Synthesis of a tetrasaccharide containing n-acetyllactosamine

Abstract

The present invention relates to the synthesis of the tetrasaccharide of formula (I) and novel intermediates used in the synthesis.

1 . A method for the preparation of Galpβ1-4GlcNAcpβ1-3Galpβ1-4Glc (LNnT) comprising the steps of:
 a) reaction of a donor characterized by general formula 5 
 
       
         
           
           
               
               
           
         
         
           wherein R 4  is optionally substituted acyl, 
           —NR 5 R 6  is —NH-haloacyl, and 
           X is selected from halogen, —OC(═NH)CCl 3 , —OAc, —OBz and —SR 7 , 
           wherein R 7  is selected from alkyl and optionally substituted phenyl, 
         
         with an acceptor of general formula 6 
       
       
         
           
           
               
               
           
         
         
           wherein R 1  is selected from optionally substituted benzyl, optionally substituted benzhydryl, optionally substituted trityl and optionally substituted naphthylmethyl, 
           R 2  is optionally substituted acyl, and 
           R 3  is selected from optionally substituted acyl or H, 
         
         to yield a compound of general formula 4 
       
       
         
           
           
               
               
           
         
         
           wherein R 1 , R 2 , R 3 , R 4  and —NR 5 R 6  are as defined above, 
         
         b) converting the compound of general formula 4 into a compound of general formula 1 
       
       
         
           
           
               
               
           
         
         
           wherein R 1  is as defined above, 
         
         c) crystallizing the compound of general formula 1, and 
         d) subsequently subjecting the compound of general formula 1 to catalytic hydrogenolysis. 
       
     
     
         2 . The method according to  claim 1 , wherein in step a) in the donor of general formula 5, X is —SR 7 , wherein R 7  is selected from optionally substituted alkyl or optionally substituted phenyl; and in compound acceptor of general formula 6, R 1  is optionally substituted benzyl and R 3  is selected from H and optionally substituted benzoyl. 
     
     
         3 . The method according to  claim 1 , wherein the crystallization in step c) is carried out in a solvent comprising one or more C 1 -C 6  alcohols, and R 1  is benzyl. 
     
     
         4 . The method according to  claim 3 , wherein the crystallization in step c) is carried out in aqueous methanol or aqueous ethanol. 
     
     
         5 . The method according to  claim 1 , wherein the conversion of the compound of general formula 4 into the compound of general formula 1 in step b) comprises the steps of:
 ba) base catalyzed transesterification deprotection of the compound of general formula 4 to give a compound of general formula 3   
       
         
           
           
               
               
           
         
         
           wherein R 1  and —NR 5 R 6  are defined as above, which compound of general formula 3 is subjected to basic hydrolysis to give rise to a compound of general formula 2 
         
       
       
         
           
           
               
               
           
         
         
           wherein R 1  is as defined above, which compound of general formula 2 is converted into the compound of general formula 1, or 
         
         bb) basic hydrolysis of the compound of general formula 4 to give a compound of general formula 2 
       
       
         
           
           
               
               
           
         
         
           wherein R 1  is as defined above, which compound of general formula 2 is converted into the compound of general formula 1. 
         
       
     
     
         6 . The method according to  claim 5 , wherein the compound of general formula 2 is
 a) N-acetylated to obtain compounds of general formula 1,   or   b) peracetylated to compounds of general formula 4a   
       
         
           
           
               
               
           
         
         
           wherein R 1  is defined as above, 
           followed by based catalyzed transesterification reaction or basic hydrolysis to obtain compounds of general formula 1. 
         
       
     
     
         7 . The method according to  claim 1 , wherein the compound of general formula 1, wherein R 1  is benzyl, is subjected to catalytic hydrogenolysis. 
     
     
         8 . A method for crystallizing a polymorph of Galpβ1-4GlcNAcpβ1-3Galpβ1-4Glc (LNnT) displaying X-ray powder diffraction reflections, based on a measurement using CuKα radiation, at 20.32±0.20, 19.10±0.20, 7.98±0.20, 21.03±0.20, 20.95±0.20 and 5.66±0.20 2θ angles, from a solvent system comprising one or more C 1 -C 6  alcohols. 
     
     
         9 . The method according to  claim 8 , wherein the solvent system contains water. 
     
     
         10 . The method according to  claim 9 , wherein the solvent system is water/methanol. 
     
     
         11 . The method according to  claim 10 , comprising the steps:
 a) making an LNnT solution with water/methanol,   b) warming the LNnT solution to 50-60° C.,   c) adding hot methanol to the LNnT solution under gradual chilling to 35-45° C.,   d) optionally adding seeding crystals,   e) cooling the resulting warm suspension to 0-8° C.   
     
     
         12 . The method according to  claim 11 , wherein the water/methanol mixture used in step a) is a ≈1:1 mixture. 
     
     
         13 . The method according to  claim 11 , wherein the LNnT solution is step a) has a concentration of 140-180 g/L. 
     
     
         14 . The method according to  claim 11 , wherein, in step c), hot methanol is added up to 115-250% of the starting volume. 
     
     
         15 . The method according to  claim 11 , wherein the water/methanol mixture in step a) is a ≈1:1 mixture, the LNnT solution is step a) has a concentration of 140-180 g/L, and, in step c), hot methanol is added up to 115-250% of the starting volume. 
     
     
         16 . The method according to  claim 8 , wherein the crystallization of LNnT is preceded by the method according to  claim 1 .