B Clonezyme Using a Glycosidase Library to Build Disaccharides

Over the past few years, glycosidases have been widely used for the preparation of a broad spectrum of glycoconjugates and derivatives employing either transglyco-sylation or reverse hydrolysis reactions [56-60]. To find biocatalysts with unprece-

Figure 3 Synthesis of a1,3-gal trisaccharides achieved by using a1,3-galactosyltransferase and UDP-galactose-4-epimerase.
Figure 4 Bifunctional proteins a1,3-GT-ga/E and ga/E-a1,3-GT.

dented properties from vast untapped resources of extremophiles, Diversa Inc. developed recombinant technologies and robotic screening approaches and ultimately produced a novel glycosidase library, namely, the CLONEZYME™ library, which contains nine unique, thermostable glycosidases [61-63]. Glycosidases are attractive for the construction of disaccharides primarily because of their commercial availability, low cost, and high stability [64-70]. N-Acetyllactosamine (5) not only is one of the most fundamental oligosaccharide sequences in glycoproteins and glycolipids but also serves as an important building block for enzymatic synthesis of a-gal epitopes and other complex sequences. With O-nitrophenyl-5-galactopyranoside (17) as the glycosylation donor and N-acetylglucosamine (18) as the acceptor (Fig. 5), the enzymatic reactions were carried out at 77°C; nine different CLONEZYME glycosidases, which all exhibited ¡-galactosidase activities, were used, as well as galactosidases from other sources for comparison. The results indicated that three out of the nine thermophilic enzymes in the library, Gly001-06, -07, and -09, had superior activity in catalyzing the glycosylation. No other regio- or stereoisomers were detected from the reactions using these three enzymes, and the hydrolysis rates of the disaccharide were considerably lower. Some of the catalytic yields were as high as 60%.

The same CLONEZYME library was examined for catalyzing the tandem trans-glycosylation of lactose (6) and glucosamine hydrochloride salt (19) to form lactos-amine (7) [71]. Enzymes Gly-06 and Gly-09 exhibited excellent regio- and stereoselectivity in the formation of lactosamine as well as moderate yields of 19 and 23%, respectively. It was discovered that when glucosamine hydrochloride (19) was used as an acceptor in a similar transgalactosylation reaction, the product lactosamine 7 could be synthesized on a multigram scale (Fig. 6).

One of the drawbacks associated with the glycosidase-catalyzed glycosylation reactions is the difficulty of separating the product from the reaction mixture, which may contain very similar compounds (molecular weight and corresponding linkages)

OH AcHN OH

17 18 5

Figure 5 Synthesis of N-acetyllactosamine with CLONEZYME™ glycosidase library.

Figure 6 Synthesis of lactosamine via glycosidase-catalyzed transgalactosylation reaction.

[70]. Various separation methodologies, such as size-exclusion chromatography (SEC), ion exchange chromatography (IEC), HPLC, and charcoal adsorption, have been evaluated for isolation efficiency [53,72-77]. SEC, which is conceivably the most used method for separating enzyme-synthesized oligosaccharides, is limited to compounds that have significant molecular weight differences. In contrast, IEC is frequently used to isolate biomolecules such as nucleic acids and proteins. The use of IEC in carbohydrate chemistry was reported for exploiting the weakly acidic character of polyhydroxyl groups of "neutral" sugars [78]. This large-scale synthesis was facilitated through the development of a resin column for cation (for purification) and anion (for neutralization) exchange. When positively charged amino sugars at neutral pH were used, separation was accomplished by means of a cation exchange resin with dilute HCl as eluent. Effective separation of the charged species from the noncharged species allowed for purification/separation from the residual by-products. The versatility of the cation and anion exchange resin column extends into the purification of a mono- and disaccharide mixture by adjusting the concentration of the eluent HCl (Fig. 7).

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