CMP-Sialic acid synthetase

A-Acetyl-9-deoxy-9-fluoroneuraminic acid (591) was prepared by treatment of a protected 6-hydroxyl precursor with A, A-diethylaminosulfur trifluoride (DAST) or through condensation of 2-acetamido-2,6-dideoxy-6-fluoro-D-mannopyranose with potassium di(/ >r/-butyl) oxaloacetate. Compound 591 is a substrate for cytidine monophosphate (CMP)-sialic acid synthetase, giving rise to CMP-5-A-acetyl-9-deoxy-9-fluoroneuraminic acid, which is cytotoxic against tumor cells. 5-A-Acetyl-3-fluoroneuraminic acids 592-594 were prepared through fluorine (or acetyl hypofluorite) addition (in AcOH) to methyl 5-acetamido-4,7,8,9-tetra-0-acetyI-2,6-anhy-dro-2,3,5-trideoxy-D- /ycm>D- a/arto-non-2-enopyranosate. Compound 592 was found to be a potent neuraminidase inhibitor. 

To HEPES buffer (100 mL, 200 mM, pH 7.5) were added ManNAc 15 (1.44 g, 6 mmol), PEP sodium salt (1.88 g, 8 mmol), pyruvic acid sodium salt (1.32 g, 12 mmol), CMP (0.64 g, 2 mmol), ATP (11 mg, 0.02 mmol), pyruvate kinase (300 U), myokinase (750 U), inorganic pyrophosphatase (3 U), /V-acctylneuraminic acid aldolase (100 U), and CMP-sialic acid synthetase (1.6 U). The reaction mixture was stirred at room temperature for 2 days under argon, until CMP was consumed. The reaction mixture was concentrated by lyophilization and directly applied to a Bio-Gel P-2 column (200-400 mesh, 3 x 90 cm), and eluted with water at a flow rate of 9 mL/h at 4°C. The CMP-NeuAc fractions were pooled, applied to Dowex-1 (formate form), and eluted with an ammonium bicarbonate gradient (0.1-0.5 M). The CMP-NeuAc fractions free of the nucleotides were pooled and lyophilized. Excess ammonium bicarbonate was removed by addition of Dowex 50W-X8 (H+ form) to the stirred solution of the residual powder until pH 7.5. The resin was filtered off and the filtrate was lyophilized to yield the ammonium salt of CMP-NeuAc 17 (1.28 g, 88%). 

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.

Scheme L Synthesis of a2,64inked sialyl-N-acetyllactosamine using a one-pot multi-enzyme system with in situ regeneration of CMP-Neu5Ac. Abbreviations for enzymes CSS, CMP-sialic acid synthetase NMK, nucleoside monophosphate kinase PK, pyruvate kinase PPase, pyrophosphatase. Abbreviations for compounds PEP, phosphoenolpyruvate ADP, adenosine 5 -diphosphate ATP, adenosine 5 -triphosphate CMP, cytidine 5-monophosphate CDP, cytidine 5 -diphosphate CTP, cytidine 5-triphosphate LacNAc, N-acetyllactosamine NeuSAc, N-acetylneuraminic acid PPi, inorganic pyrophosphate.

Table 9 A Limited Number of Sialic Acid Derivatives Are Recognized as Substrates for CMP-Sialic Acid Synthetase from Bovine Brain and Rat Liver...

Figure 22 Biosynthesis of sialosides. Sialic acid aldolases catalyze the aldol condensation of W-substituted mannosamines with pyruvate to produce sialic acids. CMP-sialic acid synthetases produce CMP-sialic acid from CTP and sialic acid. Sialic acid is transferred to acceptor molecules by sialyltransferases.

Liu JLC, Shen GJ, Ichikawa Y, Rutan JF, Zapata G, Vann WF, Wong CH. Overproduction of CMP-sialic acid synthetase for organic synthesis. J Am Chem Soc 1992 114 3901-3910. 

Karwaski M-F, Wakarchuk WW, Gilbert M. High-level expression of recombinant Neisseria CMP-sialic acid synthetase in Escherichia coli. Protein Expression Purif 2002 25 237-240. 

It is uncertain how far sialic acids are recovered for re-usage in the majority of animal tissues. In principle most sialic acids can be re-activated by CMP-sialic acid synthetase to form CMP-sialic acids. However, the enzyme is nuclear and may be unavailable, or other processes might limit access to it (Von Rinsum et aL, 1983). In any case, free sialic acids are fairly unstable and unless they were quickly trapped, appreciable spontaneous degradation might occur. Sialic acids are not direct precursors of other classes of sugars, but the various forms can be interconverted among themselves to an extent which is substantial in some tissues. 

One important exception to this is CMP sialic acid synthetase, which is largely and, probably, entirely, a soluble enzyme of the nucleoplasm (Von Rinsum et al, 1983). In this respect it is unique among the enzymes that synthesise sugar nucleotides. 

Two sulfone-based nucleoside diphosphate isosters (71a,b) has been synthesised and reported by Gervay-Hague to be inhibitors of Neisseria meningitidis CMP-sialic acid synthetase, which is a key enzyme in the biosynthesis of capsular polysaccharides required for bacterial infection. The synthetic methodology includes a condensation reaction of the nucleoside aldehydes with bisphosphonate Horner-Wadsworth-Emmons reagents (Scheme 4). The deprotection sequence was crucial for the appropriate completion of the synthetic targets. 

The final step in the activation of sialic acid to a nucleotide sugar occurs by the reaction shown above. It is clear that this reaction is unique in the sense that (1) the monosaccharide itself is the substrate rather than the more common 1-phosphate, and (2) the nucleotide sugar contains only one phosphate group rather than two. Like several of the other enzymes participating in sialic acid biosynthesis, CMP-sialic acid synthetase can utilize the N-glycolyl as well as the N-acetyl derivative. 

Neisseria meningitidis CMP-sialic acid synthetase NmCSS... 

A strategy of chemoenzymatic synthesis of C8-modified sialic acid analogues was recently reported by Withers and co-workers (Scheme 3) [83], The C8-modified sialic acid precursors 8-11 were synthesized from compounds 5-7, and each was converted to its CMP donor using a bacterial CMP-sialic acid synthetase. The Cst-1, an a 2,3-sialylatransferase from Campylobacter jejuni, was then selected and successfully used for the synthesis of sialyl thiolactoside 12-15. Notably, Cst-1 was shown to be more desirable for the synthesis of C8-modified sialyl lactose in comparison with another sialyltransferase PM0188h, which exhibited more hydrolysis activity toward natural substrates. 

The finding that CMP-sialic acid synthetase can accept many sialic acid analogs has facilitated the synthesis of CMP-sialic acid analogs for studies of the donor-specificity of these enzymes [88-90]. Both a2,3-SialT and a2,6-SialT tolerate substitutions at C-9 of CMP-sialic acid (Schemes 6 and 7) [89, 91, 92]. 

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