Enzymatic 02,6-sialyltransferase

Figure 6. Effect of CMP-NeuAc concentration (V/S), of pH (V/pH), of enzymatic protein concentration (V/protein), and of incubation time (V/t) on the activity of synaptosomal membrane-bound sialyltransferase. Calf brain cortex. Acceptor substrates for sialyltransf erase (if) lactosylceramide ( ) desialylated fetuin (%) endogenous glycoprotein (endogenous glycolipids.

Fig. 8. Enzymatic glycosylation in the solid-phase synthesis of oligosaccharides elongation of the glycopeptide with /M, 4-galactosyltrans-ferase and a-2,3-sialyltransferase. The glycopeptide is finally released by cleavage with a-chymotrypsin.

Enzymatic glycosylation on SP was first reported by Schuster et al. (157) and since then has been used to perform multiple glycosylations. Two examples are reported in Fig. 2.28. The synthesis of oligosaccharides 2.94 and 2.95 related to the sialyl Lewis X antigen was carried out via glycosylations mediated by P-l,4-galactosyltransferase and a-2,3-sialyltransferase (171) and by p-l,4-galactosyltransferase, a-2,3-sialyl-transferase, and fucosyltransferase (172), respectively. The use of unprotected saccha-... 

Sialyltransferases catalyze the transfer of a sialic acid residue from CMP-sialic acid to a galactose, GalNAc, or another sialic acid residue. Sialyltransferases from bacterial and mammalian sources have been extensively studied and used in the enzymatic synthesis of sialosides, sialoglycoconjugates, and enzymatic modiflcation of cell surface. 

A bifunctional sialyltransferase Cst-II from C. jejuni OH4384 carrying both a2,3- and a2,8-sialyltransferase activities was used in the enzymatic synthesis of ganglioside mimic GD3 (66). 

Figure 2 One-pot three-enzyme chemoenzymatic synthesis of sialosides containing sialic acid modifications. In this strategy, mannose or ManNAc derivatives are chemically or enzymatically synthesized. These compounds are then used by a recombinant coli K-12 sialic acid aldolase to obtain sialic acids and their derivatives followed by an N. meningitidis CMP-sialic acid synthetase for the formation of CMP-sialic acids. From which, sialic acids can be transferred to lactose, LacNAc, galactose, GalNAc, or their derivatives by a multifunctional P. muitocida sialyltransferase (PmSTl) or a P, damseia a2,6-sialyltransferase (Pd2,6ST) to form a2,3- or a2,6-linked sialosides in one pot without the isolation of intermediates.

Yamamoto T, Nagae H, Kajihara Y, Terada I. Mass production of bacterial alpha 2,6-sialyltransferase and enzymatic syntheses of sialyloligosaccharides. Biosci. Biotechnol. Biochem. 1998 62 210-214. 

Fig. 7. Enzymatic synthesis with integrated cof actor regeneration (Enzymes iii, vi cf. Fig. 2, viii sialate-cytidyl-transferase, ix a2-3-or a2-6-sialyltransferase,...

Since the chemical synthesis of oligosaccharides requires many synthetic steps including protection and deprotection procedures, the enzymatic approach has attracted much attention for the rapid s)uithesis of oligosaccharides. In addition, the perfect regio- and stereoselectivities of enzymatic methods with glycosyltransferases are quite attractive. Several transferases such as 8(l,4)-galactosyltransferase, a(l,3)-fucosyltransferase, and a-sialyltransferase have been used for polymer-supported enzymatic synthesis [70,71,72,73,74,75]. The selection of the pol)uner support is very important for the pol)uner-supported enz)unatic synthesis of oligosaccharides. 

Starting from lactose as donor and A -acetylglucose as acceptor substrate the -galactosidase from Bacillus circulans gives exclusively A -acetyl-lactosamine 21 [61], which has been subjected to sialyltransferase-catalyzed conversion to the trisaccharide Neu5Ac-a(2-3)Gal-y8(l-4)GlcA Ac 22 (Scheme 12). By combination of these two enzymatic steps the reaction is pushed toward the product and the problem of low yields related with the galactosidase-mediated reaction could be overcome [62]. 

A simplified one-pot two-step enzymatic approach was used by James Paulson s group to produce sialoside derivatives (23). Instead of recycling the CMP-sialic acid, the sialyltransferase donors with modifications at C-5 or C-9 position of NeuSAc were enzymatically synthesized from ManNAc or its C-2 or C-6 modified derivatives, pyruvate, and CTP using a sialic acid aldolase and a CMP-Neu5Ac synthetase/sialyltransferase fusion protein. After removing the protein by membrane filtration, the filtrate containing produced CMP-sialic acid... 

Photobacterium damsela (x2,6-siaiyltransferase (Pd2,6ST) was the first bacterial sialyltransferase which has been cloned and purified by the Yamamoto group (33, 34). This enzyme has a relaxed acceptor specificity (35, 36). For example, it has been applied for the enzymatic sialylation of Tn glycopeptides with GalNAc a-linked to either serine or threonine residue) (37). It was also shown to be able to transfer sialic acid to both N- and 0-linkM glycoproteins (38). 

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