A new class of human insulin analogues has been developed and produced by biosynthetic and semi synthetic methods. These analogues are characterized by the replacements or deletion of single amino acid residue to the C-terminal of the β chain. The analogues have been evaluated by measuring the in vitro biological potency in mouse fat cells, by osmometric determination of association state in solution at natural pH and the blood glucose lowering effect found after subcutaneous injection in pigs.
Our plan is to synthesis Nitro-Tyrosine (B16)-insulin as single chain with deleting of Thr-B30
Systematic procedure is as follows
· Zinc free insulin and enzymatic cleavage of B23-30 to give des-octa peptide (DOP) insulin
· Sulfitolysis of (DOP) insulin to give A(SSO3)4+B(SSO3)-DOP to give A(SS)2+B(SS)2-DOP after reduction-oxidation
· Synthesis of heptapeptide B23-29 by automatic peptide synthesizer
· Cleavage of heptapeptide B23-29 from resin, purification, and protection of the N-terminal heptapeptide by Boc-group
· Coupling of Boc-B23-29 to A(SS)2 by chemical coupling with Dicyclohexyl carbodiimide (DCCI) method
· Nitration of Tyr(B16)DOP insulin with tetranitromethan
· Protection of Boc-B23-29 A(SS)2 (cleavage of Boc group)
· Coupling of NO2-Tyr(B16)DOPBSS+B23-29-A(SS)2 to give single chain as des Thr-B30 insulin by using trypsine as single chain des Thr-B30 insulin
Characterization of the single Des (Thr-B30) insulin NO2-Tyrosine was held by HPLC, capillary electrophoresis amino acid analysis