Biological Activity of Synthetic Polypeptide Derivatives Related to the Structure of Hypothalamic TRF

Abstract

Based on our recent observation that as much as 81 % of the weight of the molecule of ovine TRF could be accounted for, following 6N HC1 hydrolysis, by the presence of His, Pro, Glu in equimolar ratio, tripeptide isomers containing His, Pro, Glu were synthesized. The tripeptides proved to be devoid of TRF activity. Following treatment of the tripeptides by acetic anhydride in an effort to protect the N-terminus as in natural TRF, only the acetylation mixture of H-Glu-His-Pro-OH showed biological activity qualitatively indistinguishable from that of natural TRF in in vivo and in vitroassays specific for TRF. The major product byweight of this acetylation procedure was shownto be PCA-His-Pro-OH (PCA is the abbreviationfor 2-pyrrolidone-5-carboxylic acid). PCAHis-Pro-OH isolated from the acetylation mixtureor prepared by total synthesis proved tohave TRF activity (10 U/mg, M.E.D. 10 μg/animal). N-acetyl-Glu-His-Pro-OH obtained bytotal synthesis had no TRF activity when injectedup to 250 μg/animal. Treatment of PCAHis-Pro-OH by methanolic HC1 yielded a preparationidentified as PCA-His-Pro-OMe, withTRF activity of 28,600 ±6700 U/mg (M.E.D.≤10 ng/animal). Treatment of PCA-His-Pro-OMe by ammonolysis in methanol yielded apreparation identified as PCA-His-Pro-NH2,with TRF activity of 44,000 ±1100 U/mg(M.E.D. ≤ 5 ng/animal). Similar results wereobtained with PCA-His-Pro-NH2 prepared bytotal synthesis (highly purified ovine TRF has aspecific activity of 57,200 ±9900 U/mg). PCAHis-OMe obtained by total synthesis has noTRF activity. It would appear that a structuresuch as R-Glu-His-Pro-R′ where R- designates ablocked N-terminus (most likely as PCA) mayrepresent the minimal active core of the ovinehypothalamic hypophysiotropic hormone TRFas isolated in this laboratory. (Endocrinology86: 573, 1970)