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TRAUMATIC SHOCK. II. THE PREPARATION OF CYSTINE, METHIONINE, AND HOMOCYSTINE CONTAINING RADIOACTIVE SULFUR Arnold M. Seligman, … , Alexander M. Rutenburg, Henry Banks J Clin Invest. 1943;22(2):275-279. https://doi.org/10.1172/JCI101393. Research Article Find the latest version: https://jci.me/101393/pdf TRAUMATIC SHOCK. II. THE PREPARATION OF CYSTINE, METHIONINE, AND HOMOCYSTINE CONTAINING RADIOACTIVE SULFUR By ARNOLD M. SELIGMAN, ALEXANDER M. RUTENBURG, AND HENRY BANKS 1 (From the Surgical Research Department of the Beth Israel Hospital and the Department of Surgery, Harvard Medical School, Boston) (Received for publication November 12, 1942) In order to prepare, from radioactive sulfur, the benzyl mercaptan (I) (0.6 moles), reported by sulfur-containing amino acids of a high order of Wood and du Vigneaud (4) in 23 per cent yield, specific activity for biological experiments such was found to give a 21.5 per cent yield when 0.06 as those described in the foregoing publication, it mole was used. Reduction of benzylcysteine was necessary (owing to the cost of radioactive (VIII) to cysteine with sodium and butyl alcohol sulfur) to investigate the efficiency of utilization did not give good yields; therefore, sodium and of small amounts of sulfur. The synthetic meth- liquid ammonia were used. Since radioactive ods utilized are not novel, but are described be- benzyl mercaptan is necessary for the synthesis of low because of the data obtained on yields in all three amino acids, a method of preparation of numerous small scale preparations. Since pres- the mercaptan from hydrogen sulfide, other than ent methods of preparing radioactive sulfur from that described by Tarver and Schmidt, in 70 per neutron bombardment of carbon tetrachloride in- cent yield, was investigated. Elementary sulfur volve its isolation and purification as BaSO, (1), was prepared from hydrogen sulfide by oxidation the schema of synthesis used for the preparation with iodine, and this was treated with benzylmag- of three sulfur containing amino acids begins with nesium chloride. The yield of benzyl mercaptan barium sulfate (Figure 1). was 80 per cent. It was found that the yield by The synthesis of radioactive methionine (VI) the method of Tarver and Schmidt could be raised from radioactive sulfur (0.05 moles) was reported 4 per cent if the small amount of dibenzylsulfide by Tarver and Schmidt (2), using a modification formed in their reaction was reduced to benzyl of the synthesis of homocystine (V) reported by mercaptan with sodium in liquid ammonia. Patterson and du Vigneaud (3). The same The Fischer synthesis (5) of cystine from f8- yields as they report were obtained in preparing chloroaminopropionic acid with barium hydro- homocystine from 0.06 moles of sulfur. How- sulfide was investigated under various conditions. ever, in the methylation of homocysteine, we were Much poorer yields than the 5 to 10 per cent re- unable to obtain the anticipated yields. It was ported on the basis of chloroaminopropionic acid found that for preparing homocystine, the more were obtained when sulfur was not used in large convenient reduction of benzylhomocysteine (IV) excess. with sodium and butyl alcohol gave as good yields Attempts to prepare radioactive cystine by re- as reduction with sodium and liquid ammonia. placement reactions of cysteine and radioactive For the preparation of methionine, treatment of hydrogen sulfide have been described by Tuck the resultant sodium salt of homocysteine with (6) and Tarver and Schmidt (2). In a rein- methyl iodide did not give as good yields as re- vestigation of the problem, no more than 0.3 per ported by Tarver and Schmidt, whether reduction cent of available radioactivity was incorporated in was conducted in liquid ammonia or butyl alcohol. cystine (two crystallizations) obtained after heat- Attempted methylation with dimethyl sulfate and ing cysteine hydrochloride with radioactive hy- diazomethane did not proceed well. drogen sulfide in aqueous solution at 1050 C. for The method of synthesis of cystine (IX) from 10 hours. Heating for longer intervals, at higher temperatures, or at alkaline pH, resulted in de- 1 Part of this work was done in partial fulfillment of struction of a large fraction of the cysteine. the Bachelor's Degree with honors in biochemistry, Har- The enzymatic preparation of cysteine from vard University. radioactive hydrogen sulfide has been reported by 275 -276 ARNOLD M. SELIGMAN, ALEXANDER M. RUTENBURG, AND HENRY BANKS S 98% ° 3a504- 13P04 Ea ~ % 15 UR PI 0~ .U off?,(X2--a 68%' (010)a 4Rol ciM2s m1201 laO-COD 0~~~~FS ..' i t. Tit C02K. .tI 42% .* 28 2II Na82 c M t .11s w 0 as tc 0 It J I o -so"C 'IT 's2 12E 75% *a8 CO COO COOK 01201,I 4EI .8- CE2¢S Co0X 8t-0Iw .0001 2ECa 8-I' O )932 VI .M; 2 V Ix FIG. 1. SCHEME FOR SYNTHESIS OF CYSTINE, METHIONINE, AND HOMOCYSTINE FROM RADIOACTrIVE SULFUR Smythe and Halliday (7). The isolation of electric oven 15 inches in length. The temperature was glutathione, from yeast grown on media contain- determined once by means of a thermocouple. Escaping gas was bubbled through ammoniacal cadmium sulfate ing radioactive magnesium sulfate, has been re- solution, protected by a trap to eliminate the danger of ported by Franklin (8). sucking back. The barium sulfate was spread over the bottom of a porcelain boat, avoiding a layer more than EXPERIMENTAL 3 mm. in depth. In a characteristic run, 3.14 grams of barium sulfate were reduced for 5 hours. The loss of Reduction of BaSO4 was accomplished by passing H, weight due to water formed amounted to 85 per cent of over finely ground barium sulfate at 7500 C. The rate the theoretical amount. The sintered barium sulfide was of hydrogen flow was determined by bubbling the gas ground to a powder and the reduction continued for an- through concentrated sulfuric acid. A Vicor glass tube, other 3 hours. The additional water loss amounted to 5 1 inch in diameter, was heated by means of an encircling per cent of the theoretical amount, and the cadmium sul- PREPARATION OF AMINO ACIDS CONTAINING RADIOACTIVE SULFUR 277 fide formed from the escaping gas amounted to 7.8 per reported a yield of 70 per cent on the basis of the titration cent; total yield of both sulfides, 97.8 per cent. The of an aliquot. oxidation and hydrolysis of barium sulfide was prevented by sealing under glass. SYNTHESIS OF HOMOCYSTINE Hydrogen sulfide production occurred more rapidly at the beginning of reduction when water was seen condens- Benzylthioethyl chloride (II). To the concen- ing in the cold part of the tube. trated reaction mixture containing benzyl mer- Sulfur. Hydrogen sulfide was generated from barium captan in 74 per cent yield as described above, was sulfide with 30 per cent phosphoric acid and from cad- added an equivalent of sodium methylate in the mium sulfide with concentrated hydrochloric acid. The cold. Ten moles of ethylene dichloride (50 gas was swept out of the reaction flask with nitrogen and bubbled through two absorption tubes, containing slightly grams) were added and the mixture was allowed more than. an equivalent of iodine dissolved in 20 per cent to stand overnight. It was then heated to the potassium iodide. The excess iodine was reduced with a boiling point, cooled, acidified, and washed with little sodium bisulfite and the mixture was neutralized water. The washings were extracted with a small with ammonium hydroxide. The mixture was then heated amount of ethylene chloride. The combined or- at 1000 C. for 1 hour to convert the amorphous sulfur to the crystalline form. It was ground to a powder, col- ganic layer was dried with anhydrous sodium sul- lected, and washed with water, alcohol, and ether. The fate, and the ethylene dichloride distilled at at- yield was quantitative. mospheric pressure. The pressure was lowered Benzyl mercaptan (I) from sulfur. Benzyl magnesium and the fraction boiling at 100 to 1100 C. at 3 mm. chloride, prepared from 0.12 mole of benzyl chloride and was taken. The yield was 8 grams (90 per cent) 0.13 mole of magnesium, was allowed to remain in the presence of the excess magnesium overnight to ensure of pale yellow oil. complete reaction of the benzyl chloride. To this was Benzylthioethyl phthalimidomalonic ester (III). added 2.1 grams (0.065 mole) of sulfur. The mixture The above oil was heated with 18 grams of sodium was agitated for 24 hours until the sulfur had disappeared. phthalimidomalonate (9), at 1700 C. for 5 hours. The reaction product was then decomposed with ice and The product was obtained by extraction with ammonium chloride, extracted with ether, washed, dried, and distilled. Benzyl mercaptan (6.5 grams, 80 per cent toluene. yield) distilled as a colorless liquid (b.p. 1940). The S-Benzylhomocysteine (IV). The oily prod- residue, 2.2 grams (b.p. 2 mm. 1300 C.), after reduction uct was refluxed with 20 cc. alcohol and 35 cc. with sodium in liquid ammonia failed to give further 5 N NaOH for 30 minutes. Water (100 cc.) mercaptan. and 20 cc. concentrated HC1 were added and re- Benzyl mercaptan (I) from hydrogen sulfide. Hydro- was continued for 30 minutes. After the gen sulfide, passed over a small amount of anhydrous fluxing calcium chloride, was absorbed in an equivalent of cold addition of 50 cc. concentrated HCI, refluxing was 2 N alcoholic potassium hydroxide (0.065 mole). An continued for 1 hour. The solution was then equivalent of benzyl chloride dissolved in 2 volumes of cooled and extracted with ether, and the aqueous dry ether was added at once. A fluffy precipitate of portion was evaporated to dryness at reduced potassium hydrosulfide formed, and was gradually re- pressure.

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