Enzymatic synthesis of sulfated polysaccharides

Abstract

A method of sulfating a polysaccharide is provided. The method can include providing a reaction mixture comprising at least one O-sulfotransferase (OST) enzyme and 3′-phosphoadenosine 5′-phosphosulfate (PAPS); incubating a polysaccharide substrate with the reaction mixture, wherein production of the sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to adenosine 3′,5′-diphosphate (PAP); and providing a reaction condition which modifies PAP to reduce an inhibitory effect of PAP on the polysaccharide sulfation.

1 . A method of sulfating a polysaccharide, comprising:
 (a) providing a reaction mixture comprising:
 at least one O-sulfotransferase (OST) enzyme; and 
 3′-phosphoadenosine 5′-phosphosulfate (PAPS); 
   (b) incubating a polysaccharide substrate with the reaction mixture, wherein production of the sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to adenosine 3′,5′-diphosphate (PAP); and   (c) providing a reaction condition which modifies PAP to reduce an inhibitory effect of PAP on the polysaccharide sulfation.   
     
     
         2 . The method of  claim 1 , wherein the at least one OST enzyme is selected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST, and combinations thereof. 
     
     
         3 . The method of  claim 2 , wherein the at least one OST enzyme is a recombinant OST enzyme. 
     
     
         4 . The method of  claim 3 , wherein the recombinant OST enzyme is produced in a bacterial expression system. 
     
     
         5 . The method of  claim 3 , wherein the at least one OST enzyme is a fusion protein. 
     
     
         6 . The method of  claim 5 , wherein the fusion protein is a maltose-binding protein (MBP)-2-OST fusion protein or a MBP-6-OST fusion protein. 
     
     
         7 . The method of  claim 1 , wherein the OST enzyme is immobilized on a substrate. 
     
     
         8 . The method of  claim 7 , wherein the substrate is an agarose bead. 
     
     
         9 . The method of  claim 1 , wherein providing the reaction condition comprises providing a PAPS regeneration system comprising a PAPS regenerating enzyme and a sulfur donor compound, wherein the PAPS regenerating enzyme catalyzes regeneration of the PAPS from the PAP utilizing the sulfur donor compound as a substrate. 
     
     
         10 . The method of  claim 9 , wherein the PAPS regenerating enzyme is an arylsulfotransferase. 
     
     
         11 . The method of  claim 10 , wherein the arylsulfotransferase is AST-IV. 
     
     
         12 . The method of  claim 9 , wherein the sulfur donor compound is an aryl sulfate compound. 
     
     
         13 . The method of  claim 12 , wherein the aryl sulfate compound is p-nitrophenol sulfate (PNPS). 
     
     
         14 . The method of  claim 1 , wherein providing the reaction condition comprises providing a phosphatase enzyme, wherein the phosphatase enzyme modifies the PAP. 
     
     
         15 . The method of  claim 1 , wherein the polysaccharide substrate is a chemically desulfated N-sulfated (CDSNS) heparin. 
     
     
         16 . The method of  claim 1 , wherein the polysaccharide substrate is partially sulfated prior to reaction mixture incubation. 
     
     
         17 . The method of  claim 1 , wherein the sulfated polysaccharide is a glycosaminoglycan (GAG). 
     
     
         18 . The method of  claim 17 , wherein the GAG is a heparan sulfate (HS). 
     
     
         19 . The method of  claim 18 , wherein the HS is an anticoagulant-active HS. 
     
     
         20 . The method of  claim 18 , wherein the HS is selected from the group consisting of an antithrombin-binding HS, a fibroblast growth factor (FGF)-binding HS, and a herpes simplex virus envelope glycoprotein D-binding HS. 
     
     
         21 . A method of sulfating a polysaccharide, comprising:
 (a) providing a reaction mixture comprising adenosine 3′,5′-diphosphate (PAP), a PAPS regenerating enzyme and a sulfur donor compound;   (b) incubating the reaction mixture for a time period sufficient to catalyze the production of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) from the PAP by the PAPS regenerating enzyme utilizing the sulfur donor compound as a substrate; and   (c) incubating a polysaccharide substrate and at least one O-sulfotransferase (OST) enzyme with the reaction mixture, wherein production of a sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to PAP and wherein the PAPS regenerating enzyme catalyzes regeneration of the PAPS from the PAP utilizing the sulfur donor compound as a substrate.   
     
