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

Abstract

To provide early diagnostic methods for starting treatment before the onset of irreversible symptoms of MPS syndromes.SOLUTION: Provided herein are reagents, methods and kits for assaying enzymes associated with lysosomal storage disease MPS I, II, IIIA, IIIB, IVA, VI and VII. In one aspect, the invention relates to a method for assaying β-glucuronidase comprising: (a) forming a solution containing one or more lysosomal enzymes by contacting a sample to a first solution; (b) contacting the solution containing one or more lysosomal enzymes to a β-glucuronidase enzyme substrate and incubating the solution containing one or more lysosomal enzymes for a time sufficient to form a solution containing glucuronidase enzyme product; and (c) determining the amount of β-glucuronidase.SELECTED DRAWING: None

Description

FIELD OF THEINVENTION

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 874,293, filed September 5, 2013; U.S. Provisional Patent Application No. 61 / 874,331, filed September 5, 2013; U.S. Provisional Patent Application No. 61 / 960,102, filed September 9, 2013; U.S. Provisional Patent Application No. 61 / 960,113, filed September 9, 2013; U.S. Provisional Patent Application No. 61 / 949,970, filed March 7, 2014; U.S. Provisional Patent Application No. 61 / 968,021, filed March 20, 2014; and U.S. Provisional Patent Application No. 62 / 012,020, filed June 13, 2014, the entire contents of each of which are expressly incorporated herein by reference. Government License Rights Provisions This invention was made with Government support under Grant No. DK67859 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention.

[0002] The present invention relates to reagents, methods and kits for screening for MPS I, II, IIIA, IIIB, IVA, VI and VII.

[0003] Treating some lysosomal storage diseases (LSDs) is now possible, and early initiation of therapy often leads to clinical improvement. These encouraging results have led to widespread interest in newborn screening for LSDs.

[0004] Newborn screening programs have been established to quantify the levels of metabolites associated with these treatable diseases. Currently, New York state screens for Krabbe disease, several other states have current legislation for LSD that has led to newborn screening, and Taiwan screens newborns for Pompe and Fabry diseases.

[0005] MPS (MPS I-VII) is a group of metabolic diseases / syndrome caused by the deficiency of one of the lysosomal enzymes that degrade glycosaminoglycans (such as heparan, dermatan, keratan, or chondroitin sulfates). The appropriate enzymes include five sulfatases, four exoglycosidases, and one nonhydrolytic acetyl-N-transferase. These syndromes result in undegraded or partially degraded glycosaminoglycans, which accumulate in the lysosomes, resulting in irreversible multisystem organ damage.

[0006] Although treatments are now available for some MPS syndromes, the optimal effect of these treatments is believed to be achieved when they are initiated before irreversible symptoms appear. As early detection of MPS syndromes maximizes the potential benefit of treatment, suitable tests for early diagnosis need to be developed. Similarly, rapid, inexpensive and reliable diagnostic methods need to be developed that use dried blood spots (DBS) as a sample source, e.g., those submitted to newborn screening laboratories.

[0007] Thus, a need exists for methods and reagents for newborn screening of lysosomal enzyme activity, particularly methods and reagents that allow for improved screening of MPS I, II, IIIA, IIIB, IVA, VI, and VII. The present invention fulfills this need, and further provides related advantages. Summary of the Invention

[0008] The present invention provides reagents for screening for MPS I, II, IIIA, IIIB, IVA, VI, and VII, methods for screening for MPS I, II, IIIA, IIIB, IVA, VI, and VII, and kits containing the reagents.

[0009] In one aspect, the invention provides a method of assaying one or more enzymes associated with a lysosomal storage disease.

[0010] In a first aspect, a method comprises: (a) contacting the sample with a first solution to produce a solution comprising one or more lysosomal enzymes; (b) contacting one or more lysosomal enzymes in solution with an enzymatic substrate for each lysosomal enzyme to be analyzed and incubating the substrate with the enzyme for a time sufficient to produce a solution containing an enzymatic product of each lysosomal enzyme present in the sample; wherein the enzyme substrate for each lysosomal enzyme has a carbohydrate moiety and an aglycone moiety and has the formula:

[0011] [ka]

[0012] is a compound having the formula [wherein S, when covalently attached to the aglycone moiety, is (i) α-L-iduronidase, (ii) iduronate 2-sulfatase, (iii) heparan N-sulfatase, (iv) N-acetyl-α-D-glucosaminidase, (v) N-acetylgalactosamine 6-sulfate sulfatase, (vi) N-acetylgalactosamine 4-sulfate sulfatase, and (vii) β-glucuronidase a carbohydrate moiety which provides a substrate for an enzyme selected from the group consisting of L2 is a linker containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O and S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; L3 is a linker containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; L4 is an optional linker, when present, containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; R1 is C1~C 10 Alkyl group or C1-C 10 is an alkoxy group, R2, in each occurrence, 10 Alkyl groups, C1-C 10 are independently selected from an alkoxy group, a halogen, a nitro, -C(=O)NHR, or -C(=O)OR, where R is a C1-C8 alkyl group; R3 is C1~C 10 Alkyl group or substituted or unsubstituted C6-C 10 is an aryl group, n is 0, 1, 2, 3 or 4. (c) determining the amount of one or more enzyme products; Includes.

[0013] In certain embodiments, the method further comprises contacting the enzyme product with a glycohydrolase to produce a second enzyme product, hi certain embodiments, the method further comprises adding an inhibitor to block endogenous glycohydrolase enzyme activity acting on a substrate of N-acetylgalactosamine 6-sulfate sulfatase or N-acetylgalactosamine 4-sulfate sulfatase.

[0014] In a second aspect, the method comprises: (a) contacting the sample with a first solution to produce a solution comprising one or more lysosomal enzymes; (b) contacting one or more lysosomal enzymes in solution with an enzymatic substrate for each lysosomal enzyme to be analyzed and incubating the substrate with the enzyme for a time sufficient to produce a solution containing a first enzymatic product of each lysosomal enzyme present in the sample; (c) subjecting the first enzyme products to a glycohydrolase to produce a second enzyme product of each first enzyme product that is susceptible to further enzymatic action by the glycohydrolase; (d) determining the amount of the one or more first enzyme products and / or the one or more second enzyme products; Includes.

[0015] In the above method, certain first enzyme products are not susceptible to further enzymatic action by glycohydrolases. The first enzyme product that is not susceptible to further enzymatic action by glycohydrolases is produced by the action of an enzyme selected from:

[0016] (a) α-L-iduronidase, (b) N-acetyl-α-D-glucosaminidase, and (c) β-glucuronidase.

[0017] The first enzymatic product, susceptible to further enzymatic action by a glycohydrolase, is produced by the action of an enzyme selected from:

[0018] (a) iduronate 2-sulfatase, (b) heparan N-sulfatase, (c) N-acetylgalactosamine 6-sulfate sulfatase, and (d) N-acetylgalactosamine 4-sulfate sulfatase.

[0019] In certain embodiments, the method further comprises adding an inhibitor to block endogenous glycohydrolase enzyme activity acting on a substrate of N-acetylgalactosamine 6-sulfate sulfatase or N-acetylgalactosamine 4-sulfate sulfatase, wherein the inhibitor does not significantly inhibit the activity of the glycohydrolase in step (c).

[0020] In the above method, the one or more lysosomal enzymes include an enzyme selected from the following:

[0021] (a) α-L-iduronidase, (b) iduronate 2-sulfatase, (c) heparan N-sulfatase, (d) N-acetyl-α-D-glucosaminidase, (e) N-acetylgalactosamine 6-sulfate sulfatase, (f) N-acetylgalactosamine 4-sulfate sulfatase, and (g) β-glucuronidase.

[0022] In certain embodiments of the above methods, an internal standard for each lysosomal enzyme to be analyzed is added before, after, or simultaneously with contacting the lysosomal enzyme with the substrate.

[0023] In certain embodiments of the above methods, the amount of one or more enzyme products is determined after the enzymatic reaction is inactivated.

[0024] In embodiments of the above methods, the sample is a blood or tissue sample. In certain embodiments, the sample is a dried blood spot.

[0025] Representative glycohydrolases include human hexosaminidase A, bacterial N-acetylhexosaminidase, bacterial β-N-acetylgalactosaminidase, α-L-iduronidase, β-galactosidase (Aspergillus), and α-glucosidase (yeast).

[0026] Representative inhibitors include (Z)-O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino N-phenylcarbamate, 1-deoxynojirimycin, castanospermine, swainsonine, calystegine B2, isofagamine, Tamiflu, gluconohydroxymolactone, glucuronic acid and its lactones and lactams, Relenza, miglitol, phenethyl-substituted gluco- and galacto-imidazoles, N-hydroxyethyldehydronojirimycin, GalNAc thiazoline, and GlcNAc thiazoline.

[0027] In certain embodiments of the above methods, determining the amount of the enzyme products (e.g., the first and / or second enzyme products) comprises mass spectrometry. In certain embodiments, determining the amount of the enzyme products comprises determining the ratio of each product to its internal standard by mass spectrometry. In certain embodiments, determining the amount of the enzyme products comprises tandem mass spectrometry, in which parent ions of the products and their internal standards are generated, isolated, and subjected to collision-induced dissociation to generate product fragment ions and internal standard fragment ions. In certain embodiments, determining the amount of the enzyme products comprises comparing the peak intensities of the product fragment ions and the internal standard fragment ions to calculate the amount of the product. In certain embodiments, determining the amount of the enzyme products comprises directing the products to a mass spectrometer by liquid chromatography or flow injection.

[0028] In certain embodiments of the above methods, determining the amount of the enzyme product comprises fluorimetric analysis.

[0029] In certain embodiments of the above methods, the methods further include using the amount of enzyme product to determine whether the sample is from a candidate for treating a condition associated with one or more lysosomal enzyme deficiencies.

[0030] In a second embodiment of the above method, in certain embodiments, the substrate has a carbohydrate moiety and an aglycone moiety and has the formula:

[0031] [ka]

[0032] have [In the formula, When S is covalently attached to the aglycone moiety, (a) α-L-iduronidase, (b) iduronate 2-sulfatase, (c) heparan N-sulfatase, (d) N-acetyl-α-D-glucosaminidase, (e) N-acetylgalactosamine 6-sulfate sulfatase, (f) N-acetylgalactosamine 4-sulfate sulfatase, and (g) β-glucuronidase a carbohydrate moiety which provides a substrate for an enzyme selected from the group consisting of L2 is a linker containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O and S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; L3 is a linker containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; L4 is an optional linker, when present, containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; R1 is C1~C 10 Alkyl group or C1-C 10 is an alkoxy group, R2, in each occurrence, 10 Alkyl groups, C1-C 10 are independently selected from an alkoxy group, a halogen, a nitro, -C(=O)NHR, or -C(=O)OR, where R is a C1-C8 alkyl group; R3 is C1~C 10 Alkyl group or substituted or unsubstituted C6-C 10 is an aryl group, n is 0, 1, 2, 3 or 4].

[0033] Exemplary substrates useful in the methods of the present invention include the substrates of the present invention described here and below.

[0034] Exemplary internal standards useful in the methods of the present invention include the internal standards of the present invention described herein.

[0035] In another aspect of the invention, reagents (substrates and internal standards) for assaying one or more enzymes associated with lysosomal storage diseases are provided.

[0036] Representative substrates have a carbohydrate moiety and an aglycone moiety and have the formula:

[0037] [ka]

[0038] Compounds having the formula [wherein S, when covalently attached to the aglycone moiety, is (a) α-L-iduronidase, (b) iduronate 2-sulfatase, (c) heparan N-sulfatase, (d) N-acetyl-α-D-glucosaminidase, (e) N-acetylgalactosamine 6-sulfate sulfatase, (f) N-acetylgalactosamine 4-sulfate sulfatase, and (g) β-glucuronidase a carbohydrate moiety which provides a substrate for an enzyme selected from the group consisting of L2 is a linker containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O and S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; L3 is a linker containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; L4 is an optional linker, when present, containing 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be substituted by a C1-C6 alkyl group or a halogen; R1 is C1~C 10 Alkyl group or C1-C 10 is an alkoxy group, R2, in each occurrence, 10 Alkyl groups, C1-C 10 are independently selected from an alkoxy group, a halogen, a nitro, -C(=O)NHR, or -C(=O)OR, where R is a C1-C8 alkyl group; R3 is C1~C 10 Alkyl group or substituted or unsubstituted C6-C 10 is an aryl group, n is 0, 1, 2, 3 or 4].

[0039] In certain embodiments, L2 is -(CH2) n -, where n is 1 to 6.

[0040] In certain embodiments, L3 is -(CH2) m - (wherein m is 1 to 12).

[0041] In certain embodiments, L4 is -(CH2) n - (wherein n is 1 to 6).

[0042] In certain embodiments, L4 is absent.

[0043] In certain embodiments, R1 is C1-C5 alkyl.

[0044] In certain embodiments, R2 is C1-C8 alkyl.

[0045] In certain embodiments, R3 is C1-C6 alkyl.

[0046] In certain embodiments, R3 is phenyl.

[0047] Representative substrates are shown below.

[0048] In certain embodiments, the substrate has the formula:

[0049] [ka]

[0050] has.

[0051] In one embodiment, the substrate has the formula:

[0052] [ka]

[0053] has.

[0054] In certain embodiments, the substrate has the formula:

[0055] [ka]

[0056] has.

[0057] In one embodiment, the substrate has the formula:

[0058] [ka]

[0059] has.

[0060] In certain embodiments, the substrate has the formula:

[0061] [ka]

[0062] has.

[0063] In one embodiment, the substrate has the formula:

[0064] [ka]

[0065] has.

[0066] In certain embodiments, the substrate has the formula:

[0067] [ka]

[0068] has.

[0069] In one embodiment, the substrate has the formula:

[0070] [ka]

[0071] has.

[0072] In certain embodiments, the substrate has the formula:

[0073] [ka]

[0074] has.

[0075] In one embodiment, the substrate has the formula:

[0076] [ka]

[0077] has.

[0078] In certain embodiments, the substrate has the formula:

[0079] [ka]

[0080] has.

[0081] In one embodiment, the substrate has the formula:

[0082] [ka]

[0083] has.

[0084] In certain embodiments, the substrate has the formula:

[0085] [ka]

[0086] has.

[0087] In one embodiment, the substrate has the formula:

[0088] [ka]

[0089] has.

[0090] In a further aspect of the present invention, a kit is provided for assaying one or more enzymes associated with lysosomal storage diseases. In one embodiment, the kit comprises one or more reagents (e.g., substrates and internal standards) of the present invention. In certain embodiments, the enzymes that can be assayed by the kit comprise one or more of α-L-iduronidase (MPS-I), iduronate 2-sulfatase (MPS-II), heparan N-sulfatase (MPS-IIIA), N-acetyl-α-D-glucosaminidase (MPS-IIIB), N-acetylgalactosamine 6-sulfate sulfatase (MPS-IVA), N-acetylgalactosamine 4-sulfate sulfatase (MPS-VI), and β-glucuronidase (MPS-VII). [Brief description of the drawings]

[0091] [Figure 1] 1 is a graph illustrating the amount of aglycone released as a function of the amount of added hexosaminidase A in a representative MPS-VI assay of the invention. Aglycone was detected by UHPLC-MS / MS. [Diagram 2] FIG. 2 is a schematic diagram of the preparation of a representative MPS-IVA substrate of the present invention. [Diagram 3] FIG. 3 is a schematic diagram of the preparation of a representative MPS-VI substrate of the invention. [Figure 4] FIG. 4 is a schematic diagram of the preparation of a representative MPS-VI substrate of the invention. [Diagram 5] FIG. 5 is a schematic diagram of the preparation of a representative MPS-VI product. Detailed Description of the Invention

[0092] The present invention provides reagents for screening for mucopolysaccharidoses I, II, IIIA, IIIB, IVA, VI, and VII (MPS-I, II, IIIA, IIIB, IVA, VI, and VII, respectively), methods for screening for MPS-I, II, IIIA, IIIB, IVA, VI, and VII, and kits containing the reagents.

[0093] In one aspect, the present invention provides reagents for screening MPS-I, II, IIIA, IIIB, IVA, VI, and VII. In certain embodiments, the reagents of the present invention include a substrate (S), a product (P), and an internal standard (IS) for screening MPS I, II, IIIA, IIIB, IVA, VI, and VII.

[0094] In another aspect, the present invention provides a method for screening MPS I, II, IIIA, IIIB, IVA, VI, and VII. The method assays a specific enzyme, the deficiency of which leads to the pathology of lysosomal storage disease. The method advantageously assays one or more of α-L-iduronidase (MPS-I), iduronate 2-sulfatase (MPS-II), heparan N-sulfatase (MPS-IIIA), N-acetyl-α-D-glucosaminidase (MPS-IIIB), N-acetylgalactosamine 6-sulfate sulfatase (MPS-IVA), N-acetylgalactosamine 4-sulfate sulfatase (MPS-VI), and β-glucuronidase (MPS-VII).

[0095] reagent In one aspect, the present invention provides a reagent that can be advantageously utilized to assay an enzyme. The reagent includes an enzyme substrate (S), an enzyme product (P), and an assay internal standard (IS). In certain embodiments, one or more substrates (S) and their corresponding internal standards (IS) are incubated in a suitable buffer with a suitable enzyme source, such as a dried blood spot from a newborn test card or a urine sample, for a sufficient time to form one or more products (P), which are then detected by tandem mass spectrometry. In certain embodiments, the internal standard (IS) is chemically similar or equivalent to the enzyme-formed product, except that the standard has a different mass (e.g., a substituted homolog or a heavy isotope, such as deuterium and / or carbon-13 substitution). In other embodiments, one or more substrates (S) are incubated in a suitable buffer with a suitable enzyme source to form one or more products (P), which are then detected by fluorescence analysis.

[0096] Enzymes that are advantageously assayed with the reagents of the invention include: (a) α-L-iduronidase, which acts on the substrate MPS-IS to produce the product MPS-IP and uses the internal standard MPS-I-IS in the assay; (b) iduronate-2-sulfatase, which acts on the substrate MPS-II-S to generate the product MPS-II-P and uses the internal standard MPS-II-IS in the assay; (c) heparan N-sulfatase, which acts on the substrate MPS-IIIA-S to produce the product MPS-IIIA-P and uses the internal standard MPS-IIIA-IS in the assay; (d) N-acetyl-α-D-glucosaminidase, which acts on the substrate MPS-IIIB-S to produce the product MPS-IIIB-P and uses the internal standard MPS-IIIB-IS in the assay; (e) N-acetylgalactosamine-6-sulfate sulfatase, which acts on the substrate MPS-IVA-S to produce the product MPS-IVA-P and uses the internal standard MPS-IVA-IS in the assay; (f) N-acetylgalactosamine-4-sulfate sulfatase, which acts on the substrate MPS-VI-S to produce the product MPS-VI-P and uses the internal standard MPS-VI-IS in the assay; and (g) β-glucuronidase, which acts on the substrate MPS-VII-S to produce the product MPS-VII-P and uses the internal standard MPS-VII-IS for the assay.

[0097] Below are descriptions of the substrates (S), products (P), and internal standards (IS) of the reagents of the present invention, MPS-I, MPS-II, MPS-IIIA, MPS-IIIB, MPS IVA, MPS-VI, and MPS-VII.

[0098] Sugar-Aglycone

[0099] The substrate of the present invention is a glycoside. The term "glycoside" refers to a compound in which a sugar group (glycone) is linked through its anomeric carbon to another group (aglycone) by a glycosidic bond.

[0100] The substrates of the invention are characterized by having a sugar-aglycone structure. The sugar component of the substrate is either a natural sugar that is a substrate for a particular enzyme or a modified sugar that maintains sufficient functionality to be a substrate for the particular enzyme being assayed. The aglycone component of the substrate allows for the analysis of enzyme activity. The aglycone component of the substrate is also a component of the enzyme product, which is analyzed to determine the enzyme activity. The aglycone component includes analytical capabilities, mass spectrometry or fluorescence. When the analysis is by mass spectrometry, an internal standard may be used that has a different mass than the product. The internal standard is structurally identical to the product and contains one or more isotopes (e.g., deuterium or 13 C) or are structurally similar and have functionally equivalent structures and structural variations (e.g., homolog: -(CH2)5-v.-(CH2)6- or vice versa).

[0101] Aglycone The reagents of the present invention include an aglycone component. The nature of the aglycone may vary depending on the nature of the analytical technique utilized to assay the enzyme of interest. Representative aglycones are represented by the following formulas (I)-(VI). In the aglycone structures below, the wavy line indicates the point of attachment to the sugar anomeric carbon.

[0102] In certain embodiments, the aglycone has the formula:

[0103] [ka]

[0104] It is a type A aglycon having the formula [wherein L1 is a linker covalently connecting G1 to the coumarin moiety, and X1, X2, X3, and X4 are independently in each occurrence hydrogen or halogen (e.g., chloro).

[0105] In certain embodiments, L1 contains 1-20 carbon atoms (branched or linear), where one or more of the carbon atoms may be replaced by an ether oxygen or -C(=O)O- group, a thioether sulfur or -C(=O)S- group, NH, NR, or -C(=O)NH- or -C(=O)NR-, where R is an alkyl group of 1-6 carbons. Replacement of one or more of the carbon atoms by a hydrogen atom is optional. In certain embodiments, L1 is -CH2-C(=O)-NH-(CH2)5-G1.

[0106] G1 includes a positively charged group (e.g., a permanently positively charged group, such as a quaternary ammonium ion), such as one of the following: (a)N(R a )(R b )(R c ) + (In the formula, R a , R b and R c are each independently H or an alkyl group having 1 to 6 carbon atoms; (b) S(R a )(R b ) + (In the formula, R a and R b is as above), (c) pyridinium of the following type:

[0107] [ka]

[0108] (d) pyridinium of the following type:

[0109] [ka]

[0110] (e) pyridinium of the following type:

[0111] [ka]

[0112] (f) pyridinium of the following type:

[0113] [ka]

[0114] In certain embodiments, L1 is -CH2-C(=O)-NH-(CH2)5-C(=O)NH-CH2-C6H4-N + (C5H5), where -C6H4-N + (C5H5) is p-pyridinium phenyl.

