1. [Partial synthesis and properties of des-A1-glycine-insulin (author's transl)]
D Brandenburg, M Biela, L Herbertz, H Zahn Hoppe Seylers Z Physiol Chem. 1975 Jun;356(6):961-79.
Des-Gly-A-chain-tetra-S-sulphonate was prepared by Edman degradation following two different routes. A) Via complete reaction of A-chain from bovine insulin with 150 equivalents of phenylisothiocyanate in pyridine/water and trifluoroacetic acid cleavage of the resulting phenylthiocarbamoyl A-chain. B) Via reaction of bovine insulin with about 20 equivalents of phenylisothiocyanate until a substitution degree of 2.3-2.5 was reached, trifluoroacetic acid cleavage of the crude derivatives and oxidative sulphitolysis of the resulting desaminoacyl insulins. Preparative electrophoresis (pH 2) or ion exchange chromatography using DEAE-Sephadex gave des-Gly-A-chain in a yield of 60-65% of theory according to method B, containing less than 1% of glycine. Des-GlyA1-insulin was prepared by combination with 0.67 equivalents of B-chain-bis-S-sulphonate and isolated in yields of 5-13%, based on B-chain, after gel filtration (pH 8) and ion exchange chromatography (CM-cellulose, pH 3-2). The electrophoretically (pH 2 and 8.6) homogeneous analogue did not crystallize in the presence of zinc ions. Its blood sugar lowering potency is 10-25%, its in vitro insulin activity (fat cell assay) only 1-2%. The immunoreactivity against anti-insulin sera in different test systems is markedly reduced. There are clear differences between the CD-spectra of des-Gly-insulin and insulin, indicating a loss of ordered secondary structure. From the results it is concluded that structure-stabilizing non covalent bonds are abolished by the removal of the invariant A1-glycine. This leads to conformational alterations which cause the far-going inactivation of the molecule.
2. [Semisynthetic sheep des-A1-glycine insulin (author's transl)]
H Berndt, H G Gattner, H Zahn Hoppe Seylers Z Physiol Chem. 1975 Sep;356(9):1469-72.
The synthetic Des-1-glycine-A-chain of sheep insulin as the monomeric cyclic bisdisulfide and native bovine B-chain bissulfonate were reduced together with mercaptoethanol. They combined at pH 10.6 to yield Des-A1-glycine-insulin. This was purified by gel and ion exchange chromatography. The low insulin activity (0.4 - 0.6%) as measured by the fat cell test as well as the change in the CD spectrum indicated that the loss of the N-terminal glycine of the A-chain results in fully inactive insulin. This confirms the results obtained earlier by partial synthesis of Des-A1-glycine-insulin.
3. A novel semisynthetic route to insulin analogues modified at the N-terminus of the A-chain
D J Saunders, R E Offord Hoppe Seylers Z Physiol Chem. 1977 Nov;358(11):1469-74.
We report a novel route for the semi-synthetic production of insulin analogues modified at the amino-terminus of the A-chain. The route proceeds from insulin via [Nalpha-Boc-GlyA1]-insulin to [Nalpha-(Z-methionyl)-PheB1, Nepsilon-(Z-methionyl)-LysB29]des-GlyA1-insulin. We have replaced residue A1 with glycine, to give a fully crystalline resynthesized insulin, and with D-lysine.
