The vital roles of peptide/protein thioesters in protein chemistry, including chemical or semi-synthesis of proteins, have encouraged studies on the development of methods for the preparation of such chemical units. Biochemical protocols using intein or sortase have proved to be useful in protein chemistry as methods suitable for naturally occurring sequences, including recombinant proteins. Although chemical protocols are potential options for thioester preparation, only a few are applicable to naturally occurring sequences, because standard chemical protocols require an artificial chemical device for producing thioesters. In this context, the chemical preparation of thioesters based on a reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis was investigated. Regioselective S-cyanylation, which is required for cysteine-containing thioesters, was achieved with the aid of a zinc-complex formation of a CCHH-type zinc-finger sequence. Free cysteine residues that are not involved in complex formation were selectively protected with a 6-nitroveratryl group followed by S-cyanylation of the zinc-binding cysteine. Hydrazinolysis of the resulting S-cyanopeptide and subsequent photoremoval of the 6-nitroveratryl group yielded the desired peptide hydrazide, which was then converted to the corresponding thioester. The generated thioester was successfully used in N-to-C-directed one-pot/sequential native chemical ligation using an N-sulfanylethylanilide peptide to give a 64-residue peptide toxin. (C) 2015 Wiley Periodicals, Inc.