Description:
Horse heart cytochrome c was treated with methylsulfonylethyloxycarbonyl succinimide (Msc-ONSu) to give fully N ε -protected cytochrome c . Treatment of this derivative with a hard base for 15 sec regenerated the native tetrahectapeptide chain. CNBr degradation of the protected compound produced three fragments bearing only protective Msc functions on ε-amino groups. The fragment comprising the sequence 81-104 was isolated from the mixture and acylated with N -hydroxysuccinimidyl- t -butyloxycarbonyl-L-methioninate. The resulting pentacosapeptide derivative was partially deprotected by treatment with acid and condensed in good yield (65%) with fully synthetic N α 66 , N ε 72,73,79 - tetra-Msc-cytochrome- c -(66-79)-tetradecapeptide azide. This pathway is preferred because the penta decapeptide azide derivative 66-80 acylated the N ε -protected tetracosapeptide sequence 81-104 in an unpredictable manner. Subsequent treatment of the product with a base produced unprotected semisynthetic cytochrome- c -(66-104)-nonatriacontapeptide, which is known to undergo acylation by unprotected [Hse 65 ]cytochrome- c -(1-65)-pentahexacontapeptide lactone. The high specificity of this condensation is ascribed to “conformation direction.” Semisynthetic [Hse 65 ]cytochrome c thus prepared reacts like native cytochrome c with a succinate cytochrome c reductase preparation and with cytochrome c oxidase (ferrocytochrome c :oxygen oxidoreductase, EC 1.9.3.1). This semisynthetic strategy may provide a rapid route for the production of cytochrome c analogs modified in the highly conservative sequence 66-80.