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US 9,340,812 B2Granted
Large scale enzymatic synthesis of oligosaccharides
AssigneeAcademia Sinica
InventorsChi-Huey Wong, Tsung-I Tsai, Chung-Yi Wu
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Abstract
A novel UDP-Gal regeneration process and its combined use with a galactosyltransferase to add galactose to a suitable acceptor substrate. Also described herein are synthetic methods for generating Globo-series oligosaccharides in large scale, wherein the methods may involve the combination of a glycosyltransferase reaction and a nucleotide sugar regeneration process.
Claims32
A method for enzymatically synthesizing an oligosaccharide, comprising:
- (i) producing UDP-Gal from galactose in the presence of a set of UDP-Gal regeneration enzymes, wherein the set of UDP-Gal regeneration enzymes comprises a galactokinase, an UDP-sugar pyrophosphorylase, and a pyruvate kinase;
- (ii) converting Lactose-OR1A into Globotriose-OR1A (Gb3-OR1A) in the presence of the UDP-Gal and an alpha-1,4 galactosyltransferase, wherein R1A is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or an oxygen protecting group;
- (iii) converting the Gb3-OR1A into Globotetraose-OR1A (Gb4-OR1A) in the presence of UDP-GalNAc and a beta-1,3-N-acetylgalactosaminyltransferase;
- and
- (iv) converting the Gb4-OR1A into Globopentaose-OR1A (Gb5-OR1A) in the presence of UDP-Gal and a beta-1,3-N-galactosyltransferase,
- wherein the method is performed without purifying intermediates,
- wherein the method is performed with all enzymes free of cells.
4 more elements
Dependencies are inferred from the claim text.