| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
1)The binary complex of the trimeric calf spleen phosphorylase, which is highly homologous to human purine nucleoside phosphorylase(PNP), with the potent ground state analogue inhibitor 9-(5,5-difuoro-5-phosphonopentyl)guanine (DFPP-G) was crystallized in the cubic space group P2_13. The crystal structure confirms that DFPP-G acts as a multi-substrate analogue inhibitor as it binds to both nucleoside-and phosphate-binding sites. The analysis also indicates that the linker connecting a purine and difluoromethylenephosphonic acid moiety is surrounded by hydrophobic amino acid residues (Ala 116 and Phe 159) of PNP. On the basis of these findings, DFPP-G and its hypoxanthine analogue(DFPP-H) were modified by introducing a methyl group to all possible positions of the linker to evaluate the hydrophobic effects of the methyl group on the binding affinity to PNP. The methyl group on the linker affected the inhibition in a positional-dependent manner.
2)A series of short-chain analogues of N-pa
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lmitoylsphingosine-1-phosphate, modified by replacement of the phosphate and the long alkenyl side chain with hydrolytically stable difluoromethylene phosphonate and phenyl, respectively, were prepared to study the structure-activity relationship for inhibition of sphingomyelinase(SMase). The study revealed that inhibition is highly dependent upon the stereochemistry of the asymmetric centers of the acylamino moiety, and resulted in identification of a non-competitive inhibitor with the same level of inhibitory activity of schyphostatin, the most potent of the few known small molecular inhibitors of N-SMase. Our SMase inhibitor inhibited the enhanced N-SMase activity in the serium/gulucose-deprived PC-12 cells, and DNA fragmentation in the nuclei. Administration of the inhibitor to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared the control mice.
3)In addition of the above results, we examined modification of adenosine bisphosphates and inositol phosphates by replacement of the phosphate group with a difluoromethylene phosphoninc acid. During the synthesis of the target molecule, we have developed a highly-selective β-glycosylation reaction of 2,3-dideoxyfuranosies having diethoxyphosphoryldifluoromethyl functionality at 3α-position and a facile method for introducing a difluoromethylenephosphonate unit to the allylic position within a cyclic array in a stereo-and regioselective manner. Less
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