[CONTRIBUTION FROM m ROCKEFELLER INSTITUTE FOE
MEDICAL RESEAECH]
THE SYNTHESIS OF SEVERAL ISOLEUCYL PEPTIDES AND CERTAIN OF THEIR PROPERTIES D. THEODOROPOULOS1 AND L. C. CRAIG Received April 11,1966
In the course of the hydrolytic studies with Bacitracin A (1,2) a large number of peptides have been isolated. Some of these have unexpected properties. Others have unique linkages. It was considered worthwhile to synthesize certain of them and study their properties more in detail. The first unexpected property encountered was with the dipeptide (2, 3) isoleucylphenylalanine,It proved to be very resistant to further hydrolysis and surprisingly enough gave a very low color yield in the ninhydrin procedure for colorimetric estimation. It had little or no optical rotation but when converted to the DNP derivative an optical rotation of +loo" was found, Hydrolysis of the DNP peptide gave DNP-isoleucine and free phenylalanine. On conversion of the latter to the DNP derivative it proved to be largely racemized, indicating the peptide to be a mixture of L-isoleucyl-L-phenylalanine and L-isoleucyl-Dphenylalanine with the latter predominating. In the thought that the optical configuration in a dipeptide might have an infiuence on the reactivity of the free amino group as reflected by the ninhydrin color yield, several such peptides have been synthesized in order that the cause of the low color yield could be investigated. Although isoleucine occurs widely in proteins and several peptides containing it have been isolated from partial hydrolysates (4,5 ) the literature does not appear to contain an account of the synthesis of an unsubstituted isoleucyl dipeptide. Peptides of isoleucine with the nitrogen substituted have been prepared (6, 7) and studied as substrates for enzymatic hydrolysis. Tosylisoleucyl peptides have recently been prepared (8). In the work reported here the isoleucyl peptides were synthesized through the carbobenzoxy derivative of isoleucine rather than the tosyl derivative, because of two considerations. It waa thought that removal of the inorganic salts, formed during removal of the tosyl group by reduction with sodium in liquid ammonia, might be complicated by the solubility of the free peptides in alcohol and require that systems be worked out for either countercurrent distribution or ion exchange fractionation. Secondly, with the tosyl derivative hydrolysis of the ester was done under conditions (8) in which there was greater likelihood of racemization than under those required by the carbobenzoxy ester. Apparently the latter hydrolyzes more quickly than the former. This may be due to steric hindrance. Such a possibility is also indicated by the fact that an attempt to couple a tosylisoleucine by the mixed anhydride procedure was less satisfactory than with the carbobenzoxy derivative. The carbobenzoxy group was removed by the well known catalytic hydrogenation (9) procedure. Fellow of the Greek Fellowship Foundation. 1169
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TABLE I NINHYDRIN COLORYIELD OF ISOLEUCYL PEPTIDES I N PERCENTAGE COLORYIELDGIVEN BY LEUCINE Color Yield, %
Peptide
Isoleucylphenylalanine from Bacitracin A L-isoleucyl-D-phenylalanine L-isoleucyl-L-phenylalanine L-isoleucyl-L-leucine D ,L-isoleucyiglycine
25 28 48 59 45
An alternate synthetic route through carbobenzoxyisoleucyl chloride was avoided because this chloride is an oil. In general (6), the oily chlorides have poor stability and tend to form carbonic anhydrides with loss of benzyl chloride. Carbobenzoxyisoleucine was prepared according to the directions of Bergmann and Zervas (9). It was coupled with the appropriate ester by the mixed anhydride procedure of Boissonas (10). The L-isoleucine used in this work was "allo-isoleucine free" obtained from the Mann Research Laboratories. In the synthesis of phenylalanylhistidine and isoleucylserine the mixed anhydride procedure was successfully employed by using two moles of the free amino acid to one mole of the anhydride. This appeared to minimize the undesired side reaction with the imidazole or hydroxyl group. From the color yield of the various peptides with the ninhydrin reagent, it became clear that the configuration does affect the color yield, as the data in Table I show. However, all the isoleucyl peptides studied were found to give a low color yield. EXPERIMENTAL