[0115] In addition to the coumarin (umbelliferone) aglycones described above, it is understood that other fluorescent aglycones, such as fluoresceins, resorufins, rhodamines, nitrophenols, and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-ones, and halogenated derivatives thereof, may also be utilized.

[0116] In certain embodiments, the type A aglycone has the formula:

[0117] [ka]

[0118] [Wherein, R dis hydrogen or methyl, and X1, X2, X3, and X4 are independently hydrogen or halogen (e.g., chloro) at each occurrence. In addition to the coumarin (umbelliferone) aglycon described above, it is understood that other fluorescent aglycons can be utilized. Suitable other aglycons include fluoresceins, resorufins, rhodamines, nitrophenols, and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-ones), and halogenated derivatives thereof. In the case of fluoresceins, resorufins, nitrophenols, and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-ones), the aglycon is attached to the sugar through its hydroxy group, as in the coumarin described above. In the case of rhodamine, the aglycon is attached to the sugar through its amino group.

[0119] Type A aglycone components can be included in the reagents of the invention to impart fluorescent functionality (i.e., coumarin, umbelliferone, fluorescein, resorufin, nitrophenol, rhodamine, 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) moieties) to produce reagents amenable to analysis by fluorescence techniques.

[0120] In one embodiment, the aglycone is a type B aglycone and has the formula:

[0121] [ka]

[0122] has.

[0123] L2 contains 1-20 carbon atoms (branched or linear), one or more of which may be replaced with a heteroatom (e.g., N, O, S) and / or one or more of which may be substituted (e.g., C1-C6 alkyl, halogen). In certain embodiments, L2 is -(CH2) n-, where n is 1 to 6. In certain embodiments, L2 is -(CH2)2-.

[0124] L3 contains 1-20 carbon atoms (branched or linear), one or more of which may be replaced with a heteroatom (e.g., N, O, S) and / or one or more of which may be substituted (e.g., C1-C6 alkyl, halogen). In certain embodiments, L3 is -(CH2) m -, where m is 1 to 12. In certain embodiments, L3 is -(CH2) m -, where m is 4, 5 or 6.

[0125] R1 is C1~C 10 Alkyl groups (e.g., branched or linear) or C1-C 10 In certain embodiments, R1 is a C1-C5 alkyl group (e.g., methyl, ethyl, n-propyl, n-butyl, n-pentyl).

[0126] R2, in each occurrence, 10 Alkyl groups (e.g., branched or linear), C1-C 10Independently selected from alkoxy groups (e.g., branched or straight chain), halogen (e.g., fluoro, chloro), nitro, -C(=O)NHR, or -C(=O)OR, where R is a C1-C8 alkyl group (e.g., methyl) and n is 0, 1, 2, 3, or 4. Representative substitution patterns of R2 (relative to the phenolic oxygen) include 2-, 2,6-di, 3-, 3,5-di, and 2,3-di (i.e., the 2- and 6-positions are ortho and the 3- and 5-positions are meta). In certain embodiments, R2 is a fluoro, methyl, or methoxy group positioned either ortho or meta to the phenolic oxygen (e.g., 2-fluoro, 2-methyl, 2-methoxy, 3-fluoro, 3-methyl, 3-methoxy). In other embodiments, R2 is a fluoro, methyl, or methoxy group positioned meta to the phenolic oxygen. In certain embodiments, n is zero and the phenylene group is unsubstituted.

[0127] R3 is C1~C 10 Alkyl groups (e.g., branched or linear) or substituted or unsubstituted C6-C 10 An aryl group (e.g., phenyl). The substituent of the aryl group is C1-C 10 Included are alkyl groups (e.g., branched or straight chain), and halogens (e.g., chloro). In certain embodiments, R3 is a C1-C6 alkyl group (e.g., ethyl, n-propyl, n-butyl, n-pentyl). In other embodiments, R3 is a phenyl group.

[0128] In certain embodiments, the type B aglycone has the formula:

[0129] [ka]

[0130] have [wherein L2, L3, R1, R2, and n are as described above for formula (III), R4, at each occurrence, is independently selected from C1-C6 alkyl (e.g., methyl), and m is 0, 1, 2, 3, 4, or 5. In certain embodiments, m is 0. In other embodiments, R4 is a C1-C5 alkyl group (e.g., methyl) and m is 2].

[0131] In another embodiment, the type B aglycone has the formula:

[0132] [ka]

[0133] have [wherein L2, L3, R1, R2, R3, and n are as described above for formula (III), and L4 contains 1-20 carbon atoms (branched or linear), one or more of which may be replaced with a heteroatom (e.g., N, O, S), and / or one or more of which may be substituted (e.g., C1-C6 alkyl, halogen). In certain embodiments, L4 is -(CH2) n -, where n is 1 to 6. In certain embodiments, L4 is -(CH2)-.

[0134] In certain embodiments, the type B aglycone has the formula:

[0135] [ka]

[0136] wherein L2, L3, L4, R1, R2, R4, n and m are as described above for formula (V).

[0137] heavy atom derivatives

[0138] In certain embodiments, the reagents of the present invention include these heavy atom derivatives (i.e., derivatives containing one or more heavy atom isotopes). The heavy atom derivatives are useful as internal standards for assays utilizing mass spectrometry. In certain embodiments, type A and type B aglycones have one or more (e.g., three or more) hydrogen atoms replaced with deuterium or one or more (e.g., three or more) carbon atoms replaced with carbon-13, such that the mass of the aglycone is increased by one or more daltons. In the relationship between an enzyme product and an internal standard pair (e.g., MPS-II-P and MPS-II-IS), the reagents differ in mass, and the difference in mass is due to the use of additional (or subtracted) atoms (e.g., a change in the length of a portion of the compound by, e.g., one or more methylenes for L1, L2, L3, L4, R1, R2, R3, or R4) or a heavy atom (e.g., deuterium for hydrogen, carbon for carbon, etc. in L1, L2, L3, L4, R1, R2, R3, or R4). 13 C, for nitrogen 15 This can be achieved by incorporating

[0139] Representative aglycones for substrate / internal standard pairs for MPS-I, II, IIIA, IIIB, IVA, VI, and VII reagents are listed below: For the MPS-I substrate (see formula (IV)), R1 is methyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6-, R4 is hydrogen and m is 5; for the MPS-I internal standard, R1 is methyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6-, R4 is deuterium and m is 5.

[0140] In the case of the MPS-II substrate (see formula (IV)), R1 is n-butyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6-, R4 is hydrogen and m is 5; in the case of the MPS-II internal standard, R1 is n-butyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6-, R4 is deuterium and m is 5.

[0141] In the case of the MPS-IIIA substrate (see formula (IV)), R1 is ethyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6-, R4 is hydrogen, and m is 5; in the case of the MPS-IIIA internal standard, R1 is ethyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6-, R4 is deuterium, and m is 5.

[0142] In the case of the MPS-IIIB substrate (see formula (III)), R1 is n-butyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6-, and R3 is ethyl; in the case of the MPS-IIIB internal standard, R1 is n-butyl, R2 is deuterium, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6-, and R3 is ethyl.

[0143] In the case of the MPS-IVA substrate (see formula (III)), R1 is n-butyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)5-, and R3 is 3,5-dimethylphenyl; in the case of the MPS-IVA internal standard, R1 is n-butyl, L2 is -CH2CH2-, L3 is -(CH2)5-, R3 is 3,5-dimethylphenyl, R2 is deuterium, and n is 4, and in an alternative embodiment, for the MPS-IVA substrate, R1 is n-pentyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6- and R3 is phenyl, and for the MPS-IVA internal standard, R1 is n-pentyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6- and R3 is d5-phenyl.

[0144] In the case of the MPS-VI substrate (see formula (IV)), R1 is n-butyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)5-, R4 is hydrogen and m is 5; in the case of the MPS-VI internal standard, R1 is n-butyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)5-, R4 is deuterium and m is 5.

[0145] In the case of the MPS-VII substrate (see formula (III)), R1 is butyl, R2 is hydrogen, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6- and R3 is propyl; in the case of the MPS-VII internal standard, R1 is butyl, R2 is deuterium, n is 4, L2 is -CH2CH2-, L3 is -(CH2)6- and R3 is propyl.

[0146] salt In certain embodiments, the reagents may have charged groups (e.g., -NH3 + , -CO2 - , -OSO3 - , -NHSO3 -), carboxylic acid groups (-COH), sulfonic acid groups (-OSOH), and amidosulfonic acid groups (-NHSOH). It is to be understood that the reagents of the present invention include salts thereof (e.g., metal salts).

[0147] The preparation of representative MPS-I, II, IIIA, IIIB, IVA, and VI reagents is described in Examples 1, 4, and 5.

[0148] Below is a description of representative reagents (ie, compounds) of the present invention.

[0149] MPS-I Reagents In one aspect, the present invention provides a compound of formula:

[0150] [ka]

[0151] MPS-I reagents (S, P, and IS reagents) defined by [In the formula, R1=aglycone, R2=H or R1=H, R2=aglycone R3=H, OH, NH2, and R4=H or R4=H, OH, NH2, and R3=H R5=H, OH, NH2, and R6=H or R6=H, OH, NH2, and R5=H R7=H, OH, NH2, and R8=H or R8=H, OH, NH2, and R7=H R9=COOH, and R 10 =H or R 10 =COOH, and R9=H provided that only one of the pairs R3 and R4, R5 and R6, and R7 and R8 may have both R groups as hydrogen (i.e., the carbohydrate ring may contain only a single methylene group (-CH2-) within the ring); Salts thereof and heavy atom derivatives thereof are provided.

[0152] For the compounds shown above, the aglycone is as shown above.

[0153] In certain embodiments of the above MPS-I reagents, the carbohydrate moiety is replaced by a hydrogen atom, in which case a hydrogen atom is added to the aglycone. These reagents represent MPS-I enzyme products and internal standards.

[0154] In one embodiment, the sugar has the formula:

[0155] [ka]

[0156] has.

[0157] In certain embodiments, the compound is capable of converting a charged group (e.g., -NH3 + or -CO2 - ), and carboxylic acid groups (-COH). It is understood that the compounds of the present invention include salts thereof (e.g., metal salts).

[0158] As described above, the compounds of the present invention include these heavy atom derivatives. Heavy atom derivatives are useful as internal standards. In certain embodiments, type A and type B aglycones have one or more (e.g., three or more) hydrogen atoms replaced with deuterium, or one or more (e.g., three or more) carbon atoms replaced with carbon-13, such that the mass of the aglycone increases by one or more daltons. The enzyme product and the internal standard differ in mass, and the difference in mass can be attributed to the use of additional atoms (e.g., a change in the length of a portion of the compound by one or more methylenes) or to the addition of heavy atoms (e.g., deuterium for hydrogen, carbon for carboxylate). 13 C, for nitrogen 15 This can be achieved by incorporating

[0159] In certain embodiments of the MPS-I reagents defined above, the carbohydrate moiety is replaced by a hydrogen atom, in which case a hydrogen atom is added to the aglycone. These reagents represent MPS-I enzyme products and internal standards.

[0160] Representative MPS-I reagents include the following compounds:

[0161] In certain embodiments, the MPS-I substrate has the formula:

[0162] [ka]

[0163] wherein L2, L3, R1, R2, and n are as described above for formula (III).

[0164] In certain embodiments, the MPS-I substrate has the formula:

[0165] [ka]

[0166] wherein L2, L3, and R1 are as described above for formula (III).

[0167] A representative MPS-I substrate has the formula:

[0168] [ka]

[0169] has.

[0170] The MPS-I product formed from the above substrate (MPS-IS) has the following formula:

[0171] [ka]

[0172] wherein L2, L3, R1, R2, and n are as described above for formula (III).

[0173] A representative MPS-I product has the formula:

[0174] [ka]

[0175] has.

[0176] The MPS-I internal standard useful in assaying the product formed from the above substrate (MPS-IS) has the following formula:

[0177] [ka]

[0178] where L2, L3, R1, R2, and n are as described above for formula (III), and the mass of MPS-I-IS differs from the mass of MPS-IP, and the two can be distinguished by mass spectrometry. As noted above, MPS-I-IS can include one or more heavy atom isotopes (not shown in the structure above) or can have structural variations (e.g., one or more of L2, L3, R1, and R2 of the substrate differs from L2, L3, R1, and R2 of the internal standard).

[0179] A representative MPS-I internal standard has the formula:

[0180] [ka]

[0181] has.

[0182] A representative MPS-I product derived from a representative MPS-I substrate can be assayed using a representative MPS-I internal standard.

[0183] MPS-II Reagents In one aspect, the present invention provides a compound of formula:

[0184] [ka]

[0185] MPS-II reagents (S, P, and IS reagents) defined by [In the formula, R1=aglycone, R2=H or R1=H, R2=aglycone R3=OSO3H, NHSO3H, R4=H or R4=OSO3H, NHSO3H, R3=H R5=H, OH, NH2, R6=H or R6=H, OH, NH2, R5=H R7=H, OH, NH2, R8=H or R8=H, OH, NH2, R7=H R9=COOH, R 10 =H or R 10 =COOH, R9 =H provided that only one of the pairs R5 and R6, and R7 and R8, may have both R groups as hydrogen (i.e., the carbohydrate ring may contain only a single methylene group (-CH2-) within the ring); Salts thereof and heavy atom derivatives thereof are provided.

[0186] For the compounds shown above, the aglycone is as shown above.

[0187] In one embodiment, the sugar has the formula:

[0188] [ka]

[0189] has.

[0190] In another embodiment, the sugar has the formula:

[0191] [ka]

[0192] has.

[0193] In certain embodiments, the compound is capable of converting a charged group (e.g., -NH3 + , -CO2 - , -OSO3 -), carboxylic acid groups (-COH), and sulfonic acid groups (e.g., -OSOH). It is understood that the compounds of the invention include salts thereof (e.g., metal salts).

[0194] As described above, the compounds of the present invention include these heavy atom derivatives. Heavy atom derivatives are useful as internal standards. In further embodiments, type A and type B aglycones have one or more (e.g., three or more) hydrogen atoms replaced with deuterium, or one or more (e.g., three or more) carbon atoms replaced with carbon-13, such that the mass of the aglycone increases by one or more daltons. The enzyme product and the internal standard differ in mass, and the difference in mass can be due to the use of additional atoms (e.g., a change in the length of a portion of the compound by one or more methylenes) or heavy atoms (e.g., deuterium for hydrogen, carbon for carboxylate). 13 C, for nitrogen 15 This can be achieved by incorporating

[0195] The MPS-II enzyme products and the internal standards differ in mass, the difference in mass being due to the use of additional atoms (e.g., a change in the length of a portion of the compound by one or more methylenes) or due to the use of heavy atoms (e.g., deuterium for hydrogen, carbon for 13 C, for nitrogen 15 This can be achieved by incorporating

[0196] Representative MPS-II reagents include the following compounds:

[0197] In certain embodiments, the MPS-II substrate has the formula:

[0198] [ka]

[0199] wherein L2, L3, R1, R2, and n are as described above for formula (III).

[0200] In certain embodiments, the MPS-II substrate has the formula:

[0201] [ka]

[0202] wherein L2, L3, and R1 are as described above for formula (III).

[0203] A representative MPS-II substrate has the formula:

[0204] [ka]

[0205] has.

[0206] The MPS-II product formed from the above substrate (MPS-II-S) has the following formula:

[0207] [ka]

[0208] wherein L2, L3, R1, R2, and n are as described above for formula (III). A representative MPS-II product has the formula:

[0209] [ka]

[0210] has.

[0211] An MPS-II internal standard useful in assaying products formed from the above substrate (MPS-II-S) has the following formula:

[0212] [ka]

[0213] where L2, L3, R1, R2, and n are as described above for formula (III), and the mass of MPS-II-IS differs from the mass of MPS-II-P, and the two can be distinguished by mass spectrometry. As noted above, MPS-II-IS can include one or more heavy atom isotopes (not shown in the structure above) or can have structural variations (e.g., one or more of L2, L3, R1, or R2 of the substrate differs from L2, L3, R1, or R2 of the internal standard).

[0214] A representative MPS-II internal standard has the formula:

[0215] [ka]

[0216] has.

[0217] A representative MPS-II product derived from a representative MPS-II substrate can be assayed using a representative MPS-II internal standard.

[0218] MPS-IIIA Reagent In another aspect, the present invention provides a compound of formula:

[0219] [ka]

[0220] MPS-IIIA reagents (S, P and IS reagents) defined by [R1=aglycone, R2=H or R1=H, R2=aglycone R3=H, OH, NH2, NHSO3H, OSO3H, R4=H or R4=H, OH, NH2, NHSO3H, OSO3H, R3=H R5=H, OH, NH2, R6=H or R6=H, OH, NH2, R5=H R7=H, OH, NH2, R8=H or R8=H, OH, NH2, R7=H R9=CH2OH, CH2NH2, R 10 =H or R 10 =CH2OH, CH2NH2, and R9 =H], salts thereof and heavy atom derivatives thereof, wherein the aglycone is as defined above, However, only one of the pairs R3 and R4, R5 and R6, and R7 and R8 may have both R groups as hydrogen (i.e., the carbohydrate ring may contain only a single methylene group (-CH2-) within the ring).

[0221] In one embodiment, the sugar has the formula:

[0222] [ka]

[0223] has.

[0224] In another embodiment, the sugar has the formula:

[0225] [ka]

[0226] has.

[0227] In certain embodiments, the compound is capable of converting a charged group (e.g., -NH3 + , -NHSO3 -), which can be an amino group (e.g., -NH2) and an amidosulfonic acid group (e.g., -NHSO3H). It is understood that the compounds of the invention include salts thereof (e.g., metal salts).

[0228] The MPS-IIIA enzyme products and the internal standard differ in mass, the difference in mass being due to the use of additional atoms (e.g., a change in the length of a portion of the compound by one or more methylenes) or due to the use of heavy atoms (e.g., deuterium for hydrogen, carbon for 13 C, for nitrogen 15 This can be achieved by incorporating

[0229] Representative MPS-IIIA reagents include the following compounds:

[0230] In certain embodiments, the MPS-IIIA substrate has the formula:

[0231] [ka]

[0232] wherein L2, L3, R1, R2, and n are as described above for formula (III).

[0233] In certain embodiments, the MPS-IIIA substrate has the formula:

[0234] [ka]

[0235] wherein L2, L3, and R1 are as described above for formula (III).

[0236] A representative MPS-IIIA substrate has the formula:

[0237] [ka]

[0238] has.

[0239] The MPS-IIIA product formed from the above substrate (MPS-IIIA-S) has the following formula:

[0240] [ka]

[0241] wherein L2, L3, R1, R2, and n are as described above for formula (III). A representative MPS-IIIA product has the formula:

[0242] [ka]

[0243] has.

[0244] An MPS-IIIA internal standard useful in assaying products formed from the above substrate (MPS-IIIA-S) has the following formula:

[0245] [ka]

[0246] where L2, L3, R1, R2, and n are as described above for formula (III), and the mass of MPS-IIIA-IS differs from the mass of MPS-IIIA-P, and the two can be distinguished by mass spectrometry. As noted above, MPS-IIIA-IS can include one or more heavy atom isotopes (not shown in the structure above) or can have structural variations (e.g., one or more of L2, L3, R1, R2 of the substrate differs from L2, L3, R1, R2 of the internal standard).

[0247] A representative MPS-IIIA internal standard has the formula:

[0248] [ka]

[0249] has.

[0250] A representative MPS-IIIA product derived from a representative MPS-IIIA substrate can be assayed using a representative MPS-IIIA internal standard.

[0251] MPS-IIIB Reagents In a further aspect, the present invention provides a compound of formula:

[0252] [ka]

[0253] MPS-IIIB reagents (S, P and IS reagents) defined by [R1=aglycone, R2=H or R1=H, R2=aglycone R3=H, OH, NH2, NHR 11 [In the formula, R 11 = Formyl, Acetyl, C=O((CH2) n CH3) (wherein n=1-6)], and R4=H or R4=H, OH, NH2, NHR 11 [In the formula, R 11 = Formyl, Acetyl, C=O((CH2) n CH3) (wherein n=1-6)], and R3=H R5=H, OH, NH2, and R6=H or R6=H, OH, NH2, and R5=H R7=H, OH, NH2, and R8=H or R8=H, OH, NH2, and R7=H R9 = CH2OH, CH2NH2, and R 10 =H or R 10 =CH2OH, CH2NH2, and R 10 =H], salts thereof and heavy atom derivatives thereof, wherein the aglycone is as defined above, However, only one of the pairs R3 and R4, R5 and R6, and R7 and R8 may have both R groups as hydrogen (i.e., the carbohydrate ring may contain only a single methylene group (-CH2-) within the ring).

[0254] In certain embodiments of the above MPS-IIIB reagents, the carbohydrate moiety is replaced by a hydrogen atom, in which case a hydrogen atom is added to the aglycone. These reagents represent MPS-IIIB enzyme products and internal standards.

[0255] In one embodiment, the sugar has the formula:

[0256] [ka]

[0257] has.

[0258] In the above formula, "NHAc" means "NH-C(=O)CH3".

[0259] In certain embodiments, the compound is capable of converting a charged group (e.g., -NH3 + ) It is to be understood that the compounds of the present invention include salts thereof (e.g., metal salts).

[0260] The MPS-IIIB enzyme products and the internal standard differ in mass, the difference in mass being due to the use of additional atoms (e.g., a change in the length of a portion of the compound by one or more methylenes) or due to the use of heavy atoms (e.g., deuterium for hydrogen, carbon for 13 C, for nitrogen 15This can be achieved by incorporating

[0261] Representative MPS-IIIB reagents include the following compounds:

[0262] In certain embodiments, the MPS-IIIB substrate has the formula:

[0263] [ka]

[0264] wherein L2, L3, R1, R2, and n are as described above for formula (III).

[0265] In certain embodiments, the MPS-IIIB substrate has the formula:

[0266] [ka]

[0267] wherein L2, L3, R1, and R3 are as described above for formula (III).

[0268] A representative MPS-IIIB substrate has the formula:

[0269] [ka]

[0270] has.

[0271] The MPS-IIIB product formed from the above substrate (MPS-IIIB-S) has the following formula:

[0272] [ka]

[0273] wherein L2, L3, R1, R2, R3, and n are as described above for formula (III).

[0274] A representative MPS-IIIB product has the formula:

[0275] [ka]

[0276] has.

