Semisynthetic horse heart [65-homoserine]cytochrome c from three fragments

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Abstract
Horse heart cytochrome c was treated with methylsulfonylethyloxycarbonyl succinimide (Msc-ONSu) to give fully N.epsilon.-protected cytochrome c. Treatment of this derivative with a hard base for 15 s regenerated the native tetrahectapeptide chain CNBr degradation of the protected compound produced 3 fragments bearing only protective Msc functions on .epsilon.-amino groups. The fragment comprising the sequence 81-104 was isolated from the mixture and acylated with N-hydroxysuccinimidyl-5-butyloxycarbonyl-L-methioniate. The resulting pentacosapeptide derivative was partially deprotected by treatment with acid and condensed in good yield (65%) with fully synthetic N.alpha.66,N.epsilon.7273,79-tetra-Msc-cytochrome-c-(66-79)-tetradecapeptide azide. This pathway is preferred because the pentadecapeptide azide derivative 66-80 acylated the N.epsilon.-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 [Hse65]cytochrome-c-(1-65)-pentahexacontapeptide lactone. The high specificity of this condensation is ascribed to conformation direction. Semisynthetic [Hse65]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.