Fluorogenic substrates, methods of making the substrates, and methods of detecting glycosidase activities

Abstract

Fluorogenic substrates, methods of making the fluorogenic substrates, and methods of detecting glycosidase activities (EC3.2.1) are provided. The fluorogenic substrates are composed of a sugar covalently bonded to the 7-hydroxy position of a substituted coumarin sulfonated fluorophore is provided. The fluorogenic substrates include sodium β-D-cellobioside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate; sodium β-D-glucoside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate; sodium β-D-xyloside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate; sodium β-D-xylobioside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate; and sodium β-D-xylopolyoside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate. The fluorogenic substrates may be made by covalently bonding the sugar moiety to the 7-hydroxy position of the substituted coumarin sulfonated fluorophore by reaction in DMF. Glycosidase activities can be detected in enzymatic extracts, microorganisms, and cells by contacting the extracts, microorganisms, or cells with one of the fluorogenic substrates.

What is claimed is: 
     
         1 . A fluorogenic substrate comprising a sugar covalently bonded to the 7-hydroxy position of a substituted coumarin sulfonated fluorophore selected from the group consisting of:
 sodium β-D-cellobioside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate;   sodium β-D-glucoside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate;   sodium β-D-xyloside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate;   sodium β-D-xylobioside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate; and   sodium β-D-xylopolyoside 6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate.   
     
     
         2 . A method of making the substrate according to  claim 1 , comprising covalently bonding the sugar moiety to the 7-hydroxy position of the corresponding 7-hydroxy substituted coumarin sulfonated fluorophore by reaction in dimethylformamide. 
     
     
         3 . A method of detecting glycosidase activities (EC3.2.1) in enzymatic extracts, either purified or not, or in microorganisms or cells, comprising contacting the extracts, microorganisms, or cells with the fluorogenic substrate according to  claim 1 . 
     
     
         4 . The method according to  claim 3 , wherein said extracts or microorganisms or cells are compartmented in aqueous droplets in a suspension in an oil phase. 
     
     
         5 . The method according to  claim 4 , wherein said aqueous droplets are produced by a micro fluidic device.