Structure and activity of insulin, XVI. Semisyntheses of desheptapeptide-(B24--30)- up to destripeptide-(B28--30)-insulin with lysine or alanine in place of arginine in position B22: influence on the three-step-increase of activity in positions B24--26 (Phe-Phe-Tyr).

The desonapeptide-(B22--30)-insulin pentamethyl ester, protected with Boc- at the two N-terminal amino groups, was prepared as described in the preceding XVth communication[6]. The free carboxyl group of the glutamic acid residue B21 of this compound was coupled to the following synthetic oligopeptide esters (X = Lys or Ala): X-Gly-OMe X-Gly-Phe-OMe X-Gly-Phe-Phe-OME X-Gly-Phe-Phe-Tyr-OMe X-Gly-Phe-Phe-Tyr-Ala-OMe After coupling, the semisynthetic products were deprotected and purified. Their biological activities were determined in the mouse fall test and by measurement of blood glucose levels. There were no statistical differences between the values obtained for the lysine B22 and alanine B22 products. The three-step increase in activity due to the amino acids Phe-Phe-Tyr (B24--26) was still recognizable, but compared with the analogues containing arginine B22, the activities were very stronly diminished. These results are in contrast with the assumption that activity of insulin is dependent on the formation of a strong ionic linkage between the asparagine-A21 carboxyl group and any positive charge in B22. The results, however, demonstrate the high specificity of the arginine guanidino group in position B22.

Structure and activity of insulin, XV[1-5]. Further evidence for the importance of arginine residue B22 in the activity of insulin. Semisyntheses of despentapeptide-(B23 - 30)-insulins varied in B22 using desnonapeptide-(B22 - 30)-insulin and tetrapeptides.

Insulin hexamethyl ester was digested by trypsin. The resulting desoctapeptide-(B23 - 30)-insulin pentamethyl ester was purified. This compound was digested by carboxypeptidase B to remove the arginine residue B22 at the end of the B chain. Then the N-terminal amino groups of the remaining desnonapeptide-(B22 - 30)-insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the glutamic acid residue B21 of this product was coupled to the following synthetic tetrapeptide esters: Arg-Gly-Phe-Phe-OMe, Lys(Boc)-Gly-Phe-Phe-OMe, Orn(Boc)-Gly-Phe-Phe-OMe, Cit-Gly-Phe-Phe-OMe, Ala-Gly-Phe-Phe-OMe and Gly-Gly-Phe-Phe-OMe. The syntheses of these peptide esters are described. After removal of all protecting groups, despentapeptide-insulin (B22-Arg) and analogues of this product with variation in position B22 could be obtained. They were purified by column chromatography. The biological activities of these components were determined by the mouse fall test. In the case of despentapeptide insulin (C-terminus Arg-Gly-Phe-Phe), the activity rose to the expected value of 34%. The insulin variants with amino acid residues other than arginine in position B22 had much lower activities: with lysine 13%, with ornithine 12%, with citrulline 9%, with alanine 8% and with glycine 6%. Desnonapeptide-insulin by itself posses an activity of 3%. These results demonstrate once more the essential nature of arginine residue B22 for insulin activity.

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]).