4239
verted to oily H-His(im-Bz1)-Pro-Phe-His(im-Bz1)-LeuOBzl(N02).3HBr (VI), 95 %, Rf 0.76.8 Condensation of benzyloxycarbonyl-O-benzyl-L-tyrosineL0with Lisoleucine ethyl ester by the mixed anhydride method with isobutyl chloroformate gave Z-Tyr(0-Bz1)Ile-OEt (VII), 82%, mp 127-128', [CX]~'D 4-10.7' (acetic acid). Anal. Calcd for C32H3806N2: C, 70.31 ; H, 7.01; N, 5.13. Found: C, 70.43; H, 7.03; N, 5.18. Hydrogenolysis of VI1 yielded oily H-TyrIle-OEt .HCl (VIrI), 94%, Rf 0.77.E Condensation of benzyloxycarbonyl-L-valine with VI11 by the mixed anhydride method" gave Z-Val-Tyr-Ile-OEt in 82 D (acetic acid) yield, mp 190-191', [ C X ] ~ ~-20.2' (Anal. Calcd for C3~H1107N3:C, 64.84; H, 7.44; N, 7.56. Found: C, 64.68; H, 7.30; N, 7.79, which Larry Kevan was converted to Z-Val-Tyr-Ile-NHNHz (IX) in 97 % Department of Chemistry, Unicersity of Kansas yield, mp 278-280". Anal. Calcd for C 2 ~ H 3 g 0 6 H , . Lawrence, Kansas 66044 0 . 5 H 2 0 : C, 61.07; H, 7.32; N, 12.71. Found: C, Receiced June 13, I967 60.98; H, 7.26; N, 12.59. Condensation of the azide derived from IX with VI yielded acyloctapeptide ester, Z-Val-Tyr-Ile-His(im-Bz1)-Pro -Phe - His(im - Bzl) - LeuSynthesis of Human Angiotensin Ill2 OBzl(N02) (X), 34%, mp 130-134", [ a ] l * ~-41.0' Sir: (acetic acid). Anal. Calcd for C E I H & ? N I ~ H . 2 0: C, 65.17; H, 6.55; N, 12.20. Found: C, 65.06; Human angiotensin has recently been purified from H, 6.95; N, 12.05. Oily octapeptide trihydrobromide the incubation mixture of human serum protein and obtained from X in 97 % yield, Rf0.60,8 was condensed renin.3 The amino acid sequence of isolated human with a mixed anhydride" of benzyloxycarbonyl-nitroangiotensin appears to be identical* with that of horse angiotensin I,j i.e., L-Asp-L-Arg-L-Val-L-Tyr-L-Ile- L-arginine12 to yield Z-Arg(N02)-Val-Tyr-Ile-His(imBzl)-Pro-Phe-His(im-Bzl)-Leu-OBzl(N02) (XI), 78 %, L-His-L-Pro-L-Phe-His-L-Leu, which has not been mp 138-142', [a]"D -32.5' (acetic acid). Anal. synthesized before. We wish to report the synthesis Calcd for CS7H1~~Ol7Nl8.6H20: C, 58.57; H, 6.67; N , of this decapeptide and its identity with the human 14.13. Found: C, 58.29; H,6.57; N, 14.44. Coupling natural peptide in chemical and biological properties. of oily nonapeptide trihydrobromide, obtained from L-Leucine p-nitrobenzyl ester p-toluenesulfonate (I), XI in 96% yield, with a mixed anhydride" of benzylmp 201-202", [ c x ] ~ ~4-10.2" D (ethanol), was prepared oxycarbonyl-0-benzyl-L-aspartic acid'? yielded Z-Aspin 67% yield by heating a mixture of L-leucine, nitro(P-Bzl)-Arg(N02)-Val-Tyr- Ile - His(im - Bzl) - Pro - Phebenzyl alcohol, toluenesulfonic acid, and benzene. His(im-Bz1)-Leu-OBzl(NO2) (XII), 79 %, mp 140-144', Anal. Calcd for C ~ O H & ~ N ~ C, S : 54.78; H, 5.98; [ a ] l * ~-30.0" (acetic acid), Rf 0.90.E Anal. Calcd N, 6.39. Found: C, 54.42; H, 5.98; N, 6.41. ConC, 59.17; H, 6.54; N, 13.39. densation of benzyloxycarbonyl-im-benzyl-L-histidine for C9SH117020N1~.6H20: Found: C, 58.82; H, 6.34; N, 13.38. XI1 (200 mg) (11)6 with I by the dicyclohexylcarbodiimide method7 in a solvent of methanol-acetic acid-water (60:20: 15) gave oily Z-His(im-Bzl)-Leu-OBzl(NOz) (III), 89 %, Rf was hydrogenated with palladium black at room tem0.76.8 Debenzyloxycarbonylation of I11 with HBr in perature for 48 hr.14 The filtrate from the catalyst acetic acid yielded oily H-His(im-Bz1)-Leu-OBzl(NO?) was evaporated, and the residue, 130 mg, was collected 2HBr (IV), 95%, Rf 0.76.8 Condensation of I1 with with the aid of a mixture of acetone and ether. This H-Pro-Phe-OEt . HC19 by the dicyclohexylcarbodiimide material was purified by subsequent column chromethod gave oily Z-His(im-Bz1)-Pro-Phe-OEt in 75 % matography using carboxymethylcellulose, DEAEyield, Rf 0.73,8 which was treated with hydrazine to Sephadex A-25, and Bio-Gel P-2 which have proved afford semicrystalline Z-His( im-Bz1)-Pro-Phe-N HN Hz to be most effective in isolating natural human (V), 85 %, Rf 0.73.* Oily Z-His(&-Bz1)-Pro-Pheangiotensin I s 4 The active fractions from the BioHis(im-Bzl)-Leu-OBzl(N02), R f 0.84,s obtained in 46 Gel column with 0.1 N acetic acid as a developing solyield from the azide derived from V with IV, was convent were lyophilized, leaving a white powder, 8.6 mg; (1) This work was supported in part by U. S . Public Health Service amino acid ratios in acid hydrolysate, were Asp0.98Grant No. HE 09429-02. (2) The abbreviation followed are from J . Biol. Chem., 241, 2491 Argl .03Val1. U T Y.8~11eo ~ O .g?Hisl.gd'rol.~oPhe~ .oaLeul,06. In-
by pulse radiolysis are also given on the same relative scale. An absolute comparison of the rates in the two phases has not been made. The semiquantitative correlation of the rates for eaq- and e,,,- is extremely striking and supports the conclusion that entropic effects are responsible for the rate differences between different solutes. The correlation also implies that one may properly describe e,n- as a mobile solvated electron in ice. Acknowledgment. This research was generously supported by the Air Force Rocket Propulsion Laboratory and the U. S. Atomic Energy Commission. This is AEC Document No. COO-1528-15. We thank R. Lapple for some of the epr measurements.
e
(1966) : Z, benzyloxycarbonyl; Bzl, benzyl; OBzl(NOz), p-nitrobenzyl ester; NHNHz, hydrazide. (3) I
