Large scale enzymatic synthesis of oligosaccharides

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.

1 - 34 . (canceled) 
     
     
         35 . A method for enzymatically synthesizing an oligosaccharide, comprising:
 (i) producing UDP-GalNAc from GalNAc in the presence of a set of UDP-GalNAc regeneration enzymes, wherein the set of UDP-GalNAc regeneration enzymes comprises an N-acetylhexosamine 1-kinase, an N-acetylglucosamine 1-phosphate uridyltransferase, a pyruvate kinase, and optionally, a pyrophosphatase, and   (ii) converting Gb3-OR 1A  into Gb4-OR 1A  in the presence of the UDP-GalNAc and a beta-1,3-N-acetylgalactosaminyltransferase, wherein R 1A  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.   
     
     
         36 . The method of  claim 35 , wherein (i) and (ii) occur in a Gb4-synthesis reaction mixture comprising GalNAc, PEP, ATP, UTP, the Gb3-OR 1A , the beta-1,3-N-acetylgalactosaminyltransferase, and the set of UDP-GalNAc regeneration enzymes. 
     
     
         37 . The method of  claim 35 , wherein the beta-1,3-N-acetylgalactosaminyltransferase is LgtD from  H. influenza , the N-acetylhexosamine 1-kinase is from  B. longum , the N-acetylglucosamine 1-phosphate uridyltransferase is from  E. coli , the pyruvate kinase is from  E. coli , or the pyrophosphatase is from  E. coli.    
     
     
         38 . The method of  claim 35 , wherein the R 1A  is hydrogen, allyl, substituted alkyl, biotin, or a ceramide. 
     
     
         39 . The method of  claim 35 , further comprising isolating the Gb4-OR 1A . 
     
     
         40 . The method of  claim 35 , further comprising:
 (iii) converting the Gb4-OR 1A  into Gb5-OR 1A  in the presence of UDP-Gal and a beta-1,3-galactosyltransferase.   
     
     
         41 . The method of  claim 40 , further comprising:
 (iv) producing the 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 pyrophosphorylase, a pyruvate kinase, and optionally, a pyrophosphatase.   
     
     
         42 . The method of  claim 41 , wherein (iii) and (iv) occur in a Gb5-synthesis reaction mixture comprising galactose, PEP, ATP, UTP, the Gb4-OR 1A , the beta-1,3-galactosyltransferase, and the set of UDP-Gal regeneration enzymes. 
     
     
         43 . The method of  claim 40 , wherein the beta-1,3-galactosyltransferase is LgtD from  H. influenza ; the galactokinase is from  E. coli , the UDP-sugar pyrophosphorylase is from  A. thaliana , the pyruvate kinase is from  E. coli , or the pyrophosphatase is from  E. coli.    
     
     
         44 . The method of  claim 40 , further comprising isolating the Gb5-OR 1A . 
     
     
         45 . The method of  claim 40 , further comprising:
 (v) converting the Gb5-OR 1A  into Fucosyl-Gb5-OR 1A  in the presence of GDPFuc and an alpha-1,2-fucosyltransferase.   
     
     
         46 . The method of  claim 45 , further comprising:
 (vi) producing the GDP-Fuc from fucose in the presence of a set of GDP-Fuc regeneration enzymes, wherein the set of GDP-Fuc regeneration enzymes comprises an L-fucokinase/GDP-fucose pyrophosphorylase, a pyruvate kinase, and optionally, a pyrophosphatase.   
     
     
         47 . The method of  claim 46 , wherein (v) and (vi) occur in a Fucosyl-Gb5-synthesis reaction mixture comprising fucose, ATP, GTP, PEP, the Gb5-OR 1A , the alpha-1,2-fucosyltransferase, and the set of GDP-Fuc regeneration enzymes. 
     
     
         48 . The method of  claim 47 , wherein the Fucosyl-Gb5-synthesis reaction mixture is prepared by mixing the Gb5-synthesis reaction mixture with at least fucose, GTP, the alpha-1,2-fucosyltransferase, and the L-fucokinase/GDP-fucose pyrophosphorylase. 
     
     
         49 . The method of  claim 45 , wherein the L-fucokinase/GDP-fucose pyrophosphorylase is from  B. fragilis , or the alpha-1,2-fucosyltransferase is from  H. pylori.    
     
     
         50 . The method of  claim 45 , further comprising isolating the Fucosyl-Gb5-OR 1A . 
     
     
         51 . The method of  claim 40 , further comprising:
 (vii) converting the Gb5-OR 1A  into Sialyl-Gb5-OR 1A  in the presence of CMP-Neu5Ac and an alpha-2,3-sialyltransferase.   
     
     
         52 . The method of  claim 51 , further comprising:
 (viii) producing the CMP-Neu5Ac from Neu5Ac in the presence of a set of CMP-Neu5Ac regeneration enzymes, wherein the set of CMP-Neu5Ac regeneration enzymes comprises a cytidine monophosphate kinase, a CMP-sialic acid synthetase, a pyruvate kinase, and optionally, a pyrophosphatase.   
     
     
         53 . The method of  claim 52 , wherein (vii) and (viii) occur in a Sialyl-Gb5-synthesis reaction mixture comprising Neu5Ac, CTP, PEP, the Gb5-OR 1A , the alpha-2,3-sialyltransferase, and the set of CMP-Neu5Ac regeneration enzymes. 
     
     
         54 . The method of  claim 53 , wherein the Sialyl-Gb5-synthesis reaction mixture is prepared by mixing the Gb5-synthesis reaction mixture with at least Neu5Ac, CTP, the alpha-2,3-sialyltransferase, the cytidine monophosphate kinase, and the CMP-sialic acid synthetase. 
     
     
         55 . The method of  claim 51 , wherein the alpha-2,3-sialyltransferase is from  M. bacteria , the cytidine monophosphate kinase is from  E. coli , or the CMP-sialic acid synthetase is from  P. Multocida.    
     
     
         56 . The method of  claim 51 , further comprising isolating the Sialyl-Gb5-OR 1A . 
     
     
         57 - 99 . (canceled)