WO2012027612A1 - Mass spectrometric compositions and methods for lysosomal storage disease screening - Google Patents

Mass spectrometric compositions and methods for lysosomal storage disease screening Download PDF

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WO2012027612A1
WO2012027612A1 PCT/US2011/049224 US2011049224W WO2012027612A1 WO 2012027612 A1 WO2012027612 A1 WO 2012027612A1 US 2011049224 W US2011049224 W US 2011049224W WO 2012027612 A1 WO2012027612 A1 WO 2012027612A1
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product
acetylgalactosamine
sulfatase
internal standard
sample
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PCT/US2011/049224
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French (fr)
Inventor
Michael Gelb
Trisha Ann Duffey
Brian James Wolfe
Tanvir Khaliq
Frantisek Turecek
C. Ronald Scott
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Michael Gelb
Trisha Ann Duffey
Brian James Wolfe
Tanvir Khaliq
Frantisek Turecek
Scott C Ronald
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Publication of WO2012027612A1 publication Critical patent/WO2012027612A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/075—Benzo[b]pyran-2-ones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders

Definitions

  • the mucopolysaccharidoses are a group of metabolic diseases caused by a deficiency of one of the lysosomal enzymes degrading the glycosaminoglycans heparan, dermatan, keratan, or chondroitin sulfate (Neufeld, E. F.; Muenzer, J. The Mucopolysaccharidoses. In: Scriver, C; Beaudet, A.; Sly, W.; eds. The Metabolic and Molecular Bases of Inherited Disease. New York, NY: McGraw Hill; 2001; 3421-3452).
  • the pertinent enzymes consist of five sulfatases, four exoglycosidases, and one non-hydrolytic acetyl-N-transferase. These syndromes result in non-degraded or partially degraded glycosaminoglycans amassing in the lysosome resulting in irreversible multisystemic organ damage.
  • Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome) is caused by deficiency of /V-acetylgalactosamine 4-sulfatase (also called aryl sulfatase B, ASB, EC 3.1.56.12), which is one of several enzymes involved in step-wise degradation of chondroitin and dermatan sulfate.
  • ASB deficiency is inherited as an autosomal recessive trait. The affected children may appear normal at birth but within 1-3 years develop skeletal abnormalities causing short stature and joint stiffness, as well as other symptoms such as corneal clouding.
  • MPS VI early detection of MPS VI seems prudent to maximize the potential benefit of treatment, and thus there is the need to develop tests that are appropriate for early diagnosis. Likewise, there is a need for developing a fast, inexpensive, and reliable diagnostic procedure that uses dried blood spots (DBS) as a sample source, such as those submitted to newborn screening laboratories.
  • DBS dried blood spots
  • MPS IVA mucopolysaccharidosis IVA
  • GALNS ⁇ -acetylgalactosamine 6- sulfatase
  • the enzyme hydrolyzes the sulfate ester bond of galactose 6-sulphate present in keratin sulfate and of ⁇ -acetylgalactosamine 6-sulfate present in chondroitin C.
  • MPS rVA hydrolyzes the sulfate ester bond of galactose 6-sulphate present in keratin sulfate and of ⁇ -acetylgalactosamine 6-sulfate present in chondroitin C.
  • lysosomal storage diseases for which early recognition and treatment has been shown to be efficacious include Pompe, Fabry, and mucopolysaccharidosis I (Hurler) (MPS IH) diseases.
  • MPS IH mucopolysaccharidosis I
  • substrates Provided herein are substrates, internal standards, enzymatic assays, and screening methods pertaining to the analysis of various lysosomal storage diseases in patients, such as newborns.
  • Rj is unsubsti
  • R 2 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or; substituted arylalkyl ⁇ . ⁇ ;
  • Y2 is - OH or -NHC(0)CH 3 ; and
  • m is an integer from 2 to 12.
  • R j is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or substituted arylalkyl ⁇ . ⁇ ;
  • Xj is hydrogen or a counterion;
  • Y j is -OH or -NHC(0)CH3; and
  • n is an integer from 2 to 12; with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 6-sulfatase product when the sample comprises ⁇ -acetylgalactosamine 6-sulfatase.
