April 5 , 1963
GLYCINEANALOGS OF BRADYKININ
991
oxygen in these transition states. The values obtained A classic example of a reaction whose rate follows are 4.8 when B is water and 4.0 when i t is acetate ion. eq. 21 is the enolization of acetone catalyzed by acetic to the rule of Swain and Thornton,' acid and by acetate ion. Swain and c o - w o r k e r ~ ~ ~ - ~According ~ changing B from water to acetate should lengthen the have presented convincing arguments that the acetic B. ' .H bond and shorten the carbon-oxygen bond in acid-catalyzed enolization proceeds through a prior the acetone moiety. This latter shortening should equilibrium protonation of the ketonic oxygen followed decrease the basicity of the oxygen as is observed. by a rate-determining abstraction of a proton from This observation provides a verification of the Swaincarbon by acetate ion, and that the acetate ionThornton rule which is independent of isotope effect catalyzed enolization involves a similar proton abstracarguments and measures the effect of changing the tion by acetate ion assisted by hydrogen bonding of base on the orbital covering the enol proper instead of water to the oxygen. The hydrogen ion-catalyzed the effect on the orbital utilized in the B...H bond and uncatalyzed rates are attributable to analogous (which is measured by the a-hydrogen isotope effect). mechanisms with water taking the place of acetate as The 0.8 pK unit shift in the acidity of the proton nucleophile. 2 4 which accompanies the change in nucleophile is of some The transition states for the enolizations catalyzed interest as a quantitative measure of the deviation from by acetic acid or by hydrogen ion thus have the structhe assumption2* that the reactivity of the electrophile ture 111, where B is, respectively, acetate ion or water. remains constant when the nucleophile is changed. 0-H This shift corresponds to a change of 1.1 kcal./mole in il B. *H.* .CH*-CCH3 I11 the free energy of the proton-oxygen bond in the The rate data of Bell and Jones27may be used to obtain transition state. This difference could produce a values of p K a * for the ionization of the proton from change in rate of a factor of six, which is small compared to the factor of more than lo4 calculated24for the (24) C . G . Swain, J. A m . Chem. Soc., 71, 4578 (1950). (25) C. G. Swain, E. C. Stivers, J. F. Reuwer, Jr., a n d L. J, Schaad, ibid., nucleophilicity of acetate relative to water using the 80, 5885 (1958). assumption that the reactivity of any electrophile re(26) C. G. Swain, A. J. DiMilo a n d J. P. Cordner, i b i d . , 80, 5983 (1958). mains constant. (27) R. P. Bell a n d P. Jones, J . Chem. SOC., 88 (1953).
(CONTRIBUTION FROM
THE
SQUIBBINSTITUTE FOR MEDICAL RESEARCH, YEW BRUNSWICK, N. J.]
Glycine Analogs of Bradykinin' BY MIKLOSBODANSZKY, JOHN T. SHEEHAN, MIGUELA. ONDETTIAND SAUL LANDE RECEIVED OCTOBER 24, 1962 The synthesis of three analogs of the nonapeptide bradykinin is described: 6-glycine bradykinin, 7-glycine bradykinin and L-arginyl-heptaglycyl-L-arginine.The preparation of the decapeptide, 7-glycine kallidin, an analog of I-L-lysyl bradykinin, is also reported. Biological activities of these peptides are tabulated.
For a study of the relationship between structure and activity of the nonapeptide bradykinin2 some analogs were prepared, in which glycine replaced one or more of the amino acids in the sequence of the naturally occurring peptide. The initial structure proposed8 for bradykinin was that of a linear octapeptide in which the proline residue in position 7 of the nonapeptide structure was omitted. When this octapeptide was synthesized4 it was found to be devoid of activity. The question whether the amino acid in position 7 had to be proline or whether the latter could be replaced by another amino acid, without loss in activity, still remained open. iGlycine bradykinin (XII) was prepared in an effort to explore this question. The second analog, 6-glycine bradykinin (VI), wherein serine is replaced by glycine, was synthesized in order to ascertain to what degree the serine side chain contributed to biological activity. This latter analog was also converted into l-~-lysyl-6glycine bradykinin (7-glycine kallidin) (XXII) the glycine analog of the biologically active decapeptide ~
(1) Some of t h e compounds described here were first discussed a t T h e Kew York Academy of Sciences, Conference on S t r u c t u r e a n d Function of Biologically Active Peptides: Bradykinin, Kallidin and Congeners, M a r c h 22-24, 1962. (2) (a) M . Rocha e Silva, W T. Beraldo a n d G. Rosenfeld, A m . J . Physioi., 1 5 6 , 261 (1949); (b) E. Werle, Angew. Chem., 79, 689 (1961). ( 3 ) D. F. Elliott, G. P. Lewis a n d E. W. H o r t o n , Biochem. J . , 76, 16 (1960). (4) ( a ) R. A. Boissonas, St. G u t t m a n n , P. A . Jaquenoud, H. K o n z e t t and E. Stiirmer, Erpevienlia, 16, 326 (1960); (b) R . Schwyzer, W. R i t t e l , I? Sieher, H. Kappeler a n d H. Zuher, Helw. Chim. A c t a , 43, 1130 (1960); (c) E. I). Nicolaides, H. A. DeWald, P. G. Shorley and H. 0 . J. Collier, dVakue, 187, 773 (1960).
