hot sulfuric acid according to Reaction 2.

4RSMe + 3H$Oa - RSSR + 2(Me)z(R)S+SOIH- + SOZ + 2H~0 (2)

The extent to which the latter reaction occurred was evaluated from the amount of homocystine formation and from the known rate of reaction (4). Qualitative and quantitative evidence of sulfonium salt formation was ob- tained by precipitation with phosphotungstic acid from dilute sulfuric acid.

As shown in Table I, methionine in boiling 15 N sulfuric acid reacts readily with simple alcohols to form sulfonium salts. tert-Butyl alcohol also reacts at room temperature, but the resulting sulfonium salt decom- poses at elevated temperatures and also on dilution. Benzyl and ally1 alcohols appear to react readily, but side reactions are indicated by pre-

* This investigation was supported in part by a research grant from the National Cancer Institute of the National Institutes of Health, United States Public Health Service, and in part by an institutional grant from the American Cancer Society.

107

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108 METHIONIXE SIJLFONIUM SALTS

TABLE I

Reaction of Methionine with Hydroxy Compounds in 16 N Xuljuric Acid at 155”

The reaction mixtures were prepared by weight and contained 50 mM of nn-methi- onine, 100 mM of hydroxy compound, and 50 ml. of 18 N HzSOI. Methionine and homocystine determinations were made on aliquots of a 1: 10 aqueous dilution of the reaction mixture. All results are expressed as per cent of methionine initially present, The determinations of homocystine and sulfonium salt were made at the end of the reaction.

None Methanol. Ethanol. Propyl alcohol. Isopropyl alcohol. tert-Butyl alcoholt

‘< ‘( $.. Benzyl alcohol.. Ally1 “ Phenol, Ethanolsmine. nn-Threonine nn-Serine

“ tt . .

Methionine

AftCr mixing

per cent

100 97

107 102 102 52 59 51 86

99

ssll 100 100

15 min.

per cent

2 11 20 10 66

0 0

99 975

W 70**

I -

1 hr.

per cent

12 0 0 0 0

91 5 0 0

99

84§ 1% 48** 21**

Homo- cystine

julfonium phospho- tungstate*

fw Gent per cent

50 48 0 95 0 106 0 95 0 99

0 0 0 0

2311 46 24** 45**

72 114

* The equivalent weight of sulfonium phosphotungstates was assumed to be 2138 plus the molecular weight of the respective hydroxy compound.

t The reaction was accompanied by darkening and gas evolution (isobutylene ?). After standing for 3 days the methionine content of the first diluted aliquot had increased from 52 to 100.6 per cent.

1 Reaction run at 25”. No methionine remained after 3 hours; after standing for 17 hours the diluted 3 hour aliquot contained 58.4 per cent methionine, while the original reaction mixture was still methionine-free.

$ Methionine determinations made on filtrate from mercuric sulfate precipitate

(4). 11 2 hour value; corresponding methionine value 57 per cent. f The reaction mixture had been heated on a water bath for 1 hour. ** These values are approximations based on the ratio of the total to the reversi-

ble iodine consumption values in the methionine determination. After 5 minutes oxidation, the ratio for homocystine is 3:l and for methionine 1:l.

tt Serine was allowed to stand for 1 hour with 18 M sulfuric acid before dilution to 18 N and addition of methionine.

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T. F. LAVINE, N. F. FLOYD, AND M. S. CAMMAROTI 101)

cipitate formation and the low yield of phosphotungstate in the former case and darkening of the reaction mixture in the second. The apparent lack of reaction with phenol and ethanolamine is contradicted by the absence of the self-decomposition of methionine, which suggests reversible sulfonium salt formation depending on temperature. With the biologically occurring hydroxy compounds, threonine and serine, the predominant reaction was the decomposition of methionine according to Reaction 2. Several var- iations in conditions were employed with serine (in order to prepare the methylsulfonium salt of cystathionine (7)), all with negative results. These included the use of phosphoric acid, 9 N’ and 36 N H&304, and (as shown in Table I) the prior formation of the sulfate derivative of serine by the use of concentrated sulfuric acid (8). /LPropiolactone readily formed a sulfonium salt with methionine. Qualitative experiments with glycolic and lactic acids showed no interaction with methionine, presumably owing to their decomposition in hot sulfuric acid. The use of halogen acids instead of sulfuric acid leads similarly to sulfonium salt formation with /3-bromopropionic acid and methanol, yielding the corresponding sulfonium salts in hydrobromic and hydrochloric acids, respectively.

