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

本研究旨在开发新的糖苷酶抑制剂，并从生物有机的角度将这些抑制剂作为糖苷酶研究的多功能研究工具。为此，我们设计并合成了f -糖苷胺衍生物，作为β-糖苷酶的高效糖选择性抑制剂。β-糖基胺是一种底物类似物，其中带正电的氨基基结合到糖替代物中，以保持糖醛基的整个结构，包括C-1的立体化学。β-糖基胺很容易由不同的糖在两步合成而不使用任何保护基团。具有各糖基片段的β-糖基胺对具有相应糖基底物特异性的β-糖苷酶具有高效的抑制作用（K_i<0.1μM），而具有不同糖基底物特异性的β-糖苷酶不受显著抑制。此外，可以在抑制剂的“agly…More con”部分引入不同的结构，以增加或调整对各种β-糖苷酶的抑制效力和选择性。因此，β-糖苷胺是一种多功能的β-糖苷酶抑制剂，其中的糖基和aglyon部分都可以根据被抑制的酶进行调整。这种性质可以成功地用作糖苷酶亲和层析的配体。因此，以β-氨基葡萄糖为配体的亲和吸附剂一步纯化茶叶β-葡萄糖苷酶，以β-氨基葡萄糖为配体的亲和层析一步纯化霉菌β-半乳糖苷酶。其他β-糖苷酶，如β-primeverosidase（一种新型的二糖苷特异性糖苷酶）也通过相应的糖氨基作为配体成功地进行了亲和纯化。对纯化得到的茶叶β-葡萄糖苷酶进行氨基酸序列分析，进行基因克隆。因此，从糖苷酶的分离、鉴定到基因克隆，β-糖基胺是糖苷酶研究中多功能的研究工具。少

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