     
         22 . The method of  claim 21 , wherein the PAPS regenerating enzyme is an arylsulfotransferase. 
     
     
         23 . The method of  claim 22 , wherein the arylsulfotransferase is AST-IV. 
     
     
         24 . The method of  claim 21 , wherein the sulfur donor compound is an aryl sulfate compound. 
     
     
         25 . The method of  claim 24 , wherein the aryl sulfate compound is p-nitrophenol sulfate (PNPS). 
     
     
         26 . The method of  claim 21 , wherein the time period is from about 1 minute to about 30 minutes. 
     
     
         27 . The method of  claim 21 , wherein the polysaccharide substrate is a chemically desulfated N-sulfated (CDSNS) heparin. 
     
     
         28 . The method of  claim 21 , wherein the polysaccharide substrate is partially sulfated prior to reaction mixture incubation. 
     
     
         29 . The method of  claim 21 , wherein the at least one OST enzyme is selected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST, and combinations thereof. 
     
     
         30 . The method of  claim 29 , wherein the at least one OST enzyme is a recombinant OST enzyme. 
     
     
         31 . The method of  claim 30 , wherein the recombinant OST enzyme is produced in a bacterial expression system. 
     
     
         32 . The method of  claim 30 , wherein the at least one OST enzyme is a fusion protein. 
     
     
         33 . The method of  claim 32 , wherein the fusion protein is a maltose-binding protein (MBP)-2-OST fusion protein or a MBP-6-OST fusion protein. 
     
     
         34 . The method of  claim 21 , wherein the OST enzyme is immobilized on a substrate. 
     
     
         35 . The method of  claim 34 , wherein the substrate is an agarose bead. 
     
     
         36 . The method of  claim 21 , wherein the sulfated polysaccharide is a glycosaminoglycan (GAG). 
     
     
         37 . The method of  claim 36 , wherein the GAG is a heparan sulfate (HS). 
     
     
         38 . The method of  claim 37 , wherein the HS is an anticoagulant-active HS. 
     
     
         39 . The method of  claim 37 , wherein the HS is selected from the group consisting of an antithrombin-binding HS, a fibroblast growth factor (FGF)-binding HS, and a herpes simplex virus envelope glycoprotein D-binding HS. 
     
     
         40 . A kit for sulfating a polysaccharide, the kit comprising:
 (a) at least one O-sulfotransferase (OST) enzyme; and   (b) a reagent which modifies adenosine 3′,5′-diphosphate (PAP) to reduce an inhibitory effect of PAP on polysaccharide sulfation.   
     
     
         41 . The kit of  claim 40 , further comprising instructions for sulfating a polysaccharide. 
     
     
         42 . The kit of  claim 40 , wherein the at least one OST enzyme is contained within a first container and the reagent is contained within a second container. 
     
     
         43 . The kit of  claim 40 , wherein the at least one OST enzyme is selected from the group consisting of 2-OST, 3-OST-1, 3-OST-3, 6-OST, and combinations thereof. 
     
     
         44 . The kit of  claim 43 , wherein the at least one OST enzyme is a recombinant OST enzyme. 
     
     
         45 . The kit of  claim 44 , wherein the recombinant OST enzyme is produced in a bacterial expression system. 
     
     
         46 . The kit of  claim 44 , wherein the at least one OST enzyme is a fusion protein. 
     
     
         47 . The kit of  claim 46 , wherein the fusion protein is a maltose-binding protein (MBP)-2-OST fusion protein or a MBP-6-OST fusion protein. 
     
     
         48 . The kit of  claim 38 , wherein the OST enzyme is immobilized on a substrate. 
     
     
         49 . The kit of  claim 48 , wherein the substrate is an agarose bead. 
     
     
         50 . The kit of  claim 40 , wherein the reagent comprises a PAPS regeneration system comprising a PAPS regenerating enzyme and a sulfur donor compound. 
     
     
         51 . The kit of  claim 50 , wherein the PAPS regenerating enzyme is an arylsulfotransferase. 
     
     
         52 . The kit of  claim 51 , wherein the arylsulfotransferase is AST-IV. 
     
     
         53 . The kit of  claim 50 , wherein the sulfur donor compound is an aryl sulfate compound. 
     
     
         54 . The kit of  claim 53 , wherein the aryl sulfate compound is p-nitrophenol sulfate (PNPS). 
     
     
         55 . The kit of  claim 40 , wherein the reagent comprises a phosphatase enzyme.