Application of C-H Activation and Sulfate Click Reaction to Chemical Biology

Abstract

Factors affecting cellular phenomena comprise a series of complicated and elaborate signal transductions. Due to the complexity of biomolecules, direct observation of a specific biomolecule is extremely difficult. Therefore conjugation of functionality onto the biomolecule is often implemented for the purpose of monitoring a bio-target. Since hydrophobic interaction between a biomolecule and a ligand is usually not strong enough to be monitored by common analytical tools, covalent bond formation of an artificial tag molecule has been developed. After Huisgens discovery of 1,3-dipolar cycloaddition between an azide and an alkyne, Sharpless group reported that the reaction can be catalyzed by a Cu(I) species. Furthermore, introduction of azidophenylalanine into proteins through expanded genetic code renders possible the conjugation of an artificial functionality such as fluorescence, and anti-cancer activity, etc. It is of particular note that the fluorescence attachment makes the spatiotemporal observation of bio-target possible. As a consequence, both the development of a new bioconjugation method, and the synthesis of functional molecules to be ligated have become significant research subjects in chemical biology field. This dissertation includes research results of two parts, 1) developing synthetic method of fluorescent molecules which can be used as a biosensor, and 2) discovery of novel tyrosine bioconjugation chemistry. The first part shows one of our approaches in utilizing novel metal assisted 1,3-cycloaddition and Pd-mediated C-H activation for efficient syntheses of indolizinone fluorophores containing a small molecule library. Since the photophysical property of indolizinone is regulated by the electronic characteristics of C7 and C9 substituents, diversification of those substituents was crucial for the construction of a fluorescent molecule library. Through the new synthetic route, synthesis of a number of fluorescent molecules was accomplished, as diversification of C9 substituent occurs at the late stage of the synthesis. Moreover, several biosensors were synthesized, which had been previously infeasible. In part 2, tyrosine selective conjugation method through sulfate click reaction is described. Since SuFEx (sulfur(VI) fluoride exchange) chemistry was known as the reaction between aryl fluorosulfate and aryl silyl ether, aryloxy anion has scarecely been recognized as a useful reaction partner for SuFEx. In this part, a reaction condition to allow for the reaction of aryl fluorosulfate with tyrosine in the presence of several nucleophilic amino acid residues was carefully selected, and a feasibility test of a new site-specific biomolecule modification was successfully carried out on a model peptide, TAT 47-57, and a physiologically important protein, erythropoietin.