Reagents and methods for screening MPS I, II, IIIA, IIIB, IVA, VI, AND VII

1 . A method for assaying enzymatic activities of one or more lysosomal enzymes, comprising:

(a) contacting a sample with a first solution to provide a solution comprising one or more lysosomal enzymes;

(b) contacting the one or more lysosomal enzymes in solution with an enzyme substrate for each lysosomal enzyme to be analyzed and incubating the substrates with the enzymes for a time sufficient to provide a solution comprising a first enzyme product for each lysosomal enzyme present in the sample;

(c) subjecting the first enzyme products to a glycohydrolase to provide a second enzyme product for each first enzyme product susceptible to further enzymatic action by the glycohydrolase; and

(d) determining the quantities of one or more of the first enzyme products and/or one or more of the second enzyme products

wherein the substrate has a carbohydrate moiety and an aglycone moiety and has a formula:

or a salt thereof,

wherein

L 2 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, and S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;

L 3 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;

L 4 is optional and when present is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S), and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen,

R 1 is a C 1 -C 10 alkyl group;

R 2 at each occurrence is independently selected from a C 1 -C 10 alkyl group, a C 1 -C 10 alkoxy group, halogen, nitro, —C(═O)NHR, or —C(═O)OR, where R is C 1 -C 8 alkyl group;

R 3 is a C 1 -C 10 alkyl group or a substituted or unsubstituted C 6 -C 10 aryl group; n is 0, 1, 2, 3, or 4; and

S is

2 . The method of claim 1 , wherein L 2 is —(CH 2 ) n —, where n is 1-6.

3 . The method of claim 1 , wherein L 3 is —(CH 2 ) m —, where m is 1-12.

4 . The method of claim 1 , wherein L 4 is —(CH 2 ) n —, where n is 1-6.

5 . The method of claim 1 , wherein L 4 is absent.

6 . The method of claim 1 , wherein R 1 is C 1 -C 5 alkyl.

7 . The method of claim 1 , wherein R 2 is C 1 -C 8 alkyl.

8 . The method of claim 1 , wherein R 3 is C 1 -C 6 alkyl.

9 . The method of claim 1 , wherein R 3 is phenyl.

10 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

11 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

12 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

13 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

14 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

15 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

16 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

17 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

18 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

19 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

20 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

21 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

22 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

23 . The method of claim 1 , wherein the substrate has a formula

or a salt thereof.

24 . The method of claim 1 , wherein the enzyme is selected from one or more of alpha-L-iduronidase (MPS-I), iduronate-2-sulfatase (MPS-II), heparan N-sulfatase (MPSIIIA), N-acetyl-alpha-D-glycosaminidase (MPS-IIIB), N-acetylgalactosamine-6-sulfate-sulfatase (MPS-IVA), N-acetylgalactosamine-4-sulfate-sulfatase (MPS-VI), and beta-glucuronidase (MPS-VII).

25 . The method of claim 1 , wherein determining the quantities of the enzyme products comprises mass spectrometric analysis.

26 . The method of claim 1 , wherein determining the quantities of the enzyme products comprises conducting the products to a mass spectrometer by liquid chromatography or by flow injection.

27 . The method of claim 1 further comprising using the quantities of the enzyme products to determine whether the sample is from a candidate for treatment for a condition associated with one or more lysosomal enzyme deficiencies.

28 . A method for assaying enzymatic activities of one or more lysosomal enzymes, comprising:

(a) contacting a sample with a first solution to provide a solution comprising one or more lysosomal enzymes;

(b) contacting the one or more lysosomal enzymes in solution with an enzyme substrate for each lysosomal enzyme to be analyzed and incubating the substrates with the enzymes for a time sufficient to provide a solution comprising a first enzyme product for each lysosomal enzyme present in the sample;

(c) subjecting the first enzyme products to reaction with an exogenous glycohydrolase to provide a second enzyme product for each first enzyme product susceptible to further enzymatic action by the glycohydrolase; and

(d) determining the quantities of one or more of the second enzyme products.

29 . The method of claim 28 , wherein the enzyme substrate has a carbohydrate moiety and an aglycone moiety and has a formula:

or a salt thereof,

wherein

L 2 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, and S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;

L 3 is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S, and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;

L 4 is optional and when present is a linker comprising 1-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, or S), and/or one or more of carbon atoms may be substituted with a C 1 -C 6 alkyl group or halogen;

R 1 is a C 1 -C 10 alkyl group;

R 2 at each occurrence is independently selected from a C 1 -C 10 alkyl group, a C 1 -C 10 alkoxy group, halogen, nitro, —C(═O)NHR, or —C(═O)OR, where R is C 1 -C 8 alkyl group;

R 3 is a C 1 -C 10 alkyl group or a substituted or unsubstituted C 6 -C 10 aryl group; n is 0, 1, 2, 3, or 4; and

S is

30 . The method of claim 28 , wherein the enzyme is selected from one or more of alpha-L-iduronidase (MPS-I), iduronate-2-sulfatase (MPS-II), heparan N-sulfatase (MPSIIIA), N-acetyl-alpha-D-glycosaminidase (MPS-IIIB), N-acetylgalactosamine-6-sulfate-sulfatase (MPS-IVA), N-acetylgalactosamine-4-sulfate-sulfatase (MPS-VI), and beta-glucuronidase (MPS-VII).

31 . The method of claim 28 , wherein determining the quantities of the enzyme products comprises mass spectrometric analysis.

32 . The method of claim 28 , wherein determining the quantities of the enzyme products comprises conducting the products to a mass spectrometer by liquid chromatography or by flow injection.

33 . The method of claim 28 further comprising using the quantities of the enzyme products to determine whether the sample is from a candidate for treatment for a condition associated with one or more lysosomal enzyme deficiencies.