Structure and Activity of Insulin, XIV. Further Studies on the Three-Step-Increase in Activity Due to the Aromatic Amino Acids B24 - 26 (-Phe-Phe-Tyr-)

Publisher Website
Google Scholar
Abstract
Using a reaction suite which was suggested by Ruttenberg for the semisynthesis of insulin variants, [bovine] insulin hexamethyl ester was digested by trypsin, then the N-terminal amino groups of the resulting desoctapeptide insulin pentamethyl ester were protected with the tert-butyloxycarbonyl residue. The free carboxyl group of the arginyl residue (B22) of this product was coupled to 2 different series of synthetic peptide methyl esters: Gly-OMe, Gly-Phe-OMe, Gly-Phe-Phe-OMe, Gly-Phe-Phe-Tyr-OMe and 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. The activity determinations by the mouse fall test showed the weak activity (about 4%) of the desoctapeptide insulin (C-terminus Arg B22). This activity increases drastically in 3 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 (crystalline insulin = 100%). The same peptides, elongated at their C-terminus with alanyl residues (see above, series II) yield higher activities. Coupling these peptides to the arginyl residue B22 increases the activity: -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. The activity of insulin proceeds from the arginyl residue (B22) and is only intensified by the aromatic amino acids (B24-26).