[0277] An MPS-IIIB internal standard useful in assaying products formed from the above substrate (MPS-IIIB-S) has the following formula:

[0278] [ka]

[0279] where L2, L3, R1, R2, R3, and n are as described above for formula (III), and the mass of MPS-IIIB-IS differs from the mass of MPS-IIIB-P, and the two can be distinguished by mass spectrometry. As noted above, MPS-IIIB-IS may contain one or more heavy atom isotopes (not shown in the structure above) or may have structural variations (e.g., L2, L3, R1, R2, or R3 of the substrate differs from L2, L3, R1, R2, or R3 of the internal standard).

[0280] A representative MPS-IIIB internal standard has the formula:

[0281] [ka]

[0282] has.

[0283] A representative MPS-IIIB product derived from a representative MPS-IIIB substrate can be assayed using a representative MPS-IIIB internal standard.

[0284] MPS-IVA Reagents In another aspect, the present invention provides a compound of formula:

[0285] [ka]

[0286] MPS-IVA reagents (S, P and IS reagents) defined by [R1=aglycone, R2=H or R1=H, R2=aglycone R3=H, OH, NH2, NHR 11 [In the formula, R 11 = Formyl, Acetyl, C=O((CH2) n CH3) (wherein n=1-6)], and R4=H or R4=H, OH, NH2, NHR 11 [In the formula, R 11 = Formyl, Acetyl, C=O((CH2) n CH3) (wherein n=1-6)], and R3=H R5=H, OH, NH2, and R6=H or R6=H, OH, NH2, and R5=H R7=H, OH, NH2, and R8=H or R8=H, OH, NH2, and R7=H R9 = CH2OH, CH2OSO3H, CH2NH2, CH2NHSO3H, and R 10 =H or R 10 =CH2OH, CH2OSO3H, CH2NH2, CH2NHSO3H, and R9=H] salts and heavy atom derivatives thereof, wherein the aglycone is as defined above, However, only one of the pairs R3 and R4, R5 and R6, and R7 and R8 may have both R groups as hydrogen (i.e., the carbohydrate ring may contain only a single methylene group (-CH2-) within the ring).

[0287] In one embodiment, the sugar has the formula:

[0288] [ka]

[0289] has.

[0290] In certain embodiments, the compound is capable of converting a charged group (e.g., -NH3 + , -OSO3 - ), which can be an amino group (e.g., -NH2) and a sulfonic acid group (e.g., -OSO3H). It is understood that the compounds of the present invention include salts thereof (e.g., metal salts).

[0291] The MPS-IVA enzyme products and the internal standards differ in mass, the difference in mass being due to the use of additional atoms (e.g., a change in the length of a portion of the compound by one or more methylenes) or due to the use of heavy atoms (e.g., deuterium for hydrogen, carbon for 13 C, for nitrogen 15 This can be achieved by incorporating

[0292] Representative MPS-IVA reagents include the following compounds:

[0293] In certain embodiments, the MPS-IVA substrate has the formula:

[0294] [ka]

[0295] where L2, L3, R1, R2, and n are as described above for formula (III), R4 in each occurrence is independently selected from C1-C6 alkyl (e.g., methyl), and m is 0, 1, 2, 3, 4, or 5.

[0296] In other embodiments, the MPS-IVA substrate has the formula:

[0297] [ka]

[0298] wherein L2, L3, R1, R2, R3, and n are as described above for formula (III).

[0299] In certain embodiments, the MPS-IVA substrate has the formula:

[0300] [ka]

[0301] wherein L2, L3, R1, R4, and m are as described above for formula (IV).

[0302] In other embodiments, the MPS-IVA substrate has the formula:

[0303] [ka]

[0304] wherein L2, L3, R1, and R3 are as described above for formula (III).

[0305] A representative MPS-IVA substrate has the formula:

[0306] [ka]

[0307] has.

[0308] Another representative MPS-IVA substrate has the formula:

[0309] [ka]

[0310] has.

[0311] The MPS-IVA product formed from the above substrate (MPS-IVA-S1) has the following formula:

[0312] [ka]

[0313] have [wherein L2, L3, R1, R2, R4, n, and m are as defined above in formula (IV)].

[0314] In another embodiment, the MPS-IVA product formed from the above substrate (MPS-IVA-S2) has the formula:

[0315] [ka]

[0316] wherein L2, L3, R1, and R3 are as described above for formula (III).

[0317] A representative MPS-IVA product has the formula:

[0318] [ka]

[0319] has.

[0320] Another representative MPS-IVA product has the formula:

[0321] [ka]

[0322] has.

[0323] The MPS-IVA internal standard useful in assaying products formed from the above substrates (MPS-IVA-S1 and MPS-IVA-S2) has the following formula:

[0324] [ka]

[0325] and

[0326] [ka]

[0327] where L2, L3, R1, R2, R3, R4, n, and m are as defined above, and the masses of MPS-IVA-IS1 and MPS-IVA-IS2 are different from the masses of MPS-IVA-P1 and MPS-IVA-P2, respectively, and the two can be distinguished by mass spectrometry. As noted above, MPS-IVA-IS1 and MPS-IVA-IS2 may contain one or more heavy atom isotopes (not shown in the structures above) or may have structural variations (e.g., one or more of L2, L3, R1, R2, R3, or R4 of the substrate are different from the L2, L3, R1, R2, R3, or R4 of the internal standard).

[0328] A representative MPS-IVA internal standard has the formula:

[0329] [ka]

[0330] has.

[0331] Another representative MPS-IVA internal standard has the formula:

[0332] [ka]

[0333] has.

[0334] A representative MPS-IVA product derived from a representative MPS-IVA substrate can be assayed using a representative MPS-IVA internal standard.

[0335] Further representative MPS-IVA reagent sets are described below.

[0336] Further representative MPS-IVA substrates have the formula:

[0337] [ka]

[0338] has.

[0339] A further representative MPS-IVA product standard has the formula:

[0340] [ka]

[0341] has.

[0342] A further representative MPS-IVA internal standard has the formula:

[0343] [ka]

[0344] has.

[0345] In the above formula, X is selected from fluoro, methyl, and methoxy.

[0346] MPS-VI Reagents In another aspect, the present invention provides a compound of formula:

[0347] [ka]

[0348] MPS-VI reagents (S, P and IS reagents) defined by [R1=aglycone, R2=H or R1=H, R2=aglycone R3=H, OH, NH2, NHR 11 [In the formula, R 11 = Formyl, Acetyl, C=O((CH2) n CH3) (wherein n=1-6)], and R4=H or R4=H, OH, NH2, NHR 11 [In the formula, R 11 = Formyl, Acetyl, C=O((CH2) n CH3) (wherein n=1-6)], and R3=H R5=H, OH, NH2, and R6=H or R6=H, OH, NH2, and R5=H R7=CH2OH, CH2OSO3H, CH2NH2, CH2NHSO3H, and R8=H or R8=CH2OH, CH2OSO3H, CH2NH2, CH2NHSO3H, and R7=H R9 = CH2OH, CH2NH2, and R 10 =H or R 10 =CH2OH, CH2NH2, and R9=H], salts thereof and heavy atom derivatives thereof, wherein the aglycone is as defined above, However, only one of the pairs R3 and R4, R5 and R6, and R7 and R8 may have both R groups as hydrogen (i.e., the carbohydrate ring may contain only a single methylene group (-CH2-) within the ring).

[0349] In one embodiment, the sugar has the formula:

[0350] [ka]

[0351] has.

[0352] In another embodiment, the sugar has the formula:

[0353] [ka]

[0354] has.

[0355] In the above formula, "AcNH" means "CH3C(=O)NH".

[0356] In certain embodiments, the compound is capable of converting a charged group (e.g., -NH3 + , -OSO3 - ), which can be an amino group (e.g., -NH2) and a sulfonic acid group (e.g., -OSO3H). It is understood that the compounds of the present invention include salts thereof (e.g., metal salts).

[0357] The MPS-VI enzyme products and the internal standards differ in mass, the difference in mass being due to the use of additional atoms (e.g., a change in the length of a portion of the compound by one or more methylenes) or due to the use of heavy atoms (e.g., deuterium for hydrogen, carbon for 13 C, for nitrogen 15 This can be achieved by incorporating

[0358] Representative MPS-VI reagents include the following compounds:

[0359] In certain embodiments, the MPS-VI substrate has the formula:

[0360] [ka]

[0361] wherein L2, L3, R1, R2, and n are as described above for formula (III).

[0362] In certain embodiments, the MPS-VI substrate has the formula:

[0363] [ka]

[0364] wherein L2, L3, and R1 are as described above for formula (III).

[0365] A representative MPS-VI substrate has the formula:

[0366] [ka]

[0367] has.

[0368] The MPS-VI product formed from the above substrate (MPS-VI-S) has the following formula:

[0369] [ka]

[0370] wherein L2, L3, R1, R2, and n are as described above for formula (III).

[0371] A representative MPS-VI product has the formula:

[0372] [ka]

[0373] has.

[0374] An MPS-VI internal standard useful in assaying products formed from the above substrate (MPS-VI-S) has the following formula:

[0375] [ka]

[0376] where L2, L3, R1, R2, and n are as described above for formula (III), and the mass of MPS-VI-IS differs from the mass of MPS-VI-P, and the two can be distinguished by mass spectrometry. As noted above, MPS-VI-IS may contain one or more heavy atom isotopes (not shown in the structure above) or may have structural variations (e.g., one or more of L2, L3, R1, or R2 of the substrate differs from L2, L3, R1, or R2 of the internal standard).

[0377] A representative MPS-VI internal standard has the formula:

[0378] [ka]

[0379] has.

[0380] A representative MPS-VI product derived from a representative MPS-VI substrate can be assayed using a representative MPS-VI internal standard.

[0381] MPS-VII Reagents In one aspect, the present invention provides a compound of formula:

[0382] [ka]

[0383] MPS-VII reagents (S, P and IS reagents) defined by [In the formula, R1=aglycone, R2=H or R1=H, R2=aglycone R3=H, OH, NH2, and R4=H or R4=H, OH, NH2, and R3=H R5=H, OH, NH2, and R6=H or R6=H, OH, NH2, and R5=H R7=H, OH, NH2, and R8=H or R8=H, OH, NH2, and R7=H R9=COOH, and R 10 =H or R 10 =COOH, and R9=H provided that only one of the pairs R3 and R4, R5 and R6, and R7 and R8 may have both R groups as hydrogen (i.e., the carbohydrate ring may contain only a single methylene group (-CH2-) within the ring); Salts thereof and heavy atom derivatives thereof are provided.

[0384] For the compounds shown above, the aglycone is as shown above.

[0385] In certain embodiments of the above MPS-VII reagents, the carbohydrate moiety is replaced by a hydrogen atom, in which case a hydrogen atom is added to the aglycone. These reagents are representative of MPS-VII enzyme products and internal standards.

[0386] In one embodiment, the sugar has the formula:

[0387] [ka]

[0388] has.

[0389] In certain embodiments, the compound is capable of converting a charged group (e.g., -NH3 + or -CO2 - ), and carboxylic acid groups (-COH). It is understood that the compounds of the present invention include salts thereof (e.g., metal salts).

[0390] As described above, the compounds of the present invention include these heavy atom derivatives. Heavy atom derivatives are useful as internal standards. In certain embodiments, type A and type B aglycones have one or more (e.g., three or more) hydrogen atoms replaced with deuterium, or one or more (e.g., three or more) carbon atoms replaced with carbon-13, such that the mass of the aglycone increases by one or more daltons. The enzyme product and the internal standard differ in mass, and the difference in mass can be attributed to the use of additional atoms (e.g., a change in the length of a portion of the compound by one or more methylenes) or to the addition of heavy atoms (e.g., deuterium for hydrogen, carbon for carboxylate). 13 C, for nitrogen 15 This can be achieved by incorporating

[0391] In certain embodiments of the above MPS-VII reagents, the carbohydrate moiety is replaced by a hydrogen atom, in which case a hydrogen atom is added to the aglycone. These reagents are representative of MPS-VII enzyme products and internal standards.

[0392] Representative MPS-VII reagents include the following compounds:

[0393] In certain embodiments, the MPS-VII substrate has the formula:

[0394] [ka]

[0395] wherein L2, L3, R1, R2, R3, and n are as described above for formula (III).

[0396] In certain embodiments, the MPS-VII substrate has the formula:

[0397] [ka]

[0398] wherein L2, L3, R1, and R3 are as described above for formula (III).

[0399] A representative MPS-VII substrate has the formula:

[0400] [ka]

[0401] has.

[0402] The MPS-VII product formed from the above substrate (MPS-VII-S) has the following formula:

[0403] [ka]

[0404] wherein L2, L3, R1, R2, R3, and n are as described above for formula (III).

[0405] A representative MPS-VII product has the formula:

[0406] [ka]

[0407] has.

[0408] An MPS-VII internal standard useful in assaying the product formed from the above substrate (MPS-VII-S) has the following formula:

[0409] [ka]

[0410] where L2, L3, R1, R2, R3, and n are as described above for formula (III), and the mass of MPS-VII-IS differs from the mass of MPS-VII-P, and the two can be distinguished by mass spectrometry. As noted above, MPS-VII-IS can include one or more heavy atom isotopes (not shown in the structure above) or can have structural variations (e.g., one or more of L2, L3, R1, R2, or R3 of the substrate differs from the L2, L3, R1, R2, or R3 of the internal standard).

[0411] A representative MPS-VII internal standard has the formula:

[0412] [ka]

[0413] has.

[0414] A representative MPS-VII product derived from a representative MPS-VII substrate can be assayed using a representative MPS-VII internal standard.

[0415] Reagent Kit The reagents of the present invention can advantageously be assembled in a kit to perform an enzymatic assay. The reagent kit for a particular assay includes an appropriate enzyme substrate and internal standard pair (e.g., MPS-II-S and MPS-II-IS). In certain embodiments, the kit includes more than one substrate / internal standard pair and can be used to assay more than one enzyme (i.e., a multiplex assay in which two, three, four, five or six enzymes can be assayed in one screen). In other embodiments, the kit further includes a buffer for performing the assay. In other embodiments, the kit further includes an enzyme product that can be used to calibrate the mass spectrometer. In other embodiments, the kit further includes a quality control dried blood spot. Instructions for performing the assay can also be included in the kit.

[0416] Enzymatic assays The reagent of the present invention can be advantageously used to assay enzymes associated with lysosomal storage diseases. In the assay, one or more substrates (S) are incubated in a suitable buffer with a suitable enzyme source, such as a dried blood spot from a newborn screening card or a urine sample, for a sufficient time to form one or more products (P), which are then detected by tandem mass spectrometry. In some embodiments, the assay also uses an internal standard (IS) that is chemically identical to the enzyme-formed product, but differs in mass (e.g., heavy isotopes substituted, such as deuterium and / or carbon-13 substitution). Incubation is performed in a suitable buffer to allow the enzyme reaction to proceed (e.g., 100 mM ammonium formate pH 4.5 containing 5 mM barium acetate and 7.5 mM cerium acetate).

[0417] Enzymes that are advantageously assayed with the reagents of the invention include: (a) α-L-iduronidase, which acts on a substrate for MPS-I to generate an MPS-I product and uses an MPS-I internal standard in the assay; (b) iduronate 2-sulfatase, which acts on a substrate for MPS-II to generate an MPS-II product and uses an MPS-II internal standard in the assay; (c) heparan N-sulfatase, which acts on a substrate for MPS-IIIA to generate an MPS-IIIA product and uses an MPS-IIIA internal standard in the assay; (d) N-acetyl-α-D-glucosaminidase, which acts on a substrate for MPS-IIIB to produce a product, MPS-IIIB product, and which uses an MPS-IIIB internal standard in the assay; (e) N-acetylgalactosamine-6-sulfate sulfatase, which acts on a substrate for MPS-IVA to generate an MPS-IVA product and uses an MPS-IVA internal standard in the assay; (f) N-acetylgalactosamine-4-sulfate sulfatase, which acts on a substrate for MPS-VI to generate an MPS-VI product and uses an MPS-VI internal standard in the assay; and (g) β-glucuronidase, which acts on a substrate for MPS-VII to generate an MPS-VII product and uses an MPS-VII internal standard in the assay.

[0418] Representative methods for assaying the above enzymes are described in WO2009 / 026252 (PCT / US2008 / 073516), WO2010 / 081163 (PCT / US2010 / 020801), WO2012 / 027612 (PCT / US2011 / 049224), and WO2013 / 070953 (PCT / US2012 / 064205), the entire contents of which are expressly incorporated by reference herein. The reagents of the present invention may be advantageously utilized in these methods.

[0419] Representative assays using the MPS-I, II, IIIA, IIIB, IVA, and VI reagents of the invention are described in Examples 1-11.

[0420] The assay of the present invention may include modifications that do not depart from the invention. Some modifications are described below.

[0421] In a first embodiment, the substrate and internal standard are incubated with the enzyme source in an assay buffer, followed by quenching (e.g., addition of acetonitrile) and then mass spectrometric analysis (e.g., LC / MSMS) and quantification of the sugar-aglycone product and internal standard (note that for MPS-I, MPS-IIIB, and MPS-VII, the product is the aglycone (and added hydrogen)).

[0422] In a second embodiment, the assay is as described in the first embodiment, except that the enzyme reaction mixture is extracted (optionally without quenching) with an organic solvent suitable for extracting the product and internal standard (e.g., ethyl acetate), and the extracted mixture is concentrated to dryness and then taken up in a solvent suitable for flow injection mass spectrometry analysis (e.g., FIA / MSMS).

[0423] In a third embodiment, the assay is as described in the second embodiment, except that a suspension of anion exchange resin is added to capture the substrate during quenching.

[0424] In a fourth embodiment, the assay is as described in the first embodiment, except that a second enzyme (e.g., glycohydrolase, e.g., bacterial β-N-acetylgalactaminidase) suitable for cleaving the initial sulfatase product (sulfate-removed sugar-aglycone) without cleaving the substrate is added to the assay cocktail (substrate and internal standard). After extraction, concentration, and resolubilization, mass spectrometry (e.g., FIA / MSMS) is performed to quantify the aglycone product and the internal standard. In some cases, there may be an enzyme that is endogenous in the dried blood spot sample that can act on the sulfated sugar-aglycone substrate to cleave the glycosidic bond (i.e., human hexosaminidase A). In this case, an inhibitor of this endogenous enzyme can be added to block the action of the endogenous enzyme on the added substrate. The inhibitor is selected so as not to block the action of the glycohydrolase added to the assay.

[0425] In a fifth embodiment, the assay is as described in the second embodiment, except that a second enzyme (e.g., a glycohydrolase) suitable for cleaving the selective sulfatase sugar-aglycone substrate is added to the assay cocktail (substrate and internal standard). After quenching, the aglycone product and the internal standard are quantified using mass spectrometry (e.g., LS / MSMS). In a variation of this embodiment, an inhibitor of endogenous action (e.g., human hexosaminidase A) is also added to the assay cocktail.

[0426] In a sixth embodiment, the substrate and internal standard are incubated with the enzyme source in an assay buffer, then buffer is added to shift the pH (e.g., to pH 6) to optimize the action of the second enzyme (e.g., glycohydrolase), after which the glycohydrolase (e.g., bacterial β-N-acetylgalactaminidase) is added and incubated (e.g., 1-2 hours). The sample is then quenched and the aglycone product and internal standard are quantified using mass spectrometry (e.g., LS / MSMS). In a variation of this embodiment, an inhibitor of endogenous enzyme activity (e.g., human hexosaminidase A) is also added to the assay cocktail.

[0427] In a seventh embodiment, the assay is as described in the sixth embodiment, except that the enzyme reaction mixture is extracted (optionally without quenching) with an organic solvent suitable for extracting the product and internal standard (e.g., ethyl acetate), and the extracted mixture is concentrated to dryness and then taken up in a solvent suitable for flow injection analysis (e.g., FIA / MSMS). In a variation of this embodiment, an inhibitor of endogenous enzyme activity (e.g., human hexosaminidase A) is also added to the assay cocktail.

[0428] In an eighth embodiment, for the embodiments above utilizing organic solvent extraction to isolate the product and internal standard, after removal of the solvent, a solution of a suitable acylating agent (e.g., acetic anhydride) and a suitable base (e.g., triethylamine) in a suitable solvent is added, and the resulting combination is incubated (1-2 hours) to produce the acylated (e.g., acetylated) aglycone product and internal standard with improved sensitivity in MS analysis.

[0429] In a ninth embodiment, the assay is as described in the eighth embodiment, except that an acylating agent and base are included in the extraction (e.g., ethyl acetate) solvent to acylate (e.g., acetylate) the aglycone and internal standard during the extraction process or after extraction and incubation (e.g., 1-2 hours).

[0430] In a tenth embodiment, the substrate and second enzyme (glycohydrolase) are incubated with the enzyme source in an assay buffer, then inactivated, and the fluorescent product is then quantified by fluorimetry. In a variation of this embodiment, an inhibitor of the endogenous enzyme activity (e.g., human hexosaminidase A) is also added to the assay cocktail. In this embodiment, a substrate with a type A aglycone is used.

[0431] In an eleventh embodiment, the substrate is incubated with the enzyme source in an assay buffer, then buffer is added to shift the pH (e.g., to pH 6) to optimize the action of the second enzyme (e.g., glycohydrolase), after which the glycohydrolase (e.g., bacterial β-N-acetylgalactaminidase) is added, incubated (e.g., for 1-2 hours), then inactivated and the fluorescent product quantified fluorimetrically. In a variation of this embodiment, an inhibitor of endogenous enzyme activity (e.g., human hexosaminidase A) is also added to the assay cocktail. In this embodiment, a substrate with a type A aglycone is used.

[0432] In certain embodiments, additional assay options are included within the scope of the methods of the invention. Two options are described below.

