A-2,6-Sialyl transferase

In this technique, the enzyme solution is put inside a dialysis bag which is then immersed in a solution of substrate, or cofactors. Small molecules can diffuse through the wall of the bag and react in the presence of the enzyme, while products, if also small molecules, diffuse into the outside solution, where they may be recovered. This technique has been used in syntheses with sialyl aldolase, Kdo-synthetase, the common aldolase, a mixture of hexokinase and pyruvate kinase, a-(2— 6) sialyl transferase,26 a mixture of pyruvate kinase and adenylate kinase,27 and CMP-Neu5Ac synthetase.28... 

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... 

Paulson, J. C., Weinstein, J., and de Souza-E-Silva, U., 1984, Biosynthesis of a disialated sequence in N-linked oligosaccharides Identification of an N-acetylglucosaminide a(2-6) sialyl transferase in Golgi apparatus from rat liver, Eur. J. Biochem. 140 523-530. 

McCaffrey, G. and Jamieson, J.C. (1993). Evidence for the role of a cathepsin D-like activity in the release of gal-beta-1 -4glcnac-alpha-2-6-sialyl transferase from rat and mouse-liver in whole-cell systems. Comp. Biochem. Physiol. B-Biochem. Mol. Biol., 104,... 

Introduction of a fluoride at the 3-position of neuraminic acid yielded a compound (57) that was a competitive inhibitor of the a-(2,6)-sialyl-trans-ferase. Base and sugar-modified analogues of CMP-Neu5Ac have also been prepared to investigate the tolerance of oc-(2,6)-sialyl-transferase to base exchange (58) and modification of the 5-, 8- or 9-position of neuraminic acid (59), (60). While base-exchange was not tolerated, modifications of the acid moiety yielded compounds that were substrates for the enzyme. 

The 9-amino derivative was used to attach a fluorescenyl photolabel [83]. All the donor substrates listed in the table have been probed on various a(2-6) and a(2-3) sialyl transferases [84]. 

N.m.r.-spectroscopic studies have furthennore shown that sialyl-transferase from bovine colostrum preferentially incorporates sialyl residues into a-(2— 6) linkages of glycopeptides from oq-acid glycoprotein.162... 

Glycosyl transferases are quite powerful for stepwise addition of glycosyl units to existing (neo)glycoproteins. Paulson et al. [36] used a-2,3- and a-2,6-specific sialyl transferases to resialylate red blood cells that had been extensively desialylated enzymatically. Since... 

Efficient, stereoselective sialylations are still a cumbersome challenge for synthetic carbohydrate chemists due to the lack of neighboring group participation of the sialic acid [29]. Transferase-catalyzed sialylations therefore offer a welcome synthetic alternative. To date, eight different sialic acid linkage types have been identified. Out of the more than a dozen different sialyltransferases - SiaT - that have been found and cloned [99] a rat liver a(2-6)SiaT (E.C.2.4.99.1) and porcine a(2-3)SiaT (E.C.2.4.99.4) are commercially available. The synthesis of the natural CMP-sialic acid donor and that of various derivatives have been described [39, 48, 100, 101]. The capability for transfer of donor analogs by a(2-6)SiaT and rat liver a(2-3)SiaT (E.C.2.4.99.6) has recently been compiled [28,48]. 

Scheme 10.2 Oligosaccharide synthesis and a-chymotrypsin catalyzed release from the solid support, a) Z-Phe-NH-(CH2)6-OH (7), CSA, (CHCljjz, 70°C b) Hj, Pd/C, MeOH, 50°C c) CH2 = CHCONH(CHjjsCOOH (8), EtOH-CeHe d) MeONa (cat.), MeOH/THF, CH2 = CHCONH2, TMEDA, APS, DMSO-H2O, SOT e) Galactosyl transferase, Sialyl transferase g) CT, Tris-HCI buffer, pH 7.8, 48 °C, 72% from (10).

Sialyl- and L-fucosyl-transferases have been shown to transfer their respective sugars to the terminal /S-D-Gal/ -(l 4)-D-Glc/7iVAc sequence of L-asparagine-linked oligosaccharides forming a-Neu/7VAc-(2 6)-j8-D-Gal/>-(l 4)-d-... 

In the earliest stage when the neurons are perikaryoform cells without real neurites, only gangliosides of the short-chain lactosyl and gangliotriosyl species (GM3, GM2, GD3, and GD2) are sialylated. Hence, at this stage there is a productive expression of activity only of sialyl transferases 1 and 2 (see Chapter 3) which respectively synthesize GM3 and GD3. GM2 (not shown) and GD2 are the products of V-acetyl galactosaminylation of GM3 and GD3 (see Chapter 6). 

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