P-3 エンドポリガラクツロナーゼ反応機構解明を目指した硫黄置換オリゴガラクツロン酸の設計と合成(ポスター発表の部)

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Endo-polygalacturonase (endo-PGs) hydrolyzes the internal glycosidyl linkages of polygalacturonic acids (pectic acids) into oligomers. Because oligogalacturonic acids (low-molecular-sized pectic acids) have recently attracted attention as functional supplemental foods, such as Ca^<2+> carriers, dietary fiber and so on, investigating in detail the mechanism of endo-PG has gained importance in last decade. A phyto-pathogenic fungi Stereum purpureum produces endo-PG 1 of which single crystalline provide an X-ray crystallographic structure of extremely high quality (less than 0.96A). However, soaking experiments have not provided the complex of endo-PG 1 with the oligogalacturonate as a reactant, because enzymatic hydrolysis occurred in the crystalline to afford only the complex with the hydrolysates. Thus, we planned to investigate in detail the reaction mechanism of this enzyme employing stable oligogalacturanate mimics. Based on the information accumulated in the enzymatic studies, we designed sulfur-substituted analogue of trigalacturonic acid (3) as a stable mimic of natural substrate. This analogue evaluated by molecular dynamic calculations based on expanded COSMOS90. was designed with the expectation that it would be a stable analogous substrate for endo-polygalacturonase isolated from Stereum purpureum based on computer modeling experiments. The synthesis features a effective Williamson type thioglycoside formation by an activation of 3-cyano-3-tert-butyldimethylsilylpropyl (CSP group) thioether with TBAF in the presence of MS4A. This finding brought us successful preparation of the compound 3 in sufficient amount for the next enzymatic/X-ray crystallography analysis. As expected, preliminary enzymatic studies revealed that 3 inhibited hydrolysis of oligogalacturonic acid by endo-PG1 and was stable under these conditions. The experiments employing surface plasmon resonance revealed the K_D value of 3 to be 0.2μmol/L.
天然有機化合物討論会の論文
2005-09-15

P-3 エンドポリガラクツロナーゼ反応機構解明を目指した硫黄置換オリゴガラクツロン酸の設計と合成(ポスター発表の部)

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