Processes for chemical synthesis of lipochitooligosaccharides

Abstract

Processes for the synthesis of lipochitooligosaccharides were developed. A fully acylated oligoglucosamine precursor is prepared and reacted with a glucosamine monomer that has an amine protecting phthaloyl group. With removal of the phthaloyl group, a fatty acid may be added on the terminal glucosamine unit, forming a lipochitooligosaccharide. The processes can be adapted for use on a commercial scale.

1 . A process for synthesizing a lipochithooligosaccharide compound having the structure:  
     
       
         
         
             
             
         
       
     
     where individual groups R 1 , R 2  and R 3  are independently selected from H and C 1  to C 20  alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di or polyalkynyl, groups; R 4  is selected from a monosaccharide, sulfate and phosphate; and n is from 0 to about 20;  
     comprising: 
 b) combining a compound of structure C  
                     
 wherein R 1  is selected from H, C 1  to C 20  alkyl, aryl, and aralkyl groups, with a compound of structure B  
                     
 where individual groups R 1  and R 2  are independently selected from H and C 1  to C 20  alkyl, aryl, and aralkyl groups; R 4  is selected from a monosaccharide, a sulfate and a phosphate; and n is from 0 to about 19;  
 in an aprotic solvent and agitating the solution at a temperature between about 0° C. to about −78° C. to form a first mixture;  
 b) adding to the mixture of a) a first activating agent selected from N-haloimides to form a second mixture;  
 c) adding a second activating agent selected from perfluoroalkyl sulfonic acids, and optionally adding a reagent selected from methyl perfluroalkyl sulfonates, to the second mixture to form a third mixture;  
 d) reacting the third mixture at a temperature between about 0° C. and about −78° C. to form a product comprising ester groups and an N-phthalimido group;  
 e) isolating the product of d);  
 f) removing the ester groups and the N-phthalimido group from the isolated product of e) forming a de-esterified and de-N-phthalimido product;  
 g) isolating the product of f);  
 h) selectively reacting the amino group of the terminal sugar unit of the isolated product of g) with an acid or acid halide of the formula R 1 COX:  
 where X=OH or a halide, for acids and acid halides, respectively, and R 1  is selected from H, C 1  to C 20  alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di or polyalkynyl groups; to form a lipochitooligosaccharide; and  
 i) isolating the lipochitooligosaccharide.  
 
   
   
       2 . The process of  claim 1  wherein the first activating agent is an N-halosuccinimide.  
   
   
       3 . The process of  claim 2  wherein the N-halosuccinimide is N-iodosuccinimide.  
   
   
       4 . The process of  claim 1  wherein the perfluoroalkyl sulfonic acid is in at least about a 0.25 molar equivalent amount to the compound of formula B.  
   
   
       5 . The process of  claim 4  wherein the perfluoroalkyl sulfonic acid is in about an equimolar equivalent amount to the compound of formula B.  
   
   
       6 . The process of  claim 1  wherein the perfluoroalkyl sulfonic acid is triflic acid.  
   
   
       7 . The process of  claim 1  wherein the methyl perfluroalkylsulfonate is methyltriflate.  
   
   
       8 . The process of  claim 1  wherein the temperature of a) and d) is about −20° C. to about −70° C.  
   
   
       9 . The process of  claim 1  wherein the temperature of a) and d) is about −50° C. to about −60° C.  
   
   
       10 . The process of  claim 1  wherein the ester groups of the product of e) are removed by transesterification with metal alkoxides in alcohol and the N-phthalimido group of the product of e) is removed by reacting with amines or diamines under refluxing conditions.  
   
   
       11 . The process of  claim 1  wherein the reacting of h) is carried out using a base catalyst and an acid halide, or using a fatty acid activated with a carbodiimide and N-hydroxybenztriazole.  
   
   
       12 . A process for synthesizing a lipochithooligosaccharide compound having the structure:  
     
       
         
         
             
             
         
       
     
     where individual groups R 1 , R 2  and R 3  are independently selected from: H, and C 1  to C 20  alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di or polyalkynyl, groups; R 4  is selected from monosaccharides, sulfates and phosphates; and n is from 0 to about 20;  
     comprising: 
 a) providing a compound of structure D  
                     
 wherein R 1  is selected from H, and C 1  to C 20  alkyl, aryl, and aralkyl groups;  
 b) removing the ester groups and the internal N-phthalimido groups of the compound of structure D;  
 c) selectively reacting the amino groups on the internal sugar units of the compound of structure D with an acylating reagent to make an N-acyl derivative product;  
 d) removing the silyl group and the ester and the N-phthalimido group on the terminal sugar unit of the N-acyl derivative product of (c) by reacting the N-acyl derivative product with tetra-N-alkyl ammonium fluoride followed by reacting with amines or diamines under refluxing conditions to produce a de-silylated and de-N-phthalimidated product;  
 e) acylating the terminal amino group of the de-N-phthalimidated product of (d) with fatty acids activated with carbodiimide and N-hydroxylbenztriazole, or an acid halide of the formula R 1 COX, in the presence of a base catalyst,  
 where X is a halide, and R 1  is selected from H and C 1  to C 20  alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di or polyalkynyl groups; to form a lipochitooligosaccharide; and  
 f) isolating the lipochitooligosaccharide.  
 
   
   
       13 . The process of  claim 12  wherein the acylating agent of (c) is acetic anhydride.  
   
   
       14 . The process of  claim 12  wherein the tetra-N-alkyl ammonium fluoride of (d) is tetra-n-butyl ammonium fluoride and the diamine is ethylene diamine attached to Merrifield resin.  
   
   
       15 . A compound having the structure:  
     
       
         
         
             
             
         
       
     
     where independent groups R 1  and R 2  are independently selected from H and C 1  to C 20  alkyl, aryl, and aralkyl groups; R 4  is selected from a monosaccharide, a sulfate and a phosphate; and n is from 0 to about 19.  
   
   
       16 . A composition comprising a chemically synthesized lipochitooligosaccharide represented by the structure:  
     
       
         
         
             
             
         
       
     
     where individual groups R 1 , R 2  and R 3  are independently selected from H and C 1  to C 20  alkyl, aryl, aralkyl, mono, di or polyalkenyl, mono, di or polyalkynyl, groups; R 4  is selected from a monosaccharide, sulfate and phosphate; and n is from 0 to about 20.  
   
   
       17 . A composition comprising a chemically synthesized lipochitooligosaccharide represented by the structure:  
     
       
         
         
             
             
         
       
     
   
   
       18 . A composition comprising a chemically synthesized lipochitooligosaccharide represented by the structure: