Kengo Miyoshi

Synthesis and application of N-Boc-L-2-amino-4-(diethylphosphono)-4,4-difluorobutanoic acid for solid-phase synthesis of nonhydrolyzable phosphoserine peptide analogues

Abstract Synthesis of N-Boc-L-2-amino-4-(diethylphosphono)-4,4-difluorobutanoic acid is reported. This analogue was utilized for the solid-phase synthesis of a peptide containing a nonhydrolyzable phosphoserine mimetic.

A convenient one-pot synthesis of cystine-containing peptides from protected peptidyl resins using the trimethylsilyl chloride–dimethyl sulfoxide–trifluoroacetic acid system

Cystine-containing peptides are obtained in a one-pot manner by treatment of protected peptidyl resins with trimethylsilyl chloride (TMSCl)–dimethyl sulfoxide (DMSO)–trifluoroacetic acid (TFA). The TMSCl–DMSO–TFA system simultaneously cleaves peptides from the resins, with deprotection of all side chain protecting groups and disulfide bond formation.

Practical synthesis of phosphopeptides using dimethyl-protected phosphoamino acid derivatives

A practical synthesis of phosphopeptides was achieved using a combination of dimethyl-protected phosphoamino acids and two-step deprotection protocols consisting of high acidic (SN1/SN2) and low acidic (SN2) reagent systems.

Phospho-Azatyrosine, a less effective protein-tyrosine phosphatase substrate than phosphotyrosine.

Azatyrosine (AzaTyr, 4) is a natural product isolated from Streptomyces chibanesis, whose structure is characterized by a nitrogen atom in the aryl ring of a tyrosyl residue. This seemingly minor modification to the tyrosyl residue results in profound physiological effects, as AzaTyr has been shown to promote permanent reversion of ras-dependent transformed cells to the normal phenotype in culture and to inhibit chemical induction of carcinogenesis in transgenic mice bearing oncogenic human ras. The mechanisms underlying these effects are not known, however ras-pathways involve an intricate balance between both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). The present study was undertaken to examine the general utility of AzaTyr as a structural motif for PTP inhibitor design by examining the phospho-azatyrosine (pAzaTyr)-containing peptide Ac-Asp-Ala-Asp-Glu-pAzaTyr-Leu-amide (8) in a PTP1 enzyme system. Kinetic analysis indicated that 8 binds with a Km value of 210 microM and a catalytic turnover rate, kcat of 52 s(-1). This represents a greater than 50-fold reduction in binding affinity relative to the parent phosphotyrosine-containing peptide, indicating that the aryl nitrogen adversely affects binding affinity. The much lower PTP affinity of the pAzaTyr-containing peptide reduces the potential utility of the AzaTyr pharmacophore for PTP inhibitor design. These results are discussed from the point of view that incorporation of AzaTyr residues into proteins could result in perturbation of protein-tyrosine phosphorylation,dephosphorylation cascades that control signal transduction processes, including ras-dependent pathways.

Convenient preparation of phosphopeptide fragments using High TFMSA system and its application to synthesis of long chain phosphopeptides

Protein phosphorylation provides structural and functional changes for proteins involved in intracellular signal transduction pathways [1]. Due to the important role of phosphorylation/dephosphorylation in biology, long chain phosphopeptides have received much attention as useful biological and biochemical tools to elucidate various cellular processes including signal transduction. Previously we reported a efficient synthetic methodology for phosphopeptides [2, 3]. In the present study, we tried to develop a new synthetic methodology for long chain phosphopeptides and found that partially protected phosphopeptide fragments [ClZ(2-chlorobenzyloxycarbonyl) group for lysine and methyl groups for phosphoamino acids] can be obtained by treatment of protected peptidyl resins with High TFMSA system with efficient cleavage and removal of the other protecting groups. This finding prompted us to apply the High TFMSA system to the preparations of the useful partially protected phosphopeptide fragments as the building blocks in thioester fragment condensation strategy [4]. Here, we report the convenient synthetic method of phosphopeptide fragments using High TFMSA system.