After it has been shown that removal of residues B26-B30 leaves insulin with full biological activity, provided the new C-terminus is amidated (Fischer et al. (1985) Biol. Chem. Hoppe-Seyler 366, 521-525), it is demonstrated here that it does not even preclude enhancement of potency. 7 analogues of des-(B26-B30)-insulin-B25-amide were prepared by trypsin-mediated semisynthesis, the replacements being D-Phe B24 ; His B2S , D-Phe B2S , Trp B25 ,Tyr B25 ;D-Phe B24 ' B2S and D-Phe B24 , Tyr B2S . Mere conversion of the configuration of B25-phenylalanine reduces in vitro potency to 0.5%. If B25-phenylalanine is, however, substituted by histidine or tyrosine activity is in- Verkürzte Insuline mit erhöhter AktivitätZusammenfassung: Nachdem sich erwiesen hatte, daß die Abspaltung der Reste B26 bis B30 ein biologisch voll aktives Insulin hinterläßt, vorausgesetzt der neue C-Terminus ist amidiert (Fischer et al. (1985) Biol Chem. Hoppe-Seyler 366, 521-525), wird jetzt gezeigt, daß sie sogar mit erhöhter Aktivität vereinbar ist. Durch Trypsin-vermittelte Semisynthese wurden 7 Analoge von Des-(B26-B30)-insulin-B25-amid hergestellt, und zwar mit den Substitutionen D-Phe B24 ; His B2S , D-Phe B25 , Trp B2S , Tyr B2S ; D .p he B24,B25 und D -Phe B24 , Tyr B25 ßloße Um _

In order to study the role of the amino acid in position B25 and its environment in shortened insulins, a series of analogues was prepared with the following modifications; 1, Stepwise shortening of the B‐chain including replacements of TyrB26 and ThrB27 by glycine; 2, substitutions at the carboxamide nitrogen of des‐(B26‐B30)‐insulin‐B25‐amide by apolar, polar or charged residues of various chain lengths; 3, replacement of PheB25 by asparagine‐amide, phenylalaninol or a series of alkyl and aralkyl residues. Trypsin‐catalyzed semisyntheses were performed with Boc‐protected or unprotected des‐octapeptide‐(B23‐B30)‐insulin and synthetic peptides. Relative receptor binding and in vitro bioactivity of [AsnB25]‐des‐(B26‐B30)‐insuIin‐B25‐amide was 227 and 292% (on insulin), other activities ranged between 1 and ca. 200%. We make the following conclusions. An l‐amino acid is essential in position B25. The B25‐carbonyl and NH groups favour high binding and “superpotency”, but are not indispensible for receptor contacts. For high affinity receptor interaction, the planarity at the C2‐atom and the distance of B25‐side‐chain branching in position B25 are important, but an aromatic ring is not necessary.