Compounds and methods relating to lysosomal storage disorders

Abstract

Substrates are provided that include compounds suitable for detecting the activity of an enzyme such as a lysosomal storage enzyme where the substrates include: a sugar moiety; a linker moiety allowing the conjugation of sugar moiety with the remaining structure of the substrate; and two or more fatty acid chains or derivatives thereof at least one of which is sufficiently structured to provide improved solubility in aqueous or organic solvent systems. Also provided are methods for using substrates for detecting enzymatic activity using the inventive substrates.

1 . A substrate for analysis of an enzyme having formula: 
       
         
           
           
               
               
           
         
         where A is a monosaccharide or a disaccharide linked to O by a glycosidic bond; 
         B1 is selected from the group consisting of: a C 1 -C 20  alkyl; a heteroatom containing C 1 -C 20  alkyl, C 2 -C 20  alkenyl; a heteroatom containing C 2 -C 20  alkenyl, a substituted or unsubstituted C 6 -C 20  aryl; 
         B2 is selected from the group consisting of: a C 2 -C 7  urethane; a C 2 -C 7  amido, a C 2 -C 7  ester, a C 2 -C 7  uriedo; a C 2 -C 7  carbamato; a C 2 -C 7  carbonyl; a C 1 -C 7  alkyl; a heteroatom containing C 1 -C 7  alkyl; a C 1 -C 7  alkyl having a substituent of N, O, or S; a C 2 -C 7  alkenyl; a heteroatom containing C 2 -C 7  alkenyl; a C 2 -C 7  alkenyl having a substituent of N, O, or S; and 
         B3 is selected from the group consisting of: a C 1 -C 20  alkyl, a heteroatom containing C 1 -C 20  alkyl; C 1 -C 20  alkyl having a substituent of N, O, or S; C 4 -C 20  ether; C 1 -C 20  ester; C 1 -C 20  alkenyl; a heteroatom containing C 1 -C 20  alkenyl; C 2 -C 20  alkenyl having a substituent of N, O, or S; C 1 -C 20  alkynl; a heteroatom containing C 1 -C 20  alkynl; C 2 -C 20  alkynl having a substituent of N, O, or S C 6 -C 20  aryl; and C 6 -C 20  heterocyclic containing a heteroatom of N, O or S. 
       
     
     
         2 . The substrate of  claim 1  wherein A is an aldohexose or a ketohexose. 
     
     
         3 . The substrate of  claim 1  wherein A is a D-glucose or a D-galactose. 
     
     
         4 . The substrate of  claim 1  wherein B1 is a methylene. 
     
     
         5 . The substrate of  claim 1  where B2 is a C 2 -C 7  amido. 
     
     
         6 . The substrate of  claim 1  wherein B3 is a C 2 -C 20  alkenyl having a substituent of N, O, or S. 
     
     
         7 . The substrate of  claim 1  wherein A is a D-glucose or a D-galactose; B1 is a C 1 -C 2  alkyl or a C 6  aryl; and B2 is a C 2 -C 7  amido. 
     
     
         8 . The substrate of  claim 7  wherein B3 is a C 2 -C 20  alkenyl having a substituent of N, O, or S. 
     
     
         9 . The substrate of  claim 1  comprising: 
       
         
           
           
               
               
           
         
         where: A is an aldohexose or ketohexose; R 1  is a C 1 -C 6  alkyl or a C 2 -C 20  alkenyl; and R 2  is a C 1 -C 20  alkyl, a C 1 -C 20  alkyl having a substituent of N, O, or S, a C 1 -C 20  alkenyl, or a C 1 -C 20  alkenyl having a substituent of N, O or S. 
       
     
     
         10 . The compound of  claim 9  wherein A is a D-glucose or D-galactose, R 1  is a C 4 -C 6  alkyl, and R 2  is a C 13 -C 20  alkyl, a C 13 -C 20  alkyl having a substituent of N, O, or S, a C 13 -C 20  alkenyl, or a C 13 -C 20  alkenyl having a substituent of N, O or S. 
     
     
         11 . The compound of  claim 10  wherein R 2  is a C 1-3  alkyl. 
     
     
         12 . A substrate for analysis of an enzyme comprising: 
       
         
           
           
               
               
           
         
         where A is a monosaccharide or a disaccharide linked to O by a glycosidic bond; 
         B1 is selected from the group consisting of: a C 1 -C 20  alkyl; a heteroatom containing C 1 -C 20  alkyl, C 2 -C 20  alkenyl; a heteroatom containing C 2 -C 20  alkenyl; and a substituted or unsubstituted C 6 -C 20  aryl; 
         R 1 ′ is a substituted or unsubstituted C, or N; 
         R 2 ′ is a substituted or unsubstituted C, a substituted or unsubstituted N, O, or S; 
         R 3 ′ is a substituted or unsubstituted C, N, or O; 
         R 4 ′ is a nullity, a substituted or unsubstituted C 1 -C 2 , O, or S; 
         R 1  is: a C 1 -C 6  alkyl; a heteroatom containing C 1 -C 7  alkyl: a C 1 -C 7  alkyl having a substituent of N, O, or S; a C 2 -C 7  alkenyl; a heteroatom containing C 2 -C 7  alkenyl; a C 2 -C 7  alkenyl having a substituent of N, O, or S; 
         R 5 ′ is a nullity, a substituted or unsubstituted C 1 -C 2 ; O, or S; and 
         R 6 ′ is a C 1 -C 20  alkyl, a heteroatom containing C 1 -C 20  alkyl; C 1 -C 20  alkyl having a substituent of N, O, or S; C 4 -C 20  ether; C 1 -C 20  ester; C 1 -C 20  alkenyl; a heteroatom containing C 1 -C 20  alkenyl; C 2 -C 20  alkenyl having a substituent of N, O, or S; C 6 -C 20  aryl; and C 6 -C 20  heterocyclic containing a heteroatom of N, O or S. 
       
     
     
         13 . The substrate of  claim 13  wherein A is an aldohexose or ketohexose. 
     
     
         14 . The substrate of  claim 14  wherein A is a D-glucose or D-galactose. 
     
     
         15 . The substrate of  claim 12  selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       where R 2 ′ is a substituted or unsubstituted C or a substituted or unsubstituted N; 
       
         
           
           
               
               
           
         
       
       where R 2 ″ is a H or a methyl and R 2 ′″ is a H or a methyl; and 
       
         
           
           
               
               
           
         
       
       where R 3 ′ is a substituted or unsubstituted C; and
 R 4 ′ is a nullity, a substituted or unsubstituted C 1 -C 2 , O, or S. 
 
     
     
         16 . The substrate of  claim 15  wherein A is an aldohexose or ketohexose. 
     
     
         17 . The substrate of  claim 15  wherein A is a D-glucose or D-galactose. 
     
     
         18 . The substrate of  claim 12  selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       where R 2 ′ is a substituted or unsubstituted C or a substituted or unsubstituted N; and R 6 ′ is a C 1 -C 20  alkyl, a heteroatom containing C 1 -C 20  alkyl; C 1 -C 20  alkyl having a substituent of N, O, or S; C 4 -C 20  ether; C 1 -C 20  ester; C 1 -C 20  alkenyl; a heteroatom containing C 1 -C 20  alkenyl; C 2 -C 20  alkenyl having a substituent of N, O, or S; C 6 -C 20  aryl; and C 6 -C 20  heterocyclic containing a heteroatom of N, O or S; 
       
         
           
           
               
               
           
         
       
       where R 2 ′ is a substituted or unsubstituted C or a substituted or unsubstituted N; R 2  is a C 13 -C 20  alkyl, a heteroatom containing C 13 -C 20  alkyl, a C 13 -C 20  alkyl having a substituent of N, O, or S; a C 13 -C 20  alkenyl, a heteroatom containing C 13 -C 20  alkenyl; or a C 13 -C 20  alkenyl having a substituent of N, O, or S; and 
       
         
           
           
               
               
           
         
       
       where R 2  is a C 13 -C 20  alkyl, a heteroatom containing C 13 -C 20  alkyl, a C 13 -C 20  alkyl having a substituent of N, O, or S; a C 13 -C 20  alkenyl, a heteroatom containing C 13 -C 20  alkenyl; or a C 13 -C 20  alkenyl having a substituent of N, O, or S. 
     