  • a further method provided is a method for determining the presence or absence of ⁇ -acetylgalactosamine 6-sulfatase enzymatic activity in a sample, comprising: (a) incubating an N- acetylgalactosamine 6-sulfatase substrate with sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 6-sulfatase product when the sample comprises N- acetylgalactosamine 6-sulfatase; and (b) determining the presence or absence of the N- acetylgalactosamine 6-sulfatase product, wherein the presence of N-acetylgalactosamine 6-sulfatase product indicates the presence of N-acetylgalactosamine 6-sulfatase enzymatic activity, and wherein the absence of N-acetylgalactosamine 6-sulfatas
  • a method for determining the quantity of an N-acetylgalactosamine 6-sulfatase product in a blood sample comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating (i) an ⁇ -acetylgalactosamine 6-sulfatase substrate of formula (I):
  • Rj is unsubsti
  • X j is hydrogen or a counterion
  • Y j is -OH or -NHC(0)CH3
  • n is an integer from 2 to 12; and (ii) an ⁇ -acetylgalactosamine 6-sulfatase internal standard, with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 6-sulfatase product when the sample comprises ⁇ -acetylgalactosamine 6-sulfatase; and (c) determining the quantity of the N-acetylgalactosamine 6-sulfatase product by tandem mass spectrometric analysis.
  • R3 is unsubst
  • R 4 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or; substituted arylalkyl ⁇ . ⁇ ; and q is an integer from 2 to 12. Also provided is a method for providing an N- acetylgalatosamine 4-sulfatase product, comprising incubating an N-acetylgalactosamine 4-sulfatase substrate of formula (III):
  • R3 is unsubst
  • X2 is hydrogen or a counterion; and
  • p is an integer from 2 to 12; and with a sample for a predetermined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 4-sulfatase product when the sample comprises ⁇ -acetylgalactosamine 4-sulfatase.
  • a method for determining the presence or absence of N-acetylgalactosamine 4-sulfatase enzymatic activity in a sample comprising: (a) incubating an ⁇ -acetylgalactosamine 4-sulfatase substrate with sample for a pre-determined time sufficient to effect enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 4-sulfatase product when the sample comprises ⁇ -acetylgalactosamine 4-sulfatase; and (b) determining the presence or absence of the ⁇ -acetylgalactosamine 4-sulfatase product, wherein the presence of N-acetylgalactosamine 4-sulfatase product indicates the presence of N-acetylgalactosamine 4-sulfatase enzymatic activity, and wherein the absence of N- acetylgalactosamine 4-sulfatase product indicates the
  • a method for determining the quantity of an ⁇ -acetylgalactosamine 4-sulfatase product in a blood sample comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating (i) an ⁇ -acetylgalactosamine 4-sulfatase substrate of formula (III):
  • R 3 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or; substituted arylalkyl ⁇ . ⁇ :
  • X2 is hydrogen or a counterion; and p is an integer from 2 to 12; and (ii) an N- acetylgalactosamine 4-sulfatase internal standard, with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 4-sulfatase product when the sample comprises N-acetylgalactosamine 4-sulfatase; and (c) determining the quantity of the N- acetylgalactosamine 4-sulfatase product by tandem mass spectrometric analysis.
  • a further embodiment provides a method for providing a solution comprising an a-glucosidase product, an a-galactosidase product, and an a-L-iduronidase product, comprising incubating an a-glucosidase substrate, an a-galactosidase substrate, and an a- L-iduronidase substrate with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an a- glucosidase product, an ⁇ -galactosidase product, and an a-L-iduronidase product when the sample comprises ⁇ -glucosidase, ⁇ -galactosidase, and ⁇ -L-iduronidase, wherein the buffer comprises an aqueous solution of acarbose.
  • a method for determining the presence or absence of ⁇ -glucosidase enzymatic activity, a-galactosidase enzymatic activity, and ⁇ -L-iduronidase enzymatic activity in a sample comprising: (a) incubating an ⁇ -glucosidase substrate, an ⁇ -galactosidase substrate, an a-L-iduronidase substrate with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -glucosidase product, an ⁇ -galactosidase product, and an ⁇ -L-iduronidase product when the sample comprises a- glucosidase, ⁇ -galactosidase, and ⁇ -L-iduronidase; and (b) determining the presence or absence of the a-glucosidase product, the ⁇ -galactosidase product, and the a
  • Also provided is a method for determining the quantity of a ⁇ -glucosidase product, a ⁇ -galactosidase product, and a a-L- iduronidase product in a blood sample comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating an ⁇ -glucosidase substrate, an a- glucosidase internal standard, an ⁇ -galactosidase substrate, an ⁇ -galactosidase internal standard, an ⁇ -L-iduronidase substrate, and an ⁇ -L-iduronidase internal standard with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -glucosidase product, an a- galactosidase product, and an ⁇ -L-iduronidase product when the sample comprises a- glucosidase
  • FIGURE 1 Structures of Maroteaux-Lamy substrate 1 and internal standard 3 and Morquio A substrate 2 and internal standard 4.
