Compounds and methods relating to lysosomal storage disorders
Methods are provided for detecting enzymatic activity of various lysosomal storage enzymes using substrates that 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 internal standards, and inhibitors for use in detecting or reducing enzymatic activity using the inventive substrates.
1. A method for detecting enzymatic activity, comprising:
contacting a sample containing a target enzyme with a composition comprising a first substrate and a second substrate, under conditions wherein the target enzyme is capable of acting on the first substrate or the second substrate to produce an enzymatic product; and
detecting said enzymatic product;
the first substrate having the formula:
where A is a galactose 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;
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; and 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 a C 6 -C 20 heterocyclic containing a heteroatom of N, O or S; and
the second substrate having the formula:
2. The method of claim 1 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.
3. The method of claim 1 wherein A is a a D-galactose.
4. The method of claim 1 wherein B1 is a methylene.
5. The method of claim 1 where B2 is a C 2 -C 7 amido.
6. The method of claim 1 wherein B3 is a C 2 -C 20 alkenyl having a substituent of N, O, or S.
7. The method of claim 1 wherein: A is 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 method of claim 7 wherein B3 is a C 2 -C 20 alkenyl having a substituent of N, O, or S.
9. The method of claim 1 wherein the first substrate comprises:
where: A is galactose; R 1 is a C 1 -C 6 alkyl or a C 2 -C 6 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 method of claim 9 wherein A is a 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 method of claim 9 wherein R 2 is a C 13 alkyl.