[0433] Assay Option 1 After incubating the desired set of substrates and enzyme source in a suitable buffer, the reaction is subjected to liquid-liquid extraction with a suitable solvent, e.g., ethyl acetate. If MPS-II is assayed to produce aglycones without the use of a second enzyme (glycohydrolase), the mixture must be acidified with a suitable acid, e.g., citric acid, to a pH of about 2-3, which protonates the carboxylate groups of MPS-II-P and allows them to be better extracted into ethyl acetate. If a second enzyme is used to remove sugars in the assay, the aglycones do not need to be acidified and are extracted into the solvent because of the lack of a carboxy group. The purpose of the liquid-liquid extraction step is twofold: (1) the extraction removes most of the buffer salts that are believed to interfere with the ionization process in the mass spectrometer, and (2) the extraction extracts most of the enzyme product while minimizing extraction of the enzyme substrate. This is useful because the loss of sulfate in the ionization source of the mass spectrometer results in partial decomposition of the substrate to form a product, which is the only enzymatically generated product that one wishes to quantify. After liquid-liquid extraction, the ethyl acetate layer is transferred to a new container and the solvent is removed by evaporation. The residue is taken up in a solvent suitable for injection into a mass spectrometer. An exemplary solvent is an aqueous ammonium formate / methanol mixture. The products and internal standards are detected in a multiple reaction monitoring mode, where precursor ions are isolated in the first quadrupole and then undergo collision-induced dissociation to form one or more product ions. One such product ion is isolated in the third quadrupole and detected by an ion detector (tandem mass spectrometry). Each fragmentation reaction is monitored separately in a duty cycle manner, one reaction for each product and internal standard, to quantify the entire set of products and internal standards. To obtain the number of moles of product, the mass spectrometry signal (ion count) of the product is divided by that of the internal standard, and this ratio is multiplied by the number of moles of internal standard added to the assay.

[0434] Assay Option 2 A modification of the above assay uses a modified pre-mass spectrometry sample work-up. After incubation to enzymatically generate the product from the substrate, a small aliquot of a suitable anion exchange resin is added to the mixture. An exemplary resin is DE52 from Whatmann. It is well known that anions bind to cations on anion exchange resins by electrostatic interactions, in which case all anionic analytes bind to the resin. Substrates MPS-IS, MPS-II-S, MPS-IIIA-S, MPS-IVA-S, MPS-VI-S, and MPS-VII-S contain either a carboxylate (MPS-IS and MPS-VII-S) or a sulfate ester to bind to the resin. MPS-IP, MPS-I-IS, MPS-IVA-P, MPS-IVA-IS, MPS-VI-P, MPS-VI-IS, MPS-VII-P, and MPS-VII-IS lack a charge or contain a positive charge (MPS-IIIA-P and MPS-IIIA-IS) and therefore do not bind to the resin. MPS-IIIB-S, MPS-IIIB-P, and MPS-IIIB-IS also lack a negative charge and do not bind to the resin. MPS-II-S, MPS-II-P, and MPS-II-IS are all anionic and therefore all bind to the resin. In this assay option, the assay buffer contains recombinant α-L-iduronidase, which acts on MPS-II-P and MPS-II-IS, but not on MPS-II-S, to remove iduronic acid residues from the aglycon, leaving a free aglycon lacking a charge. Therefore, the resulting analytes derived from MPS-II-P and MPS-II-IS do not bind to the anion exchange resin. If recombinant α-L-iduronidase is included, the assay cannot include MPS-IS because the enzyme acts on MPS-IS to create MPS-IP. The use of α-L-iduronidase is not limited to those MPS-II assays that add an anion exchange resin. The addition of this enzyme is applicable to all other MPS-II assays that do not use an anion exchanger.

[0435] After addition of the anion exchange resin, the mixture is extracted with ethyl acetate, and any analytes not bound to the resin are extracted into the ethyl acetate, as in Assay Option 1. The ethyl acetate layer is then processed as described in Assay Option 1 for analysis by tandem mass spectrometry.

[0436] Multiplex Assays The method of the present invention provides for the analysis of one or more of MPS-I, MPS-II, MPS-IIIA, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII, and any combination thereof. In the embodiment of using mass spectrometry to quantify the assay products, in certain embodiments, the products for each assay are different in mass. The mass of each product is different so that one assay can be used to quantify all the assay products. Mass discrimination is achieved by the selection of the substrate.

[0437] The representative substrates for MPS-I, MPS-II, MPS-IIIA, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII above generate products of different masses (i.e., no two products are identical in mass), which, when used in conjunction with corresponding internal standards (see representative internal standards for MPS-I, MPS-II, MPS-IIIA, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII above), results in the ability to run and analyze more than a single assay at a time.

[0438] In other embodiments, more than one product ion may have the same mass. In these embodiments, quantification is obtained as long as the fragment masses derived from these isobaric products are different (i.e., the parent ion mass / fragment ion mass combination is unique for quantifying each species in a mixture).

[0439] In one aspect, the invention provides a method for simultaneously assaying MPS-I, MPS-II, MPS-IIIA, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII, or any subset thereof, using the reagents described herein.

[0440] Sulfatase Assay: MPS II, IIIA, IVA, and VI Screening In another embodiment, the present invention provides assays, reagents and kits for the detection of sulfatases associated with lysosomal storage diseases (MPS II, IIIA, IVA and VI) for newborn screening.

[0441] Assays for lysosomal enzymes using tandem mass spectrometry are useful for newborn screening for lysosomal storage diseases. The assays utilize substrates of the general structure: sulfate-sugar-aglycone. These substrates are acted upon by lysosomal sulfatases to yield products lacking the sulfate: sugar-aglycone. In some assays, the sugar-aglycone products were detected using tandem mass spectrometry.

[0442] The present invention provides an alternative to such sulfatase assays. In certain aspects of the assays of the present invention, a second enzyme is added to the assay cocktail to remove sugars to produce aglycones. Detection of the aglycones by tandem mass spectrometry is more sensitive than detection of sugar-aglycones. The second enzyme that removes sugars does not act on sulfated sugars (i.e., the second enzyme does not act on a substrate for sulfatase). Thus, the present invention provides methods that include the additional step of adding a suitable glycohydrolase or a suitable lyase to the assay cocktail to produce aglycones as the final enzymatic product, which are then detected by tandem mass spectrometry.

[0443] As used herein, the term "glycohydrolase" refers to an enzyme that hydrolyzes a glycoside. The term "lyase" refers to an enzyme that removes a proton from the sugar C2, eliminating the glycosidic oxygen (e.g., the aglycone leaving group) and generating an unsaturated sugar derivative.

[0444] Suitable second enzymes (e.g., glycohydrolases and lyases) are characterized in that they cleave sugars from the aglycones of the enzyme products described herein (e.g., they only cleave sugars if the sulfate is removed), they do not act on the enzyme substrate itself, and they are not inhibited by inhibitors added to the assays of the invention to block the action of endogenous enzymes present in dried blood spots, such as hexosaminidase A, which may cleave sulfated substrates to produce the aglycones.

[0445] Suitable second enzymes include glycohydrolases and lyases.

[0446] Representative glycohydrolases include human hexosaminidase A, bacterial N-acetylhexosaminidase, bacterial β-N-acetylgalactosaminidase (eg, Paenibacillus TS12), α-L-iduronidase, β-galactosidase (Aspergillus), and α-glucosidase (yeast).

[0447] Representative lyases include heparin lyase (heparinase) and heparanase.

[0448] Assay Method In one aspect, the present invention provides a method for screening MPS II, IIIA, IVA, and VI, which assays specific enzymes whose defects lead to lysosomal storage disease conditions. The method advantageously assays one or more of iduronate 2-sulfatase (MPS-II), heparan N-sulfatase (MPS-IIIA), N-acetylgalactosamine-6-sulfate sulfatase (MPS-IVA), and N-acetylgalactosamine-4-sulfate sulfatase (MPS-VI).

[0449] As mentioned above, in the method of the invention, a second enzyme is utilized to improve the sensitivity of mass spectrometric embodiments and to generate a fluorophore in fluorometric embodiments. According to the method, a suitable sulfatase substrate (i.e., sulfate-sugar-aglycone) is contacted with the sample to be evaluated for sulfatase activity. If the sample contains sulfatase, the substrate is enzymatically converted to the initial enzyme product (i.e., sugar-aglycone). In the method of the invention, a second enzyme (e.g., glycohydrolase) acts on the initial enzyme product to generate a secondary enzyme product (i.e., aglycone). Analysis of the secondary enzyme product (i.e., aglycone) by tandem mass spectrometry shows high sensitivity compared to previous assays that do not include the second enzyme and rely on analysis of the sugar-aglycone of the initially formed enzyme product. In the case of substrates containing type A aglycone, the second enzyme acts to release the fluorescent aglycone only after the sulfate is removed. This allows the assay of sulfatase by fluorometric analysis.

[0450] For fluorescence analysis, quenching may involve buffering with an increased pH to about 10, which deprotonates the phenolic hydroxy of the aglycone, thereby making the product (aglycone) more fluorescent.

[0451] In the assay, one or more substrates (S) are incubated in a suitable buffer with a suitable enzyme source, e.g., a dried blood spot from a newborn screening card or a urine sample, for a sufficient time to form one or more products (P1), which are subsequently subjected to a second enzyme (i.e., a glycohydrolase) to generate a secondary enzyme product (P2), which is detected by tandem mass spectrometry. In certain embodiments, the assay also uses an internal standard (IS) that is chemically identical to the enzyme-formed product, but has a different mass (e.g., a substituted heavy isotope, e.g., deuterium and / or carbon-13 substitution). In certain assays, the internal standard is also acted upon by the second enzyme to generate a final internal standard, which is detected by tandem mass spectrometry. Incubation is performed in a suitable buffer to allow the enzymatic reaction to proceed. A suitable buffer is, for example, 100 mM ammonium formate pH 4.5 containing 7.5 mM barium acetate and 5 mM cerium acetate.

[0452] Advantageously, enzymes to be assayed together with the reagents of the invention include: (a) iduronate 2-sulfatase, which acts on a substrate for MPS-II to generate an MPS-II product and uses an MPS-II internal standard in the assay; (b) heparan N-sulfatase, which acts on a substrate for MPS-IIIA to generate an MPS-IIIA product and uses an MPS-IIIA internal standard in the assay; (c) N-acetylgalactosamine-6-sulfate sulfatase, which acts on a substrate for MPS-IVA to generate an MPS-IVA product and uses an MPS-IVA internal standard in the assay; (d) N-acetylgalactosamine-4-sulfate sulfatase, which acts on an MPS-VI substrate to generate an MPS-VI product and uses an MPS-VI internal standard in the assay.

[0453] In certain embodiments, additional assay options are also included within the scope of the methods of the invention. Assay options 1 and 2 above may be utilized in these assay methods.

[0454] reagent The reagents for screening MPS II, IIIA, IVA, and VI include substrates (S), products (P), and internal standards (IS) for screening MPS II, IIIA, IVA, and VI.

[0455] Reagents can be advantageously utilized to assay enzymes. The reagents include enzyme substrates (S), enzyme products (P), and assay internal standards (IS). In certain embodiments, one or more substrates (S) and their corresponding internal standards (IS) are incubated in a suitable buffer with a suitable enzyme source, such as a dried blood spot from a newborn screening card or a urine sample, for a sufficient time to form one or more products (P), which are subsequently subjected to a second enzyme (e.g., glycohydrolase) to generate a second enzyme product, which is detected by tandem mass spectrometry. In certain embodiments, the internal standards (IS) are chemically identical to the enzyme-formed products, except for the mass of the standard (e.g., heavy isotopes substituted, such as deuterium and / or carbon-13 substitutions).

[0456] Reagents useful in assaying these sulfatases include substrates (S), products (P), and internal standards (IS) for MPS-II, MPS-IIIA, MPS IVA, and MPS-VI, including those described herein.

[0457] Representative assays for MPS-II, MPS-IIIA, MPS IVA, and MPS-VI are described in Examples 7-11.

[0458] Representative Assays and Results of MPS IVA and VI Screening As described above, glycohydrolase and lyase enzymes are used to improve the sensitivity of the sulfatase assay using mass spectrometry and to generate a fluorescent aglycone product for fluorometric analysis. In a related aspect, a method is provided which further comprises adding an inhibitor to block endogenous glycohydrolase activity.

[0459] A suitable inhibitor blocks endogenous glycohydrolase activity but does not significantly inhibit the action of added glycohydrolase. Dried blood spots contain, for example, hexosaminidase A, which can act on sulfated substrates in one step to form aglycones. In this case, it is optimal to add an inhibitor of hexosaminidase when measuring aglycones by tandem mass spectrometry or fluorimetry to quantify sulfatase enzymes. The added inhibitor should not significantly inhibit the second enzyme added to the assay to convert the initial sulfatase product to aglycone.

[0460] A suitable inhibitor blocks the hexosaminidase in the biological sample but does not completely block the second enzyme. The inhibitor can partially block the second enzyme, but not completely, which may convert most if not all of the initial sulfatase product to its aglycone. A suitable inhibitor inhibits human hexosaminidase A, human hexosaminidase B, and / or human hexosaminidase X.

[0461] Representative inhibitors include (Z)-O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenylcarbamate (Z-PUG-NAc), 1-deoxynojirimycin, castanospermine, swainsonine, calystegine B2, isofagamine, Tamiflu, gluconohydroxymolactone, glucuronic acid and its lactones and lactams, Relenza, miglitol, phenethyl-substituted gluco- and galacto-imidazoles, N-hydroxyethyldehydronojirimycin, GalNAc thiazoline, and GlcNAc thiazoline.

[0462] Representative assays for MPS-IVA and MPS-VI are described below.

[0463] Representative Assays for Mucopolysaccharidosis IVA

[0464] In one embodiment, an assay for MPS-IVA, also known as Morquio A syndrome, is performed to detect N-acetylgalactosamine-6-sulfatase (also known as GALNS, and used interchangeably herein), an enzyme that is deficient in MPS-IVA. The GALNS substrate is N-acetylgalactose-6-sulfate attached to an aglycon, where R1 is n-butyl, L2 is -CH2CH2-, L3 is -(CH2)5-, and R3 is phenyl (see below). The assay involves adding an enzyme that removes N-acetylgalactosamine from the aglycon only after the 6-sulfate has been removed. The enzyme that removes N-acetylgalactosamine from the aglycon is a β-hexosaminidase (e.g., human hexosaminidase A), which cleaves β-glycosides into N-acetylgalactosamine and N-acetylglucosamine residues. The biological sample may contain human hexosaminidase A and other human hexosaminidases. Human hexosaminidase A can act on MPS-IVA substrates to generate aglycones. As shown in Table 1, human hexosaminidase A can cleave glycosides even when the sugar is sulfated at the 6-position. See the row where Z-PUG-NAc is omitted.

[0465] Table 1 also shows that the amount of endogenous hexosaminidase A in dried blood spots poses a problem in the GALNS assay using dried blood spots for newborn screening for Morquio A syndrome.

[0466] [Table 1]

[0467] In certain embodiments, the bacterial enzyme, β-N-acetylgalactosaminidase (β-NGA) from Paenibacillus sp. TS12 (J. Biol. Chem. 2011, 286, 14065-14072), is used in the assay. This enzyme is structurally unrelated to human hexosaminidase, and the data shown in Table 1 indicate that the bacterial enzyme cleaves β-glycosides to N-acetyl-galactosamine when not sulfated at the 6-position, and the enzyme acts less on the substrate if it carries a sulfate.

[0468] In certain embodiments, an inhibitor of human hexosaminidase A is used in the assay to block the action of human hexosaminidase A on the GALNS substrate. In certain embodiments, the inhibitor does not significantly inhibit β-NGA.

[0469] The GALNS substrate used in the above assay has the following structure:

[0470] [ka]

[0471] Representative assays use dried blood spots from newborn screening cards as a source of GALNS. These assays also use β-NGA and an inhibitor of human hexosaminidase A: Z-PUG-NAc ((Z)-O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino N-phenylcarbamate).

[0472] The GALNS enzyme in the dried blood spot acts on the GALNS substrate shown above and liberates the product (i.e., the GALNS substrate without 6-sulfate). The bacterial β-NGA in the assay cocktail acts on the GALNS substrate without 6-sulfate to liberate the aglycone, but does not act on the GALNS substrate. The addition of Z-PUG-NAc to the assay cocktail blocks the ability of endogenous human hexosaminidase A to act on the GALNS substrate to generate the aglycone. The aglycone is then detected by tandem mass spectrometry. Thus, the formation of the aglycone marks the action of GALNS, and sensitivity is gained by detecting the aglycone rather than the desulfation product initially formed.

[0473] A representative sulfatase assay for MPS-IVA is described in Example 10.

[0474] Representative assays for MPS-VI

[0475] In another aspect, the present invention provides an assay developed for MPS-VI to detect arylsulfatase B (ASB), the enzyme deficient in MPS-VI, also known as Maroteaux-Lamy syndrome.

[0476] The following ASB (MPS-VI) substrates were used in the assay:

[0477] [ka]

[0478] ASB removes the 4-sulfate from the N-acetylgalactosamine-4-sulfate group of the ASB substrate. Prior to the present invention, no data were available regarding the ability of human hexosaminidase A to act on N-acetylgalactosamine-4-sulfate. Recombinant human hexosaminidase A acts on the ASB substrate to release the aglycone. The aglycone was detected by UHPLC-MS / MS (see FIG. 1).

[0479] Figure 1 shows that the amount of this aglycone increases with the addition of increasing amounts of hexosaminidase A to the mixture. The mixtures contain 1 mM ASB substrate in 100 mM ammonium formate buffer at pH 5.6 containing the indicated amounts of hexosaminidase. After 5 h of incubation at 37°C, the mixtures are analyzed by UHPLC-MS / MS. The aglycone signal does not indicate contamination of the substrate with aglycone, since there is no aglycone when hexosaminidase A is omitted. The amount of aglycone increases with increasing hexosaminidase A (Figure 1), and since the UHPLC retention period of the aglycone coincides with that of the observed aglycone and differs from that of the ASB substrate, the aglycone is not generated by cleavage of ASB in the electrospray ionization source of the mass spectrometer. Thus, hexosaminidase A acts on the ASB substrate to generate the aglycone. Since hexosaminidase A acts on the ASB substrate to generate an aglycone, this enzyme cannot be used in the coupled analysis of ASB, and furthermore, it is important to inhibit hexosaminidase A so that it does not generate an aglycone directly from the ASB substrate.

[0480] To establish the action of human hexosaminidase A on the ASB substrate, an ASB assay was developed using the same procedure as that developed for GALNS, i.e., adding β-NGA and Z-PUG-NAc. The results are shown in Table 2 below. The experiment for the GALNS assay (see Example 10) was performed, but the GALNS substrate was replaced with 1 mM ASB substrate.

[0481] [Table 2]

[0482] Comparison of experiment 1 (all assays with blood) and experiment 2 (control without blood) shows that in both experiments, the presence of β-NGA converts most of the ASB internal standard to its aglycone. The amount of aglycone with blood, 1,570,000, greatly exceeds the 58,300 formed in the control without blood. Experiment 3, with blood and Z-PUG-NAc but without β-NGA, compares the amount of ASB product, 371,000, with the signal for its aglycone in the presence of β-NGA, 1,570,000, again showing the advantage of sensitivity in converting the product to the aglycone. Finally, experiment 4, without blood, β-NGA or Z-PUG-NAc, shows that the ASB substrate is contaminated by small amounts of the product, 3,340, and its aglycone, 33,800.

[0483] The present invention provides for the use of any enzyme having the properties described herein below to carry out assays of GALNS and ASB enzyme activity: (a) the enzymes used in the assays described herein should cleave sugars to produce aglycones only if the sugars do not possess sulfates at the 4- or 6-positions; and (b) The enzymes used in the assays described herein should not be significantly inhibited by inhibitors added to the assay mixture that significantly inhibit human hexosaminidase A present in the biological sample (e.g., dried blood spots) that is the source of the lysosomal enzyme being assayed.

[0484] Fluorescence detection

[0485] In some embodiments, certain assays of the invention are also useful in assaying the enzymes described herein using fluorescent methods. In one aspect, the aglycones are fluorescent, but are weakly fluorescent when they form glycosidic bonds with sugars. Thus, in one aspect, the combined action of arylsulfatase B (ASB) and bacterial N-acetylgalactosaminidase forms a highly fluorescent aglycone, thus increasing the fluorescent signal. Fluorescent glycosides suitable for use in the assays of the invention are listed below.

[0486] [ka]

[0487] The fluorescent glycoside shown above is a fluorescent ASB substrate containing a glycoside bound to a 7-hydroxycoumarin bearing an additional substituent (R group). The subsequent action of bacterial N-acetylgalactosaminidase followed by ASB results in a substituted 7-hydroxycoumarin, which is more fluorescent than the glycoside. Thus, ASB is assayed by observing an increase in the fluorescent signal. Other fluorophores can be used as long as the glycoside is a substrate for ASB and N-acetylgalactosaminidase, and the fluorescence intensity of the fluorophore changes when the glycoside is cleaved. It should be appreciated that the fluorescence intensity is greatly enhanced at high pH when the phenol becomes a phenoxide as a result of deprotonation. Experimentally, in certain embodiments, the assay is quenched with a pH 10.5 buffer to reveal fluorescence.

[0488] Previously, this method of assaying ASB, nor the use of an enzyme to selectively cleave glycosides, was of no use unless they were sulfated. This method can also be used to assay GALNS using a suitable substrate that contains sulfate at the 6-position of the sugar residue. The present invention provides an assay that uses an N-acetyl-galactosamine-6-sulfate-based substrate, which acts more rapidly on GALNS than other substrates, such as 4-methylumbelliferone, which is linked to galactose-6-sulfate as a glycoside.

[0489] The following examples are offered by way of illustration and not by way of limitation of the present invention.

[0490] [Example] Example 1 Synthesis of Representative MPS-I and MPS-II Reagents In this example, the synthesis of representative MPS-I (MPS-IS-acetyl-C6 and MPS-I-IS-acetyl-C6) and MPS-II (MPS-II-S-pentanoyl-C6 and MPS-II-IS-pentanoyl-C6) reagents is described.

[0491] Synthesis of 1-F-2,3,4-triacetoxy-iduronic acid methyl ester The synthesis of 1-F-2,3,4-triacetoxy-iduronic acid methyl ester is described below.