  • FIGURE 2 Preparation of MPS IVA substrate 2 and internal standards 3 and 4.
  • FIGURE 3 Preparation of MPS VI substrate 1. Reagents and conditions: (a) benzoyl chloride (2.1 equiv), pyridine, 0°C, 3 h, 58%; (b) triflic anhydride, pyridine, DCM, -20° C, 3 h; (c) sodium nitrite, DMF, overnight, 51% over two steps; (d) sulfur trioxide-pyridine complex, pyridine, 3 h; DOWEX 50WX8 (Na+ form), 50%; (e) sodium methoxide, MeOH, 5 d, 80%.
  • benzoyl chloride 2.1 equiv
  • pyridine 0°C, 3 h, 58%
  • triflic anhydride pyridine, DCM, -20° C, 3 h
  • sodium nitrite, DMF overnight, 51% over two steps
  • sulfur trioxide-pyridine complex pyridine, 3 h
  • DOWEX 50WX8 Na+ form
  • FIGURE 4 Structures of aryl sulfatase B (ASB-S), aryl sulfatase B product (ASB-P), and aryl sulfatase B internal standard (ASB-IS) for the ASB reaction.
  • ASB-S aryl sulfatase B
  • ASB-P aryl sulfatase B product
  • ASB-IS aryl sulfatase B internal standard
  • FIGURE 5 Amount of aryl sulfatase B (ASB)-generated product measured in dried blood spots (DBS) as a function of the concentration of substrate. Reactions were carried out at 37 °C for 16 h using the standard assay described herein using solid phase extraction method. Error bars are shown for triplicate analyses. The solid line shows the regression fit of the data to the Michaelis-Menten equation.
  • ASB aryl sulfatase B
  • FIGURE 6 Distribution of ASB activities in DBS from humans. Black bars show data for 89 unaffected newborns obtained with the liquid-liquid extraction method. White bars show data for 10 unaffected newborns obtained with the solid phase extraction method. The hashed bar is data for 1 MPS VI patient. ASB activity values for each sample are given in Tables 1 and 2.
  • FIGURES 7 A and 7B FIGURE 7 A: Structures of N- acetylgalactosamine 6- sulfatase substrate (GALNS-S), N-acetylgalactosamine 6-sulfatase product (GALNS-P), and ⁇ -acetylgalactosamine 6-sulfatase internal standard (GALNS-IS) for the GALNS reaction.
  • GALNS-S N- acetylgalactosamine 6- sulfatase substrate
  • GALNS-P N-acetylgalactosamine 6-sulfatase product
  • GALNS-IS ⁇ -acetylgalactosamine 6-sulfatase internal standard
  • GALNS-P and GALNS-IS were quantified by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in positive-ion multiple-reaction-monitoring mode as the sodiated species (M + Na)+.
  • FIGURE 7B GALNS activities were measured in DBS by the standard assay described herein with ethyl acetate extraction workup.
  • FIGURE 8 Typical work-flow of the triplex assay run in a newborn screening laboratory. Assay results are obtained within 48 h of the beginning of the procedure.
  • FIGURES 9A, 9B, and 9C Activity distribution of cc-galactosidase A (GLA) (FIGURE 9A), cc-glucosidase (GAA) (FIGURE 9B), and a-L-iduronidase (IDUA) (FIGURE 9C) from 5,990 anonymous newborn blood spots. Assays were performed in the Washington State Newborn Screening laboratory using the triplex assay.
  • mucopolysaccharidosis VI Maroteaux-Lamy Syndrome
  • mucopolysaccharidosis IVA Mossaccharidosis I
  • Fabry Syndrome Fabry Syndrome
  • Pompe Syndrome mucopolysaccharidosis I
  • MPS IH mucopolysaccharidosis I
  • Substrates that can be used to assay N-acetylgalactosamine 4-sulfatase, the enzyme deficient in Maroteaux-Lamy Syndrome, and galactose 6-sulfatase, the enzyme deficient in Morquio A syndrome, are provided herein.
  • the substrates are readily prepared, specific for the enzymes, and tagged for detection in the newborn screening laboratory.