The isolation of sulfonium salts in the solid state, apart from occasional inherent instability, frequently depends on the choice of a suitable anionic component and on the ionic state of polar groups in the sulfonium moiety. The methionine carboxyethylsulfonium salt, for example, may be ionized as shown, depending on the acidity of the solution. The bromide hydro- bromide (I) was obtained as an oil, the bromide (II) as an unstable solid, and the thetin (III) as a solid admixed with II; the isolation of IV was not attempted. Methionine isopropylsulfonium bromide was obtained as a solid but the n-propyl derivative yielded a syrup; the instability of the tert-butyl derivative has been mentioned.

R(NH,+)COOH R(NHa+)COO- R(NHa+)COO- I I I

CH:$- S+ 2Br- CHr-S+ 13~ CHr-S+ I I I

R’COOH R’COOH R’COO-

(I) (II) (III)

R(NHJCOO- I

CHs---S+ I

R’COO-Nn’

(IV)

1 In I) N I&SO,, the decomposition of methionine (Reaction 2) does not take place; however, the rate of reaction with methanol (Reaction 1) is also decreased (33 per cent sulfonium salt formation in 1 hour at 104”).

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110 METHIONINE SULFONIUM SALTS

The reactivity of sulfonium salts is manifested by displacement of one of the groups contributing to the sulfonium function. Which group is elim- inated is determined by the relative electron release effects among the three substituents which, in turn, often vary with changes in the acidity of the medium. Decomposition can thus lead to a variety of products and the biochemical significance of methionine sulfonium salts, accordingly, would not appear to be limited to transmethylation phenomena alone.

The simplest case is presented by the methylsulfonium salt of methionine which has only two alternatives for decomposition.

RSMez+ + RSMe + Me+ (3)

RSMe2+ - Me8 + R+ (4)

Previous investigations of methionine sulfonium salts have revealed that, in general, the pathway of decomposition is related to the acidity (9), that the methylsulfonium salt evolves dimethyl sulfide from alkaline solution at temperatures of 90” or higher (lo), and that, on boiling aqueous solutions of the sulfonium salt derived from methionine and “mustard gas,” products corresponding to both Reactions 3 and 4 are formed (11).

Our results show that the methionine methylsulfonium salt in the form Me$S+R(NH,+)COOH decomposes according to Reaction 3, while MezS+R- (NH3+)COO- and Me&+R(NH2)COO- d ecompose according to Reaction 4. Reaction 3 is the reverse of formation of the sulfonium salt (Reaction 1) and takes place in hot acid solution. The rate of reaction varied with the nature of the acid as follows: 7 N HI > 6 N HCI > 6 N HBr > 16 to 18 N HzS04, no decomposition being detected in 16 to 18 N H&04.2 The acid HX, by its influence on the equilibrium MeOH + HX 4 MeX + HzO, determines the reactivity and volatility of the ester MeX and hence the rate and extent of Reaction 3. With HI, Reaction 3 is followed by similar decomposition of methionine (12) and both methyl groups of the sulfonium salt are quantitatively eliminated as methyl iodide. Accord- ingly, the results of demethylation procedures for the estimation of methi- onine must be taken advisedly if conditions admit of sulfonium salt formation (13). In concentrated HCl, methanol formed a sulfonium salt with methionine, but the reaction ceased at about 70 per cent conversion and decomposition ensued owing presumably to the volatility of methyl chloride. The reversibility of Reaction 3 in concentrated HCl and the

2 Dr. J. A. Stekol (unpublished data) of this Institute has established that CP- methyl-labeled methionine, when refluxed for 30 minutes in 18 N H2S04 with 1, 2, 4, and 8 moles of methanol, yields the methylsulfonium salt (isolated both as the phos- photungstate and bromide) without any loss of radioactivity; i.e., there is no dilution of methyl groups of the sulfonium salt and consequently little tendency for Reaction 3 in sulfuric acid.

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T. F. LAVINE, N. F. FLOYD, AND M. S. CAMMAROTI 111

preferential elimination of the methyl group have long been utilized for the preparation of S-substituted homocysteine derivatives from methionine and organic halides (14, 15). Decreasing the concentration of acid de- creases the rate of Reaction 3, 6 per cent methionine being formed on refluxing for 4 hours in N HCl in comparison with approximately 40 per cent in 6 N HCl (see Fig. 1).

