Synthesis and use of glycodendrimer reagents

Abstract

The present invention relates to a chemically modified mutant protein including a cysteine residue substituted for a residue other than cysteine n a precursor protein, the substituted cysteine residue being subsequently modified by reacting the cysteine residue with a glycosylated thiosulfonate. Also a method of producing the chemically modified mutant protein is provided. The present invention also realtes to a glycosylated methanethiosulfonate. Another aspect of the present invention is a method of modifying the functional characteristics of a protein including providing a protein and reacting the protein with a glycosylated methanethiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein. In addition, the present invention relates to methods of determining the structure-function relationships of chemically modified mutant proteins. The present invention also relates to synthetic methods for producing thio-glycoses, the thio-glycoses so produced, and to methods for producing glycodendrimer reagents.

1 . A chemically modified mutant protein, said mutant protein comprising a cysteine residue substituted for a residue other than cysteine in a precursor protein, the substituted cysteine residue being subsequently modified by reacting said cysteine residue with a glycosylated thiosulfonate.  
     
     
         2 . A method for producing a carbohydrate alkylthiosulfonate, comprising the steps of: 
 a) providing a carbohydrate, an alkylthiosulfonate, and a phase transfer catalyst;    b) reacting said carbohydrate and said alkylthiosulfonate in an organic solvent in the presence of said phase transfer catalyst; and    c) refluxing said organic solvent to produce a carbohydrate alkylthiosulfonate.    
     
     
         3 . The method of  claim 2 , wherein said phase transfer catalyst comprises a quaternary ammonium salt.  
     
     
         4 . The method of  claim 3 , wherein said quaternary ammonium salt is tetrabutylammonium iodide.  
     
     
         5 . The method of  claim 2 , wherein said organic solvent comprises a non-polar organic solvent.  
     
     
         6 . The method of  claim 5 , wherein said non-polar organic solvent comprises toluene.  
     
     
         7 . The method of  claim 2 , wherein said alkylthiosulfonate is methanethiosulfonate.  
     
     
         8 . The method of  claim 7 , wherein said methanethiosulfonate is a salt.  
     
     
         9 . The method of  claim 2 , wherein said carbohydrate comprises a monosaccharide.  
     
     
         10 . The method of  claim 9 , wherein said monosaccharide is selected from the group consisting of galactose, glucose and mannose.  
     
     
         11 . The method of  claim 2 , wherein said carbohydrate alkylthiosulfonate is a β-anomer.  
     
     
         12 . The method of  claim 2 , wherein said carbohydrate alkylthiosulfonate is an α-anomer.  
     
     
         13 . A composition of matter having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         14 . A composition of matter having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         15 . A composition of matter having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         16 . A glycodendrimer reagent composition having the structure:  
       
         
           
           
               
               
           
         
       
       wherein a, b, c, and d are individually the same or different and are independently selected from the group consisting of integers from 0 to 10, wherein X═SR or R, and wherein R is a monosaccharide selected from the group consisting of galactose, glucose, mannose and lactose.  
     
     
         17 . The composition of  claim 16 , wherein said monosaccharide is galactose.  
     
     
         18 . The composition of  claim 16 , wherein said monosaccharide is glucose.  
     
     
         19 . The composition of  claim 16 , wherein said monosaccharide is mannose.  
     
     
         20 . The composition of  claim 16 , wherein X is  
       
         
           
           
               
               
           
         
       
     
     
         21 . The composition of  claim 16 , wherein X is  
       
         
           
           
               
               
           
         
       
     
     
         22 . The composition of  claim 20 , wherein a=1, b=0, c=1, and d=1.  
     
     
         23 . The composition of  claim 21 , wherein a=1, b=0, c=1, and d=1.  
     
     
         24 . The composition of  claim 16 , wherein a=3, b=0, c=1, d =1, X is R, and R is  
       
         
           
           
               
               
           
         
       
     
     
         25 . A glycodendrimer reagent composition having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         26 . A glycodendrimer reagent composition having the structure:  
       
         
           
           
               
               
           
         
       
     
     
         27 . A method for inhibiting adhesin or lectin activity, comprising the steps of: 
 a) providing a modified protease, said modified protease having a thiol side chain comprising a carbohydrate moiety;    b) contacting said modified protease with a composition having an adhesin or lectin activity; and    c) incubating said modified protease with said composition such that the adhesin or lectin activity of said composition is inhibited.    
     
