Substrate-specific Inhibitors of Glycosidases as Tools for Bioorganic Chemical Studies on Glycosidases

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This study aims to develop novel glycosidase inhibitors and to use these inhibitors as versatile research tools in glycosidases studies from a bioorganic point of view. For this purpose, we have designed and synthesized f -glycosylamidine derivatives as highly potent and glycon-selective inhibitors of β-glycosidases. β-Glycosylamidines are a substrate analog in which a positively charged amidino group is incorporated into a sugar surrogate in such a way as to maintain the whole structure of the glycon, including the stereochemistry at C-1. The β-glycosylamidines are readily synthesized from various sugars n two steps without using any protective groups. The β-glycosylamidines with each glycon moiety serve as highly potent inhibitors (K_i<0.1μM) toward the β-glycosidases with the corresponding glycon substrate specificity, while the β-glycosidases with different glycon substrate specificity are not inhibited significantly. Furthermore, varying structures can be introduced into the "agly … More con" moiety of the inhibitor to increase or tune the inhibition potency and selectivity towards various β-glycosidases. Therefore the β-glycosyamidines are versatile β-glycosidase inhibitors in which both the glycon and aglyon moieties can be adjusted according to the enzyme to be inhibited. This property can be successfully used as a ligand for affinity chromatography of glycosidases.. Thus, tea leaf β-glucosidases were purified to homogeneity in one step by the affinity adsorbent with β-glucosylamidine as ligand, while a β-galactosidase from mold was also purified in one step by an affinity chromatography with β-galactosylarnidine as ligand. Other β-glycosidases such as β-primeverosidase, a novel diglycoside-specific glycosidase, was also affinity-purified successfully by using the corresponding glycosylamidine as ligand. The purified tea leaf β-glucosidases were subjected to amino acid sequence analysis to clone the genes. Hence the β-glycosylamidines are versatile research tools in glycosidase studies spanning from the isolation, the characterization and the gene cloning. Less