Synthesis And Enzyme Inhibitory Activity Study Of Natural Phosphorus-Containing Glycocongujates

Glycopetides are a family of conjugates which contain a covalent linkage between glycosyl and peptide moieties. These molecules possess a lot of biological fonctions in vivo. Natural glycosidic linkages mainly include N-glycosides,O-glycosides, and uncommon C-glycosides, S-glycosides and P-glycosides. Among them, P-glycosides have important research value in the realm of biology and medicine. Their synthetic methods have been one of the hotpots in the field of chemical synthesis.Phosphoramidon is a natural phosphorus-linked glycopetide isolated from a strain of Streptomyces tanashiensis. It has potent inhibitory activity against thermolysin (TLN, Ki=32nM), a zinc endopeptidase widely used in the production of aspartame and peptide sequencing. As a biochemical reagent, commercial phosphoramidon is expensive. There are only a few methods reported for the chemical synthesis of phosphoramidon and its analogs. Therefore, it is of great significance to develop efficient chemical methods to obtain highly pure phosphoramidon for practical applications.In this thesis, phosphoramidon was synthesized efficiently via the optimized H-phosphonate method at5gram scale. Nine novel glycosyl phosphoramidate conjugates were obtained by the new H-phosphonate method. Further research on the biological activity of the synthetic phosphoramidon and its isomer determined their inhibition constants (Ki) and dissociation constants (Kd) against TLN. The main research work of this thesis is described as follows.1. The H-phosphonate method for the synthesis of phosphoramidon was optimized and improved. The preparation of the glycosyl-1-H-phosphonate, oxidative coupling, and deprotection procedures were systematically investigated. Phosphoramidon and nine analogs were synthesized efficiently by this method.2. The biological activities of phosphoramidon and its β anomer were evaluated. A simple and convenient route was developed for the chemical synthesis of3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA), the fluorescent substrate of TLN. The Ki values of phosphoramidon and its β anomer were determined by Henderson’s plot, and their Kd values were calculated by fluorescence titrimetric method.3. On the basis of1D (1H,13C, and31P) and2D (1H-1H COSY, HSQC, and NOESY) NMR spectral data, all of the NMR signals were assigned for both phosphoramidon and its β anomer. The anomeric configurations of these two isomers were also determined by their one-bond C-H coupling constants and nuclear overhauser effect (NOE).