     
         28 . The method of  claim 27 , wherein said modified protease is a modified serine protease.  
     
     
         29 . The method of  claim 28 , wherein said modified serine protease is a modified subtilisin.  
     
     
         30 . The method of  claim 29 , wherein said modified subtilisin is a modified  Bacillus lentus  subtilisin.  
     
     
         31 . The method of  claim 28 , wherein said modified subtilisin is a modified  Bacillus amyloliquefaciens  subtilisin.  
     
     
         32 . The method of  claim 27 , wherein said carbohydrate moiety comprises a monosaccharide.  
     
     
         33 . The method of  claim 32 , wherein said monosaccharide is selected from the group consisting of glucose, mannose, and galactose.  
     
     
         34 . The method of  claim 27 , wherein said thiol side chain is selected from the group consisting of —S-β-Glc, -Et-β-Gal, —S-Et-β-Glc, —S-Et-α-Glc, —S-Et-α-Man, —S-Et-Lac, —S-β-Glc(Ac), —S-β-Glc(AC) 2 , —S-β-Glc(Ac) 3 , —S-β-Glc(Ac) 4 , S-Et-αGlc(Ac), —S-Et -α-Glc(Ac) 2,  —S-Et-α-Glc(Ac) 3 , —S-Et-α-Glc(Ac) 4 , —S-Et-β-Glc(Ac), —S-Et-β-Glc(Ac) 2 , —S-Et-β-Glc(Ac) 3 , —S-Et-β-Glc(Ac) 4 , —S-Et-α-Man(Ac), —S-Et-αMan(Ac) 2 , —S-Et-α-Man(Ac) 3 , —S-Et-α-Man(Ac) 4,  —S-Et-β-Gal(Ac), —S-Et-βGal(Ac) 2 , —S-Et-β-Gal(Ac) 3 , —S-Et-β-Gal(Ac) 4 , —S-Et-Lac(Ac) 5,  —S-Et-Lac(Ac) 6,  —S-Et-Lac(Ac) 7 , —S-β-Gal, —S-β-Gal(Ac), —S--β-Gal(Ac) 2 , —S-βGal(Ac) 3 , —S-β-Gal(Ac) 4 , —S-β-Man, —S-β-Man(Ac), —S-β-Man(Ac) 2 , —S-β-Man(Ac) 3 , —S-β-Man(Ac) 4 , —S-α-Man, —S-α-Man(Ac), —S-α-Man(Ac) 2 , —S-α-Man(Ac) 3 , and —S-α-Man(Ac) 4 .  
     
     
         35 . The method of  claim 27 , wherein said composition comprises an adhesin or lectin from a bacteria.  
     
     
         36 . The method of  claim 35 , wherein said bacteria are  A. naeslundii.    
     
     
         37 . The method of  claim 30 , wherein said modified  Bacillus lentus  subtilisin is S156C—SS-ethyl-2-(β-D-galactopyranose).  
     
     
         38 . The method of  claim 37 , wherein said composition comprises an adhesin or lectin from a bacteria.  
     
     
         39 . The method of  claim 38 , wherein said bacteria are  A. naeslundii.    
     
     
         40 . The method of  claim 27 , wherein said carbohydrate, moiety is a dendrimer moiety.  
     
     
         41 . The method of  claim 40 , wherein said modified protease is a modified serine protease.  
     
     
         42 . The method of  claim 41 , wherein said modified serine protease is a modified subtilisin.  
     
     
         43 . The method of  claim 42 , wherein said modified subtilisin is a modified  Bacillus lentus  subtilisin.  
     
     
         44 . The method of  claim 41 , wherein said modified subtilisin is a modified  Bacillus amyloliquefaciens  subtilisin.  
     
     
         45 . The method of  claim 40 , wherein said dendrimer moiety comprises mesitylene.  
     
     
         46 . The method of  claim 43 , wherein said modified  Bacillus lentus  subtilisin is S156C-mes(SS-β-Gal) 2.    
     
     
         47 . The method of  claim 46 , wherein said composition comprises an adhesin or lectin from a bacteria.  
     
     
         48 . The method of  claim 47 , wherein said bacteria are  A. naeslundii .