Introduction

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Carbohydrates are organic molecules made of sugars and their polymers. One of the main functions of carbohydrates in animals is to provide an energy source. Besides their role in energy storage, a variety of carbohydrates are distributed in cells and tissues. A part of carbohydrates conjugated with lipids have been isolated from many kinds of plants as bioactive ingredients and were known to play a crucial role in various cellular processes, including bacterial and viral infection, cancer metastasis, modulation and activation of the immune system, tissue differentiation and development and many other intercellular recognition events.1 Among them, there are many P-D-glucopyranosides possessing a primary alcohol moiety as an aglycone part in nature. The development of stereoselective methods for the synthesis of glycosidic linkages presents a considerable challenge to synthetic chemists.2 3 Although well-developed chemical synthesis of the glycosidic structure is increasingly being established, several steps of selective protection, activation and coupling are necessary. Especially in the absence of participating neighboring groups, it leads to an a and P anomeric mixture and requires tedious chromatographic purification (Fig. 1, conventional approach). This problem in chemical synthesis has promoted the development of enzymatic approaches.

Figure 1: Glucosidation method based on conventional approach.

4) Separation

Figure 1: Glucosidation method based on conventional approach.

The enzymatic formation of glycosidic linkages is being recognized as an efficient method to synthesize a variety of carbohydrates.4 The major advantages of enzymatic approach is that the method could directly produce completely controlled stereochemistry at the newly formed anomeric center due to specific reactivity and substrate recognition of the enzyme. It follows that protection-deprotection sequences could be avoidable in the case of enzymatic approach (Fig. 1, enzymatic approach). Lipase-catalyzed synthesis of acyl sugar is reported,5 whereas much less is known about glycosidase-catalyzed synthesis of alkyl glycosides.6 Glycosidases are responsible for the formation of the glycosidic linkage and are increasingly being used in carbohydrate synthesis. There are two basic ways of using glycosidases to provide glycosides: kinetically controlled transglycosilation and equilibrium-controlled reverse hydrolysis (Fig. 2).

In kinetically controlled transglycosilation, a reactive intermediate from an activated glycosyl donor (e.g., 4-nitrophenyl O-P-D-glycoside, glycosyl fluoride) reacts with exogenous nucleophiles in the reaction medium to generate a new glycosidic bond. This approach depends on the more rapid trapping of the reactive intermediate by the glycosyl acceptor than by water. Indeed, enhanced rates of glycosidase-catalyzed glycosyl cleavage have been observed in the presence of alcohols. This could be due to more effective binding of the alcohol at the active site relative to water. Another proposed rationalization is that the mechanism involves a solvent-separated ion pair toward which an alcohol is a better nucleophile than water. While under proper conditions glycoside synthesis may be favored kinetically, hydrolysis is favored thermodynamically. On the other hand, the reverse hydrolysis procedure is based on the shift of the reaction equilibrium, normally in favor of hydrolysis of the glycosidic bond in aqueous medium, toward synthesis (Fig. 2). In efforts to shift the equilibrium toward desired products, the addition of water-miscible organic co-solvents and the use of high substrate concentrations have been explored.

Enzymatic formation of a glycosidic bond is thought to be mechanistically similar to the acid-catalyzed formation of glycosides.4 The active site of p-glucosidase was constructed with two carboxylic acid parts which play the important role of catalyzing the hydrolysis of glycosidic linkages. One is the carboxylate ion which acts as a general base and the other is carboxylic acid which acts as a general acid. When the substrate is brought close to the active site of the enzyme, the oxocarbenium ion with a-configuration at the anomeric carbon as shown in Fig. 3 was formed. This oxonium ion or the enzyme-bound glycosyl cation was stabilized by an ion pair intermediate or covalent bonding and can be captured by an alcohol to yield a glycoside. Nucleophilic alcohol presumably attacks the anomeric carbon from the p-side to afford exclusively p-glucopyranoside.

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