kallidin,5 by adding a lysine residue to the N-terminal arginine. Finally, a glycine analog embodying two characteristic features of bradykinin, namely, the terminal arginine residues separated by a peptide chain of seven amino acid residues, L-arginyl-heptaglycyl-Larginine (XX) was prepared on the premise that these features might represent the minimum structural requirements for biological activity. The schemes for the synthesis of 6-glycine- and 7glycine bradykinin are shown in Fig. 1 and 2 , respectively. These schemes differ, of course, in t h a t they require two different protected C-terminal pentapeptide moieties I11 and IX, respectively, but follow parallel courses from there on. Thus the protected C-terminal pentapeptide ester, methyl benzyloxycarbonyl-L-phenylalanylglycyl - L - propyl - L - phenylalanylnitro - L - argininate (JII), required for 6-glycine bradykinin was obtained in crystalline form and in good yield by allowing the @-nitrophenyl ester of the protected tripeptide benzyloxycarbonyl-L-phenylalanylglycyl-L-proline (11) to react with the dipeptide ester methyl-L-phenylalanyl-nitro-L-argininate. The p-nitrophenyl ester of I1 was prepared b y coupling benzyloxycarbonyl-L-phenylalanine-p-nitrophenyl ester with glycyl-L-proline in aqueous pyridine followed by esterification with p nitrophenol. The protected C-terminal pentapeptide of 7-glycine bradykinin, methyl benzyloxycarbonyl-L-phenylalanylL-serylglycyl-L-phen ylalan yl-nitro-L-arginine (IX), was secured by coupling the protected dipeptide benzyl( 5 ) J V. Pierce and hZ. E. Webster, Biochem. Bioph,~. Res. Commun., 5 , 353 (1961).
992
M. BODANSZKY, J . T. SHEEHAN, 11.-4. ONDETTIASD S. LASDE
+
Z - L - ~ ~ ~ - O C ~ H gly-L-pro-OH ~KO~ d 2-L-phe-gly-L-pro-OH
NI 0
Z-L-phe-gly-L-pro-OCsH4K02(11)
2
+ L-phe-L-arg-OCH1
-
Vol. 8.5
(I) I
so2 i
Z-L-phe-gly-L-pro-L-phe-L-arg-OCHl (111)
/,
? - ~ ~ ~ ~ ~ $ O CKO2 ~ H ~ ~ O Z
I
Z-~-pro-gly-~-phe-gly-~-pro-~-phe-~-arg-OCHa (IV)
I HBr-HOAc NO9
SO?
-
I I.SaOH Z - ~ - a r g - ~ - p r o - ~ - p r o - g l y - ~ - p h e - g l y - ~ - p r ~ - ~ - p h e - ~ - a r g6-glycine - O C H ~ bradykinin 2.H9 I
I
1 J -
VI1 HBr-HOAc
Z-N02-~-arg-~-pro-OCsHlr\'02 ,4c
r\'0
2
1,h'aOH Z - ~ - a r ~ - ~ - p r o - ~ - p r o - g l y - ~ - p h e - ~ - ~ e r - g l y - ~ - p h e - ~- a+ r g - O C H7-glycine 3 bradykinin 2 = C6HsCHzOCO 2,H? XI1 Figure 2 I
oxycarbonyl-L-phenylalanyl-L-serine (VIII) with the tripeptide derivative methyl glycyl-L-phenylalanylnitro-L-nrgininate. N-Ethyl 5-phenylisoxazolium-3'sulfonate was used as the coupling agent6 in this step because of the presence of the unprotected alcoholic hydroxyl group. I n all other cases, the nitrophenyl ester method' was used for the formation of peptide bonds. The tripeptide partner for the above coupling reaction was prepared from the dipeptide L-phenylalanyl-nitro-L-arginine methyl ester and the nitrophenyl ester of benzyloxycarbonylglycine to give the amorphous protected tripeptide ester, methyl benzyloxycarbonyl - glycyl - L - phenylalanyl - nitro - L - argininate (VII), which was subsequently converted into the free tripeptide methyl ester by treatment with hydrogen bromide in acetic acid. The dipeptide partner benzyloxy-carbonyl-L-phenylalanyl-L-serine (VIII) was (6) R . B. Woodward, R. A. Olofson a n d H. Mayer, J . A m . Chem .So
