DEVELOPMENT OF EFFICIENT 2-STEP DEPROTECTION METHODOLOGY FOR DIMETHYL-PROTECTED PHOSPHOAMINO ACID-CONTAINING PEPTIDE RESINS AND ITS APPLICATION TO THE PRACTICAL SYNTHESIS OF PHOSPHOPEPTIDES

A protocol has been developed for the synthesis of peptides containing O-phosphorylated tyrosines, serines, and/or threonines. The procedure involves incorporation of dimethyl-protected O-phosphorylated amino acid derivatives (1-3) into peptides using standard Boc chemistry and subsequent removal of Me groups using a two-step deprotection method consisting of high-acidic and low-acidic treatments. Optimized deprotection conditions for the protected resins (4-6) were established, which consist of a combination of the first-step reagent (1 M TMSOTf-thioanisole in TFA (100), m-cresol (5), EDT (5), (v/v)) and the second-step reagent (first-step reagent (110) + DMS-TMSOTf(30:20 to 40:10), (v/v)). The two-step deprotection protocol can be conducted in one pot by appropriate modification of the first-step reagent. The second deprotection step proceeds by an S(N)2 mechanism with little tendency to induce side reactions resulting from harsh acid treatment. A 19-residue MAP-kinase peptide 10 possessing not only two phosphoamino acids but also Met and Trp was subjected to this synthetic procedure and was obtained in 24% yield based on the protected resin. The present synthetic method afforded phosphoamino acid-containing peptides in high yield without significant accompanying side reactions (e.g., loss of phosphate groups, migration of phosphate groups, or alkylation of Met and Trp residues).