Various models have been proposed for the reaction mechanism of glycosylase in the transition state, 1 4) but a reasonable model remains to be established. The oxocarbonium ion model has been applied to interpret the reaction mechanism of many glycosylases such as lysozyme, 5,6) glucoamylase 7) and glucosidase. 8) In this mechanism, when the glycosidic bond is cleaved, a glycosyl oxocarbonium ion intermediate is formed for catalysis. Evidence of the role of the transition state intermediates came from inhibition studies involving validamine, 9) N substituted valiolamine derivatives, 10) acarbose, 11) nojirimycin, 12) 1 deoxy nojirimycin 13) and N substituted derivatives of 1 deoxynojirimycin. 14) These inhibitors have been designed, synthesized and purified from plant extracts or the culture broth of microorganisms as the analogues of the charge and or the shape of the oxocarbonium ion like transition state intermediates. 15) They have an easily pro-tonated basic nitrogen atom in the position of the anomeric oxygen or the ring oxygen atom. However, the incorporation of a nitrogen positive center into a pyranose ring requires a relatively long reaction sequence. 16) As its structural similarity with the oxocarbonium ion like transition state intermediates, many researchers have performed their inhibition studies on glycosylases using glucono 1,5 lactone. 17 19) This compound is a relatively strong inhibitor toward glucosidases and has no nitrogen atom in a pyranose ring. However, it has been pointed out that their tendency to undergo hydrolytic ring opening under mildly basic conditions and the conversion into glucono 1,4 lactone (inactive) are the great disadvantage of the measurement of inhibition constants. 18,19) We therefore attempted to prepare more convenient glucosidase inhibitors by enzymatic method, which have rather simple chemical structures without a nitrogen atom in the position of the anomeric oxygen or the ring oxygen atom, and are quite stable in an aqueous solution. MATERIALS AND METHODSMaterials. Cyclopropanemethanol (CPAM) was purchased from Kanto Kagaku Co., Ltd. (Japan). Cyclopentanol (CPE) and cyclopentanemethanol (CPEM) were obtained from Lancaster Co., Ltd. (England). p Nitorophenyl glucopyranoside (pNP G) was a product of Nihon Shokuhin Kako Co., Ltd. (Tokyo). The highly purified glucosidases from Aspergillus niger and Trichoderma viride were generous gifts from Dr. T. Unno, Nihon Shokuhin Kako Co., Ltd. (Japan) and Dr. G. Okada, Shizuoka University (Japan), respectively. The purified sweet almond glucosidase was purchased from Sigma Aldrich (USA). These purified glucosidases were used for the inhibitory studies. On the other hand, glucosidases from Trichoderma reesei (Cellcrust) and A. niger (Novozyme 188) were obtained from Novo Nordisk A S, and the enzymes from T. viride (Meicelase) and sweet almond (BGH 101) were purchased from Meiji Seika Kaisha, Ltd. (Japan) and Toyobo Co., Ltd. (Japan), respectively. These enzymes were used for the preparation of cycloalkyl D J. Appl. Glycosci., 5...

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