Cytidine Monophosphate-Sialic Acid Synthetase

Cytidine monophosphate-sialic acid synthetase (EC 2.7.7.43) catalyzes the activation of /V-acetylneuraminic acid (NANA) by CTP to form CMP-NANA. 

The HPLC assay method developed for this activity involves the separation of the three compounds NANA, CTP, and CMP-NANA. However, at the wavelength used for detection, only the last two are detected. 

The synthetase was prepared from calf brains by homogenization, centrifugation (1500g for 20 min), and lyophilization of the supernatant solution. 

Succinyl-CoA synthetase participates in the Krebs cycle by catalyzing the reaction between sucdnyl-CoA, phosphate, and a nucleoside diphosphate to 

SURVEY OF ENZYMATIC ACTIVITIES ASSAYED BY THE HPLC METHOD 

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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. 

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.

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