Masaya Denda

Development of a traceable linker containing a thiol-responsive amino acid for the enrichment and selective labelling of target proteins

A traceable linker that is potentially applicable to identification of a target protein of bioactive compounds was developed. It enabled not only thiol-induced cleavage of the linker for enrichment of the target protein but also selective labelling to pick out the target from contaminated non-target proteins for facile identification.

Development of a Chemical Methodology for the Preparation of Peptide Thioesters Applicable to Naturally Occurring Peptides Using a Sequential Quadruple Acyl Transfer System

Peptide thioesters are very useful in protein chemistry, and chemistry- and biochemistry-based protocols are used for the preparation of thioesters. Among such protocols, only a few biochemistry-based approaches have been use for naturally occurring peptide sequences. The development of chemistry-based protocols applicable to natural sequences remains a challenge, and the development of such methods would be a major contribution to protein science. Here, we describe the preparation of peptide thioesters using innovative methodology that features nickel(II)-mediated alcoholysis of a naturally occurring peptide sequence, followed by O−N and N−S acyl transfers. This protocol involves sequential quadruple acyl transfer, termed SQAT. Notably, the SQAT system consists of sequential chemical reactions that allow naturally occurring peptide sequences to be converted to thioesters without requiring an artificial chemical unit.

Development of caged non-hydrolyzable phosphoamino acids and application to photo-control of binding affinity of phosphopeptide mimetic to phosphopeptide-recognizing protein

The design and synthesis of caged non-hydrolyzable phospho-serine, -threonine, and -tyrosine derivatives that generate parent non-hydrolyzable phosphoamino acids, containing a difluoromethylene unit instead of the oxygen of a phosphoester, after UV-irradiation are described. The caged non-hydrolyzable amino acids were incorporated into peptides by standard Fmoc solid-phase peptide synthesis, and the obtained peptides were successfully converted to the parent non-hydrolyzable phosphopeptides by UV-irradiation. Application of the caged non-hydrolyzable phosphoserine-containing peptide to photo-control the binding affinity of the peptide to 14-3-3β protein is also reported.

Development of Chemistry-Based Protocol for Sequence-Dependent Thioesterification

One of the useful methods for exploration of the function of proteins is utilization of the synthesized proteins which include artificial units such as fluorescence dye. For the synthesis of such proteins, a peptide thioester and an N-terminal cysteinyl peptide prepared by Solid Phase Peptide Synthesis (SPPS) are coupled to each other using Native Chemical Ligation (NCL) [1]. However, due to the limitation of the number of amino acid residues applicable to SPPS, multistep NCLs are required for chemical synthesis of large proteins. On the other hand, in semi-synthesis of proteins, only single NCL of protein thioester prepared from expressed protein with chemically synthesized N-terminal cysteinyl peptide enables to incorporate functional moieties to large proteins. However, there are only a few methods for preparation of protein thioesters applicable to naturally occurring sequences [2]. Therefore, new protocols for preparation of protein thioesters has been required.