Refluxing a solution of methionine methylsulfonium bromide in water (pH 4.0) led, after 1 hour, to 64 per cent decomposition according to

FIG. 1. Decomposition of methionine methylsulfonium salts by refluxing in con- stant boiling halogen acids. 0, 0.9 M methionine methylsulfonium bromide in 6 N hydrochloric acid; A, 0.9 M methionine methylsulfonium bromide in 6 N hydrobromic acid. Methionine was determined iodometrically (16).

Reaction 4, a. From the pH of the solution and the dissociation constant

RSMes+Br- + Hz0 - MezS + ROH + HBr (4, a)

pK 7.7 (see the next paragraph), this decomposition represents the be- havior of the ion Me&+R(NH3+)COO- and it may be concluded that dissociation of the carboxyl group makes the amino acid moiety the more labile group. Elimination of the charge on the amino group intensifies this effect: the sulfonium salt in the presence of 2 equivalents of alkali was found to be completely decomposed after refluxing for 1 hour; i.e.

RSMezf + OH- - Me&S + ROH (4, b)

Heating of methionine methylsulfonium bromide in the solid state also results in dimethyl sulfide formation.

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112 MBTHIONTNE STJLFONIUM S.1LTS

The titration curve (Fig. 2) shows that methionine methylsulfonium bromide (pKz 7.7) is a stronger acid than methionine (pKz 9.2), which may be attributed to the effect of the positive sulfonium group on the amine acid moiety. In practice about 90 per cent of the acid is titrated at the turning point of thymol blue.

EXPERIMENTAL

AnaZy2icaZ-Methionine was estimated iodometrically (16); if homo- cystine was also present, methionine was determined on the filtrate from

FIG. NaOH.

0.5 0.5 ,CQL//VALE/VTS Of AL/CAL/ ,CQL//VALE/VTS Of AL/CAL/

2. Titration of 0.05 M methionine methylsulfonium bromide with 2. Titration of 0.05 M methionine methylsulfonium bromide with The mid-point of the titration occurs at pH 7.7. The mid-point of the titration occurs at pH 7.7.

0.05 M

mercuric sulfate precipitation (4). Homocystine was estimated colori- metrically (17). The form01 titration and the Volhard procedure were used for the routine estimation of amino nitrogen and bromide. A modifi- cation of the Baernstein apparatus was used for the estimation of methyl groups (18, 19). Digestion for 4 hours with 1 ml. of solution and 10 ml. of constant boiling hydriodic acid yielded the following results expressed as per cent of the theoretical methyl group content: 0.1 M methionine, 93.0, 93.6; 0.1 M N-acetylmethionine, 94.2; 0.05 M methionine methylsulfonium bromide, 96.2, 96.7. No correction for a blank was found necessary. The methionine value is in essential agreement with the result obtained by Kassell and Brand (20). About 90 per cent of the methyl groups of the sulfonium salt were eliminated in 1 hour.

Sulfonium salts were precipitated by the addition of a slight excess of

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T. F. LAVINE, N. F. FLOYD, AND M. 8. CAMMAROTI 113

phosphotungstic acid to a lo-fold aqueous dilution of the reaction mixture. On the basis of results with the methylsulfonium salt (4) the equivalent weights per nitrogen mere calculated as 2138 plus the molecular weight of the hydrosy compound. The methyl- and ethylsulfonium phosphotung- states wcrc soluble in hot acid-alcohol mixtures (1 to 2 N H&O* or HCI made up in 50 per cent ethanol or methanol) but insoluble in the cold. The remaining phosphotungstates of Table I were soluble in the cold al- coholic mixture. Sulfonium bromides were prepared from the phospho- tungstates by the procedure previously described (4). m-Methionine was used throughout.

DL-Methionine Ethylsulfonium Bromide-Over-all yield, 42.5 per cent; equivalent weight from Br-, 260, from NHZ-N, 259; theory, 258; m.p., 146-147’ with decomposition.

DL-Methionine Isopropylsulfonium Bromide-Over-all yield, 68 per cent; equivalent weight from Br-, 282, from NHz-N, 263; theory, 272; m.p., 131-131.5” with decomposition. A strong odor of presumably isopropyl- methyl sulfide developed on storage.

