Discovery of new bioactive compounds from marine actinomycete and development of new LC/MS-based analytical methods for the absolute configurations of amino acids-derived natural products

Abstract

During my doctoral course, reseach works had been divided into two parts and performed simultaneously. One was for discovering the new bioactive compounds, and the other was for developing new analytical methods to determine the absolute configurations of amino acids-derived natural products. According to these, this thesis were composed of two contents, part A and B. Part A. Microorganisms inhabiting relatively extreme and thus uninvestigated environments have attracted attention as new resources for bioactive compounds. As a part of continuing efforts to explore the chemical diversity of microorganisms from extreme habitats for drug discovery, I have been focusing on bacterial strains inhabiting the Arctic Ocean, tidal mudflats and estuary. I isolated bacterial strains from these extreme habitats, cultured them, and analyzed their secondary metabolites by LC/MS. Based on the bacterial chemical profiles obtained by LC/MS analysis, I prioritized several strains for further chemical investigation and finally discovered 6 new bioactive natural products which had different moieties for example, benzoxazine rings, petacyclic rings and polyol macrorides. Part B. α-hydroxy acids and N,N-dimethyl amino acids are commonly found in natural bioactive molecules. For convenient determination of the absolute configurations of α-hydroxy acids and N,N-dimethyl amino acids, I developed two simple methods, named the O-Marfey method and PGME method. O-Marfey method was developed using a chiral derivatization strategy with (1-fluoro-2,4-dinitrophenyl-5)-L-alanine amide (L-FDAA). The liquid chromatography-mass spectrometry (LC/MS) analysis of the derivatives clearly determined the absolute configurations of various α-hydroxy acids based on their elution times and sequences. The absolute configuration of a natural despsipeptide bearing an α-hydroxy acid and α-amino acids was successfully determined by O-Marfey application. This new method has advantageous of short reaction time and remarkable sensitivity for application at a sub-milligram scale without any purification. This method also enables the simultaneous chirality analysis of α-hydroxy acids and α-amino acids. In addition, PGME method was modified utilizing phenylglycine methyl ester (PGME) to the carboxylic acid of N,N-dimethyl amino acids. This method enabled the determination of the absolute configurations of various N,N-dimethyl amino acids, based on the elution times and sequences of derivatives via liquid chromatography-mass spectrometry (LC/MS) analysis.