[0492] Process 1

[0493] [ka]

[0494] A 50.05 g (133.0 mmol) portion of methyl 1,2,3,4-tetra-O-acetyl-α,β-glucopyranosyluronic acid (Carbosynth, UK) was suspended in 295 mL of 33% HBr / acetic acid (Acros Cat. No. 123180010) at 0°C under nitrogen and stirred at 0°C for 15 min. The reaction was allowed to warm to room temperature and stirring was continued for 3 h. The reaction was diluted with 295 mL of toluene and then concentrated on a rotary evaporator (30-35°C water bath and water aspirator suction). The residue was dissolved in 800 mL of EtOAc, washed with 500 mL of ice-cold saturated aqueous NaHCO3, washed with ice-cold brine, then dried over anhydrous Na2SO4. The solution was filtered and the solvent removed by rotary evaporation as above. The compound was placed under high vacuum at room temperature for 1 h. Proton-NMR (CDCl3) is consistent with the product. TLC run on silica using 20% ​​EtOAc / Hexanes (charring with 5% H2SO4 in MeOH) shows starting material at Rf ∼0.5 and product at Rf ∼0.6. Two small spots were detected just above the origin.

[0495] The crude bromide from above was dissolved in 600 mL of anhydrous acetonitrile and stirred under nitrogen for about 5 minutes to dissolve the bromide. The flask was wrapped in aluminum foil to protect from light. 20.27 g of AgF (Oakwood Cat. No. 002862) was added in portions. The reaction was stirred under nitrogen for 24 hours in the dark at room temperature. TLC performed on silica with 20% EtOAc / Hexanes (charred with 5% H2SO4 in MeOH) shows starting material with an Rf of about 0.6 and product with an Rf of about 0.5. The mixture was filtered through a Celite pad by suction and the filtrate was then concentrated by rotary evaporation (water bath at 30-35°C and water aspirator for suction). 180 g silica was loaded into a glass column and hexane was passed through the column. The compound was dissolved in EtOAc and the minimum amount of dry silica was added. The EtOAc was removed by rotary evaporation and the resulting compound / silica mixture was loaded onto the top of the column. The column was operated at low pressure. First, 1 L of 100% hexane was passed through the column and no product was eluted. Then, 2 L of 20% EtOAc / hexane was passed through the column and no product was eluted. 1 L of 25% EtOAc / hexane also did not elute the product. Finally, 3 L of 30% EtOAc / hexane was passed through the column and all the product was eluted. Fractions were pooled and the solvent was removed by rotary evaporation to give a white crystalline solid. The product was dried under high vacuum at room temperature for 1 hour to give 27.0 g of product (60.4% yield). Proton-NMR (CDCl3) was consistent with the product.

[0496] Process 2

[0497] [ka]

[0498] 6.0 g of the above fluoride and 6.0 g of NBS (Aldrich Cat. No. B81255-500G, recrystallized from boiling water and dried overnight under high vacuum) were dissolved in 240 mL of CCl4 (JT Baker Cat. No. 1512-3) and stirred under nitrogen in a round bottom flask placed next to a UV lamp (450 W, Ace Glass 7825-34 lamp, quartz jacket, lamp cooled by water circulation). Another round bottom flask containing the same reaction was placed on the opposite side of the UV lamp. Two reactions were carried out simultaneously in this manner.

[0499] The outside of the hood was covered with aluminum foil to protect the chemist. The lamp was turned on and the reactions were refluxed at 78°C for 2 hours. The lamp was turned off and a 6g portion of NBS was added to each reaction and the UV lamp was turned back on and the reactions were allowed to continue refluxing. After an additional 2 hours, an additional 6g portion of NBS was added as above and reflux continued for another 3 hours in the presence of the UV lamp. After a total of 7 hours, the lamp was turned off and the reactions were allowed to cool to room temperature. Each reaction was filtered through glass wool and the wool was washed with 50 mL of CCl4. Solvent was removed by rotary evaporation (35°C water bath and water aspirator for suction). TLC using 30% EtOAc / Hexanes showed a product spot with an Rf of approx. 0.6 that migrated just above the starting material (charring with 5% H2SO4 in MeOH). 4 x 6 g scale reactions were purified simultaneously on columns loaded with 180 g of silica. The compound was dissolved in EtOAc, a minimal amount of dry silica was added, the EtOAc was removed by rotary evaporation, and the silica / compound mixture was then loaded onto the top of the column. The column was flushed with 1 L of 100% hexane, which did not elute the product. The column was flushed with 2 L of 10% EtOAc / hexane, which did not elute the product. The column was flushed with 3 L of 20% EtOAc / hexane, which eluted the product with two slower eluting spots visible by TLC. Fractions (700 mL-1300 mL of the 3 L addition) that contained the by-product (faster eluting TLC spot) were discarded. Fractions (1400 mL-2500 mL of the 3 L addition) that contained all the product were combined and the solvent removed by rotary evaporation to give a viscous yellow liquid that was left overnight to give a crystalline solid. The solid was placed under high vacuum for 12 hours. A total of 18.90 g of product was obtained from 4 x 6 g of reaction (63.3% yield).

[0500] Process 3

[0501] [ka]

[0502] 9.92 g of the above bromide was dissolved in 154 mL of toluene (non-dried, Macron Chemicals Cat. No. 4483-4L) and stirred under nitrogen. 10.0 mL of Bu3SnH (either Aldrich 234188-10G or Acros 215730500) was added and the mixture was refluxed at 110° C. for 1 h. TLC (10% EtOAc in toluene) indicated some starting material remained, so reflux was continued for an additional hour. TLC was repeated, indicating the reaction was complete. The round-bottom flask was allowed to cool to room temperature and the solvent was removed by rotary evaporation (water bath at approximately 40° C. and water aspirator for suction).

[0503] 180 g of silica was loaded onto the column. The crude residue was absorbed onto the silica and loaded onto the column as before. The column was flushed with 1 L of 100% toluene, which did not elute the product. The column was flushed with 1 L of 10% EtOAc / toluene, which did not elute the product. The column was flushed with 2.5 L of 20% EtOAc / toluene, which eluted the product and gave two slower eluting spots at Rf .about.0.6 (TLC in 10% EtOAc / toluene). The faster eluting spot at Rf .about.0.7 was the by-product glucuronic acid. The fractions containing the faster eluting TLC spots (600 mL-1000 mL of the 2.5 L) were discarded. Fractions containing the product (1100 mL-2100 mL of 2.5 L) were pooled, concentrated by rotary evaporation (water bath at approximately 40° C. and water aspirator for suction), and placed under high vacuum for 3 hours to give 5.2 g of product (64.7% yield), which upon purification is a clear, viscous liquid.

[0504] Preparation of aglycones The aglycone can be made by two methods. The first method utilizes the Michael addition of commercially available mono-BOC-1,6-hexanediamine to HP-Ph-NHCO-CH=CH2, followed by acetylation of the secondary amine, removal of the BOC, and benzoylation of the primary amine. Some benzoylation of the phenol-OH occurs, but this ester is cleaved by saponification before processing of the sample. The second method involves treating mono-BOC-1,6-hexanediamine with benzoyl chloride and removal of the BOC to give monobenzoyl-1,6-hexanediamine, which is used in the Michael addition followed by acetylation of the secondary amine. Both benzoylation and BOC removal are nearly quantitative, so each route is essentially equivalent.

[0505] Monobenzoylation of symmetric diamines (Tang, W.; Fang, S. Tetrahedron Lett. 2008, 49, 6003)

[0506] [ka]

[0507] To methyl benzoate (1.0 g, 7.34 mmol) was added pentane-1,5-diamine (0.75 g, 7.34 mmol) and water (0.37 mL) and the mixture was heated to 100° C. for 24 hours with constant stirring. The reaction mixture was cooled to room temperature and loaded directly onto a short silica column (which had been previously flushed with 4% triethylamine in chloroform followed by 100% chloroform prior to loading of the reaction mixture). Elution with 30% methanol in chloroform gave the desired monobenzoylated product (0.80 g, 53%) as a pale yellow oil. 1 H NMR (300 MHz, MeOD) δ 7.83 (d, J = 7.4 Hz, 2H), 7.58 - 7.31 (m, 4H), 3.41 (t, J = 7.0 Hz, 2H), 2.76 (t, J = 7.2 Hz, 2H), 1.74 - 1.21 (m, 6H). [M+H] +MS (ESI + ), calculated: 207.1, observed: 207.2.

[0508] Method 1 A solution of 4-aminophenol (50 g, 458 mmole) in CHCl (400 mL) and saturated NaHCO in water (400 mL) was stirred at room temperature for 10 min, then acryloyl chloride (40.9 mL, 503.8 mmole) was added dropwise and the reaction was stirred at room temperature for an additional 6 h. The resulting solid was collected by filtration, washed with water and dried under vacuum (oil pump) to give 75 g of 4-acrylamidophenol.

[0509] 4-Acrylamide phenol (163 mg, 1 mmol) and mono-BOC-1,6-hexanediamine (Ark Pharm Inc.) (237 mg, 1.1 mmol) were dissolved in a solution of isopropanol (9 mL) and water (1 mL) and heated in an oil bath at 65° C. for 48 h. The reaction mixture was concentrated by rotary evaporation to give the Michael addition reaction product, which was used in the next step without further purification.

[0510] To the residue obtained in the above step was added CH2Cl2 (4 mL) and 4 mL of saturated aqueous sodium bicarbonate. Acetyl chloride (0.21 mL, 3 mmole) was added dropwise with stirring at room temperature, and the mixture was stirred for an additional 3 h at room temperature. The layers were separated, and the CH2Cl2 layer was concentrated by rotary evaporation.

[0511] The residue was dissolved in 4 mL of CH2Cl2 and 2 mL of 4M HCl in dioxane was added dropwise with stirring. Stirring was continued at room temperature for 1 hour. The resulting solid was collected by filtration and the solid was dried under vacuum (oil pump).

[0512] To the above solid was added 10 mL of CH2Cl2 and 10 mL of saturated aqueous sodium bicarbonate. Benzoyl chloride (0.23 mL, 2 mmole) was added dropwise with stirring and the mixture was stirred at room temperature for an additional 3 h. The layers were separated and the CH2Cl2 layer was concentrated by rotary evaporation. The residue was dissolved in 2 mL of MeOH and 2 mL of 5% aqueous NaOH was added. The mixture was stirred at room temperature for 30 min (this step is necessary to completely remove the benzoylated phenol). The mixture was neutralized with 1 M HCl and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered and the solvent was removed by rotary evaporation. The residue was chromatographed on silica gel using 5% MeOH in CH2Cl2 to give 170 mg of pure product (40% overall yield).

[0513] Method 2

[0514] To an ice-cold dry solution of mono-BOC-1,6-hexanediamine·HCl (20 g, 79.12 mmol) in dichloromethane (350 mL) was added dropwise with stirring under nitrogen atmosphere anhydrous triethylamine (33 mL, 237.4 mmol). After 10 min, benzoyl chloride (9.64 mL, 83 mmol) was added dropwise at 0 °C and the resulting mixture was stirred at room temperature overnight. Water (200 mL) was added and the aqueous layer was extracted twice with 200 mL portions of CHCl, the organic extracts were combined, washed with water, brine, dried over anhydrous NaSO, filtered and concentrated by rotary evaporation. The resulting solid was washed with hexane to remove low polarity impurities and the solid was used in the next step without purification.

[0515] The above solid was dissolved in 100 mL of CH2Cl2, 300 mL of 20% TFA in CH2Cl2 was added dropwise with stirring at 0 °C, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated by rotary evaporation. The resulting residue was dissolved in 300 mL of water and washed with two 200 mL portions of CH2Cl2 to remove less polar impurities. The resulting aqueous layer was neutralized with 5% aqueous NaOH and extracted four times with 200 mL portions of CH2Cl2. The organic layers were combined, dried over Na2SO4, filtered, and the solvent was removed by rotary evaporation. The resulting crude product (14.6 g, 66.4 mmol, 84%) was used in the next step without further purification. In this manner, the free amine was obtained, which is required for the next step (Michael addition).

[0516] 4-Acrylamidophenol (8.43 g, 51.6 mmol) and mono-benzoyl-1,6-hexanediamine (12.5 g, 56.8 mmol) were dissolved in a solution of isopropanol (450 mL) and water (50 mL) and heated in an oil bath at 65° C. for 48 h. The reaction mixture was concentrated by rotary evaporation to give the Michael addition product, which was divided into two portions and used in the next step without further purification.

[0517] Acetylation

[0518] To the residue obtained in the above step, CHCl (100 mL), DMF (10 mL) and 100 mL of saturated aqueous sodium bicarbonate were added. Acetyl chloride (3.7 mL, 52 mmol) was added dropwise with stirring at room temperature, and the mixture was stirred at room temperature for an additional 6 h. The organic layer was separated and the aqueous layer was extracted twice with 50 mL portions of 5% MeOH in CHCl. ​​The organic layers were combined and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography (1–5% MeOH in CHCl) to give MPS-I aglycone (4.5 g, 10.6 mmol) in 41% yield. Pentanoylation

[0519] To the residue obtained in the above step, CH2Cl2 (100 mL), DMF (10 mL) and 100 mL of saturated aqueous sodium bicarbonate were added. Pentanoyl chloride (6.17 mL, 52 mmol) was added dropwise with stirring at room temperature, and the mixture was stirred at room temperature for another 6 h. The organic layer was separated and the aqueous layer was extracted twice with 50 mL portions of 5% MeOH in CH2Cl2. The organic layers were combined and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography (1-5% MeOH in CH2Cl2) to give MPS-II aglycone (3.5 g, 7.5 mmol) in 29% yield.

[0520] MPS-I-IS-acetyl-C6 The internal standard MPS-IP-acetyl-C6 was prepared similarly to that described for the MPS-I aglycone, except that d5-benzoyl chloride (Aldrich) was used. The enzyme product MPS-IP-acetyl-C6 is the MPS-I aglycone (i.e., identical to IS, but the benzoyl is not deuterated).

[0521] Conjugation of the aglycone with iduronyl-F, deacetylation, and methyl ester hydrolysis The procedure for conjugation to MPS-II aglycone is described below, and the procedure for conjugation to MPS-I aglycone is similar.

[0522] [ka]

[0523] MPS-II aglycone (1.9 g, 4.06 mmol, 1 equiv.), 2,3,4-trihydroxy-iduronosy-1-methyl (1.23 g, 3.66 mmol, 0.9 equiv.) and 2,6-di-tert-butyl-4-methylpyridine (2.5 g, 12.2 mmol, 3 equiv.) were dried under high vacuum (oil pump) for 1 h and dissolved in dry CHCl (80 mL, 0.05 M). All MPS-II aglycone was dissolved before adding BF-etherate. For the reaction with MPS-I aglycone, more CHCl was used to obtain 0.02 M aglycone (not all was dissolved even after adding BF-etherate).

[0524] BF3·Et2O (5.1 mL, 40.6 mmol, 10 equiv.) was added dropwise with stirring at room temperature under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 2.5 h, after which 150 mL of saturated aqueous NaHCO3 was added. The aqueous layer was extracted with CHCl2, and the organic extracts were combined, washed with water, brine, and dried over anhydrous NaSO4. The solution was filtered and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography (CHCl2, then 1–4% MeOH in CHCl2) to give the product (1.87 g, 2.38 mmol) in 65% yield.

[0525] Deacetylation

[0526] To a solution of the coupled product (2.7 g, 3.44 mmol, 1 equiv) in 75 mL of dry methanol (Aldrich), 0.5 M sodium methoxide in methanol (2.75 mL, 1.38 mmol, 0.4 equiv) was added dropwise with stirring under nitrogen atmosphere at 0 °C. The reaction mixture was stirred at 0 °C for 3 h. The reaction mixture was neutralized with AG 50W-X8 resin (H+) and filtered. The filtrate was concentrated by rotary evaporation. Column chromatography on silica gel (1-6% MeOH in Ch2Cl2) afforded the product (2.1 g, 3.19 mmol) in 92% yield.

[0527] Methyl ester hydrolysis

[0528] MPS-IS-acetyl-C6 is made by demethylating the methyl ester. In the case of MPS-II-S-pentanoyl-C6, the deacetylated compound is sulfated and then the methyl ester is hydrolyzed. MPS-II-P-pentanoyl-C6 is made by saponification of the methyl ester, without sulfation as in the case of MPS-IS-acetyl-C6. MPS-II-IS-pentanoyl-C6 is made similarly to MPS-II-P-pentanoyl-C6, but with the aglycone containing a d5-benzoyl group.

[0529] The deacetylated compound (1.5 g, 2.44 mmol, 1 equiv.) was dissolved in 150 mL of water / methanol (1:1) at room temperature. 0.1 M aqueous sodium hydroxide was added in increments of 0.1 equiv. NaOH until the pH of the solution reached approximately 8 (pH paper). The pH was maintained by incremental addition of 0.1 M NaOH solution as the reaction progressed (approximately 2 equiv. NaOH was added). The reaction mixture was stirred overnight. The reaction mixture was neutralized with 1 M HCl and concentrated by rotary evaporation. The residue was purified by column chromatography on silica (5% MeOH and 1% AcOH in Ch2Cl2, then 10% MeOH and 2% AcOH in Ch2Cl2) to give the product MPS-IS-acetyl-C6 (1.45 g, 2.41 mmol) in 98% yield.

[0530] It is important to remove as much of the MPS-I enzyme product from the substrate as possible, otherwise the assay blank will be higher. The product is easy to extract, so the substrate may be dissolved in water at pH 7 and extracted with EtOAc. However, the substrate, which is negative due to its carboxylate, will remain in the water. Dissolve 1.5 g of MPS-IS-acetyl-C6 in 200 mL of distilled water and adjust the pH to near 7 with KOH using a pH meter. Extract three times with 200 mL portions of EtOAc. Transfer the aqueous layer to a round-bottom flask and place on a rotary evaporator equipped with water aspiration and a 30°C water bath and rotovapour for approximately 20 minutes to remove all the EtOAc in the water. The aqueous layer is then lyophilised to obtain the final product, the sodium salt of MPS-IS-acetyl-C6. This procedure produced a substrate containing the MPS-I product. An alternative purification was investigated.

[0531] In one alternative, 50 mg of MPS-IS-Ac-C6 was dissolved in 5 mL of water, adjusted to pH 7 (pH meter) with dilute aqueous NaOH, extracted with 8 mL of ethyl acetate by vortexing, then centrifuged to separate the layers, and repeated four more times (i.e., a total of 40 mL of ethyl acetate). The aqueous layer was lyophilized. The amount of MPS-I product was very small and acceptable for this purification.

[0532] In another alternative, 120 mg of MPS-IS-Ac-C6 was purified by flash Si column (10 g silica) using a 1-10% MeOH / CH2Cl2 linear gradient over 10 min, then up to 20% MeOH / 2% water / CH2Cl2. The peak of material was pooled in three batches: one third, two thirds and the last third of the peak. Solvent was removed by rotary evaporation with a 30°C water bath and water aspirate. Assays performed with three separate batches showed that the middle and last third peak material contained acceptable amounts of MPS-I-product.

[0533] Sulfation and methyl ester hydrolysis

[0534] The deacetylated compound (2 g, 3.04 mmol, 1 equiv.) was solubilized in anhydrous MeOH (120 mL) and dibutyltin(IV) oxide (1.13 g, 4.56 mmol, 1.5 equiv.) was added. The reaction mixture was heated under reflux under nitrogen for 1 h, after which the dibutyltin oxide was completely dissolved. The reaction mixture was allowed to cool and concentrated under vacuum. The residue was coevaporated once with anhydrous toluene (100 mL) to remove traces of water. The residue was solubilized in anhydrous N,N-dimethylformamide (120 mL). Sulfur trioxide-trimethylamine complex (633.8 mg, 4.56 mmol, 1.5 equiv.) was added and the reaction mixture was stirred at room temperature under nitrogen atmosphere for 24 h. The reaction mixture was quenched with MeOH (20 mL). The mixture was then concentrated under vacuum. The residue was purified by column chromatography on silica gel (10% MeOH and 1% H2O / CH2Cl2, then 20% MeOH and 2% H2O / CH2Cl2) to give the sulfate compound (1.2 g, 1.63 mmol) in 53.6% yield.

[0535] The sulfate compound (1 g, 1.35 mmol) was solubilized in 1:1 methanol-water (100 mL) at room temperature. 0.1 M aqueous NaOH was added in increments of 0.1 equivalents of NaOH until the pH of the solution reached approximately 8 (pH paper). The pH was maintained by adding increasing amounts of 0.1 M NaOH solution as the reaction progressed (every 15-30 min). It is probably important not to increase the pH because this may result in some hydrolysis of the sulfate ester. The reaction mixture was stirred overnight (approximately 2 equivalents of NaOH added) and then concentrated under vacuum to remove the methanol and water. The residue was purified by column chromatography on silica gel (10% MeOH and 1% H2O / CH2Cl2, then 20% MeOH and 2% H2O / CH2Cl2) to give MPS-II-S (0.77 g, 1.06 mmol) in 79% yield.

[0536] Removal of the non-sulfated material from the sulfated material can be accomplished by extraction or ion exchange chromatography as described below.

[0537] Extract Cleanup

[0538] The compound (0.77 g, 1.06 mmol) from the silica gel was dissolved in purified water (200 mL) and 1 M HCl was added dropwise to adjust the pH to 2.7 by pH meter. The aqueous layer was extracted with EtOAc (5 x 200 mL). The remaining aqueous layer was transferred to a round bottom flask, which was placed on a rotary evaporator with a 30°C water bath and water aspiration for 30 min to remove traces of EtOAc from the aqueous layer. The aqueous layer was lyophilized. LC-MSMS of five extractions with EtOAc showed approximately 100-fold drop in non-sulfated material in the third extraction compared to the first. The amounts in the fourth and fifth extractions were smaller and similar to those in the third extraction.

[0539] Purification by ion exchange

[0540] AKTA uses solvent A (MeOH) and solvent B (MeOH + 1M ammonium formate). A commercially available Pharmacia HiLoad26 / 10 Q-Sepharose column (3 mL / min) is used. Approximately 10 mg of MPS-II-IS-Pent-C6-d5 was injected onto the column in approximately 0.5 mL, held at 100% A for 20 min, then a linear gradient was run from 0 to 100% B over 30 min, held at 100% B (eluted at 51 min). 22 mg of MPS-II-S-Pent-C6 in 1.5 mL MeOH was injected using the above program (2 mL loop). The substrate eluted at 100 min. MeOH was removed by rotary evaporation (water aspirator, 25-30 °C water bath). The residue was dissolved in about 10 mL of water and loaded onto a Waters C18 Sep-Pak (size 50 g) that had previously been washed with about 100 mL of MeOH, then about 100 mL of water, and washed with about 200 mL of water (OD280nm close to 0). The compound was eluted with about 200 mL of MeOH, additional MeOH had an OD280 close to 0, and the methanol was removed by rotary evaporation (water aspirator, 30° C. water bath). The residue was dissolved in a few mL of MeOH, transferred to 2×5 mL vials (more MeOH was used to complete the transfer), and placed in a Speed-Vac overnight without heat.

[0541] Example 2 Representative Assays Using MPS-I Reagents In this example, representative assays using the MPS-I reagents of the present invention are described, and the results of these reagents are compared with other MPS-I reagents.

[0542] The original MPS-I reaction is shown below (Blanchard, Sophie, Sadilek, Markin, Scott, C. Ronald, Turecek, Frantisek, and Gelb, Mechael H. (2008) "Tandem mass spectrometry for the direct assay of Lysosomal enzymes in dried blood spots: Application to screening newborns for Mucopolysaccharidosis I." Clin. Chem., 54: 2067-2070). Note that S, P, and IS have BOC groups, and P and IS are not chemically identical (P has four CH2 groups in the bond, while IS has three).

[0543] [ka]

[0544] Alternative MPS-I reactions are shown below:

[0545] [ka]

[0546] Note that the different aglycone has an N-acetyl group and no BOC carbamate group. Also note that the internal standard is not chemically identical to the product, but does have five deuteriums in the benzoyl group.

[0547] The MPS-I original and surrogate substrates were compared in the following enzymatic assay: 0.5 mM substrate, 3.5 uM internal standard in 30 uL of buffer (100 mM ammonium formate, pH 4.0). A 3 mm punch of dried blood spot was added and the mixture was incubated at 37°C for 16 hours with shaking. The reaction was quenched by adding 120 uL of acetonitrile. The wells were centrifuged and 120 uL of the supernatant was transferred to a new well. The sample was diluted by adding 120 uL of water and 10 uL was injected onto the LC / MSMS system. LC and MS / MS conditions were as published (Spacil, Z., Tatipaka, H., Barcenas, M., Scott, CR, Turecek, F., Gelb, MH (2012) "High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening." Clinical Chemistry., 59(3), 1530-8561). A blank assay was also performed, where a 3 mm blood-free filter paper punch replaced the dried blood spot. The blank was incubated and processed as above.

[0548] [Table 3]

[0549] 1 Enzyme activity is expressed as umoles of product formed per hour per liter of blood.

[0550] 2 Coefficients of variation (CV) are based on six assays, each performed on a different punch from the same dried blood spot.

[0551] 3 MSMS response is the amount of ion counts measured in a tandem mass spectrometry channel per pmole of analyte.

[0552] 4The blood-no blood assay ratio is the ratio of the enzyme activity measured in an assay using a dried blood spot punch to the enzyme activity measured in a blood-free punch.

[0553] It can be seen from the above table that while both MPS-I substrates have similar effects on the MPS-I enzyme (umoles of product produced per hour per liter of blood), the alternative substrate induces products (ion counts detected per pmole of sample) that are approximately 5 times more sensitive for MSMS detection. Similar results were obtained by flow injection-MSMS. The MSMS response to the alternative MPS-I products and internal standard was approximately 5 times higher than the original (not shown).

[0554] The MSMS response of the MPS-I product was compared to the Fabry assay product (structure shown below).

[0555] [ka]

[0556] Note that the MPS-I product has an acetyl on the amine, while the Fabry product has a BOC carbamate. The BOC carbamate partially decomposes in the electrospray ionization source of the MSMS instrument due to loss of isobutylene and CO2, producing a decomposition product in which the BOC is replaced by H. The Fabry product gives an ion count of 211 per pmole, while the MPS-I product gives an ion count of 500 per pmole.

[0557] Example 3 Representative Assays Using MPS-II Reagents In this example, representative assays using the MPS-II reagents of the invention are described, and the results for these reagents are compared with other MPS-II reagents.

[0558] The original MSP-II reaction is shown below (Wolfe, BJ, Blanchard, S., Sadilek, M., Scott, CR, Turecek, F., Gelb, MH (2011) "Tandem mass spectrometry for the direct assay of Lysosomal enzymes in dried blood spots: Application to screening newborns for Mucopolysaccharidosis II (Hunter Syndrome)" Anal. Chem., 83: 1152-1156). Note that S, P and IS have BOC groups.

[0559] [ka]

[0560] An alternative MPS-II reaction is shown below:

[0561] [ka]

[0562] Note the different aglycone, with an N-pentanoyl group and without the BOC carbamate group. Also note that the internal standard is chemically identical to the product but with five deuteriums in the benzoyl group.

[0563] The MPS-II original and surrogate substrates were compared in the following enzymatic assay: 1 mM substrate, 5 uM internal standard in 30 uL of buffer (100 mM ammonium formate, pH 4.0, 7.5 mM barium(II) acetate, 5.0 mM cerium(III) acetate). A 3 mm punch of dried blood spot was added and the mixture was incubated at 37°C for 16 hours with shaking. The reaction was quenched by adding 200 uL of 44 mM citric acid, followed by 400 uL of ethyl acetate and 100 uL of water. After mixing by pipetting up and down several times, the samples were centrifuged (10 min at 3000 rpm) to separate the liquid layers. 200 uL of the top ethyl acetate layer was transferred to a new well at once and the solvent was removed by evaporation with an oil-free air stream. The residue was taken up in 100 uL of methanol / 5 mM aqueous ammonium formate (80 / 20, v / v) and injected into the tandem mass spectrometer. Barium and cerium salts are present to precipitate free sulfate and phosphate present in the dried blood spot, since these anions cause inhibition of MPS-II enzyme production. Citric acid is used to acidify the mixture, so that the carboxylates of the iduronic acid moiety of the MPS-II product and the internal standard are protonated and extracted into ethyl acetate. A blank assay is also performed, where a 3 mm punch of blood-free filter paper replaces the dried blood spot. The blank is incubated and processed as above.

[0564] [Table 4]

[0565] 1 Enzyme activity is expressed as umoles of product formed per hour per liter of blood.

[0566] 2 Coefficients of variation (CV) are based on six assays, each performed on a different punch from the same dried blood spot.

[0567] 3MSMS response is the amount of ion counts measured in a tandem mass spectrometry channel per pmole of analyte.

[0568] 4 The blood-no blood assay ratio is the ratio of the enzyme activity measured in an assay using a dried blood spot punch to the enzyme activity measured in a blood-free punch.

[0569] It can be seen from the above table that while both MPS-II substrates have similar effects on the MPS-II enzyme (umoles of product produced per hour per liter of blood), the alternative substrate induces products that are approximately 10 times more sensitive for MSMS detection (ion counts detected per pmole of sample).

[0570] Example 4 Synthesis of representative MPS-IVA substrates and enzyme products In this example, the synthesis of a representative MPS-IVA substrate reagent is described. A general scheme for the synthesis of MPS-IVA substrates is shown in FIG.

[0571] (2R,3R,4R,5R,6S)-5-Acetamido-2-(acetoxymethyl)-6-(4-nitrophenoxy)tetrahydro-2H-pyran-3,4-diyldiacetic acid (1)

[0572] [ka]

[0573] Pyridine (60 mL) was added to a nitrogen-flooded flask containing D-galactosamine hydrochloride (5 g, 23.2 mmol) and the resulting slurry was cooled on an ice bath. Acetic anhydride (25 g, 245 mmol) was added dropwise to the cooled mixture and allowed to warm to room temperature and then stirred at this temperature for 16 hours. Methanol (15 mL) was added to quench the reaction mixture and allowed to stir for 20 minutes. The resulting mixture was concentrated under reduced pressure, the mixture was allowed to warm and the residue was dissolved in 20% methanol in chloroform. This solution was washed with 1N HCl solution followed by brine solution. The resulting organic layer was dried using anhydrous sodium sulfate and concentrated under reduced pressure. The residue was taken up in a nitrogen-flooded flask equipped with a dropping funnel. Anhydrous dichloromethane (100 mL) was added to the residue and the resulting slurry was cooled on an ice bath. In a dropping funnel, titanium chloride (6.5 g, 42.1 mmol) was dissolved in anhydrous dichloromethane (40 mL) and the resulting solution was added dropwise to the cooled solution. The reaction mixture was warmed to 50° C. in an oil bath and allowed to stir at this temperature for 48 hours. The reaction mixture was cooled again on an ice bath and saturated sodium bicarbonate solution was added dropwise with vigorous shaking. The resulting mixture was extracted between dichloromethane and saturated sodium bicarbonate solution. The organic layer was dried using anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was dissolved in acetone (60 mL) and slowly added to a solution of 4-nitrophenol (16.1 g, 116 mmol) in acetone (130 mL) and 4N aqueous KOH (23.2 mL). The reaction was allowed to stir at room temperature for 48 hours and concentrated under reduced pressure to less than 20 mL. The solution was extracted between 1N NaOH and chloroform. The organic layer was dried using anhydrous sodium sulfate and concentrated under reduced pressure. The crude product thus obtained was purified by silica flash chromatography using 3% methanol in dichloromethane as the eluent mixture. Fractions containing the desired compound as determined by TLC were combined and concentrated under reduced pressure to give 1 (3.29 g, 30%). 11H NMR (300 MHz, CDCl3) δ 8.20 (d, J = 9.1 Hz, 2H), 7.09 (d, J = 9.1 Hz, 2H), 5.61 (d, J = 8.0 Hz, 1H), 5.56 - 5.39 (m, 3H), 4.32 - 4.07 (m, 4H), 2.18 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H). [M+Na] + MS (ESI for + ), calculated: 491.1, observed: 491.2.

[0574] N-(5-(N-(3-((4-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-3-oxopropyl)pentanamido)pentyl)benzamide (2)

[0575]

Chemical Structure

[0576] To a solution of 1 (3.5 g, 7.47 mmol) in anhydrous methanol (90 mL) was added dropwise a solution of 0.5 M sodium methoxide in methanol (3 mL, 1.50 mmol) cooled on an ice bath and allowed to warm to room temperature. After 2 hours, formic acid (0.1 mL) was added to the reaction mixture and concentrated to dryness under reduced pressure. To the resulting residual methanol (135 mL) was added water (15 mL) and 10% palladium on activated carbon (125 mg) and allowed to stir at room temperature under hydrogen atmosphere for 16 hours. Water was added dropwise to the reaction mixture until all the white residue was completely dissolved. The reaction mixture was filtered and the filtrate was cooled on an ice bath. To this was added pyridine (2 mL) followed by a solution of acryloyl chloride (2.1 g, 23.2 mmol) in dichloromethane (50 mL) dropwise. The reaction was allowed to stir on the ice bath for 30 minutes, then allowed to warm to room temperature and proceed for 2 hours. Sodium carbonate powder (3.0 g) was added to the reaction mixture, allowed to stir for 15 min and filtered. The filtrate was concentrated under reduced pressure and further dried under high vacuum. The residue was dissolved in a mixture of 2-propanol (50 mL) and water (6.6 mL) to which N-(5-aminopentyl)benzamide (2.0 g, 9.69 mmol) was added and allowed to stir at 65° C. for 40 h. The reaction mixture was cooled to room temperature and methanol (25 mL) was added to it. The mixture was cooled on an ice bath and triethylamine (2.5 mL) was added followed by dropwise addition of a solution of pentanoyl chloride (2.7 g, 22.4 mmol) in dichloromethane (50 mL). The reaction was allowed to stir on an ice bath for 30 min and then warmed to room temperature and continued for 16 h. The reaction mixture was concentrated under reduced pressure and subjected to purification by silica flash chromatography using 15% methanol in dichloromethane as the eluting mixture to give 2 (2.96 g, 60%). 1H NMR (300 MHz, MeOD) δ 7.84 (d, J = 7.1 Hz, 2H), 7.59 - 7.35 (m, 5H), 7.09 - 6.91 (m, 2H), 5.00 (d, J = 8.4 Hz, 1H), 4.28 - 4.09 (m, 1H), 3.97 - 3.55 (m, 7H), 3.46 - 3.24 (m, 4H), 2.72 - 2.51 (m, 2H), 2.49 - 2.28 (d, J = 7.3 Hz, 2H), 2.02 (s, 3H), 1.78 - 1.49 (m, 6H), 1.49 -1.22 (m, 4H), 0.94 (t, J = 7.1 Hz, 3H).[M+H] + MS (ESI + ), calculated: 657.3, observed: 657.5.

[0577] Sodium ((2R,3R,4R,5R,6S)-5-acetamido-6-(4-(3-(N-(5-benzamidopentyl)-pentanamido)propanamido)phenoxy)-3,4-dihydroxytetrahydro-2H-pyran-2-yl)methyl sulfate (3)

[0578] [ka]

[0579] To compound 2 (32 mg, 0.048 mmol) was added anhydrous pyridine (1 mL) under nitrogen. To this solution was added sulfur trioxide pyridine complex (18 mg, 0.113 mmol) and allowed to stir at room temperature for 5 h. The reaction was quenched by adding methanol (0.3 mL) and stirred for 30 min. The reaction mixture was concentrated under reduced pressure, redissolved in water, and subjected to reverse phase (C18) HPLC purification using a water-methanol gradient system to give 3 (23 mg, 63%). 11H NMR (300 MHz, MeOD) δ 7.81 (d, J = 7.3 Hz, 2H), 7.57 - 7.39 (m, 5H), 7.00 (dd, J = 9.0, 2.4 Hz, 2H), 4.93 (d, J = 8.5 Hz, 1H), 4.38 - 4.12 (m, 3H), 3.95 (t, J = 4.5 Hz, 2H), 3.83 - 3.61 (m, 3H), 3.49 - 3.34 (m, 4H), 2.72 - 2.54 (m, 2H), 2.50 - 2.30 (m, 2H), 1.98 (s, 3H), 1.78 - 1.49 (m, 6H), 1.49 - 1.24 (m, 4H), 1.02 - 0.84 (m, 3H). [M - Na + - MS (ESI - ) for [M - Na], calculated: 735.3, observed: 735.5.

[0580] N-(6-(N-(3-((4-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-3-oxopropyl) Hexanamide )Hexyl)benzamide (4)

[0581]

Chemical Structure

[0582] ​To a solution of 1 (0.27 g, 0.576 mmol) in anhydrous methanol (9 mL) was added dropwise a solution of 0.5 M sodium methoxide in methanol (0.3 mL, 0.15 mmol) cooled in an ice bath and allowed to warm to room temperature. After 2 h, formic acid (10 μL) was added to the reaction mixture and concentrated to dryness under reduced pressure. To the resulting residual methanol (13.5 mL) was added water (1.5 mL) and 10% palladium on activated carbon (12.5 mg) and allowed to stir at room temperature under hydrogen atmosphere for 16 h. Water was added dropwise to the reaction mixture until all the white residue was completely dissolved. The reaction mixture was filtered and the filtrate was cooled on an ice bath. To this was added pyridine (0.16 mL) followed by a solution of acryloyl chloride (0.16 g, 1.76 mmol) in dichloromethane (4 mL) dropwise. The reaction was allowed to stir on the ice bath for 30 min, then allowed to warm to room temperature and proceed for 2 h. Sodium carbonate powder (0.3 g) was added to the reaction mixture, allowed to stir for 15 minutes and filtered. The filtrate was concentrated under reduced pressure and further dried under high vacuum. The residue was dissolved in a mixture of 2-propanol (6.3 mL) and water (0.7 mL) to which was added N-(6-aminohexyl)benzamide (0.17 g, 0.77 mmol) and allowed to stir at 65° C. for 40 hours. The reaction mixture was cooled to room temperature and methanol (8 mL) was added to it. The mixture was cooled on an ice bath and triethylamine (0.25 mL) was added followed by dropwise addition of a solution of hexanoyl chloride (0.24 g, 1.78 mmol) in dichloromethane (4 mL). The reaction was allowed to stir on the ice bath for 30 minutes and then warmed to room temperature and continued for 16 hours. The reaction mixture was concentrated under reduced pressure and subjected to purification by flash chromatography on silica using 15% methanol in dichloromethane as the eluent mixture to give 4 (0.23 g, 58%). 1H NMR (300 MHz, MeOD) δ 7.80 (d, J = 6.9 Hz, 2H), 7.56 - 7.39 (m, 5H), 6.99 (d, J = 9.1 Hz, 2H), 4.96 (d, J = 8.4 Hz, 1H), 4.17 (dd, J = 10.7, 8.4 Hz, 1H), 3.96 - 3.51 (m, 7H), 3.44 - 3.33 (m, 4H), 2.69 - 2.50 (m, 2H), 2.48 - 2.28 (m, 2H), 1.98 (s, 3H), 1.72 - 1.49 (m, 6H), 1.49 - 1.21 (m, 9H), 0.89 (dt, J = 8.7, 4.8 Hz, 3H).[M+H] + MS (ESI + ), calculated: 685.4, observed: 685.5.

[0583] Sodium ((2R,3R,4R,5R,6S)-5-acetamido-6-(4-(3-(N-(6-benzamidohexyl)-hexanamido)propanamido)phenoxy)-3,4-dihydroxytetrahydro-2H-pyran-2-yl)methyl sulfate (5)

[0584] [ka]

[0585] To compound 4 (99 mg, 0.144 mmol) was added anhydrous pyridine (5 mL) under nitrogen. To this solution was added sulfur trioxide pyridine complex (34 mg, 0.214 mmol) and allowed to stir at room temperature for 5 h. The reaction was quenched by adding methanol (0.5 mL) and stirred for 30 min. The reaction mixture was concentrated under reduced pressure, redissolved in water, and subjected to reverse phase (C18) HPLC purification using a water-methanol gradient system to give 3 (36 mg, 32%). 1H NMR (300 MHz, MeOD) δ 7.81 (d, J = 6.9 Hz, 2H), 7.59 - 7.35 (m, 5H), 7.00 (d, J = 9.0 Hz, 2H), 4.93 (d, J = 8.4 Hz, 1H), 4.32 - 4.10 (m, 3H), 4.03 - 3.90 (m, 2H), 3.83 - 3.60 (m, 3H), 3.44 - 3.33 (m, 4H), 2.61 (q, J = 7.0 Hz, 2H), 2.49 - 2.29 (m, 2H), 1.98 (s, 3H), 1.72 - 1.49 (m, 6H), 1.48 - 1.22 (m, 9H), 0.98 - 0.81 (m, 3H).[M-Na + ] - MS (ESI - ), calculated: 763.3, observed: 763.7.

[0586] Example 5 Synthesis of representative MPS-VI substrates and enzyme products In this example, the synthesis of representative MPS-VI substrates and enzyme product reagents is described. A general scheme for the synthesis of MPS-VI substrates is shown in FIG.

[0587] MPS-VI substrate (hexanamide) The preparation of a representative MPS-VI substrate (hexanamide) is described below and illustrated in FIG.

[0588] N-(5-(N-(3-((4-hydroxyphenyl)amino)-3-oxopropyl)hexanamido)pentyl)-benzamide (3)

[0589] [ka]

[0590] A solution of N-(5-aminopentyl)benzamide (180 mg, 0.872 mmol) and N-(4-hydroxyphenyl)acrylamide (171 mg, 1.05 mmol) in i-propyl alcohol (7.8 mL) and water (0.87 mL) was heated to 65° C. for 24 h with constant stirring. The reaction mixture was cooled to room temperature and concentrated under reduced pressure and then under high vacuum. To the crude concentrate was added anhydrous N,N-dimethylformamide (DMF) (3.0 mL) and triethylamine (220 mg, 2.17 mmol) until complete dissolution. The solution was cooled to 0° C. and hexanoyl chloride (234 mg, 1.74 mmol) was added dropwise, warmed to room temperature, and stirred for 2 h. The reaction was quenched by the addition of saturated sodium bicarbonate solution and the reaction mixture was extracted with DCM / methanol (4:1). The organic layer was further washed with water and dried over anhydrous sodium sulfate. The organic layer was concentrated to dryness under reduced pressure and methanol (3.0 mL) was added to redissolve. To this solution, 5% aqueous sodium hydroxide (3.0 mL) was added dropwise and stirred at room temperature for 2 h. The reaction was acidified with 1N HCl solution as indicated by pH paper and extracted with DCM / methanol (4:1). The organic layer was concentrated under reduced pressure and the residue was subjected to silica column chromatography eluting with 5% methanol in DCM to give 3 (151 mg, 37%). 1 H NMR (300 MHz, MeOD) δ 8.47 (s, 1H), 7.81 (d, J = 7.7 Hz, 2H), 7.57 - 7.40 (m, 3H), 7.36 - 7.27 (m, 2H), 6.78 - 6.67 (m, 2H), 3.69 (dt, J = 18.7, 6.9 Hz, 2H), 3.46 - 3.33 (m, 4H), 2.60 (q, J = 7.0 Hz, 2H), 2.47 - 2.29 (m, 2H), 1.63 (ddd, J = 11.6, 10.6, 5.7 Hz, 6H), 1.48 - 1.22 (m, 6H), 0.89 (td, J = 6.6, 2.6 Hz, 3H).[M+Na] + MS (ESI + ), calculated: 490.3, observed: 490.6.

[0591] N-(5-(N-(3-((4-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-3-oxopropyl)hexanamido)pentyl)benzamide (4)

[0592] [ka]

[0593] To a solution of 3 (73 mg, 0.156 mmol) and (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-chlorotetrahydro-2H-pyran-3,4-diyl diacetate (114 mg, 0.312 mmol) in anhydrous DMF (0.7 mL) was added cesium carbonate (152 mg, 0.466 mmol) and allowed to stir at room temperature for 6 h. The reaction mixture was then extracted between water and DCM, and the organic layer was further washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified by flash silica column chromatography using 4% methanol in DCM as the eluent to give the peracetylated intermediate. NMR spectroscopy showed that the peracetylated intermediate co-eluted with the starting material 3. This mixture was used in the subsequent deacetylation step without further purification. To a solution of the above mixture in anhydrous methanol (5.0 mL) was added dropwise a solution of 0.5 M sodium methoxide in methanol (200 μL) at 0° C. and allowed to stir at room temperature for 2 h. The reaction was quenched by the addition of formic acid (100 μL) and subjected to semi-preparative reversed-phase HPLC purification (gradient water / methanol system) to give 4 (23 mg, 22%). 11H NMR (300 MHz, MeOD) δ 7.85 - 7.77 (m, 2H), 7.58 - 7.38 (m, 5H), 6.99 (dd, J = 9.1, 2.6 Hz, 2H), 4.96 (dd, J = 8.4, 1.3 Hz, 1H), 4.17 (dd, J = 10.7, 8.4 Hz, 1H), 3.90 (d, J = 3.1 Hz, 1H), 3.84 - 3.60 (m, 6H), 3.38 (dd, J = 11.1, 6.8 Hz, 4H), 2.61 (q, J = 7.1 Hz, 2H), 2.47 - 2.29 (m, 2H), 1.98 (s, 3H), 1.74 - 1.50 (m, 6H), 1.47 - 1.21 (m, 6H), 0.89 (td, J = 6.6, 3.3 Hz, 3H). [M+Na] + MS (ESI + ) for, calculated value: 693.3, observed value: 693.4.

