POLYPEPTIDES. XLVII
.Tournal of Mwlirinal Chrmistry, 2970, 1'01. 1.7, AVo.6
1099
XLVII. Effect of the Pyrazole-Imidazole Replacement on the Biological Activity of Thyrotropin-Releasing Hormone's2
Polypeptides.
KLAUSHOFMANN* AND CYRIL Y. BOWERS Protein Research Laboratory, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15213, and the Department of Medicine, Tulane University School of Medicine, S e w Orleans, I,ouiriana 70112 Received J u l y 11, 1970 ~Pyroglutamyl-~-~-(pyrazolyl-3)-alanyl-tprolinamide, an analog of thyrotropin-releasing hormone (TRH) in which the imidazole residue is replaced by ~-P-(pyrazolyl-3)-alanine,has been synthesized and evaluated biologically. The analog exhibited the characteristic biological responses of natural or synthetic TRH; its activity was approximately 5% that of the parent hormone. These results suggest that the acid-base properties of the imidazole ring are not essential for thyrotropin-releasing activity.
I n a previous communication3 it was suggested that the replacement of histidine by ~-P-(pyrazolyl-3)-alanine [Pyr(3)Ala] may provide valuable information regarding the importance for function of the characteristic imidazole pK of histidine in biologically active peptides containing this residue. p-(Pyrazolyl-3)-alanine is isosteric with histidine but the acid-base behavior of the pyrazole ring differs markedly from that of imidazole. The validity of this concept mas verified experimentally by the observation that p-(pyrazolyl3)-alunine12 S-peptidel-14 mas a powerful competitive inhibitor of R S a ~ e - S . ~ l The j analog combines with S-protein as firmly as does S-peptide but the pyrazole ring fails to function catalytically because of its inability to accept or donate protons under the condirions of assay. I n order to gain information regarding the functional importance of histidine for hormone activity the Pyr(3)Ala analogs of Val5-angiotensin I P 8 and of Glnj~-corticotropinl-zoamideg~lo mere prepared and tested for biological activity. Since both analogs possessed significant levels of biological activity it was concluded that the acid-base characteristics of the imidazole ring cannot be essential for the function of these hormones. The recent disclosure of the covalent structure of a thyrotropin-releasing hormone T R H (TRF) (I)11-14
provided another opportunity to test the importance of histidine dissociation for hormonal activity by the pyrazole-imidazole substitution approach. This communication describes a synthesis of L-pyroglutamyl-Lp- (pyrazol yl-3) -alanyl-L-prolinamide (11) [Pyr (3)Ala T R H ] and relates observations pertaining to certain biological properties of this molecule. ti I
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* T o whom correspondence should he addressed. (1) See L. Moroder a n d K . Hofmann, J . .\fed. C h e m . , 13, 839 (1970) for paper X L V I in this series. ( 2 ) Supported by grants from t h e Hoffmann-La Roche Foundation, Nutley. N. J., a n d U. S. Public Health Service Grant AM-06164-07. (3) K. Hofmann and H . Bohn, J . A m e r . C h e m . SOC.,88, 5914 (1966). (4) F . XI. Richards, Proc. N a t . A c a d . Sei. C. S . , 44, 162 (1968); RNase-& subtilisin modified beef RNase-.% (enzymically fully active) ; S-peptide, the peptide component obtained from R N a s e 4 (enzymically inactive) : Sprotein, t h e protein component obtained from RKase-S (enzymically inactive) : RKase-S', the reconstituted enzyme obtained by mixing equimolar proportions of S-peptide a n d S-protein (enzymically fully active) : Speptidel-14, a synthetic fragment of S-peptide which is equipotent t o Spept,ide, K. Hofmann, F. M. Finn, AI. Limetti, J. Xfontibelier, a n d G . Zanetti, .I. A m e r . C h e m . Soc.. 88, 3633 (1966). ( 5 ) I
