Transferases sialyltransferase

As an alternative to chemical sialylations, glycosyl transferases have been used to accomplish sialylations, particularly of N-linked glycopeptide building blocks (see also Section 6.3.1.1.5). The 2,3- and 2,6-sialyltransferases have been easily expressed in preparative amounts. Their use in glycopeptide building block synthesis is described in Section 6.3.3.6. 

Substrate-specificity studies on microsomal, frog-liver sialyltrans-ferase revealed the presence of (2—>3) and (2—>6) activities.277 This enzyme system readily sialylates oligosaccharides, but is almost inactive with asialofetuin, which is in contrast to the sialylation of oligosaccharides, as well as asialofetuin, by rat-liver sialyltransferase.278 The conclusion from this observation is that acceptor specificity of sialyl-transferases isolated from liver of evolutionary distant animals is similar for substrates of low molecular weight, but differs for compounds of high molecular weight.279... 

CMP, CDP, CTP, and synthetic derivatives of these nueleotides have been found to inhibit sialyltransferase activity.301" 02 Interest in such inhibitors is increasing, as they may be expected to serve as anticancer agents.269 901,303 Therefore, regulation of Golgi sialyltransferase activity appears possible by nucleotides as products of sialyl- and other glycosyl-transferase activities.1" 2 Interestingly, Epstein-Barr virus infection of human B, lymphoblastoid cell-lines leads to a diminution of sialyltransferase activity.304... 

Kinetic Properties of Sialyltransferases. The sialyl-transferase activities with the endogenous glycoprotein and glycolipid acceptors in the standard assays (15) were linear with time for at least 60 min, while those with the exogenously added GMi and DS-fetuin were linear with time only for about 30 min (Figure 1). Activities were directly proportional to the amount of enzyme added up to 0.75 mg protein/assay (Figure 2). 

The basis for the multiplicity of the sialyltransferase activities remains to be elucidated. We plan to purify these enzyme species to homogeneity, using isoelectric focusing columns of smaller pH ranges in conjunction with affinity chromatography which has been successfully used to purify the soluble sialyl-transferases from bovine colostrum (57). Possibility exists that the heterogeneity of sialyltransferase activities as observed is due to differences in polypeptide sequences, carbohydrate content, or non-covalent interactions with other membrane components, and these possibilities can be clarified only with highly purified enzyme preparations. 

SERPR, serine protease SialylT, sialyltransferase SLOX, soya bean 15-lipoxygenase, soya bean lipoxygenase SNF1K, SNF1 protein kinase kinase 80S PT, 80S ribosome (eukaryote) peptidyl transferase... 

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-... 

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,...

Schmidt and co-workers have been investigating a series of potent inhibitors of a(2,6)-sialyl-transferase (O Fig. 9). They developed the transition analog 42 [153], based on the proposed mechanism of the sialyl transfer, that involves the partial dissociation of the CMP and the formation of the planer oxocarbenium ion structure in the transition state. They also found that the planer neuraminyl moiety in 42 can be replaced by the aromatic groups, leading to the readily accessible aromatic inhibitors of a(2,6)-sialyltransferase from rat liver. Further library... 

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. 

The synthetic utilities of sialyltransferases as alternative methods to sialylate oligosaccharide and glycoconjugates have been further explored by Halcomb who described the synthesis of a novel anomeric sulfur analogue of CMP-sialic acid (103). The key step in the synthesis of this novel CMP-sialic acid was a tetrazole-promoted coupling of a cytidine-5 -phosphoramidite with a glycosyl thiol of a protected sialic acid. The rate of solvolysis of (103) in aqueous buffer was reported to be 50-fold slower than that of CMP-sialic acid. Thioester (103) was a substrate for the transferase with a three-fold higher than the of the... 

The relative rates of sialylation for the C5-modified CMP-NeuAc derivatives shown in Scheme 31 were also determined [37]. All the C5-modihed sialyl donors were accepted by the transferase enzymes, but the variants containing free amines were poor substrates for all the sialyltransferases examined. Generally, higher relative... 

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