Semisynthetic analogues of insulin. The use of N-substituted derivatives of methionine as acid-stable protecting groups

1. We describe the use of benzyloxycarbonylmethionine and ethoxycarbonylmethionine for the selective protection of the amino groups of glycine-A1 and lysine-B29 of pig insulin. We have used the Edman method to remove residues from the N-terminal and of the B-chain of the N^A1N^B29-di-protected derivatives. The benzyloxycarbonyl group shows slight but noticeable lability in the acid-cleavage step, but the ethoxycarbonyl group remained intact even after five cycles of degradation. 2. We have prepared the following truncated forms of insulin via the di(ethoxycarbonylmethionyl) derivative: des-Phe^B1-insulin;des-(Phe^B1-Val^B2)-insulin; des-(Phe^B1-Val^B2-Asn^B3)-insulin;des- (Phe^B1-Val^B2-Asn^B3-Gln^B4)-insulin; des-(Phe^B1-Val^B2-Asn^B3 -Gln^B4-His^B5)-insulin. 3. Insulin was re-synthesized from the di-protected des-Phe^B1-insulin by reaction with an active ester of t-butoxycarbonyl-l-phenylalanine. The product after deprotection crystallized, and the immunoreactivity of the crystalline material was identical with that of the native protein. 4. We have prepared the following analogues of insulin in a similar manner: l-Ala^B1]insulin; l-Val^B1]insulin; l-Tyr^B1]insulin; m-F-l-Phe^B1]insulin; o-F-l-Phe^B1]-insulin; o-F-l-Phe^B2]des-Phe^B1-insulin. All had between 34 and 62% of the activity of insulin in the fat-cell test. 5. We have also investigated the use of the benzyol, toluene-p-sulphonyl, p-nitrobenzyloxycarbonyl and 2,4-dinitrophenyl groups for the N-protection of the methionine active esters. Each should have had some particular advantage over the benzyloxycarbonyl and ethoxycarbonyl groups, but all proved in practice to have disadvantages that more than outweighed anything in their favour.