Issues of chemoselectivity and regioselectivity pose an enormous strain on the modern-day synthetic organic chemist. Most natural products are enantiomerically or diastereomerically pure, meaning that their conformation plays a specific role in the recognition of proteins for inhibition, suppression, or activation. Nature has developed an ingenious system to combat these problems and has allowed for an unsurpassable specificity in the biological system through the creation of enzymes. Enzymes are highly specific both in binding chiral substrates and in catalyzing reactions. This stereospecificity arises because enzymes, by virtue of their inherent chirality (proteins consist of only l-amino acids), form asymmetrical active sites. The required synthetic methodology for studying the structural features necessary for anti-Gal binding, and for the subsequent preparation of analog structures is made available through initial synthetic studies targeting the a-gal structure itself. The overall scheme of synthesis must remain flexible enough to accommodate varying structural motifs to allow for the preparation of analog structures, afford suitable quantities of material for biological analysis, and incorporate a minimum number of synthetic operations.
Traditional organic synthesis of oligosaccharides requires multiple protection and deprotection steps, including tedious separation procedures at each step. Gly-cosyltransferases play important roles in obtaining oligosaccharides, glycopeptides, and glycolipids under mild conditions. The high specificity in the formation of gly-cosides of glycotransferases makes them a viable strategic choice for the preparative synthesis of complicated oligosaccharides and glycoconjugates. Nevertheless, the limitations of quantity and purity of the glycosyltransferases available from natural sources have directed a great interest toward the cloning of the glycosyltransferase genes into convenient expression systems by recombinant DNA technology. Although several studies on cloning and characterization of a1,3-galactosyltransferase (a1,3-GT) have been reported, [25,26,46-49], no practical production of the recombinant enzyme has been accomplished.
There has recently been developed an efficient chemoenzymatic approach based on the use of a recombinant a1,3-GT for the synthesis of xenoactive a-galactosyl epitopes. A truncated bovine a1,3-GT (80-368) was cloned into pET 15b vector and subsequently transformed into an E. coli BL21 strain (Fig. 2) . This expression system efficiently produced the soluble recombinant enzyme on a large scale with high specific activity. Subsequent to the synthesis of galactosyl disaccharide acceptors, via chemical methods and enzymatic approaches involving glycosidase-cata-lyzed reactions, the recombinant enzyme was used to synthesize a variety of a1,3-galactosylated trisaccharides on a preparative scale. UDP-Glc was used as the donor, along with commercially available UDP-Gal 4-epimerase, to avoid direct use of the costly UDP-Gal. UDP-Gal-4-epimerase (galE), from E. coli, is a dimer of identical subunits, with an overall molecular weight of 79 kDa, which plays a key role in the Leloir pathway for galactose metabolism. Members of a series of glycosylation acceptors were examined, including lactose 6, 0-lactosylazide 8,  0-thiophenyl lactoside 9 , V-acetyllactosamine 5, 0-allyl-V-acetyllactosamine 10, and lactos-amine 7 (Fig. 3, p. 589). These dissaccharides served as acceptors to produce the trisaccharide a-gal epitopes and derivatives 11-16 in good yields. However, one of the main issues surrounding glycosyltransferase's commercial viability is the limited dynamics with the type of reaction it is involved in. The high cost factor of the sugar nucleotides (donor) required for biocatalysis necessitates the use of UDP-glucose 4-epimerase to alleviate some of the expense [53,54].
Genetic engineering has allowed for the generation of bifunctional proteins, which are formed from enzymes acting in sequence of cofactor regeneration. The construction of bifunctional enzymes consisting of a truncated bovine recombinant a1,3-GT (BGT) and an E. coli UDP-galactose 4-epimerase has made the a-gal ep-itope synthesis feasible by allowing for the conversion of an inexpensive sugar nu-cleotide (UDP-glucose) followed by glycosidation by a1,3-GT. The bifunctional enzyme, galE-a1,3-GT, was constructed by in-frame fusion of the Escherichia coli gene galE to the 3'-terminus of bovine a1,3-GT gene within a high-expression plas-mid. The incorporation of two enzymes into one plasmid vector reduces the amount of exhaustible materials required for the overexpression of particular proteins. This methodology allows for the reversal of genetic sequences producing two bifunctional proteins, termed galE-a1,3-GT and a1,3-GT-galE (Fig. 4), capable of catalyzing the same reaction with similar kinetic rates .
Since various immobilization techniques are available for the use of enzymes, the bifunctional protein technology can be utilized in a one-pot, enzyme-immobilized reaction system, facilitating preparation protocols. Bifunctional enzymes are also thought to have a greater reaction rate. This increase in the overall kinetics of the reaction may occur as a result of the substrate proximity to the binding sites of both enzymes. This technology can be exploited in constructing various bifunctional enzyme systems where the number of genetic sequences incorporated into the plasmid may depend solely on the product desired.
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