Heparan sulfate synthesis

Abstract

The invention provides an efficient modular chemical synthesis for heparan sulfate oligosaccharides based on orthogonal protection strategies. Modular disaccharide building blocks, themselves the product of a novel combinatorial synthesis, are combined in numerous ways to produce a range of oligosaccharides.

1 . A disaccharide comprising a hexuronic acid unit and a glucosamine unit, said disaccharide having formula (I) 
       
         
           
           
               
               
           
         
         wherein each of R 1 , R 2  and R 3  is independently a temporary protecting group or a permanent protecting group; 
         Z is —N 3  or —NHR 7 ; 
         R 4  is H or a protecting group; 
         X is —OR 5  or —SR 6 ; 
         R 5  is H, alkyl, cycloalkyl, substituted alkyl or cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl or substituted aryl; silyl or substituted silyl; benzyl; a temporary protecting group; a permanent protecting group; an anomeric spacer or linker; a fluorous tag; or a leaving group; 
         R 6  is H, alkyl, cycloalkyl, substituted alkyl or cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; 
         R 7  is H or a temporary protecting group; 
         R 8  is a permanent protecting group; 
         R 9  is a permanent protecting group; 
         wherein at least two of R 1 , R 2 , R 3 , R 4 , R 5  and R 7  are temporary protecting groups that are orthogonal. 
       
     
     
         2 . The disaccharide of  claim 1  wherein each of R 1 , R 2  and R 3  is independently a temporary protecting group or a permanent protecting group; wherein the permanent protecting group for R 1  and R 2  is selected from acetyl (Ac), benzyl (Bn), benzoyl (Bz), difluorobenzoyl, (dfBz), pivaloyl benzoyl (PivBz), pivaloyl or anisoyl; and wherein the permanent protecting group for R 3  is Ac. 
     
     
         3 . The disaccharide of  claim 1  wherein R 4  is a protecting group selected from 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), [2,2,2-trichloroethoxycarbonyl] (Troc), levulinoyl (Lev), benzoyl (Bz), difluorobenzoyl, (dfBz), pivaloyl levulinoyl (PivLev), pivaloyl benzoyl (PivBz), or a NAP ether. 
     
     
         4 . The disaccharide of  claim 1  wherein R 4  is different from R 1 , R 2  and R 3 . 
     
     
         5 . The disaccharide of  claim 1  wherein R 5  is a temporary protecting group comprising a silyl or substituted silyl; or a permanent protecting group comprising methyl (Me), benzyl (Bn), 2-naphthylmethyl (NAP) or 1-naphthylmethyl (1-NAP). 
     
     
         6 . The disaccharide of  claim 1  wherein R 5  is a leaving group comprising trichloroacetimidate —C(NH)—CCl 3 , phenyltrifluoroacetimidate —C(NPh)—CF 3 , trifluoroacetimidate —C(NH)—CF 3 ; thioformimidate, or S-glycosyl N-phenyltrifluoroacetimidate. 
     
     
         7 . The disaccharide of  claim 1  wherein R 7  is a temporary protecting group selected from 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), [2,2,2-Trichloroethoxycarbonyl] (Troc), trichloroacetyl (TCA), acetyl (Ac), phthalimido (Phthal), carbobenzyloxy (Cbz) or tert-butoxycarbonyl (Boc). 
     
     
         8 . The disaccharide of  claim 1  wherein R 8  is a permanent protecting group selected from benzyl (Bn), a substituted benzyl, 2-naphthylmethyl (NAP) or 1-naphthylmethyl (1-NAP). 
     
     
         9 . The disaccharide of  claim 1  wherein R 9  is a permanent protecting group selected from methyl (Me), benzyl (Bn), a substituted benzyl, Tert-butyl ( t Bu), 2-naphthylmethyl (NAP) or 1-naphthylmethyl (1-NAP). 
     
     
         10 . The disaccharide of  claim 1  wherein cleavage conditions for an orthogonal protecting group do not cause cleavage of other protecting groups present on the disaccharide. 
     
     
         11 . A tetrasaccharide comprising the disaccharide of  claim 1   
     
     
         12 . A hexasaccharide comprising the disaccharide of  claim 1 . 
     
     
         13 . The hexasaccharide of  claim 12  comprising at least one of an N-acetyl group, an N-sulfate group, and a free amino group. 
     
     
         14 . The hexasaccharide of  claim 12  comprising an N-acetyl group, an N-sulfate group, and a free amino group. 
     