[0594] (2R,3R,4R,5R,6S)-5-acetamido-6-(4-(3-(N-(5-benzamidopentyl)-hexanamido)propanamido)phenoxy)-4-hydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl sulfate sodium (5)

[0595]

Chemical Structure

[0596] To a cooled (0° C.) solution of 4 (22 mg, 32.8 μmol) in anhydrous pyridine (0.5 mL) was added benzoyl chloride (7.7 μL, 65.6 μmol). After 1 h at room temperature, the solution was cooled back to 0° C. and another portion of benzoyl chloride (7.7 μL, 65.6 μmol) was added and left to stir at room temperature for 2 h. The reaction was quenched by adding methanol (200 μL) and stirred for an additional 30 min. The resulting mixture was concentrated under reduced pressure and purified by flash silica column chromatography using 4% methanol in DCM as the eluent. The desired fractions were concentrated under reduced pressure and then under high vacuum. The resulting residue was dissolved in anhydrous pyridine (1.0 mL) to which sulfur trioxide pyridine complex (17 mg, 109 μmol) was added at room temperature. The resulting mixture was heated to 45° C. for 3 h, after which methanol (0.5 mL) was added and stirred for an additional 10 min. The reaction mixture was concentrated under reduced pressure and then under high vacuum. The resulting residue was redissolved in anhydrous methanol (6.0 mL) and cooled to 0° C. To this cooled solution was added dropwise a solution of 0.5 M sodium methoxide in methanol (0.8 mL) and allowed to stir for 16 h. The reaction was quenched by the addition of a 1 M aqueous solution of basic sodium phosphate (1.0 mL) and subjected to semi-preparative reverse-phase HPLC purification (gradient water / methanol system) to give 5 (12 mg, 47%). 1 H NMR (300 MHz, MeOD) δ 7.85 - 7.76 (m, 2H), 7.58 - 7.38 (m, 5H), 7.05 - 6.92 (m, 2H), 5.00 (dd, J = 8.4, 1.2 Hz, 1H), 4.75 (d, J = 3.1 Hz, 1H), 4.15 (dd, J = 10.9, 8.4 Hz, 1H), 3.95 - 3.60 (m, 6H), 3.38 (dt, J = 11.2, 5.6 Hz, 4H), 2.62 (dd, J = 15.9, 6.9 Hz, 2H), 2.47 - 2.29 (m, 2H), 1.97 (s, 3H), 1.76 - 1.51 (m, 6H), 1.47 - 1.21 (m, 6H), 0.89 (td, J = 6.6, 3.4 Hz, 3H).[M-Na + ]- MS (ESI - ), calculated: 749.3, observed: 749.5.

[0597] MPS-VI substrate (pentanamide).

[0598] The preparation of a representative MPS-VI substrate (pentanamide) is described below and illustrated in FIG.

[0599] N-(5-(N-(3-((4-hydroxyphenyl)amino)-3-oxopropyl)pentanamido)pentyl)-benzamide.

[0600] A solution of N-(5-aminopentyl)benzamide (207 mg, 1.00 mmol) and N-(4-hydroxyphenyl)acrylamide (197 mg, 1.21 mmol) in i-propyl alcohol (9.0 mL) and water (1.0 mL) was heated to 65° C. for 24 h with constant stirring. The reaction mixture was cooled to room temperature and concentrated under reduced pressure and then under high vacuum. To the crude concentrate was added anhydrous N,N-dimethylformamide (DMF) (3.0 mL) and triethylamine (253 mg, 2.50 mmol) until complete dissolution. The solution was cooled to 0° C. and valeryl chloride (241 mg, 2.00 mmol) was added dropwise, warmed to room temperature, and stirred for 2 h. The reaction was quenched by the addition of saturated sodium bicarbonate solution and the reaction mixture was extracted with DCM / methanol (4:1). The organic layer was further washed with water and dried over anhydrous sodium sulfate. The organic layer was concentrated to dryness under reduced pressure and methanol (3.0 mL) was added to redissolve. To this solution, 5% aqueous sodium hydroxide (3.0 mL) was added dropwise and stirred at room temperature for 2 hours. The reaction was acidified with 1N HCl solution (as indicated by pH paper) and extracted with DCM / methanol (4:1). The organic layer was concentrated under reduced pressure and the residue was subjected to silica column chromatography eluting with 5% methanol in DCM to give the title compound (203 mg, 45%). 11H NMR (300 MHz, MeOD) δ 8.45 (s, 1H), 7.83 - 7.78 (m, 2H), 7.56 - 7.40 (m, 3H), 7.34 - 7.26 (m, 2H), 6.76 - 6.68 (m, 2H), 3.69 (dt, J = 19.4, 6.9 Hz, 2H), 3.44 - 3.37 (m, 4H), 2.60 (dd, J = 14.7, 7.4 Hz, 2H), 2.47 - 2.30 (m, 2H), 1.75 - 1.49 (m, 6H), 1.47 - 1.26 (m, 4H), 0.91 (t, J = 7.3 Hz, 3H). [M+Na] + MS (ESI + ) for, calculated value: 476.3, observed value: 476.5.

[0601] N-(5-(N-(3-((4-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-3-oxopropyl)pentanamido)pentyl)benzamide.

[0602] To a solution of N-(5-(N-(3-((4-hydroxyphenyl)amino)-3-oxopropyl)pentanamido)pentyl)-benzamide (148 mg, 0.326 mmol) and (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-chlorotetrahydro-2H-pyran-3,4-diyl diacetate (239 mg, 0.653 mmol) in DCM (0.4 mL) was added tetrabutylammonium hydrogen sulfate (110 mg, 0.324 mmol) and 2M aqueous sodium hydroxide (0.4 mL) and allowed to stir at room temperature for 3 hours. To the reaction mixture was added another portion of the diacetate (90 mg, 0.246 mmol) and allowed to stir for an additional 13 hours. The reaction mixture was then extracted between water and DCM and the organic layer was further washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude product was purified by flash silica column chromatography using 4% methanol in DCM as the eluent to give the peracetylated intermediate. NMR spectroscopy showed that the peracetylated intermediate co-eluted with starting material 3. This mixture was used in the next deacetylation step without further purification. A solution of 0.5 M sodium methoxide in methanol (200 μL) was added dropwise to a solution of the above mixture in anhydrous methanol (5.0 mL) at 0° C. and allowed to stir at room temperature for 2 h. The reaction was quenched by the addition of formic acid (100 μL) and subjected to semi-preparative reversed-phase HPLC purification (gradient water / methanol system) to give the title compound (29 mg, 14%). 11H NMR (300 MHz, MeOD) δ 7.83 (d, J = 7.1 Hz, 2H), 7.59 - 7.35 (m, 5H), 7.06 - 6.90 (m, 2H), 4.99 (d, J = 8.4 Hz, 1H), 4.27 - 4.11 (m, 1H), 3.96 - 3.55 (m, 7H), 3.46 - 3.35 (m, 4H), 2.71 - 2.51 (m, 2H), 2.49 - 2.27 (m, 2H), 2.01 (s, 3H), 1.76 - 1.49 (m, 6H), 1.37 (dd, J = 14.6, 7.2 Hz, 4H), 0.93 (t, J = 7.2 Hz, 3H). [M+Na] + M for S(ESI + ), calculated: 679.3, observed: 679.7.

[0603] Sodium (2R,3R,4R,5R,6S)-5-acetamido-6-(4-(3-(N-(5-benzamidopentyl)-pentanamido)propanamido)phenoxy)-4-hydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl sulfate.

[0604] To a cooled (0° C.) solution of N-(5-(N-(3-((4-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-3-oxopropyl)pentanamido)pentyl)benzamide (25 mg, 38.1 μmol) in anhydrous pyridine (0.5 mL) was added benzoyl chloride (4.9 μL, 41.9 μmol). After 1 h at room temperature, the solution was cooled again to 0° C. and another portion of benzoyl chloride (9.4 μL, 80.4 μmol) was added and allowed to stir at room temperature for 2 h. The reaction was extracted between 1 M HCl solution and chloroform. The chloroform layer was further washed with a mixture of water and brine solution (1:1). The organic layer was concentrated and purified by flash silica column chromatography using 5% methanol in DCM as the eluent. The desired fractions were concentrated under reduced pressure and then under high vacuum. The resulting residue was dissolved in anhydrous pyridine to which sulfur trioxide pyridine complex (8.3 mg, 52.1 μmol) was added at room temperature. The resulting mixture was heated to 45° C. for 3 h, after which methanol (0.5 mL) was added and stirred for an additional 10 min. The reaction mixture was concentrated under reduced pressure and then under high vacuum. The resulting residue was redissolved in anhydrous methanol (5.0 mL) and cooled to 0° C. To this cooled solution was added dropwise a solution of 0.5 M sodium methoxide in methanol (0.5 mL) and allowed to stir for 16 h. The reaction was quenched by the addition of a 1 M aqueous solution of basic sodium phosphate (1.0 mL) and subjected to semi-preparative reverse phase HPLC purification (gradient water / methanol system) to give the title compound (5.8 mg, 20%). 1H NMR (300 MHz, MeOD) δ 7.80 (dd, J = 7.0, 1.2 Hz, 2H), 7.58 - 7.38 (m, 5H), 7.03 - 6.94 (m, 2H), 5.01 (dd, J = 8.4, 1.1 Hz, 1H), 4.75 (d, J = 3.1 Hz, 1H), 4.13 (dd, J = 10.9, 8.4 Hz, 1H), 3.95 - 3.61 (m, 6H), 3.45 - 3.34 (m, 4H), 2.61 (dd, J = 16.1, 7.0 Hz, 2H), 2.47 - 2.31 (m, 2H), 1.97 (s, 3H), 1.73 - 1.49 (m, 6H), 1.43 - 1.23 (m, 5H), 0.91 (td, J = 7.3, 2.2 Hz, 3H).[M-Na + ] - MS (ESI - ), calculated: 735.3, observed: 735.4.

[0605] MPS-VI product

[0606] The preparation of a representative MPS-VI product is described below and illustrated in FIG.

[0607] (2R,3R,4R,5R,6S)-5-Acetamido-2-(acetoxymethyl)-6-(4-nitrophenoxy)tetrahydro-2H-pyran-3,4-diyldiacetic acid (1)

[0608] [ka]

[0609] Pyridine (60 mL) was added to a nitrogen-flooded flask containing D-galactosamine hydrochloride (5 g, 23.2 mmol) and the resulting slurry was cooled on an ice bath. Acetic anhydride (25 g, 245 mmol) was added dropwise to the cooled mixture and allowed to warm to room temperature and then stirred at this temperature for 16 hours. Methanol (15 mL) was added to quench the reaction mixture and allowed to stir for 20 minutes. The resulting mixture was concentrated under reduced pressure, the mixture was allowed to warm and the residue was dissolved in 20% methanol in chloroform. This solution was washed with 1N HCl solution followed by brine solution. The resulting organic layer was dried using anhydrous sodium sulfate and concentrated under reduced pressure. The residue was taken up in a nitrogen-flooded flask equipped with a dropping funnel. Anhydrous dichloromethane (100 mL) was added to the residue and the resulting slurry was cooled on an ice bath. In a dropping funnel, titanium chloride (6.5 g, 42.1 mmol) was dissolved in anhydrous dichloromethane (40 mL) and the resulting solution was added dropwise to the cooled solution. The reaction mixture was warmed to 50° C. in an oil bath and allowed to stir at this temperature for 48 hours. The reaction mixture was cooled again on an ice bath and saturated sodium bicarbonate solution was added dropwise with vigorous shaking. The resulting mixture was extracted between dichloromethane and saturated sodium bicarbonate solution. The organic layer was dried using anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was dissolved in acetone (60 mL) and slowly added to a solution of 4-nitrophenol (16.1 g, 116 mmol) in acetone (130 mL) and 4N aqueous KOH (23.2 mL). The reaction was allowed to stir at room temperature for 48 hours and concentrated under reduced pressure to less than 20 mL. The solution was extracted between 1N NaOH and chloroform. The organic layer was dried using anhydrous sodium sulfate and concentrated under reduced pressure. The crude product thus obtained was purified by silica flash chromatography using 3% methanol in dichloromethane as the eluent mixture. Fractions containing the desired compound as determined by TLC were combined and concentrated under reduced pressure to give 1 (3.29 g, 30%). 11H NMR (300 MHz, CDCl3) δ 8.20 (d, J = 9.1 Hz, 2H), 7.09 (d, J = 9.1 Hz, 2H), 5.61 (d, J = 8.0 Hz, 1H), 5.56 - 5.39 (m, 3H), 4.32 - 4.07 (m, 4H), 2.18 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H). [M+Na] + MS (ESI + ) for, calculated value: 491.1, observed value: 491.2.

[0610] N-(5-(N-(3-((4-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-3-oxopropyl)pentanamido)pentyl)benzamide (2)

[0611]

Chemical Structure

[0612] To a solution of 1 (3.5 g, 7.47 mmol) in anhydrous methanol (90 mL) was added dropwise a solution of 0.5 M sodium methoxide in methanol (3 mL, 1.50 mmol) cooled on an ice bath and allowed to warm to room temperature. After 2 h, formic acid (0.1 mL) was added to the reaction mixture and concentrated to dryness under reduced pressure. To the resulting residual methanol (135 mL) was added water (15 mL) and 10% palladium on activated carbon (125 mg) and allowed to stir at room temperature under hydrogen atmosphere for 16 h. Water was added dropwise to the reaction mixture until all the white residue was completely dissolved. The reaction mixture was filtered and the filtrate was cooled on an ice bath. To this was added pyridine (2 mL) followed by a solution of acryloyl chloride (2.1 g, 23.2 mmol) in dichloromethane (50 mL) dropwise. The reaction was allowed to stir on the ice bath for 30 min, then allowed to warm to room temperature and proceed for 2 h. Sodium carbonate powder (3.0 g) was added to the reaction mixture, allowed to stir for 15 min and filtered. The filtrate was concentrated under reduced pressure and further dried under high vacuum. The residue was dissolved in a mixture of 2-propanol (50 mL) and water (6.6 mL) to which N-(5-aminopentyl)benzamide (2.0 g, 9.69 mmol) was added and allowed to stir at 65° C. for 40 h. The reaction mixture was cooled to room temperature and methanol (25 mL) was added to it. The mixture was cooled on an ice bath and triethylamine (2.5 mL) was added followed by dropwise addition of a solution of pentanoyl chloride (2.7 g, 22.4 mmol) in dichloromethane (50 mL). The reaction was allowed to stir on an ice bath for 30 min and then warmed to room temperature and continued for 16 h. The reaction mixture was concentrated under reduced pressure and subjected to purification by silica flash chromatography using 15% methanol in dichloromethane as the eluting mixture to give 2 (2.96 g, 60%). 1H NMR (300 MHz, MeOD) δ 7.84 (d, J = 7.1 Hz, 2H), 7.59 - 7.35 (m, 5H), 7.09 - 6.91 (m, 2H), 5.00 (d, J = 8.4 Hz, 1H), 4.28 - 4.09 (m, 1H), 3.97 - 3.55 (m, 7H), 3.46 - 3.24 (m, 4H), 2.72 - 2.51 (m, 2H), 2.49 - 2.28 (d, J = 7.3 Hz, 2H), 2.02 (s, 3H), 1.78 - 1.49 (m, 6H), 1.49 -1.22 (m, 4H), 0.94 (t, J = 7.1 Hz, 3H).[M+H] + MS (ESI + ), calculated: 657.3, observed: 657.5.

[0613] Example 6 Representative Assays Using MPS-VI Reagents In this example, representative assays using the MPS-VI reagents of the invention are described, and the results of these reagents are compared with other MPS-VI reagents.

[0614] The original MSP-VI reaction is shown below (Duffey,TA, Sadilek,M., Scott,CR, Turecek,F., Gelb,MH (2010) "Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: Application to screening newborns for Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome)" Anal. Chem., 82: 9587-9591). Note that S, P and IS have BOC groups, and P and IS are not chemically identical (P has six CH2 groups in the bond, while IS has five).

[0615] [ka]

[0616] An alternative MPS-VI reaction is shown below:

[0617] [ka]

[0618] Note the different aglycone, which has an N-pentanoyl group but no BOC carbamate group. Also note that the internal standard is chemically identical to the product but has five deuteriums in the benzoyl group.

[0619] The MPS-VI original and surrogate substrates were compared in the following enzymatic assay: 1 mM substrate, 10 uM internal standard in 30 uL of buffer (100 mM ammonium formate, pH 4.0, 7.5 mM barium(II) acetate, 5.0 mM cerium(III) acetate). A 3 mm punch of dried blood spot was added and the mixture was incubated at 37°C for 16 hours with shaking. The reaction was quenched by adding an aqueous suspension (1:1) of DEAE cellulose (Whatmann DE52) (100 uL) followed by the addition of 400 uL of ethyl acetate. The mixture was mixed by pipetting up and down several times and then centrifuged (10 min at 3000 rpm) to separate the liquid layers and pellet the DE52. A 300 uL portion of the top ethyl acetate layer was transferred to a new well and the solvent was removed by evaporation with an oil-free air stream. The residue was taken up in 100 uL of methanol / 5 mM aqueous ammonium formate (80 / 20, v / v) and injected into the tandem mass spectrometer. Barium and cerium salts were provided to precipitate the free sulfate and phosphate present in the dried blood spot, as these anions cause inhibition of the production of the MPS-VI enzyme. DE52 was added to capture the remaining substrate, so that only the product and internal standard (charge neutral) were extracted into ethyl acetate. A blank assay was also performed, where a 3 mm punch of blood-free filter paper replaced the dried blood spot. The blank was incubated and processed as above.

[0620] [Table 5]

[0621] 1 Enzyme activity is expressed as umoles of product formed per hour per liter of blood.

[0622] 2 Coefficients of variation (CV) are based on six assays, each performed on a different punch from the same dried blood spot.

[0623] 3MSMS response is the amount of ion counts measured in a tandem mass spectrometry channel per pmole of analyte.

[0624] 4 The blood-no blood assay ratio is the ratio of the enzyme activity measured in an assay using a dried blood spot punch to the enzyme activity measured in a blood-free punch.

[0625] It can be seen from the above table that while both MPS-VI substrates have similar activity against the MPS-VI enzyme (umoles of product generated per hour per liter of blood), the alternative substrate induces products (ion counts detected per pmole of sample) that are approximately 10 times more sensitive for MSMS detection. The improvement in response of the blood-no blood test is likely due to the smaller amount of product that is an impurity in the MPS-VI alternative substrate, as the new substrate is more likely to be generated in a product-free form.

[0626] Example 7 Representative sulfatase assay for MPS-II In this example, a representative assay of the invention is described, an assay for iduronate 2-sulfatase, the enzyme defective in Hunter syndrome (MPS-II).

[0627] The first step in the reaction is as described above (see also WO2009 / 026252 (PCT / US2008 / 073516), WO2010 / 081163 (PCT / US2010 / 020801), WO2012 / 027612 (PCT / US2011 / 049224), and WO2013 / 070953 (PCT / US2012 / 064205)).

[0628] In the assay, a second enzyme, α-L-iduronidase (glycohydrolase), is added to the assay cocktail, which converts the initial MPS-II product to the final MPS-II product by removing the iduronic acid residue, leaving the aglycone. Iduronidase may be present in the assay cocktail incubated with the dried blood spot, or it may be added after a second incubation period following the first incubation with the dried blood spot. The amount of iduronidase added is sufficient to convert all of the initial MPS-II product to the final MPS-II product. The assay cocktail also contains an initial MPS-II internal standard (identical to the initial MPS-II product, but with, for example, five deuteriums in the benzoyl group), which is converted to the final MPS-II internal standard by iduronidase. Both the final MPS-II product and the final MPS-II internal standard are detected by tandem mass spectrometry, allowing the amount of the final MPS-II product to be quantified. Typically, the reaction mixture is extracted with an organic solvent, and the final MPS-II internal standard and the final MPS-II product are partitioned into the organic solvent phase in a relatively salt-free form. The organic solvent is removed by evaporation, the residue is dissolved in a solvent, and the solvent is injected into a tandem mass spectrometer. The analytes are detected by multiple reaction monitoring.

[0629] Using an assay that does not contain a second enzyme (i.e., glycohydrolase) and using a punched 3 mm dried blood spot from a newborn screening card, typically 10,000-30,000 ion counts are observed in the initial MPS-II product after a 12-18 hour incubation period. Using the assay of the present invention using iduronidase, typically 1,000,000-5,000,000 ion counts are observed in the final MPS-II product. Thus, the sensitivity of the assay has been improved by about 100-fold. A second advantage of the method of the present invention is that in the preceding assay, the residual MPS-II substrate that enters the mass spectrometer electrospray ionization source undergoes some degree of desulfation due to heating in the source. This increases the assay background by enhancing the product signal that is independent of the action of iduronate 2-sulfatase. With the assay of the present invention, this desulfation is irrelevant since the product detected is the aglycone (the final MPS-II product).

[0630] The iduronidase used is a human enzyme obtained by overexpression in mammalian cells. Any iduronidase can be used as long as it does not act on the MPS-II substrate (i.e., it cleaves the glycosidic bond only after the sulfate has been removed from iduronic acid).

[0631] Example 8 Representative sulfatase assay for MPS-VI A preferred second enzyme for use in a representative assay for MPS-VI is bacterial N-acetylgalactosaminidase, which is used to release N-acetylgalactosamine from the aglycone after the MPS-VI enzyme removes the sulfate from the 4-position of the sugar, improving assay sensitivity by approximately 20-fold. Other suitable enzymes include bacterial N-acetylhexosaminidase.

