Insulin synthesis from natural chains by means of reversible bridging compounds

Synthesis and properties of carbonylbis(methionyl)insulin, a proinsulin analog which is convertible to insulin by cyanogen bromide cleavage

“…All exhibit hormonal activities which are about 4-10% of that of in- sulin while retaining about 90% of the immunological activity. These observations, coupled with the facts that (1) the CD spectra of insulin and the cross-linked insulins, although not identical, are quite similar (Figure 5) and ( 2) the ability to crystallize the zinc complex of some of the insulin derivatives which are cross-linked with decarboxylic acids (Lindsay, 1972;Brandenburg, 1972;Geiger and Obermeier, 1973;Brandenburg et al, 1973a) suggest (Gliemann and Gammeltoft, 1974) that the cross-linking residues cover a portion of the molecule which is essential for hormonal activity without causing a major change in the tertiary structure. Attempts to crystallize CBM-insulin under a variety of conditions have been unsuccessful.…”

“…This disadvantage has been overcome in the present studies in which it has been demonstrated that the disulfide bonds in CBM-insulin can be reduced and then reoxidized to give, under certain conditions, a 68-85% recovery of the CBM-insulin (with correct pairing of disulfide bonds) which in turn can be converted back to insulin by CNBr cleavage (Figure 1). Another solution to this problem has been presented by Geiger and Obermeier (1973) and Brandenburg et al (1973b) both of whom used the di(Boc)a,a'-diaminosuberoyl moiety as a cross-linking residue. After reduction-reoxidation of the cross-linked insulin, the Boc groups were removed in anhydrous trifluoroacetic acid and the resulting diaminosuberoyl residue was cleaved by an Edman degradation.…”

“…After reduction-reoxidation of the cross-linked insulin, the Boc groups were removed in anhydrous trifluoroacetic acid and the resulting diaminosuberoyl residue was cleaved by an Edman degradation. Geiger and Obermeier (1973) have also presented procedures by which the separate A and B chain ^-sulfonates are specifically cross-linked between glycine Al and lysine B29 with the suberoyl derivative, followed by reduction, reoxidation, and cleavage to yield the hormone. A different strategy to achieve the correct pairing of the disulfide bridges has recently been presented by Sieber et al (1974) in pioneering work involving the directed stepwise formation of the disulfide bonds at different stages of the synthesis.…”

Design and Folding of [GluA4(OβThrB30)]Insulin (“Ester Insulin”): A Minimal Proinsulin Surrogate that Can Be Chemically Converted into Human Insulin

“…Most of the previously studied chemically tethered insulin precursors have involved covalent linking of the N-terminal of the insulin A chain to the side chain of Lys B29 , near the C-terminal of the insulin B-chain. [15][16][17][18][19][20] With the goal of an efficient total synthesis of human insulin and analogues, a variety of different length chemical tethers between these two functionalities has been explored, using both non-cleavable [15,16] and cleavable [17][18][19][20] tethers. The shortest tether reported to be effective in promoting high yield folding/disulfide formation contained 8 carbon atoms.…”

Intramolecular Enzymatic Peptide Synthesis: Trypsin-mediated Coupling of the Peptide Bond between B22-Arginine and B23-Glycine in A Split Crosslinked Insulin

“…Inouye et al! 7 l reported the use of trypsin for peptide bond formation bet ween N aA l , N aB lBoc 2 -des[(B23-30)-] octapeptide insulin (derived from native porcine insulin) and synthetic human octapeptide B (23)(24)(25)(26)(27)(28)(29)(30), allowing the semisynthesis of human insulin from porcine insulin. The most straightforward conversion of porcine to human insulin was accomplished by coupling thieonine (methyl or feri-butylester) to des-Ala B3°-insulin by meansof trypsin!…”