D A Unique Chemoenzymatic Synthesis of gal Epitope

Amino sugars (e.g., glucosamine, galactosamine, mannosamine) are widely distributed in living organisms, where they constitute building blocks of glycoconjugates such as glycopeptides and glycolipids [80,81]. They are found in milk, in blood group substances, and in lipopolysaccharide antigens, where they serve as part of the cell surface antigenic determinants (epitopes) [1,32,82-85]. The chemical synthesis of oligosaccharides containing amino sugar moieties (amino oligosaccharides) relies on glycosylation employing the oxazoline method or phthalimido protecting scheme [86]. Enzyme-catalyzed synthesis of oligosaccharides has evolved into a powerful shortcut for those chemical strategies [53,72-74]. Thus we have focused our attention on the chemoenzymatic synthesis of a-gal analogs containing versatile handles, such as free amino (NH2) groups, which can be used for further synthetic manipulation to generate carbohydrate diversity [50]. These derivatives would present the possibility of identifying unnatural ligands with enhanced binding affinity toward anti-Gal antibodies. The synthesis of the intermediate 24 was accomplished by both enzymatic and chemical methods. The azido group was purposefully introduced for its synthetic flexibility in the solid phase synthesis of glycopeptides, gly-copolymers, and glycodendrimers [87,88]. Another benefit for using the azido functionality is the ease with which functional group interconversion can allow for a variety of useful glycosylation functionalities such as glycosyl fluorides for orthogonal oligosaccharide synthesis. Compound 23 did not undergo glycosylation with UDP-Gal and ยก1,4-galactosyltransferase; however, it still served as a substrate for the thermophilic enzyme Gly001-09 to yield the important intermediate amino tetra-saccharide 24 in 11% after purification with IEC (Fig. 9).

In comparison, tetrasaccharide 27 was also prepared on the basis of chemical transformation, which could be thought of as a precursor of 24. The various chemical steps introduced led to the disaccharide acceptor 26, which was utilized in conjunction with donor 25 to form the important tetrasaccharide via a Schmidt-type glycosylation procedure (Fig. 10) [89]. The relevance of this methodology allows for a chemoenzymatic approach to the synthesis of an important intermediate.

Bifunctional protein (galE-a1,3-GT) containing both uridine-5'-diphospho-ga-lactose 4-epimerase (galE) and a1,3-galactosyltransferase (a1,3-GT) was used as a catalyst with UDP-glucose (UDP-Glc) as a glycosylation donor for the enzymatic approach to pentasaccharide 28 (Fig. 11). The important characteristic to 28 is the unprotected amino group, which was synthesized in an attempt to determine the inhibition activities toward human anti-Gal IgG. Bifunctional protein galE-a1,3-GT

Figure 8 Synthesis of a-D-Galp-(1-3)-0-D-Galp-(1-4)-0-D-GlcpNAc-(1-3)-0-D-Galp-(1-4)-0-D-Glci>N3 pentasaccharide 22 with in situ cofactor regeneration.

2 steps

Figure 9 Utilizing CLONEZYME library for the enzymatic synthesis of intermediate 24.

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