Carbobenzozy-L-isoleucine. A solution containing 0.5 g. of L-isoleucine, 5 ml. of water, and 2 ml. of 2 N sodium hydroxide in a n ice-bath was treated at intervals with small portions of carbobenzoxy chloride. At the end of an hour 2 moles (1.2 9.) had been added. During this time the solution was maintained slightly alkaline by addition of sodium hydroxide. The solution was extracted twice with ether and acidified with 1 N HCl. The product was extracted with ether, and the ether solution was washed twice with water and dried over sodium sulfate. After evaporation of the ether an oil remained which was The yield was 0.8 g. or 80% of the theoretical. further dried over Pz05. Carbobenzoxy-L-isoleucyl-L-phenylalanine. A solution containing 0.8 g. of carbobenzoxyL-isoleucine, 10 ml. of dry chloroform, and 0.3 g. of dry triethylamine was cooled t o 0" and 0.325 g. of ethyl chlorocarbonate was added. After ten minutes 0.8 g. of phenylalanine methyl ester dissolved in chloroform was added. Coupling proceeded with evolution of COz. The solution was permitted to stand a t room temperature for 30 minutes and then was washed successively with 1 N HC1, sodium bicarbonate, and water. It was dried over sodium sulfate and evaporated t o dryness in vacuo. The yield was 1 g. The above residue (1 g.) in 15 ml. of 90% ethanol was treated a t room temperature with 2.5 ml. of 1 N sodium hydroxide added in portions with continuous shaking over a period of 15 minutes. The ester which at the beginning was only partly in solution, dissolved as the hydrolysis proceeded. The alcohol was evaporated in vacuo and 40 ml. of water was added. After washing with ether t o remove the unhydrolyzed ester the solution was acidified. The precipitated carbobenzoxy acid weighed 0.8 g. When recrystallized from aqueous ethanol, i t was found t o melt a t 166-167'. Anal. Calc'd for CzaHz8N20s:C, 66.97; H, 6.84; N, 6.79. Found: C, 67.13; H , 6.66; N, 6.83.
SEPT. 1955
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SYNTHESIS OF ISOLEUCYL PEPTIDES
TABLE I1 ANALYTICAL DATAOF SEVERALPEPTIDES AND DERIVATIVES
I
Peptide ~
~
_
_
_
_
_
~~
_
Carbobenzoxy-D, L-isoleucylglycine" I),L-Isoleucylglycine L-Isoleucyl-D-phenylalanine Carbobenzoxy-L-isoleucyl-Lleucineb L-Isoleucyl-L-leucine 0
M.p. 154-156".
* M.p.
I
Calc'd _
~
~
~
Found
~
6.87
8.69
60.00
6.68
8.65
51.04 64.72 63.47
8.56 7.96 7.98
14.87
51.02 64.77 63.60
8.43 7.96 8.01
14.73
58.99
9.9
11.45
58.70
9.94
11.55
130-131".
L-Isoleucyl-L-phenylalanine. The above carbobenzoxy derivative (0.4 g.) was dissolved in 10 ml. of absolute ethanol and 6 ml. of water was added. Hydrogenolysis was carried out with palladium-on-charcoal according t o the directions of Bergmann and Zervas (9). After removal of the catalyst the solution was evaporated t o dryness i n vacuo and the solid residue was washed with acetone. It weighed 0.22 g. Anal. Calc'd for C ~ ~ H ~ Z NC,Z64.72; O ~ : H, 7.96;N, 10.06. Found: C, 64.32;H, 7.97;N, 9.98. DNP-L-isoleucyl-L-phenylalanine. The peptide (25 mg.) was dissolved in a few ml. of 60% ethanol and an excess of fluor0-2~4-dinitrobenzene was added. The solution was maintained slightly alkaline with 10% potassium bicarbonate while i t was heated t o 40" for a few minutes. After evaporation of the ethanol the excess FDNP was extracted with ethyl ether. The aqueous layer was treated with a slight excess