Permethylation Of Oligosaccharides

Abstract

A solid-phase permethylation procedure is described. For example, solid-phase permethylation can be utilized to prepare permethylated linear and branched, neutral and sialylated oligosaccharides, which can be analyzed by MALDI-MS.

1 . A microreactor comprising:
 a) a container;   b) a solid base disposed within said container; and   c) a solvent disposed within said container, wherein said solvent is a polar, aprotic solvent.   
     
     
         2 . The microreactor of  claim 1 , wherein the container is selected from the group consisting of a spin column and a fused-silica capillary. 
     
     
         3 . The microreactor of  claim 1 , wherein the container is a fused-silica capillary. 
     
     
         4 . The microreactor of  claim 1 , wherein the base is an inorganic base. 
     
     
         5 . The microreactor of  claim 4 , wherein the inorganic base is selected from the group consisting of LiOH, NaOH, KOH, Mg(OH) 2 , and Ca(OH) 2 . 
     
     
         6 . The microreactor of  claim 4 , wherein the inorganic base is NaOH. 
     
     
         7 . The microreactor of  claim 1 , wherein the solvent is selected from the group consisting of DMSO, sulfolane, acetonitrile, HMPA, DMF, DMA, and NMP. 
     
     
         8 . The microreactor of  claim 1 , wherein the solvent is DMSO. 
     
     
         9 . A microreactor for conducting solid-phase permethylation of oligosaccharides, the microreactor comprising:
 a) a container;   b) a solid base disposed within said container; and   c) a polar, aprotic solvent disposed within said container, wherein said solvent includes an oligosaccharide and a source of methyl groups.   
     
     
         10 . The microreactor of  claim 9 , wherein the container is selected from the group consisting of a spin column and a fused-silica capillary. 
     
     
         11 . The microreactor of  claim 9 , wherein the container is a fused-silica capillary. 
     
     
         12 . The microreactor of  claim 9 , wherein the base is an inorganic base. 
     
     
         13 . The microreactor of  claim 12 , wherein the inorganic base is selected from the group consisting of LiOH, NaOH, KOH, Mg(OH) 2 , and Ca(OH) 2 . 
     
     
         14 . The microreactor of  claim 12 , wherein the inorganic base is NaOH. 
     
     
         15 . The microreactor of  claim 9 , wherein the solvent is selected from the group consisting of DMSO, sulfolane, acetonitrile, HMPA, DMF, DMA, and NMP. 
     
     
         16 . The microreactor of  claim 9 , wherein the solvent is DMSO. 
     
     
         17 . The microreactor of  claim 9 , wherein the source of methyl groups is selected from the group consisting of methyl iodide, methyl bromide, and methyl triflate. 
     
     
         18 . The microreactor of  claim 9 , wherein the source of methyl groups is methyl iodide. 
     
     
         19 . The microreactor of  claim 9 , wherein the oligosaccharide is selected from the group consisting of N-linked oligosaccharide and O-linked oligosaccharide. 
     
     
         20 . A method for conducting solid-phase permethylation of oligosaccharides, the method comprising:
 a) infusing a polar, aprotic solvent through a packed inorganic base, wherein the solvent includes an oligosaccharide and a source of methyl groups;   b) contacting the oligosaccharide with the source of methyl groups; and   c) collecting a permethylated oligosaccharide.   
     
     
         21 . The method of  claim 20 , wherein said method is conducted in the microreactor of  claim 9 . 
     
     
         22 . The method of  claim 21 , wherein the infusing solvent is diposed within the microreactor container during permethylation. 
     
     
         23 . The method of  claim 21 , wherein the infusing solvent is delivered to the microreactor container during permethylation. 
     
     
         24 . The method of  claim 21 , wherein the container is a fused-silica capillary. 
     
     
         25 . The method of  claim 21 , wherein the base is NaOH. 
     
     
         26 . The method of  claim 21 , wherein the solvent is DMSO. 
     
     
         27 . The method of  claim 21 , wherein the source of methyl groups is methyl iodide. 
     
     
         28 . The method of  claim 21 , wherein infusing is carried out by centrifugation. 
     
     
         29 . The method of  claim 21 , wherein infusing is carried out by a means including a syringe. 
     
     
         30 . A method for analyzing oligosaccharides, the method comprising:
 a) conducting solid-phase permethylation of oligosaccharides to afford permethylated oligosaccharides, and   b) analyzing said permethylated oligosaccharides by mass spectrometry.   
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 30 , wherein mass spectrometry is selected from the group consisting of MALDI-TOFMS and ESI-MS. 
     
     
         33 . The method of  claim 30 , further comprising the step of separating the permethylated oligosaccharides by RPLC prior to analyzing the permethylated oligosaccharides by mass spectrometry. 
     
     
         34 . An apparatus for interfacing permethylation of oligosaccharides on-line with separation and analysis of permethylated oligosaccharides, the apparatus comprising:
 a) a microreactor for conducting solid-phase permethylation of oligosaccharides;   b) a RPLC column; and   c) a mass spectrometer, wherein said microreactor, said RPLC column, and said mass spectrometer are interconnected.   
     
     
         35 . (canceled) 
     
     
         36 . The apparatus of  claim 34  wherein the RPLC column is a C 18  column. 
     
     
         37 . The apparatus of  claim 34 , wherein the mass spectrometer is selected from the group consisting of a MALDI-TOFMS and ESI-MS mass spectrometer.