Further studies on the three-step-increase in activity due to the aromatic amino acids B24-26 (-Phe-Phe-Tyr-)

Using a reaction suite which was suggested by Ruttenberg [5] for the semisynthesis of insulin variants, insulin hexamethyl ester was digested by trypsin, then the N-terminal amino groups of the resulting desoctapeptide insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the arginyl residue (B22) of this product was coupled to two different series of synthetic peptide methyl esters: I) Gly-OMe, Gly-Phe-OMe, Gly-Phe-Phe-OMe, Gly-Phe-Phe-Tyr-OMe and II) Gly-Ala-OMe, Gly-Phe-Ala-OMe, Gly-Phe-Phe-Ala-OMe, Gly-Phe-Phe-Tyr-Ala-OMe. Removal of all protecting groups yielded the corresponding insulin variants. The syntheses of these peptide methyl esters are described. Following the original prescription of Ruttenberg[5], we were not able to prepare the desired variants. That is why we were forced to change some important details of the Ruttenberg[5] recipe. The activity determinations by the mouse fall test showed the weak activity (ca. 4%) of the desoctapeptide insulin (C-terminus Arg B22). This activity increases drastically in three steps, when the amino acids Phe, Phe, Tyr (B24-26) are added successively to the insulin trunk. Coupling of Gly-Phe yields 14%, -Gly-Phe-Phe 36%, and -Gly-Phe-Phe-Tyr 61% of the biological activity (cryst. insulin=100%). The same peptides, elongated at their C-terminis with an alanyl residues (see above, series II) yield higher activities. Coupling these peptides to the arginyl residue B22 increases the activity as follows: -Gly-Phe-Ala, 36%, -Gly-Phe-Phe-Ala, 59%, and -Gly-Phe-Phe-Tyr-Ala, 91%. Comparing the activities of the variants with the C-termini-Gly-Phe-Phe (36%) and -Gly-Phe-Ala (36%) or -Gly-Phe-Phe-Tyr (61%) and -Gly-Phe-Phe-Ala (59%), it becomes clear that the aromatic amino acids Phe (B25) and Tyr (B26) can be substituted by Ala without loss of activity. In our preceding work (published 1969-1973 [3, 6-8]), we synthesized successively shortened insulin B-chains which yielded, after combination with natural A-chain, practically the same activity values as we have now obtained with the Ruttenberg semisynthesis. As we have already mentioned l.c.[1-4], it is obvious that the activity of insulin proceeds from the arginyl residue (B22) and is only intensified by the aromatic amino acids (B24-26). We[2,3] observed the same three-step increase in activity in the case of our synthetic oligopeptides Arg-Gly-Phe, Arg-Gly-Phe-Phe and Arg-Gly-Phe-Phe-Tyr (B22-26), which we assume to be the active region of insulin (1971[2]).