of HCl. On standing in the ice box the oil which had precipitated crystallized, After recrystallization from ethanol it melt8eda t 175477". Anal. Calc'd for C Z I H ~ ~ NC, ~O 56.74; ~ : H, 5.44. Found: C, 56.70;H, 5.42. [a]: f113" (c, 0.53 in glacial acetic acid) The other peptides listed in Table I were prepared in the same way as L-isoleucyl-Lphenylalanine. The analytical data are given in Table 11. DNP-L-isoleucyl-&phenylalanine. This derivative prepared as above from the L , D peptide melted at 170-171". Anal. Found: C, 56.97;H, 5.61. [CY]: +312" (c, 0.56 in glacial acetic acid) Carbobenzoxy-D,L-isoleucyl-o,L-serine. Carbobenzoxy-n,L-isoleucine (0.6 g.) was dissolved in a solution of 10 ml. of dry chloroform and 0.228 g. of triethylamine. After cooling t o O", 0.244 g. of ethyl chlorocarbonate was added. The reaction was complete in 10 minutes. A previously cooled mixture made from 0.6 g. of serine methyl ester hydrochloride, 10 ml. of chloroform, and 0.4 g. triethylamine was added. At the end of three minutes the mixture was brought to room temperature for 30 minutes. The chloroform solution then was washed with dilute hydrochloric acid, bicarbonate, and finally water. The chloroform was evaporated. A sample of 0.57 g. of this residue was suspended in 3 ml. of ethanol and 1.8 ml. of 1 N sodium hydroxide was added in portions during a period of 20 minutes a t room temperature. The alcohol was evaporated in vacuo and 4 ml. of water was added. Ether extraction removed the unhydrolyzed ester. Acidification with hydrochloric acid in the cold gave an oily precipitate which crystallized on standing and weighed 0.49 g. After recrystallization from hot water it melted at 138-139".
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Anal. Calc'd for C X ~ H W N ZC, O ~57.94; : H, 6.86. Found: C, 57.75; H, 6.61. n,L-IsoleucyE-D,L-serine. The carbobenzoxy group from 0.1 g. of material was removed by catalytic hydrogenation aa described above. After removal of the catalyst the peptide could not be induced t o crystallize. It was purified by counter-current distribution in the system 2-butanol-water. After 21 transfers the solute in a major band in tubes 1 to 9 ww recovered and freeze-dried from water. It weighed 70 mg. Anal. Calc'd for CoHlsN~04:C, 49.5; H, 8.28. Found: C, 49.67; H, 7.99. Carbobenzozy-L-phenylalanyl-&-histidine.Carbobenzoxyphenylalanine (1 9.) wm treated with 0.34 g. of triethylamine, 0.36 g. of ethyl chlorocarbonate, and then 1.6 g. of histidine methyl ester in the same way as was done with the serine derivative except for the washing operation with HC1. This step was omitted. The carbobenzoxy-phenylalanylhistidine methyl ester did not crystallize but was recovered in 80yoyield as an oil. The ester was hydrolyzed as described above with the serine derivative except that acidification to pH 4.6 was cautiously done with acetic acid. The precipitated acid would redissolve on further acidification. It was filtered, washed with water, and recrystallized from ethanol. Thus 0.9 g. of crystals melting a t 198-2OOo (decomp.) was obtained. Anal. Calc'd for CZSHZ,N,O~: C, 63.28; H, 5.54. Found: C, 63.00; H, 5.60.
Acknowledgment. We are indebted to Mr. Rigakos for the analysis. NEW YORK21, N. Y. REFERENCES
(1) BARRY, GREGORY, AND CRAIG,J. Biol. Chem., 176,485 (1948). (2) HAUSYANN, WEISIGER,A N D CRAIG,J. Am. Chem. SOC.,77, 723 (1955). AND BARRY, Cold Spring Harbor Symposia on Quantitative Biology, (3) CRAIG,GREGORY, XIV, 24, (1950). (4) ABDERHALDEN, HIRSCH,AND SCHULER, Ber., 42, 2331 (1909). A N D THOMPSON, Biochem. J., 63, 353 (1953). (5) SANGER (6) BERGMANN AND FRUTON, J. Biol. Chem., 164, 753 (1946). (7) SMITH, J . Biol. Chem., 199,801 (1952). (8) KATSOYANNIS AND DU VIGNEAUD, J. Am. Chem. SOC.,76, 3113 (1954). AND ZERVAS,Ber., 66, 1192 (1932). (9) BERGMANN (10) BOISSONAS, Helu. Chim. Acta, 34,874 (1951).
