SYNTHESIS OF A PROTECTED C-TERMINAL DODECAPEPTIDE OF THE A-CHAIN OF INSULIN

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Dodecapeptide ester Ⅰ has been saponified to give a protected C-terminal dodecapeptide (Ⅱ) of the A-Chain of insulin. Further experiments showed that the saponification of ester Ⅰ under various conditions yielded preparations of dodecapeptide Ⅱ varying somewhat in melting points and solubility. These saponification products have been used for the partial synthesis of bovine insulin and all the crude hormone products obtained showed biological activity of 1—4％ of natural insulin in mouse convulsion test. In order to avoid the saponification dodecapeptide Ⅱ has been directly synthesized by two alternative routes. By route 1, the known tetrapeptide Ⅲ was first converted into its hydrazide (Ⅳ), which was then coupled by the azide method with pentapeptide V_b to form nonapepfide Ⅵ (C_(66)H_(86)N_(12)O_(19)S·2H_2O, m.p. 235—236° (decomp.), [α]_D~(15)—44° (c 1.0, DMF)). Nonapeptide Ⅵ was decarbobenzoxylated and combined with the known tripeptide hydrazide Ⅶ through the azide method to give dodecapeptide Ⅱ(C_(84)H_(111)N_(15)O_(23)S_2·2H_2O, m.p. 246—248°, [α]_D~(15)—34° (c 1.0, HCOOH)). Pentapeptide V_b was prepared from pentapeptide V which was obtained by the following ways: (1) The known dipeptide ⅩⅢ was decarbobenzoxylated and the N-deblocked product was combined with dipeptide hydrazide ⅩⅣ by the azide method to form tetrapeptide XV (C_(35)H_(40)N_6O_(10)S·H_2O, m.p. 191—193°, [α]_D~(21)-41° (c 1.0, DMF)). The same product has also been synthesized by coupling N-carbobenzoxy-L-asparagine p-nitrophenyl ester with N-deblocked tripeptide ⅩⅧ. Compound ⅩⅧ was in turn prepared from the condensation of N-carbobenzoxy-L-tyrosyl hydrazide with N-deblocked dipeptide ⅩⅢ (m.p. 188—190°, [α]_D~(30)-34° (c 1.0, DMF)). Tetrapeptide ⅩⅣ was further decarbobenzoxylated and the resulting product was condensed with α-p-nitrophenyl-γ-tert-butyl N-carbobenzoxy-L-glutamate (ⅩⅦ) to yield pentapeptide Ⅴ (C_(44)H_(55)N_7O_(13)S·H_2O, m.p. 185—186°, [α]_D~(22)-37° (c 2.0, DMF)). Both compounds ⅩⅧ and ⅩⅤ have been prepared by another method. (2) N-deblocked dipeptide ⅩⅥ was first coupled with ⅩⅦ to give tripeptide ⅩⅪ (m.p. 179—180°, [α]_D~(32)+7° (c 1.0, DMF)), which was then converted into its hydrazide ⅩⅫ (m.p. 219—220°,[α]_D~(32)—16.5° (c 1.0, DMF)). Hydrazide ⅩⅫ was finally condensed by the azide method with dipeptide ⅩⅢ to yield pentapeptide V. It seems that way (1) led to better overall yield and higher quality of the pentapeptide. By route 2, dodecapeptide Ⅱ was synthesized by condensing heptapeptide hydrazide ⅩⅩⅦ with pentapeptide V_b through the azide method. Hydrazide ⅩⅩⅦ (C_(52)H_(74)N_(10)O_(12)S·H_δO, m.p. 253—254° (decomp.), [α]_D~(26)-36° (c 0.93, HCOOH)) was prepared from heptapeptide ester ⅩⅩⅥ. Ester ⅩⅩⅥ was synthesized by two alternative methods. In one of these methods tripeptide hydrazide Ⅶ was transformed into its azide and condensed with decarbobenzoxylated dipeptide ester ⅩⅩⅢ to form pentapeptide ester ⅩⅩⅣ (C_(43)H_(57)N_5O_(10)S·H_2O, m.p. 220—222°,[α]_D~(15)-21° (c 1.0, DMF)), which was then converted into its hydrazide ⅩⅩⅤ and finally coupled through the azide method with N-deblocked dipeptide Ⅹ to give heptapeptide ester ⅩⅩⅥ (C_(53)H_(74)N_8O_(13)S·H_2O, m.p. 249—250° (decomp.), [α]_D~(15)-34° (c 1.0, DMF)). In the other method, ester ⅩⅩⅥ was obtained from tripeptide hydrazide Ⅶ and N-deblocked tetrapeptide Ⅲ by the azide method. All synthetic peptide intermediates have been subjected to elementary analyses, as well as paper chromatographic and paper electrophoretic examinations, and dodecapeptide Ⅱ has been analysed for its amino acid composition. The results showed that all synthetic peptides are chemically pure and homogeneous. Dodecapeptide Ⅱ prepared by the above routes has higher melting point and levorotatory power than that obtained by the saponification of ester Ⅰ. It has been used for the partial as well as total synthesis of crystalline bovine insulin.