A- -sialyltransferase

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). 

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. 

Highly efficient chemoenzymatic synthesis of naturally occurring and non-natural a-2,6-linked sialosides a P. damsela a-2,6-sialyltransferase with extremely flexible donor-substrate 26. 

Attachment of neuraminic acid to )3-LacNAc-elymoclavine (11) yielding 19 was accomplished by the use of a-2,6-sialyltransferase from rat liver (EC 2.4.99.1) (Scheme 10). It was interesting that the affinity of the sialyltrans-ferase towards 11 was approximately 20% higher than that towards N-acetyl-lactosamine, the natural substrate of this enzyme. 

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... 

Muller, B., Martin, T.J., Schauh, C., and Schmidt, R.R., Synthesis of phosphonate analogues of CMP-Neu5Ac. Determination of a(2-6)-sialyltransferase inhihition, Tetrahedron Lett., 39, 509, 1998. 

NeuAc synthetase route [37]. The relative rates of these C9-modified CMP-NeuAc derivatives shown in Scheme 30 were compared against the natural donor CMP-NeuAc in sialyltransferase assays that utilized enzymes from different sources with their appropriate natural acceptors. The rat liver a-2,6-sialyltransferase tolerated a wide range of functional groups without signihcant decreases in the relative rates. Synthetically useful relative rates were observed for most of the CMP-NeuAc analogs with porcine sialyltransferase and rat liver a-2,3-sialyltransferase. The exception was the 9-amino analog, which was a poor substrate for both enzymes. Overall, these assays demonstrated that a wide variety of modihcations at the C9 position are tolerated by these sialyltransferases. 

The cascade begins with stoichiometric amounts of phosphoenolpyruvate (PEP), 8-allyl-A-acetyl lactosamine 120, NeuAc 1, and catalytic quantities of ATP and CMP. Initially, CMP is converted to CDP by nucleoside monophosphate kinase (NMK) in the presence of ATP. The CDP produced reacts with PEP under pyruvate kinase (PK) catalysis to form CTP. Next, CMP-NeuAc synthetase catalyzes the in situ formation of the sialyl donor from NeuAc and CTP. The pyrophosphate byproduct is decomposed to inorganic phosphate by inorganic pyrophosphatase (PPase). Subsequently, the a-2,6-sialyltransferase accomplishes the sialyation of the lactosamine acceptor 120 and produces the ttansferase inhibitor CMP as a by-product. The CMP concentrations are kept low by conversion to CDP, and in so doing the problem of product inhibition is minimized. The cycle afforded 21% of the sialylated ttisac-charide 121, which is remarkable considering the complexity of the system and number of synthetic steps that can be avoided. 

We are only beginning to understand the regulation of the expression of sialyltransferase activity. The higher level of sialylation of many tumor cells and increased sialyltransferase activities are well known (see chapter 3 of Vol. 30 and section 10.5 below]. Transformation of FR3T3 cells with the c-Ha-ras oncogene resulted in a marked increase of the expression of 3-galactoside a-2,6-sialyltransferase activity and. 

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