5866 241-243"; [ c Y ] ~ ~-26.4" D (c 1, acetic acid [lit.za mp 230-233; [ ( u ] 2 2 ~ After 30 min the precipitated material was removed by filtration: -27" (c 0.5, formic acid)]; after hydrogenation in methanol con22 mg. Amino acid analysis of an acid hydrolysate showed the taining acetic acid, Rfl0.32, Rr2 2.35 X His; single ninhydrinfollowing composition expressed in molar ratios : Hiso.8Aspl,opositive spot. Amino acid ratios after acid hydrolysis are: Hisl.oSero ,2Glul.oGlyo.*Vall.oLeuo.zPhel.oS-benzylcysteineo.laindicating Aspl,oGlul.oVall.oPhel.o. that this material is predominantly unreacted pentapeptide derivaAnal. Calcd for C38HasN9010: C, 57.6; H, 6.23; N, 15.9. tive. The above filtrate was diluted further with methanol (80 ml) Found: C, 57.8; H,6.50; N, 15.8. and allowed to stand at room temperature overnight. The preAfter catalytic hydrogenation and digestion with leucine amino cipitated product was isolated by filtration and washed with methpeptida~e,~' amino acid ratios are: Hisl.oVall.lPhel.l. Glutamine anol and ether: 737 mg (59%); mp 239-240". A sample for and asparagine emerge at the same position and were not deteranalysis was reprecipitated from dimethyl sulfoxide-methanol: mined. melting point unchanged; [a]"D -27.7" (c 1, DMSO); after HBr N-Benzyloxycarbonyl-~-phenylalanyl-~-valy~-~-asparaginyl-~in trifluoroacetic acid treatment, Rrl0.62, Rr2 4.2 X His. glutaminyl-L-histidine Hydrazide (XV). To a suspension of XIV Anal. Calcd for CjIIH79N13015S:C, 57.0; H, 6.41; N, 14.7. (5 g) in D M F (200 ml) containing hexamethylphosphoramide Found: C, 57.2; H,6.32; N, 14.7. (10 ml) which was heated t o 60" for a few minutes and then cooled Amino acid analysis after acid hydrolysis showed the following t o 40" was added hydrazine hydrate (10 ml). The resulting solucomposition expressed in molar ratios: Hisl,oAspl.oSero.sGlul.otion was stirred at 40" for 5 hr and at room temperature for 25 hr. OLeuo.oPhel,oS-benzylcysteineo. 7 . G lyo. During this period the reaction mixture thickened considerably N-Benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-asparaginy1-Land it was necessary t o dilute it with methanol (220 ml). The glutaminyl-L-histidyl-L-leucyl-S-benzyl-L-cys tein y lgly c y I-L-se rine precipitated hydrazide was subsequently filtered off and washed Hydrazide (XVII). The protected nonapeptide ester XVI (20 successively with methanol and water and dried: 4.8 g (96%); g) was dissolved in D M F (600 ml) and treated with hydrazine mp 245". A sample for analysis was reprecipitated from dimethyl hydrate (40 ml). After 96 hr at room temperature, the reacsulfoxide-methanol: mp 248-250'; [alZ7~ - 18.6" (c 1, DMSO) tion mixture was diluted with ice-cold water (4 1.) and the precipi(c 0.5, formic acid)]. [lit.2smp 238-240'; [ ( u l z 2-36" ~ tated hydrazide was filtered quickly and washed with water. On Anal. Calcd for C37H49N1108:C, 56.1; H , 6.23; N, 19.5. reprecipitation from dimethyl sulfoxide-water the product was Found: C , 5 6 . 0 ; H,6.43; N, 19.4. obtained in analytically pure form: 16 g (80%); mp 250" dec; N-Benzyloxycarbonyl-L-phenylalanyl-L-valyl-L-asparaginy1-L- [~tl''D - 2 9 " ( ~ 1,DMSO). glutaminyl-L-histidyI-L-leucyl-S-benzyl-L-cysteinylglycyl-L-serine Anal. Calcd for C5sH79N15014S:C, 56.1; H, 6.41; N , 16.9. Methyl Ester (XVI). To a solution of compound XV (790 mg) Found: C,55.8; H,6.56; N, 16.5. in a mixture of DMSO (6 ml) and D M F (6 ml) was added 8.3 Amino acid analysis after acid hydrolysis showed the following N HCl in freshly distilled tetrahydrofuran (0.5 ml). T o this solucomposition expressed in molar ratios: Hisl.oAspl,~Ser~.~G1ul,~tion cooled t o - 20' (Dry Ice-acetone), tert-butylnitrite (0.14 ml) Gly, .0Leul,oPhei,oS-benzylcysteineo,D. For evaluation of was added and the reaction was allowed to proceed for 6 min. T o stereochemical homogeneity a sample of the decarbobenzoxylated the reaction mixture, cooled to -3O", was then added a solution of (HBr in trifluoroacetic acid) derivative was digested with APM. L-leucyl-S-benzyl-L-cysteinylglycyl-L-serine methyl ester hydroAmino acid analysis of the digest gave the following amino acid bromide, prepared from its N-benzyloxycarbonyl derivative (1.3 g) composition expressed in molar ratios: Hisl.oGlyo.~Vall.lLeuo,~as described previously, in the synthesis of compound IX, in D M F Phel.lS-benzylcysteineo,~. Glutamine, asparagine, and serine (4 ml) containing triethylamine (0.28 ml). After 24 hr at 2" and emerge at the same position on the chromatogram and were not 24 hr at room temperature the reaction mixture (its pH being kept determined. at about 8.0 by the addition of triethylamine) was diluted with Acknowledgment. The authors wish to express their methanol (15 ml) and its p H was adjusted t o 6 with acetic acid (27) R. L. Hill and E. L. Smith, J . Biol. Chem., 228,577 (1957),
appreciation to Miss Karen Scheibe for the amino acid analyses and the enzymatic analyses.
Insulin Peptides. XX. The Synthesis of a Heneicosapeptide Derivative Corresponding to the C-Terminal Sequence of the Human Insulin B Chain'3z Panayotis G. Katsoyannis,* James Ginos, and Manohar Tilak Contribution from the Department of Biochemistry, Mount Sinai School of Medicine, the City University of New York, New York, New York 10029. Received March 2, 1971 Abstract: T h e synthesis of a heneicosapeptide derivative embodying the amino acid sequence found at the carboxyl terminus of t h e h u m a n insulin B chain is described. The desired product was obtained by interaction of the C-terminal hexadecapeptide fragment with the azide of the N-terminal nonapeptide fragment.
I n one of
the preceding papers we have reported the synthesis of a partially protected heneicosapeptide embodying the amino acid sequence found at the terminus of the B chain of bovine, sheep, or porcine in(1) This work was supported by the National Institute for Arthritis and Metabolic Diseases, U. S.Public Health Service (AM-12925). (2) A preliminary report of part of the results described in this paper has auoeared: P. G . Katsoyannis. A. Tometsko. J. 2. Ginos, and M. Tilak; j . Amer. Chem. SOC.,88, 16411966).
Journal of the American Chemical Society
s u h 3 In the present paper we describe the synthesis of a heneicosapeptide derivative with the amino acid sequence corresponding to the carboxyl terminal portion of the human insulin B chain. The two heneicosapeptide derivatives differ only in the C-terminal amino acid residue; instead of alanine present in the bovine (sheep (3) P. G . Katsoyannis, M. Tilak, and I