     
         15 . A disaccharide comprising a hexuronic acid unit and a glucosamine unit, said disaccharide having formula (I) 
       
         
           
           
               
               
           
         
         wherein each of R 1 , R 2  and R 3  is independently a temporary protecting group or a permanent protecting group; 
         Z is —N 3  or —NHR 7 ; 
         R 4  is a permanent protecting group; 
         X is —OR 5  or —SR 6 ; 
         R 5  is H, alkyl, cycloalkyl, substituted alkyl or cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl or substituted aryl; a temporary protecting group; a permanent protecting group; an anomeric spacer or linker; a fluorous tag; or a leaving group; 
         R 6  is H, alkyl, cycloalkyl, substituted alkyl or cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; 
         R 7  is H or a temporary protecting group; 
         R 8  is a permanent protecting group; 
         R 9  is a permanent protecting group; 
         wherein at least two of R 1 , R 2 , R 3 , R 4 , R 5  and R 7  are temporary protecting groups that are orthogonal. 
       
     
     
         16 . A method for making an oligosaccharide comprising:
 providing one or more disaccharides according to  claim 1 ;   forming one or more glycosyl donors from one or more of the disaccharides;   forming one or more glycosyl acceptors from one of more of the disaccharides; and   reacting the one or more glycosyl donors and the one or more glycosyl acceptors to form an orthogonally protected oligosaccharide.   
     
     
         17 . The method of  claim 16  wherein forming the glycosyl acceptor comprises deprotecting the disaccharide at position C-4′ to form a free hydroxyl group. 
     
     
         18 . The method of  claim 16  wherein the glycosyl acceptor has formula (I) wherein R 4  is H and X is —OR 5 . 
     
     
         19 . The method of  claim 16  wherein forming the glycosyl donor comprises deprotecting the disaccharide at position C-1 and installing a leaving group at the C-1 position. 
     
     
         20 . The method of  claim 16  wherein the glycosyl donor has formula (I) wherein R 4  is temporary protecting group and X is —OR 5 , wherein R 5  is H or a leaving group. 
     
     
         21 . The method of  claim 16  wherein the glycosyl donor has formula (I) wherein R 4  is permanent protecting group and X is —OR 5 , wherein R 5  is H or a leaving group. 
     
     
         22 . The method of  claim 16  wherein the orthogonally protected oligosaccharide is a tetrasaccharide comprising four monosaccharide units or a hexasaccharide comprising six monosaccharide units. 
     
     
         23 . The method of  claim 16  further comprising
 removing a protecting group from position C-4 of the terminal monosaccharide unit at the non-reducing end of the orthogonally protected oligosaccharide; or 
 removing a protecting group from position C-1 of the terminal monosaccharide unit at the reducing end of the orthogonally protected oligosaccharide. 
 
     
     
         24 . The method of  claim 16  further comprising sequentially removing different protecting groups from the orthogonally protected oligosaccharide. 
     
     
         25 . The method of  claim 24  further comprising:
 removing a hydroxyl protecting group from one or more positions on the oligosaccharide to form a free hydroxyl; and 
 sulfating the free hydroxyl. 
 
     
     
         27 . The method of  claim 24  comprising selectively O-sulfating a primary hydroxyl group of a glucosamine unit of the oligosaccharide. 
     
     
         28 . The method of  claim 27  wherein the primary hydroxyl group is at position C-6 of the glucosamine unit. 
     
     
         29 . The method of  claim 27  further comprising selectively O-desulfating a primary O-sulfate group of a glucosamine unit of the oligosaccharide. 
     
     
         30 . The method of  claim 29  where the primary O-sulfate group is at position C-6 of the glucosamine unit. 
     
     
         31 . The method of  claim 24  further comprising:
 removing an amine protecting group from the C-2 position of a glucosamine unit of the oligosaccharide to form a free amine; and 
 sulfating the free amine. 
 
     
     
         32 . The method of  claim 24  further comprising:
 removing an amine protecting group from the C-2 position of a first glucosamine unit of the oligosaccharide to form a free amine; 
 sulfating the free amine; and 
 acetylating a second glucosamine unit of the oligosaccharide at the C-2 position. 
 
     
     
         33 . The method of  claim 24  further comprising removing permanent protecting groups to form an unprotected, sulfated oligosaccharide. 
     
     
         34 . The method of  claim 33  wherein the sulfated oligosaccharide is sulfated at one or more of a C-2 position on a glucosamine unit, a C-3 position on a glucosamine unit, a C-6 position on a glucosamine unit, and a C-2 position on a hexuronic acid unit. 
     
     
         35 . The method of  claim 33  wherein all amines on the sulfated oligosaccharide are N-sulfated. 
     
     
         36 . The method of  claim 33  wherein the sulfated oligosaccharide comprises an N-sulfated amine and an N-acetylated amine. 
     
     
         37 . The method of  claim 33  wherein the sulfated oligosaccharide comprises an N-sulfated amine, an N-acetylated amine and a free amino moiety. 
     
     
         38 . The method of  claim 33  wherein the sulfated oligosaccharide is a heparan sulfate oligosaccharide. 
     
     
         39 . An oligosaccharide formed by the method of  claim 16 . 
     
     
         40 . A library of oligosaccharides formed by the method of  claim 1 .