Alkynyl sugar analogs for labeling and visualization of glycoconjugates in cells

Abstract

The present disclosure relates to a method for metabolic oligosaccharide engineering that incorporates derivatized alkyne-bearing sugar analogs as “tags” into cellular glycoconjugates. The disclosed method incorporates alkynyl derivatized Fuc and alkynyl derivatized ManNAc sugars into a cellular glycoconjugate. A chemical probe comprising an azide group and a visual probe or a fluorogenic probe is used to label the alkyne-derivatized sugar-tagged glycoconjugate. In one aspect, the chemical probe binds covalently to the alkynyl group by Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition and is visualized at the cell surface, intracellularly, or in a cellular extract. The labeled glycoconjugate is capable of detection by flow cytometry, SDS-PAGE, Western blot, ELISA or confocal microscopy, and mass spectrometry.

1 .- 78 . (canceled) 
     
     
         79 . A composition comprising: an azido-derivatized sugar comprising an azido functional group, wherein the sugar is capable of incorporation into a cellular glycan. 
     
     
         80 . The composition of  claim 79 , wherein the azido-derivatized sugar is an azido-derivatized fucose or an azido-derivatized fucose derivative. 
     
     
         81 . The composition of  claim 80 , wherein the azido-derivatized sugar is a 1,2,3,4-tetraacetyl azido fucose 2: 
       
         
           
           
               
               
           
         
       
     
     
         82 . The composition of  claim 79 , further comprising:
 an alkynyl-derivatized probe coupled to the azido functional group.   
     
     
         83 . The composition of  claim 82 , wherein the probe is coupled to the azido functional group by Cu(I) catalyzed [3+2] azide-alkyne cycloaddition. 
     
     
         84 . The composition of  claim 82 , wherein the probe comprises a directly or indirectly detectable moiety. 
     
     
         85 . The composition of  claim 84 , wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety, a fluorogenic moiety, a moiety detectable by biotin-avidin interaction, a moiety detectable by antigen-antibody interaction, and a coumarin. 
     
     
         86 . The composition of  claim 85 , wherein the probe comprises a fluorogenic detectable moiety which turns fluorescent upon Cu(I) catalyzed [3+2] azide-alkyne cycloaddition with the alkynyl functional group. 
     
     
         87 . The composition of  claim 85 , wherein the probe comprises a 4-ethynyl-N-ethyl-1,8-naphthalimide fluorogenic detectable moiety 
       
         
           
           
               
               
           
         
       
     
     
         88 . The composition of  claim 81 , wherein the 1,2,3,4-tetraacetyl azido fucose 2: 
       
         
           
           
               
               
           
         
       
       is prepared by a process comprising:
 (a) providing L(+)-galactonic acid γ-lactone; 
 (b) converting L(+)-galactonic acid γ-lactone to 1,2:3,4-Di-O-isopropylidene-α-L-galactose by treating with strongly acidic cation exchange resin and NaBH 4 ; 
 (c) converting the hydroxyl group at position 6 of 1,2:3,4-Di-O-isopropylidene-α-L-galactose to an azido group by treatment with TsCl and NaN 3  to create 6,7-deoxy-1,2:3,4-di-O-isopropylidene-α-L-Fucose-6-azide (6-azidofucose diacetonide); 
 (d) removing the diacetonide protecting groups from 6-azidofucose diacetonide to form 6-azido fucose; and 
 (e) acetylating the resultant deprotected product 6-alkynyl fucose to form 1,2,3,4-tetraacetyl azido fucose, wherein the 1,2,3,4-tetraacetyl azido fucose is present as a mixture of pyranoside and furanoside forms. 
 
     
     
         89 . A composition comprising: an alkynyl-derivatized sugar comprising an alkynyl functional group, wherein the sugar compound has the structure: