Regeneration CMP-NeuAc

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. 

CMP-NeuAc regeneration has also been coupled to P-galactosidase (EC 3.2.1.23) activity (Scheme 9). [31] The biological role of glycosidases is the hydro-... 

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). 

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>... 

I. Combined Use of (3-Galactosidase and a2,6-Sialyltransferase with Regeneration of CMP-NeuAc [35]... 

D. Formation of the sialyl a2,3-linkage using a2,3-sialyltransferase without regeneration of CMP-NeuAc 497... 

F. Formation of the sialyl o2,6-linkage using a2,6-sialyltransferase with regeneration of CMP-NeuAc 499... 

Scheme 29 Large-scale synthesis of sialylated lactose using a CMP- ialir acid svnthetase/a2,3-sialyltra enzyme with in situ regeneration of CMP-NeuAc.

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