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

本研究旨在开发新型糖苷酶抑制剂，并从生物有机的角度将这些抑制剂作为糖苷酶研究的通用研究工具。为此目的，我们设计并合成了f -糖基脒衍生物作为β-糖苷酶的高效和糖基选择性抑制剂。β-糖基酰胺是一种底物类似物，其中带正电荷的脒基以维持糖基的整个结构（包括C-1的立体化学）的方式并入糖替代物中。β-葡糖基酰胺容易地由各种糖分两步合成，而不使用任何保护基团。具有不同糖基的β-葡糖胺对具有相应糖基底物特异性的β-葡糖苷酶有很强的抑制作用（Ki <0.1μM），而对具有不同糖基底物特异性的β-葡糖苷酶无明显抑制作用。此外，可以将不同的结构引入到“agly”中， ...更多信息 抑制剂的“con”部分以增加或调节对各种β-糖苷酶的抑制效力和选择性。因此，β-糖基脒是通用的β-糖苷酶抑制剂，其中糖基和苷元部分都可以根据待抑制的酶进行调节。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-葡萄糖脒为配基的亲和吸附剂一步纯化了茶叶β-葡萄糖苷酶，以β-半乳糖脒为配基的亲和层析一步纯化了霉菌β-半乳糖苷酶。其他β-糖苷酶如β-樱草糖苷酶（一种新的双糖苷特异性糖苷酶）也成功地用相应的糖基酰胺作为配基进行了亲和纯化。对纯化的茶叶β-葡萄糖苷酶进行氨基酸序列分析，克隆基因。因此，β-葡糖胺是从分离、表征到基因克隆的糖苷酶研究中的通用研究工具。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