Method and composition for synthesizing sialylated glycosyl compounds
AssigneeScripps Clinic and Research Foundation
InventorsChi-Huey Wong, Yoshitaka Ichikawa, Gwo-Jenn Shen
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Abstract
The abstract isn’t available for this document.
Claims24
A method of synthesizing a sialylated glycosyl compound comprising reacting in an aqueous buffered solvent free of chelators that bind Mg.sup.+2 or Mn.sup.+2 at a temperature of about 15.degree. C. to about 40.degree. C. in the presence of each other:
- a sialic acid;
- a glycosyl compound:
- a CMP-sialic acid regenerating system that comprises CMP, a nucleoside triphosphate, a phosphate donor and catalytic amounts of an isolated and purified kinase to transfer a phosphate group from said phosphate donor and an isolated and purified nucleoside monophosphate kinase;
- a pyrophosphate scavenger and;
- catalytic amounts of an isolated and purified CMP sialic acid synthetase and an isolated and purified sialyl transferase having substrate specificity for said glycosyl compound.
A method of synthesizing sialyl N-acetyllactosamine comprising mixing N-acetyllactosamine, neuraminic acid, CMP, ATP, phosphoenolpyruvate, KCl, MgCl.sub.2, MnCl.sub.2, and catalytic amounts of nucleoside mono-phosphate kinase (EC 2.7.4.4), pyruvate kinase (EC 2.7.1.40), inorganic pyrophosphatase (EC 3.6.1.1), CMP-NeuAc synthetase (EC 2.7.7.43) and Sia.alpha.2.fwdarw.6Gal transferase (EC 2.4.99.1) with an aqueous buffer at pH 7.5 to form a reaction mixture and maintaining the reaction mixture under argon at about 25.degree. C. for a period of time sufficient to deplete said neuraminic acid.
A composition for use in sialylating a glycosyl compound comprising a buffered aqueous solution free of chelators that bind mg.sup.+2 or Mn.sup.+2, said solution also containing:
- a sialic acid;
- a CMP-sialic acid regenerating system that comprises CMP, a nucleoside triphosphate, a phosphate donor and catalytic amounts of an isolated kinase to transfer a phosphate group from said phosphate donor and an isolated purified nucleoside monophosphate kinase;
- a pyrophosphate scavenger;
- and
- a catalytic amount of an isolated and purified CMP sialic acid synthetase.
Dependencies are inferred from the claim text.