[0632] Example 9 Representative sulfatase assay for MPS-IVA A preferred second enzyme for use in a representative assay for MPS-IVA is β-galactosidase from an Aspergillus species, which is used to release galactose from the aglycone after the MPS-IVA enzyme removes the sulfate from the 6-position of the sugar. This improves assay sensitivity by about 20-fold. The Aspergillus enzyme preferably exceeds, for example, the E. coli enzyme, as it retains high activity at pH 4-5, the pH of the MPS-IVA assay. Alternatively, an MPS-IVA substrate bearing N-acetyl-galactosamine-6-sulfate may be used, with the initial MPS-IV product being acted upon to produce the aglycone by the same enzyme as used in the MPS-VI assay (see above).

[0633] Example 10 Representative sulfatase assay for MPS-IIIA A preferred second enzyme for use in a representative assay for MPS-IIIA is yeast α-glucosidase from baker's yeast, which is used to release glucosamine from its aglycone after the MPS-IIIA enzyme removes the sulfate from the amino group of glucosamine-N-sulfate. Alternatively, acetyl-CoA:glucosamine N-acetyltransferase can be used to acetylate the free amino group after the MPS-IIIA enzyme removes the sulfate. Both mammalian and bacterial acetyltransferases can be used.

[0634] Example 11 Representative assay for MPS-IVA In this example, a representative assay for MPS-IVA is described using the bacterial enzyme β-N-acetylgalactosaminidase (β-NGA), which cleaves β-glycosides to N-acetylgalactosamine if not sulfated at the 6-position, and the inhibitor of human hexosaminidase A, (Z)-O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino N-phenylcarbamate (Z-PUG-NAc), which blocks human hexosaminidase A on the GALNS substrate.

[0635] The GALNS substrate used in this assay has the following structure:

[0636] [ka]

[0637] The GALNS internal standard used in the assay has the following structure:

[0638] [ka]

[0639] Experiment 1

[0640] A 3 mm punch dried blood spot from a substrate random newborn is incubated with 0.03 mL of an assay cocktail consisting of 1 mM GALNS substrate in 50 mM ammonium formate, pH 4.0, containing 7.5 mM barium acetate, 5.0 mM cerium acetate, 1 mM Z-PUG-NAc, and 0.01 mg of bacterial β-N-acetylgalactosaminidase. The mixture also contains 0.005 mM internal standard. After 16 h at 37°C with shaking, the mixture is quenched with 0.12 mL of acetonitrile and the sample plate is centrifuged to pellet the sediment. The supernatant (0.12 mL) is transferred to a new plate and 0.12 mL of water is added. The plate is placed on the autosampler of a UHPLC-MS / MS instrument (Waters Xevo TQ MS / MS with Waters Acquity UHPLC system). A portion of the sample (0.01 mL) is injected and the GALNS product (substrate minus sulfate), internal standard, and aglycone derived from the GALNS product and internal standard are quantified by multiple reaction monitoring (MS / MS). Note that the added internal standard, e.g., the GALNS product, is converted to its aglycone by the addition of bacterial β-N-acetylgalactosaminidase. Table 1 above shows the MS / MS ion peak areas for each analyte.

[0641] Experiment 1 shows the results of a complete assay using blood-containing punch, GALNS substrate, internal standard, 1 mM Z-PUG-NAc, and 0.01 mg of β-NGA in assay buffer. The aglycone signal at 411,000 ion peak area is significantly higher than that of the early formed GALNS product, indicating that β-NGA converts most of the product to aglycone. Experiment 1 also shows that most of the internal standard is converted to aglycone. The amounts of aglycone and internal standard aglycone are used to determine the amount of GALNS enzyme activity.

[0642] Experiment 2

[0643] Experiment 2 is the same as experiment 1, but uses punched filter paper (no blood). Most of the internal standard is converted to the aglycone, indicating that the added β-NGA is working. The amount of aglycone is only 42,000, about 10-fold less than that seen in the presence of blood. This high blood to no blood ratio indicates that the GALNS assay is working. Furthermore, when using dried blood spots from confirmed Morquio A patients, the amount of aglycone is similar to that seen in the absence of blood (45,000), indicating that the assay is working to detect only GALNS present in the blood of unaffected individuals.

[0644] Experiment 3

[0645] Experiment 3 shows the full assay but without β-NGA. As expected, most of the internal standard is not converted to its aglycone because there is no β-NAG and all blood-borne hexosaminidase A is blocked by Z-PUG-NAc. The amount of GALNS product is 121,000 and the amount of aglycone is 42,900.

[0646] Experiment 4

[0647] Run 4 is the same as run 3, but without blood. Run 4 shows only 753 for GALNS product. Thus, most of the product count of 121,000 in run 3 is due to GALNS. Since only β-NGA produces aglycone, the product count in run 3 cannot be due to hexosaminidase A. The amount of aglycone in runs 3 and 4 is similar, representing aglycone present as an impurity in the GALNS substrate.

[0648] Experiments 1-4 also show the benefit of converting the GALNS product to its aglycone, with an aglycone signal of 411,000 in the presence of blood and β-NGA, compared to only 121,000 for the GALNS product when not converted to its aglycone by β-NGA, thus providing a 4-fold improvement in GALNS assay sensitivity.

[0649] Experiment 5

[0650] Experiment 5 shows the full assay but lacking the hexosaminidase A inhibitor Z-PUG-NAc. Because β-NGA is present, most of the internal standard is converted to its aglycone, as expected. The aglycone signal is 1,350,000, considerably higher than in experiment 1. This indicates that hexosaminidase A produces substantial amounts of aglycone from the GALNS substrate when not inhibited.

[0651] Experiment 6.

[0652] Experiment 6 contains blood but does not contain β-NGA and Z-PUG-NAc. The amount of GALNS product is 110,000, unlike 25,100 in experiment 5, due to the action of GALNS on the GALNS substrate. Since β-NGA is absent, only a small amount of GALNS product is converted to aglycone, but the amount of aglycone remains high at 1,110,000. This is because hexosaminidase A in blood acts on the GALNS substrate in the absence of Z-PUG-NAc.

[0653] Experiments 7 and 8

[0654] Experiments 7 and 8 use filter paper (without blood). Experiment 7 contains β-NGA, whereas experiment 8 does not. As expected, most of the internal standard is converted to aglycone in experiment 7, but not in experiment 8. Experiment 8 shows that the GALNS substrate contains small amounts of product (820) and aglycone (14,500) as impurities. These can be removed by further purification steps of the GALNS substrate.

[0655] The results in Table 1 clearly show that the action of uninhibited human hexosaminidase A endogenously present in dried blood spots produces excess aglycones from the GALNS substrate, resulting in a useful GALNS assay in the absence of hexosaminidase A inhibitors. The data also clearly show that β-NGA has the desired properties, namely, that it converts most of the GALNS products and internal standards to their aglycones, even in the presence of sufficient hexosaminidase A inhibitor Z-PUG-NAc to completely block hexosaminidase A.

[0656] While the preferred embodiment of the invention has been illustrated and described, it will be understood that various changes can be made therein without departing from the spirit and scope of the invention.

[0657] The aspects of the invention in which an exclusive ownership or license is claimed are defined as follows: The invention as originally claimed is set forth below. [1] A method for assaying for an enzyme associated with a lysosomal storage disease, comprising: (a) contacting the sample with a first solution to produce a solution comprising one or more lysosomal enzymes; (b) contacting the one or more lysosomal enzymes in solution with an enzymatic substrate for each lysosomal enzyme to be analyzed and incubating the substrate with the enzyme for a time sufficient to produce a solution containing an enzymatic product of each lysosomal enzyme present in the sample; wherein the enzyme substrate for each lysosomal enzyme has a carbohydrate moiety and an aglycone moiety and has the formula: [ka] [wherein S, when covalently attached to the aglycone moiety, is (i) α-L-iduronidase, (ii) iduronate 2-sulfatase, (iii) heparan N-sulfatase, (iv) N-acetyl-α-D-glucosaminidase, (v) N-acetylgalactosamine 6-sulfate sulfatase, (vi) N-acetylgalactosamine 4-sulfate sulfatase, and (vii) β-glucuronidase a carbohydrate moiety which provides a substrate for an enzyme selected from the group consisting of L 2 contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O and S, and / or one or more of which may be replaced by C 1 ~C 6 a linker optionally substituted with an alkyl group or a halogen; L 3 contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be replaced by C 1 ~C 6 a linker optionally substituted with an alkyl group or a halogen; L 4 is optional and, when present, contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be replaced by C 1 ~C 6 a linker optionally substituted with an alkyl group or a halogen; R 1 is C 1 ~C 10 Alkyl group or C 1 ~C 10 is an alkoxy group, R 2 In each occurrence, C 1 ~C 10 Alkyl group, C 1 ~C 10 Alkoxy groups, halogens, nitro, -C(=O)NHR, or -C(=O)OR, where R is C 1 ~C 8 alkyl group; R 3 is C 1 ~C 10 Alkyl group or substituted or unsubstituted C 6 ~C 10 is an aryl group, n is 0, 1, 2, 3 or 4. is a compound having the formula (c) determining the amount of one or more of said enzyme products; A method comprising: [2] The method of [1], further comprising contacting the enzyme product with a glycohydrolase to produce a second enzyme product. [3] (a) N-acetylgalactosamine 6-sulfate sulfatase, and (b) N-acetylgalactosamine 4-sulfate sulfatase The method according to [1], further comprising adding an inhibitor to block endogenous glycohydrolase enzyme activity acting on a substrate of one or more enzymes selected from the group consisting of: [4] A method for assaying enzymatic activity of one or more lysosomal enzymes, comprising: (a) contacting the sample with a first solution to produce a solution comprising one or more lysosomal enzymes; (b) contacting the one or more lysosomal enzymes in solution with an enzymatic substrate for each lysosomal enzyme to be analyzed and incubating the substrate with the enzyme for a time sufficient to produce a solution containing a first enzymatic product of each lysosomal enzyme present in the sample; (c) subjecting the first enzyme products to a glycohydrolase to produce a second enzyme product of each first enzyme product that is susceptible to further enzymatic action by the glycohydrolase; (d) determining the amount of one or more of the first enzyme products and / or one or more of the second enzyme products. [5] the first enzyme product, which is susceptible to further enzymatic action by the glycohydrolase, (a) iduronate 2-sulfatase, (b) heparan N-sulfatase, (c) N-acetylgalactosamine 6-sulfate sulfatase, and (d) N-acetylgalactosamine 4-sulfate sulfatase, The method according to [4], wherein the compound is produced by the action of an enzyme selected from the group consisting of: [6] The first enzyme product, which is not susceptible to further enzymatic action by the glycohydrolase, (a) α-L-iduronidase, (b) N-acetyl-α-D-glucosaminidase, and (c) β-glucuronidase The method according to [4], wherein the compound is produced by the action of an enzyme selected from the group consisting of: [7] (a) N-acetylgalactosamine 6-sulfate sulfatase, and (b) N-acetylgalactosamine 4-sulfate sulfatase The method according to [4], further comprising adding an inhibitor to block endogenous glycohydrolase enzyme activity acting on a substrate of one or more enzymes selected from the group consisting of: [8] The one or more lysosomal enzymes are (a) α-L-iduronidase, (b) iduronate 2-sulfatase, (c) heparan N-sulfatase, (d) N-acetyl-α-D-glucosaminidase, (e) N-acetylgalactosamine 6-sulfate sulfatase, (f) N-acetylgalactosamine 4-sulfate sulfatase, and (g) β-glucuronidase The method according to [1] or [4], comprising an enzyme selected from the group consisting of: [9] The method according to [1] or [4], further comprising adding an internal standard for each lysosomal enzyme to be analyzed before, after, or simultaneously with contacting the lysosomal enzyme with the substrate.

[10] The method according to [1] or [4], further comprising inactivating the enzymatic reaction prior to determining the amount of one or more of the enzyme products.

[11] The method according to [1] or [4], wherein the sample is a blood or tissue sample.

[12] The method of [1] or [4], wherein the sample is a dried blood spot.

[13] The method according to [2] or [4], wherein the glycohydrolase is selected from the group consisting of human hexosaminidase A, bacterial N-acetylhexosaminidase, bacterial β-N-acetylgalactosaminidase, α-L-iduronidase, β-galactosidase (Aspergillus), and α-glucosidase (yeast).

[14] The inhibitor is selected from the group consisting of (Z)-O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino N-phenylcarbamate, 1-deoxynojirimycin, castanospermine, swainsonine, and calystegine B. 2 , isofagamine, Tamiflu, gluconohydroxymolactone, glucuronic acid and its lactones and lactams, Relenza, miglitol, phenethyl-substituted gluco- and galacto-imidazoles, N-hydroxyethyldehydronojirimycin, GalNAc thiazoline, and GlcNAc thiazoline.

[15] The method of [1] or [4], wherein determining the amount of the enzyme product comprises mass spectrometry.

[16] The method of [1] or [4], wherein determining the amount of the enzyme product comprises determining the ratio of each product to its internal standard by mass spectrometry.

[17] The method of [1] or [4], wherein determining the amount of the enzyme product comprises tandem mass spectrometry, in which parent ions of the product and their internal standards are generated, isolated, and subjected to collision-induced dissociation to generate product fragment ions and internal standard fragment ions.

[18] The method according to [1] or [4], wherein determining the amount of the enzyme product comprises calculating the amount of the product by comparing the peak intensities of product fragment ions and internal standard fragment ions.

[19] The method of [1] or [4], wherein determining the amount of the enzyme product comprises conducting the product by liquid chromatography or flow injection into a mass spectrometer.

[20] The method of [1] or [4], wherein determining the amount of the enzyme product comprises fluorescence analysis.

[21] The method of [1] or [4], further comprising using the amount of the enzyme product to determine whether the sample is derived from a candidate for treating a disease condition associated with one or more lysosomal enzyme deficiencies.

[22] The substrate has a carbohydrate moiety and an aglycone moiety and has the formula:

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[23] The substrate has the following formula:

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[22] , comprising:

[24] The substrate has the following formula:

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[22] , comprising:

[25] The substrate has the formula:

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[22] , comprising:

[26] The substrate has the formula:

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[22] , comprising:

[27] The substrate has the following formula:

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[22] , comprising:

[28] The substrate has the following formula:

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[22] , comprising:

[29] The substrate has the following formula:

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[22] , comprising:

[30] The substrate has the following formula:

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[22] , comprising:

[31] The substrate has the following formula:

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[22] , comprising:

[32] The substrate has the formula:

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[22] , comprising:

[33] The substrate has the formula:

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[22] , comprising:

[34] The substrate has the formula:

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[22] , comprising:

[35] The substrate has the formula:

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[22] , comprising:

[36] The substrate has the formula:

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[22] , comprising:

[37] A compound having a carbohydrate moiety and an aglycone moiety, the compound having the formula:

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[38] L 2 But -(CH 2 ) n - (wherein n is 1 to 6).

[39] L 3 But -(CH 2 ) m - (wherein m is 1 to 12).

[40] L 4 But -(CH 2 ) n - (wherein n is 1 to 6).

[41] L 4 The compound according to

[37] ,

[42] R 1 But, C 1 ~C 5 The compound according to

[37] , wherein the R is an alkyl group.

[43] R 2 But, C 1 ~C 8 The compound according to

[37] , wherein the R is an alkyl group.

[44] R 3 But, C 1 ~C 6 The compound according to

[37] , wherein the R is an alkyl group.

[45] R 3

[37] , wherein R is phenyl.

[46] The following formula:

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[37] , having the formula:

[47] The following formula:

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[37] , having the formula:

[48] ​​The following formula:

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[37] , having the formula:

[49] The following formula:

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[37] , having the formula:

[50] The following formula:

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[37] , having the formula:

[51] The following formula:

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[37] , having the formula:

[52] The following formula:

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[37] , having the formula:

[53] The following formula:

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[37] , having the formula:

[54] The following formula:

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[37] , having the formula:

[55] Equation:

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[37] , having the formula:

[56] Equation:

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[37] , having the formula:

[57] The following formula:

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[37] , having the formula:

[58] The following formula:

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[37] , having the formula:

[59] The following formula:

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[37] , having the formula:

[60] A kit for assaying an enzyme associated with a lysosomal storage disease, comprising a substrate selected from one or more of the compounds described in

[37] to

[59] .

[61] The kit described in

[60] , wherein the enzyme is selected from one or more of α-L-iduronidase (MPS-I), iduronate 2-sulfatase (MPS-II), heparan N-sulfatase (MPS-IIIA), N-acetyl-α-D-glucosaminidase (MPS-IIIB), N-acetylgalactosamine 6-sulfate sulfatase (MPS-IVA), N-acetylgalactosamine 4-sulfate sulfatase (MPS-VI), and β-glucuronidase (MPS-VII).

[62] The kit of

[60] , further comprising an internal standard for each of the enzymes to be assayed.

[63] A method for assaying for an enzyme associated with a lysosomal storage disease, comprising contacting a sample with a substrate selected from one or more of the compounds described in

[37] to

[59] .

[64] The method of

[63] , wherein the enzyme is selected from one or more of α-L-iduronidase (MPS-I), iduronate 2-sulfatase (MPS-II), heparan N-sulfatase (MPS-IIIA), N-acetyl-α-D-glucosaminidase (MPS-IIIB), N-acetylgalactosamine 6-sulfate sulfatase (MPS-IVA), N-acetylgalactosamine 4-sulfate sulfatase (MPS-VI), and β-glucuronidase (MPS-VII).

[65] The method of

[63] , further comprising contacting the sample with an internal standard for each of the enzymes to be assayed.

[66] A method for assaying α-L-iduronidase (MPS-I), comprising contacting a sample with a compound according to

[46] or

[47] .

[67] A method for assaying iduronate 2-sulfatase (MPS-II), comprising contacting a sample with a compound according to

[48] or

[49] .

[68] A method for assaying heparan N-sulfatase (MPS-IIIA), comprising contacting a sample with a compound according to

[50] or

[51] .

[69] A method for assaying N-acetyl-α-D-glucosaminidase (MPS-IIIB), comprising contacting a sample with a compound according to

[52] or

[53] .

[70] A method for assaying N-acetylgalactosamine 6-sulfate sulfatase (MPS-IVA), comprising contacting a sample with a compound according to

[54] or

[55] .

[71] A method for assaying N-acetylgalactosamine 4-sulfate sulfatase (MPS-VI), comprising contacting a sample with a compound according to

[56] or

[57] .

[72] A method for assaying β-glucuronidase (MPS-VII), comprising contacting a sample with a compound according to

[58] or

[59] .

Claims

1. A method for assaying for β-glucuronidase, comprising: (a) contacting the sample with a first solution to produce a solution comprising one or more lysosomal enzymes; (b) contacting the one or more lysosomal enzymes in a solution with an enzymatic substrate for β-glucuronidase and incubating the one or more lysosomal enzymes in a solution for a period of time sufficient to produce a solution containing an enzymatic product of β-glucuronidase; wherein the enzyme substrate for β-glucuronidase has a carbohydrate moiety and an aglycone moiety and has the formula: 【Chemistry 1】 [In the formula, L ２ contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O and S, and / or one or more of which may be replaced by C １ ~C ６ a linker optionally substituted with an alkyl group or a halogen; L ３ contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be replaced by a heteroatom selected from C １ ~C ６ a linker optionally substituted with an alkyl group or a halogen; L ４ is optional and, when present, contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be replaced by a heteroatom selected from C １ ~C ６ a linker optionally substituted with an alkyl group or a halogen; R １ is C １ ~C １０ Alkyl group or C １ ~C １０ is an alkoxy group, R ２ In each occurrence, C １ ~C １０ Alkyl group, C １ ~C １０ Alkoxy groups, halogen, nitro, -C(=O)NHR, or -C(=O)OR, where R is C １ ~C ８ alkyl group; R ３ is C １ ~C １０ Alkyl group or substituted or unsubstituted C ６ ~C １０ is an aryl group, n is 0, 1, 2, 3 or 4. and (c) determining the amount of β-glucuronidase; A method comprising:

2. The compound, 【Chemistry 2】 The method of claim 1, wherein 3. The method according to claim 1, wherein L 2 is -CH 2 CH 2 -.

4. The method according to claim 1, wherein L 3 is —C 6 H 12 —.

5. The method according to claim 1, wherein R 1 is —C 4 H 9 .

6. The compound 【Chemistry 3】 The method according to claim 1 or 2,

7. The following formula: 【Chemistry 4】 [In the formula, L2 contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O and S, and / or one or more of which may be replaced by a heteroatom selected from C １ ~C ６ a linker optionally substituted with an alkyl group or a halogen; L ３ contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be replaced by a heteroatom selected from C １ ~C ６ a linker optionally substituted with an alkyl group or a halogen; L ４ is optional and, when present, contains 1 to 20 carbon atoms, one or more of which may be replaced by a heteroatom selected from N, O or S, and / or one or more of which may be replaced by a heteroatom selected from C １ ~C ６ a linker optionally substituted with an alkyl group or a halogen; R １ is C １ ~C １０ Alkyl group or C １ ~C １０ is an alkoxy group, R ２ In each occurrence, C １ ~C １０ Alkyl group, C １ ~C １０ Alkoxy groups, halogen, nitro, -C(=O)NHR, or -C(=O)OR, where R is C １ ~C ８ alkyl group; R ３ is C １ ~C １０ Alkyl group or substituted or unsubstituted C ６ ~C １０ is an aryl group, n is 0, 1, 2, 3 or 4. A compound having the formula:

8. The compound, 【Chemistry 5】 8. The compound of claim 7, wherein 9. The compound according to claim 7 or 8, wherein L 2 is -CH 2 CH 2 -.

10. The compound according to claim 7 or 8, wherein L 3 is —C 6 H 12 —.

11. The compound according to claim 7 or 8, wherein R 1 is —C 4 H 9 .

12. The compound 【Chemistry 6】 9. The compound according to claim 7 or 8,

13. A kit for assaying an enzyme associated with a lysosomal storage disease comprising a substrate selected from one or more of the compounds according to any one of claims 7 to 12.

14. The kit according to claim 13, wherein the enzyme is β-glucuronidase (MPS-VII).

15. The kit of claim 14, further comprising an internal standard of β-glucuronidase (MPS-VII).

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