Reaction with Phenol and Related Compounds-p-Phenolsulfonic acid be- haved similarly to phenol and prevented the decomposition of methionine in hot HzS04. A likely explanation of the protective action is that the phenolic compound forms a sulfonium salt in hot solution which on cooling or dilution spontaneously decomposes to the original components somewhat as the tert-butylsulfonium salt and the sulfonium salt derived from methi- onine and mustard gas3 (11). On the other hand, the presence of phenol did not affect the rate of sulfonium salt formation by methanol.

p-Methoxyphenylsulfonic acid (2 moles) was found to be an efficient methylating agent in hot 15 N HzSO+ a 90 per cent yield of methionine methylsulfonium phosphotungstate being obtained after refluxing for 2 hours.

DL-Methionine Carboxyethylsulfonium Salt. /3-Propiolactone-6.3 ml. (100 mM) of P-propiolactone were dissolved in 25 ml. of water and 7.5 gm. (50 mM) of methionine added. The methionine dissolved slowly with heat evolution over a period of about 2 hours. At this point only 16 per cent of the methionine remained. The solution was then made 15.8 N in sul- furic acid and allowed to stand overnight at room temperature, whereupon the methionine value was 2.8 per cent of that added. The mixture was diluted with 10 volumes of water and 105 gm. of phosphotungstic acid in 200 ml. of 2 N sulfuric acid were added. The precipitate was filtered, washed with dilute hydrochloric acid until sulfate-free, and dried in vacua over sodium hydroxide; yield 103 gm.

3 In this connection, it is interesting to note that iodobenaene, apparently, does not form a sulfonium salt in concentrated HCl (21).

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I14 METHIONINE SULFONIUM SALTS

P-Bromopropionic Acid-A solution (50 ml.) 1 M in methionine, 1 M in P-hrom.opropionic acid, and 6.6 M in hydrobromic acid was allowed to stand for 16 hours at room temperature and then heated on the steam bath for 1 hour. The methionine concentration at these intervals was 0.656 and 0.098 M, respectively. The addition of phosphotungstic acid yielded 99 gm. of precipitate. Phosphotungstic acid was removed from the precipi- tate by tetraethylammonium bromide in 75 per cent methanol and the filtrate evaporated to a thick syrup; weight, 18.9 gm.; water, 7.4 per cent; amino N, 42.4 mM; bromide, 93.2 mM; acid (to methyl red), 84.2 mM; methionine, 1.07 mM.* These results (on a dry basis) indicate a mixture of 42.2 IllM of methionine carboxyethylsulfonium bromide hydrobromide (theory, 50 mM) and 9 mM of tetraethylammonium bromide. The syrup was soluble in ethanol from which it was precipitated by ether as a viscous oil. Neutralization, in an alcoholic solution, of the hydrobromic acid, i.e. one-third of the total acidity, by alcoholic ammonia (0.14 M in 99 per cent ethanol) yielded a white amorphous precipitate, which after washing with absolute ethanol amounted to 55 per cent of the theory for methionine carboxyethylsulfonium bromide; molecular weight, 302; found, 307 by the Volhard titration and 313 by neutralization; no 12 consumption at pH 7.4 Oxidation by iodate in N HCl was negligible after 15 minutes but amounted to 1.5 atoms of 0 per 300 gm. after 28 hours, suggesting decomposition.6 On standing the solid material underwent a marked change in physical state, accompanied by formation of an intense odor. Attempts to produce the thetin by a similar neutralization procedure resulted in precipitation of mixtures of the thetin and sulfonium bromide which evidently reflected the equilibrium among the various ionic forms on neutralization.

Decomposition of Methionine Methylsulfonium Bromide-Demethylation by refluxing in HI has been discussed. The action of concentrated HCl and HBr is shown in Fig. 1. A solution of 0.05 M methionine methylsul- fonium bromide in N HCl yielded 2 and 5.6 per cent methionine, respec- tively, after refluxing for 2 and 4 hours. There was no dimethyl sulfide formation; i.e., the loss of methyl groups was in agreement with the amount of methionine formed. Solutions of the methylsulfonium salt in 2 N HCl were evaporated to semidryness on a water bath (time, 10 to 30 minutes) without any loss of methyl groups.

A solution of methionine methylsulfonium bromide (0.0521 M) in water was refluxed for 4 hours. The increase in acidity after 1 and 4 hours was,

4 A black periodide is formed at pH 7 which reacts with thiosulfate without loss of 1%

5 The above results also indicate the absence of S-(2-carboxyethyl)homocysteine (kindly furnished by Dr. J. A. Stekol) which behaves like methionine on oxidation by iodine and iodate.