     
         19 . The substrate of  claim 18  wherein A is an aldohexose or ketohexose. 
     
     
         20 . The substrate of  claim 19  wherein A is a D-glucose or D-galactose. 
     
     
         21 . A substrate for analysis of an enzyme comprising: 
       
         
           
           
               
               
           
         
       
       where n is 0, 1, 2, 3, 4, or 5. 
     
     
         22 . The substrate of  claim 21  wherein A is an aldohexose or ketohexose. 
     
     
         23 . The substrate of  claim 22  wherein A is a D-glucose or D-galactose. 
     
     
         24 . A molecule having formula: 
       
         
           
           
               
               
           
         
         where B1 is selected from the group consisting of: a C 1 -C 20  alkyl; a heteroatom containing C 1 -C 20  alkyl, C 2 -C 20  alkenyl; a heteroatom containing C 2 -C 20  alkenyl, a substituted or unsubstituted C 6 -C 20  aryl; 
         B2 is selected from the group consisting of: a C 2 -C 7  urethane; a C 2 -C 7  amido, a C 2 -C 7  ester, a C 2 -C 7  uriedo; a C 2 -C 7  carbamato; a C 2 -C 7  carbonyl; a C 1 -C 7  alkyl; a heteroatom containing C 1 -C 7  alkyl; a C 1 -C 7  alkyl having a substituent of N, O, or S; a C 2 -C 7  alkenyl; a heteroatom containing C 2 -C 7  alkenyl; a C 2 -C 7  alkenyl having a substituent of N, O, or S; and 
         B3 is selected from the group consisting of: a C 1 -C 20  alkyl, a heteroatom containing C 1 -C 20  alkyl; C 1 -C 20  alkyl having a substituent of N, O, or S; C 4 -C 20  ether; C 1 -C 20  ester; C 1 -C 20  alkenyl; a heteroatom containing C 1 -C 20  alkenyl; C 2 -C 20  alkenyl having a substituent of N, O, or S; C 1 -C 20  alkynl; a heteroatom containing C 1 -C 20  alkynl; C 2 -C 20  alkynl having a substituent of N, O, or S C 6 -C 20  aryl; and C 6 -C 20  heterocyclic containing a heteroatom of N, O or S. 
       
     
     
         25 . The molecule of  claim 24  including a stable secondary prevalence isotope of an element. 
     
     
         26 . The molecule of  claim 25  wherein said stable secondary prevalence isotope in each occurrence is selected from the group consisting of  2 H,  13 C,  15 N,  17 O,  18 O,  31 P and  34 S. 
     
     
         27 . The molecule of  claim 24  wherein B1 is a methylene. 
     
     
         28 . The molecule of  claim 24  where B2 is a C 2 -C 7  amido. 
     
     
         29 . The molecule of  claim 24  wherein B3 is a C 2 -C 20  alkenyl having a substituent of N, O, or S. 
     
     
         30 . A method for detecting enzymatic activity, comprising:
 contacting a sample containing a target enzyme with a substrate of  claim 1 , under conditions wherein the target enzyme is capable of acting on the substrate to produce an enzymatic product; and   detecting said enzymatic product.   
     
     
         31 . The method of  claim 30  wherein said target enzyme is acid β-glucocerebrosidase and said substrate of  claim 1  wherein said B2 is a C 2 -C 7  amido, and B3 is a C 2 -C 20  alkenyl having a substituent of N, O, or S. 
     
     
         32 . The method of  claim 30  wherein said target enzyme is acid galactocerebroside β-galactosidase or acid-β-glucocerebrosidase and said wherein said B2 is a C 2 -C 7  amido, and B3 is a C 13 -C 20  having a substituent of O present as a hydroxyl.