Solution synthesis of Na+,K(+)-ATPase inhibitor-1 (SPAI-1).

SPAI-1, a 49-amino acid peptide including eight Cys residues with Na+,K(+)-ATPase inhibitory activity, was synthesized by the solution procedure. Protecting groups, including the formyl group on the Trp residue, were cleaved simultaneously by HF treatment in the presence of a sufficient amount of thiol compound. After removal of the Acm group on the Cys residue, the resulting octa SH peptide was subjected to an oxidative folding reaction in the presence or absence of redox reagents and/or denaturant. Addition of redox reagents accelerated the reaction but was not crucial to the formation of the correct disulfide bonds in the molecule. The product was purified to homogeneity and found to have the same physicochemical properties and inhibitory potency as those reported for the natural product. Four intramolecularly linked disulfide bridges were assigned as connecting Cys8 to Cys37, Cys15 to Cys41, Cys24 to Cys36, and Cys30 to Cys45 based on results from a combination of enzymatic and synthetic approaches.