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T. F. LAVINE, N. F. FLOYD, AND M. S. CAMMAROTI 115

respectively, 63.7 and 92.1 per cent of the theory for Reaction 4, a. No methionine was found by iodine titration and the loss of methyl groups was 91 per cent of the theory.

A solution of 0.0516 M methionine methylsulfonium bromide and 0.1052 M NaOH was refluxed for 1 hour. The decrease in alkalinity and the loss

0.D. D

10

D 3.0

0.d

A s C

FIG. 3. Absorption spectra of tetraethylammonium bromide in water (Curve A), methionine methylsulfonium bromide in 0.1 N HCl (E (molar extinction coefficient) = 4.0 at 238 rnp) (Curve B), and homocysteine thiolactone hydrochloride in water (E = 4427 at 238 m,u) (Curve C). The inset shows the results obtained with 2.5 X 1OP 1~ homocysteine thiolactone hydrochloride (Curve C) and an aqueous solution of the product isolated after treatment of methionine methylsulfonium bromide with HI (Curve D) as described in the text. The measurements were made with a Beckman model DU quartz spectrophotometer.

of methyl groups were 101 and 99 per cent, respectively, of the theory for Reaction 4, b. There was no evidence of olefin formation, as indicated by the absence of bromination with iodate and bromide. The homoserine to be expected from the reaction was isolated as the lactone by utilizing the procedure of Livak et al. (22). The alkaline reaction mixture from 10 m&t of the methylsulfonium salt was neutralized with sulfuric acid and evaporated to semidryness in vacua. The residue was treated with ethanol and filtered from sodium sulfate. Alcohol was removed from the filtrate

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116 METHIONINE SULFONIUM SALTS

by distillation and the residue digested with hydrobromic acid. After removal of the acid in vacua, a crude product (bromide, 92 per cent of the theory) was obtained in nearly quantitative yield which was recrystallized from ethanol; yield, 1.0 gm. or 55 per cent of the theory; equivalent weight, 191.6 by the Volhard titration and 192.5 by neutralization (total acidity); sinters at 212’, m.p. 215-218’ with decomposition; a-amino-y-butyrolac- tone hydrobromide molecular weight, 182; sinters at 215”, m.p. 221” with decomposition (20).

Solutions of methionine methylsulfonium salts are relatively stable at ordinary temperatures and even alkaline solutions may be distilled in vacua, with a water pump, without decomposition (4).

Identification of Homocysteine Thiolactone-For identification of the thio- la&one as a product of hydriodic acid digestion, 1 gm. (4.1 mM) of methi- onine methylsulfonium bromide was refluxed with 5 ml. of constant boiling hydriodic acid for 4 hours. The solution was then diluted with 30 ml. of water and excess phosphotungstic acid added. The precipitate was centri- fuged, washed with water containing phosphotungstic acid, and extracted with hot ethanol to yield a solution of the thiolactone phosphotungstate. Phosphotungstic acid was precipitated from the solution by tetraethyl- ammonium bromide and the resulting filtrate was decolorized and diluted to volume. The results of the spectrophotometric analysis shown in the inset of Fig. 3 indicate that 32 per cent of the methylsulfonium salt was recovered as homocysteine thiolactone.

SUMMARY

The reaction of hydroxy compounds with methionine in 50 per cent sulfuric acid was investigated. Methyl, ethyl, propyl, and isopropyl al- cohols readily yielded the corresponding sulfonium salts in hot acid. tert- Butyl alcohol formed a sulfonium salt at room temperature which was de- composed on heating and also on dilution. With threonine and serine, only the self-decomposition of methionine to homocystine and methionine methylsulfonium ion occurred.

In the case of phenol and ethanolamine, the formation of a sulfonium salt was indicated in hot acid, since there was no self-decomposition of methionine; however, on dilution, reversal of the reaction evidently oc- curred. Methionine carboxyethylsulfonium bromide was prepared by sev- eral methods.

The decomposition of methionine methylsulfonium ion was investigated in some detail. In acid solutions, methionine is regenerated, the rate and extent of the reaction depending on the nature of the acid present. In hot neutral and alkaline solutions, dimethyl sulfide is evolved. The pos- sible importance of the reactions to biological systems has been discussed.

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T. F. LAVINE, N. F. FLOYD, AND M. S. CAMMAROTI 117

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Mary S. CammarotiTheodore F. Lavine, Norman F. Floyd andMETHIONINE IN SULFURIC ACID

SALTS FROM ALCOHOLS AND THE FORMATION OF SULFONIUM

1954, 207:107-118.J. Biol. Chem. 

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