Apparatus and methods for the automated synthesis of oligosaccharides

Abstract

One aspect of the present invention relates to an apparatus for the efficient synthesis of oligosaccharides on a solid support, e.g., formed by subunit addition to terminal subunits immobilized on solid-phase particles. In certain embodiments, the apparatus of the present invention is used in combinatorial methods, e.g., as described herein, of synthesizing oligosaccharides.

1 - 30 . (canceled)  
   
   
       31 . A method of forming a carbon-heteroatom bond between a glycosyl donor and a substrate, comprising the step of combining in solution, in a reaction vessel containing at least one insoluble resin bead, a glycosyl donor comprising a reactive anomeric carbon, a substrate comprising a heteroatom bearing a hydrogen, and an activating reagent, wherein said activating reagent activates said reactive anomeric carbon of said glycosyl donor, thereby forming a product comprising a carbon-heteroatom bond between said anomeric carbon of said glycosyl donor and said heteroatom of said substrate.  
   
   
       32 . The method  claim 31 , wherein said glycosyl donor comprising a reactive anomeric carbon is selected from the group consisting of glycosyl phosphates, glycosyl phosphites, glycosyl trichloroacetimidates, glycosyl halides, glycosyl sulfides, glycosyl sulfoxides, n-pentenyl glycosides, and 1,2-anhydroglycosides.  
   
   
       33 . The method  claim 31 , wherein said glycosyl donor comprising a reactive anomeric carbon is selected from the group consisting of glycosyl phosphates and glycosyl trichloroacetimidates.  
   
   
       34 . The method of  claim 31 , wherein said heteroatom bearing a hydrogen of said substrate is selected from the group consisting of oxygen, nitrogen, and sulfur.  
   
   
       35 . The method of  claim 31 , wherein said heteroatom bearing a hydrogen of said substrate is selected from the group consisting of oxygen and nitrogen.  
   
   
       36 . The method of  claim 31 , wherein said heteroatom bearing a hydrogen of said substrate is oxygen.  
   
   
       37 . The method of  claim 31 , wherein said activating reagent is a Lewis acid.  
   
   
       38 . The method of  claim 31 , wherein said activating reagent is a silyl trifluoromethanesulfonate.  
   
   
       39 . The method of  claim 31 , wherein said activating reagent is trimethylsilyl trifluoromethanesulfonate.  
   
   
       40 . The method of  claim 31 , wherein said glycosyl donor comprising a reactive anomeric carbon is selected from the group consisting of glycosyl phosphates, glycosyl phosphites, glycosyl trichloroacetimidates, glycosyl halides, glycosyl sulfides, glycosyl sulfoxides, n-pentenyl glycosides, and 1,2-anhydroglycosides; said heteroatom bearing a hydrogen of said substrate is selected from the group consisting of oxygen, nitrogen, and sulfur; and said activating reagent is a Lewis acid.  
   
   
       41 . The method of  claim 31 , wherein said glycosyl donor comprising a reactive anomeric carbon is selected from the group consisting of glycosyl phosphates and glycosyl trichloroacetimidates; said heteroatom bearing a hydrogen of said substrate is selected from the group consisting of oxygen, nitrogen, and sulfur; and said activating reagent is a silyl trifluoromethanesulfonate.  
   
   
       42 . The method of  claim 31 , wherein said glycosyl donor comprising a reactive anomeric carbon is selected from the group consisting of glycosyl phosphates and glycosyl trichloroacetimidates; said heteroatom bearing a hydrogen of said substrate is selected from the group consisting of oxygen, nitrogen, and sulfur; and said activating reagent is trimethylsilyl trifluoromethanesulfonate.  
   
   
       43 . The method of  claim 31 , wherein said substrate comprising a heteroatom bearing a hydrogen is tethered to a solid support via a covalent linker.  
   
   
       44 . The method of  claim 43 , wherein said covalent linker is —O—(CH 2 ) 3 CH═CH(CH 2 ) 3 —O—.  
   
   
       45 . The method of  claim 44 , wherein said solid support is a resin bead.  
   
   
       46 . The method of  claim 45 , wherein said substrate comprising a heteroatom bearing a hydrogen is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and glycoconjugates.  
   
   
       47 . The method of  claim 31 , wherein said glycosyl donor comprising a reactive anomeric carbon is tethered to a solid support via a covalent linker.  
   
   
       48 . The method of  claim 47 , wherein said covalent linker is —O—(CH 2 ) 3 CH═CH(CH 2 ) 3 —O—.  
   
   
       49 . The method of  claim 48 , wherein said solid support is a resin bead.  
   
   
       50 . The method of  claim 49 , wherein said substrate comprising a heteroatom bearing a hydrogen is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and glycoconjugates.  
   
   
       51 . The method of  claim 43 , further comprising the steps of applying positive pressure or a vacuum to said reaction vessel of said apparatus, thereby removing the liquid phase from said reaction vessel of said apparatus; and adding solvent to said reaction vessel of said apparatus.  
   
   
       52 . The method of  claim 47 , further comprising the steps of applying positive pressure or a vacuum to said reaction vessel of said apparatus, thereby removing the liquid phase from said reaction vessel of said apparatus; and adding solvent to said reaction vessel of said apparatus.  
   
   
       53 . The method of  claim 51 , further comprising the steps of applying positive pressure or a vacuum to said reaction vessel of said apparatus, thereby removing the liquid phase from said reaction vessel of said apparatus; and adding solvent to said reaction vessel of said apparatus.  
   
   
       54 . The method of  claim 52 , further comprising the steps of applying positive pressure or a vacuum to said reaction vessel of said apparatus, thereby removing the liquid phase from said reaction vessel of said apparatus; and adding solvent to said reaction vessel of said apparatus.  
   
   
       55 . The method of  claim 51 , further comprising the step of treating said product, in said reaction vessel of said apparatus, with a solution comprising a deprotection reagent, thereby removing from said product a protecting group to produce a second product comprising a heteroatom bearing a hydrogen, wherein said second product is tethered to a solid support via a covalent linker.  
   
   
       56 . The method of  claim 55 , further comprising the step of combining in solution, in said reaction vessel of said apparatus, a glycosyl donor comprising a reactive anomeric carbon, said second product comprising a heteroatom bearing a hydrogen, and an activating reagent, wherein said activating reagent activates said reactive anomeric carbon of said glycosyl donor, thereby forming a third product comprising a carbon-heteroatom bond between said anomeric carbon of said glycosyl donor and said heteroatom of said second product, wherein said third product is tethered to a solid support via a covalent linker.  
   
   
       57 . The method of  claim 52 , further comprising the step of treating said product, in said reaction vessel of said apparatus, with a solution comprising a converting reagent to produce a second product comprising a reactive anomeric carbon, wherein said second product is tethered to a solid support via a covalent linker.  
   
   
       58 . The method of  claim 57 , further comprising the step of combining in solution, in said reaction vessel of said apparatus, a substrate comprising a heteroatom bearing a hydrogen, said second product comprising a reactive anomeric carbon, and an activating reagent, wherein said activating reagent activates said reactive anomeric carbon of said second product, thereby forming a third product comprising a carbon-heteroatom bond between said anomeric carbon of said second product and said heteroatom of said substrate, wherein said third product is tethered to a solid support via a covalent linker.