CMP-NeuAc

A recent modification of this recycling system was in the expression of an a2,3-SiaT/CMP-NeuAc synthetase fusion protein [25]. This construct was more soluble than a2,3-SiaT alone, which is a poorly soluble transmembrane protein. The fusion 

The monosaccharide sialic acid remains relatively costly for preparative-scale synthesis. Although it has been demonstrated that NeuAc can be generated in situ by reaction of ManNAc with pyruvate, catalyzed by NeuAc aldolase [27], ManNAc is also relatively expensive and difficult to prepare. Therefore, a method for the generation of NeuAc from the inexpensive monosaccharide GlcNAc in a two-enzyme system might also prove useful in regeneration schemes. It has been shown that GlcNAc can be converted to ManNAc chemically [28], or enzymatically by the 

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N-Acetylneur-aminic acid Sialic acid (nine C atoms) NeuAc CMP-NeuAc Often the terminal sugar in both N- and 0-linked glycoproteins. Other types of sialic acid are also found, but NeuAc is the major species found in humans. Acetyl groups may also occur as 0-acetyl species as well as N-acetyl. 

The principle of the assay procedure for CMP-KDO synthetase had been developed previously, to assay for CMP-NeuAc synthetase in ex-... 

Scheme 10.3 Ceramide glycanase mediated release by transglycosylation. a) Pd/C, MeOH b) (8), EEDQ, EtOH-CgHe c) MeONa d) CH2=CHCONH2, TMEDA, APS, DMSO-H2O, 50"C, CMP-NeuAc, a-2,3-sialyltransferase, BSA, MnCb, CIAP, 50 nM sodium cacodylate buffer, pH 7.49, ceramide, Triton CF-54, sodium citrate buffer, pH 6.0, 37°C, 61%.

Scheme 2.2.5.25 Enzymatic activation of sialic acids to Leloir-type sugar nucleotides using the CMP-NeuAc synthetase of Neisseria meningitidis.

Sialosyllactosylceramide (GM3) is one of the major gangliosicles found in visceral organs. However, in the central nervous system, the concentration of this ganglioside is so low that it can only be considered to be the precursor for various complex gangliosides. A sialosyl-transferase that catalyzes the transfer of sialic acid from CMP-NeuAc to lactosylceramide was first described in the embryonic chicken-brain by Basu107 and Kaufman and coworkers,108 and in the brain of... 

The enzymatic preparation of the activated sugar nucleotide may also involve a cofactor regeneration system. An example of this is an economic one-pot procedure, in which N-acetylneuraminic acid (NeuAc) is generated in situ from IV-acetylmannosamine (ManNac) and pyruvate with sialic acid aldolase and then converted irreversibly to CMP-NeuAc ([14], see also Sec. III). 

CMP-NeuAc From NeuAc and CMP with CMP-NeuAc synthetase By chemical synthesis... 

In this one-pot procedure NeuAc 16 is generated from ManNAc 15 and pyruvic acid in situ with sialic acid aldolase and then converted irreversibly to CMP-NeuAc 17. CMP is converted to CDP with myokinase and ATP. The released ADP is converted to ATP with pyruvate kinase and PEP. CDP is then converted to CTP also with pyruvate kinase and phosphoenolpyruvate (PEP). The formed CTP reacts with NeuAc catalyzed by NeuAc synthetase to give 17. 

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%). 

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

The regeneration system for CMP-NeuAc can be employed both for a2,3-sialyltransferase-catalyzed reactions and for reactions mediated by a2,6-sialyltransferase. The system starts with NeuAc, the glycosyl acceptor, PEP, and catalytic amounts of ATP and CMP. CMP is converted to CDP by nucleoside monophosphate kinase (EC 2.7.4.4 NMK) in the presence of ATP, which is regenerated from the by-product ADP, catalyzed by PK in the presence ol PEP, then to CTP with PEP by PK. The CTP thus formed reacts with NeuAc, catalyzed b>... 

E i a2,6-sialyKransferase E2 nucleoside monophosphate kinase or adenylate kinase E3 pyruvate kinase E4 CMP-NeuAc synthetase E5 pyrophosphatase... 

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. 

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