Sialyltransferases, sialylated

Example 5 Hayakawa and Noyori group in their studies on new activators for phosphoroamidite coupling reactions have applied the most effective member of the group of acid/azole complexes AT-(phenyl)imidazolium tri-flate (N-PhIMT) in the efficient synthesis of biologically important compounds [20j]. A noteworthy example is synthesis of cytidine-5 -monophos-pho-AT-acetylneuraminic acid. This compound is a source of sialic acid in the sialyltransferase-catalysed biosynthesis of sialyl oligosaccharides [25]. 

This set of enzymes consists of those proteins that catalyze the transfer of sialyl moieties. (1) CMP-A-acetyl-neuraminate-j8-galactoside o -2,6-sialyltransferase [EC... 

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... 

A unique, membrane-associated, sialyltransferase system in E. coli has been described that catalyzes the synthesis of sialic acid polymers with the aid of sialyl-monophosphonoundecaprenol.293 In mammalian systems, no evidence was obtained lor the involvement of lipid intermediates in sialic acid transfer, or in the fonnation of sialyl-(2— -8) link-... 

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... 

D. Formation of the Sialyl 2,3-Linkage Using [Pg.497]

CMP-NeuAc synthetase (EC 2.7.7.43) to produce CMP-NeuAc. The by-product pyrophosphate (PPi) is hydrolyzed to phosphate (Pi) by inorganic pyrophosphatase (PPase). Sialyla-tion is accomplished with a2,3-sialyltransferase (< 2,3NeuAcT) or a2,6-sialyltransferase (a2,3NeuAcT), respectively. The released CMP is again converted to CDP, to CTP, and finally to CMP-NeuAc. The UDP-Gal and CMP-NeuAc regeneration schemes have been combined in a one-pot reaction and applied to the synthesis of sialyl Lewis X. 

G. F. Herrmann, Y. Ichikawa, C. Wandtey, F. C. A. Gaeta, J. C. Paulson, and C.-H. Wong, A new multi-enzyme system for a one-pot synthesis of sialyl oligosaccharides Combined use of P-galactosidase and a(2,6)-sialyltransferase coupled with regeneration in situ of CMP-sialic acid, Tetrahedron Lett. 34 3091 (1993). 

It was apparent, however, that the rat brain sialyltransferases have not been sufficiently characterized for the postulation of a biological role for the sialylation-desialylation cycle. Consequently, we concentrated our efforts to characterizing the general behaviors of the sialyltransferases in their membrane environments in the rat brain. What follows is a summary of our... 

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 clear distinction between the glycoprotein and glycolipid sialyltransferases suggests that the extent of sialylation of the two classes of complex carbohydrates in vivo may be under different metabolic controls. This notion is supported by the observation that the developmental profiles of the glycoprotein and glycolipid sialyltransferases are distinctly different. 

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. 

Compound 34 is a triantennary, sialo glycopeptide terminated with NeuAc a-(2- 6)-linked to Gal in all three branches it could be obtained by exhaustive, in vitro sialylation of compound 9 with /3-D-ga-lactoside a-(2— 6)-sialyltransferase.13,78 The Gln-Asn analog of compound 34, in admixture with compound 53 and a glycopeptide analog of 41, has been isolated from human-plasma ceruloplasmin (see compound 53). The 500-MHz, -n.m.r. spectrum of 34 is given in Fig. 26, and its pertinent spectral parameters are listed in Table X. 

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