Method for the production of sialylated oligosaccharides

Abstract

Disclosed are methods for the enzymatic synthesis of α-sialylated oligosaccharide glycosides. Specifically, in the disclosed methods, α2,3-sialyltransferase is used to transfer an analogue of sialic acid, employed as its CMP-nucleotide derivative, to the non-reducing sugar terminus of an oligosaccharide having a fucosyl group in the penultimate saccharide unit to the non-reducing sugar terminus. The analogue of sialic acid and the oligosacchairde employed in this method are selected to be compatible with the sialyltransferase employed.

1 . A method for the enzymatic synthesis of an α-sialylated fucosylated oligosaccharide containing a sialic acid or analogue thereof which method comprises the steps of: 
 a) selecting a sialyltransferase compatible with a fucosylated oligosaccharide having a fucose group in the non-reducing penultimate saccharide position;  
 b) selecting a CMP-sialic acid or an analogue thereof which is compatible with the sialyltransferase selected in step (a);  
 c) contacting said CMP-sialic acid or an analogue thereof with a fucosylated oligosaccharide of the formula  
                 
 
 wherein R 1  represents a saccharide residue, R 2  represents a saccharide residue, and R 1  and R 2  together represent an acceptor for the selected sialyltransferase; n is from 0 to about 10, Y is selected from the group consisting of O, NH and S, and R 3  is selected from the group consisting of a protein, a lipid or an aglycon moiety having at least one carbon atom, in the presence of the sialyltransferase selected in (a) above under conditions whereby the sialic acid or analogue thereof is transferred from the CMP-sialic acid or analogue thereof to the non-reducing sugar terminus of the fucosylated oligosaccharide so as to form an α-sialylated fucosylated oligosaccharide containing a sialic acid or analogue thereof.  
 
     
     
         2 . The method of    claim 1   , wherein the aglycon moiety is selected from the group consisting of —(A)—Z′ wherein A represents a bond, an alkylene group of from 2 to 10 carbon atoms, and a moiety of the form —(CH 2 CR 4 R 5 G) n (CH 2 CR 4 R 5 )— wherein n is an integer equal to 0 to 5; R 4  and R 5  are independently selected from the group consisting of hydrogen, phenyl, phenyl substituted with 1 to 3 substituents selected from the group consisting of amine, hydroxyl, halogen, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms, methyl, or ethyl; and G is selected from the group consisting of a bond, oxygen, sulphur, NH, and Z′ is selected from the group consisting of hydrogen, methyl, —OH, —SH, —NH 2 , —NHR 6 , —N(R 6 ) 2 , —C(O)OH, —C(O)OR 6 , —C(O)NHNH 2 , —C(O)NH 2 , —C(O)NHR 6 , —C(O)N(R 6 ) 2 , and —OR 7  wherein each R 6  is independently alkyl of from 1 to 4 carbon atoms and R 7  is an alkenyl group of from 3 to 10 carbon atoms.  
     
     
         3 . A method for the enzymatic synthesis of a fucosylated and α-sialylated oligosaccharide which method comprises the steps of: 
 a) selecting a sialyltransferase capable of sialylating an oligosaccharide having a fucose group in the non-reducing penultimate saccharide position;  
 b) selecting a fucosyltransferase;  
 c) selecting a CMP-sialic acid or an analogue thereof which compatible with the sialyltransferase selected in step (a);  
 d) selecting a GDP-fucose or an analogue thereof which is compatible with the fucosyltransferase selected in step (b);  
 e) contacting said CMP-sialic acid or an analogue thereof and said GDP-fucose or an analogue thereof with an oligosaccharide of the formula 
 R 1 —R 2 -(saccharide) n —Y—R 3   
 wherein R 1  represents a saccharide residue, R 2  represents a saccharide residue, and R 1  and R 2  together represent an acceptor for the selected sialyltransferase and the selected fucosyltransferase; n is from 0 to about 10, Y is selected from the group consisting of O, NH and S, and R 3  is selected from the group consisting of a protein, a lipid or an aglycon moiety having at least one carbon atom, in the presence of said sialyltransferase and said fucosyltransferase selected in (a) and (b) above under conditions whereby the sialic acid or analogue thereof and the fucose or analogue thereof are transferred from the CMP-sialic acid or analogue thereof and the GDP-fucose or analogue thereof, respectively, to the non-reducing sugar terminus of the oligosaccharide so as to form an α-sialylated fucosylated oligosaccharide.  
 
     
     
         4 . The method of    claim 3   , wherein the aglycon moiety is selected from the group consisting of —(A)—Z′ wherein A represents a bond, an alkylene group of from 2 to 10 carbon atoms, and a moiety of the form —(CH 2 CR 4 R 5 G) n (CH 2 CR 4 R 5 )— wherein n is an integer equal to 0 to 5; R 4  and R 5  are independently selected from the group consisting of hydrogen, phenyl, phenyl substituted with 1 to 3 substituents selected from the group consisting of amine, hydroxyl, halogen, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms, methyl, or ethyl; and G is selected from the group consisting of a bond, oxygen, sulphur, NH, and Z′ is selected from the group consisting of hydrogen, methyl, —OH, —SH, —NH 2 , —NHR 6 , —N(R 6 ) 2 , —C(O)OH, —C(O)OR 6 , —C(O)NHNH 2 , —C(O)NH 2 , —C(O)NHR 6 , —C(O)N(R 6 ) 2 , and —OR 7  wherein each R 6  is independently alkyl of from 1 to 4 carbon atoms and R 7  is an alkenyl group of from 3 to 10 carbon atoms.  
     
     
         5 . A method for determining the non-reducing terminus structure of an unknown oligosaccharide acceptor, which method comprises the steps of: 
 a) contacting the oligosaccharide acceptor with a sialyltransferase which is not capable of sialylating a non-reducing terminus of an oligosacchairde having a fucose group in the non-reducing penultimate saccharide position and determining whether the oligosaccharide was sialylated;    b) contacting the oligosaccharide with a sialyltransferase which is capable of sialylating a non-reducing terminus of an oligosacchairde having a fucose group in the non-reducing penultimate saccharide position and determining whether the oligosaccharide was sialylated; and    c) comparing the results to determine whether the non-reducing terminus was fucosylated.