  • Natural substrates for the sulfatases are oligosaccharides containing a sulfate on the terminal sugar. While these oligosaccharides would be selective substrates for the enzymes, they are difficult to prepare on the scale needed for worldwide newborn screening (-10 g/year).
  • aryl sulfatases are readily available but show low specificity between the sulfatase enzymes.
  • Substrate design was based on the terminal sugar of the natural substrates, i.e., ⁇ -acetylgalactosamine 4-sulfate and galactose 6-sulfate. The anomeric carbon of these sugars were coupled to an umberferryl moiety, which could be used for fluorescence assays in laboratories lacking tandem mass spectrometers.
  • a carbon chain with a fragmentable N-iert-butyl carbamate was attached to direct the fragmentation of the parent ion in the mass spectrometer along a single reaction pathway, which increases the sensitivity of the tandem mass spectrometry assay.
  • linker chain lengths were chosen such that the mass of the products and internal standards allow for the assays to be multiplexed in the mass spectrometry analysis. See Example 1 for exemplary preparations of MPS VI and MPS IVA substrates and internal standards.
  • each assay is highly specific and can be used, for example, with the substrates described herein to directly measure the relevant enzyme in a sample.
  • Mass spectrometric methods may be employed in these assays, such as electrospray ionization-tandem mass spectrometry (ESTMS/MS), which offers the capability of assaying products of several enzymes by a single infusion into the mass spectrometer for simultaneous sampling, and may provide more accurate data than fluorometric assays that are presently used. See Example 2 for an exemplary assay regarding MPS VI and Example 3 for an exemplary assay regarding MPS IVA.
  • a triplex assay to detect Fabry, Pompe, and MPS IH lysosomal storage diseases is also provided. These disorders were selected because each may be difficult to recognize clinically, and recent studies have shown enzyme replacement therapy or bone marrow transplantation to improve the natural history of the conditions.
  • the assay for the three related lysosomal enzymes cc-glucosidase, cc-galactosidase A, and cc-L-iduronidase
  • the procedure is adaptable to a newborn screening laboratory. By assaying three enzymes at once, problematic samples due, for example, to insufficient blood in the assay are readily flagged for reanalysis.
  • Example 4 presents an exemplary triplex assay.
  • R j is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or substituted arylalkyl ⁇ . ⁇ ;
  • Xj is hydrogen or a counterion;
  • Y j is -OH or -NHC(0)CH3; and n is an integer from 2 to 12.
  • R j may be unsubstituted alkyl cl _ 6 .
  • R j may be ie/t-butyl.
  • X j may be a counterion.
  • the counterion may be a metal counterion or a non-metal counterion.
  • the counterion is a metal counterion, such as a sodium counterion, a potassium counterion, or a lithium counterion.
  • the metal counterion is a sodium counterion.
  • the counterion may be a non-metal counterion, such as an ammonium counterion.
  • R2 is unsubst
  • R 2 is - OH or -NHC(0)CH3; and m is an integer from 2 to 12.
  • R 2 may be unsubstituted alkyl ⁇ j . 5.
  • R 2 may be ie/t-butyl.
  • m 2-6.
  • m 6.
  • R j is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 _i 2 ; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or substituted arylalkyl ⁇ . ⁇ ;
  • Xj is hydrogen or a counterion;
  • Y j is -OH or -NHC(0)CH 3 ; and
  • n is an integer from 2 to 12; with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an N-acetylgalactosamine 6-sulfatase product when the sample comprises N-acetylgalactosamine 6-sulfatase.
  • the sample is a blood sample, such as a dried blood sample from a newborn screening card.
  • a method may further comprise using the amount of the N- acetylgalactosamine 6-sulfatase product to determine whether the dried blood sample is from a candidate for treatment for Mucopolysaccharidosis IVA (Morquio Syndrome Type A).
  • a sample may further comprise an ⁇ -acetylgalactosamine 6-sulfatase internal standard.
  • the ⁇ -acetylgalactosamine 6-sulfatase internal standard is a compound of formul
  • R 2 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or; substituted arylalkyl ⁇ . ⁇ ;
  • Y 2 is - OH or -NHC(0)CH3; and
  • m is an integer from 2 to 12, wherein n ⁇ m. Any of the R 2 , Y2, and m descriptions provided herein for formula (II) are provided as well.
  • a method may further comprise extracting the enzyme product solution to provide the N- acetylgalactosamine 6-sulfatase internal standard and the ⁇ -acetylgalactosamine 6- sulfatase product when the sample comprises ⁇ -acetylgalactosamine 6-sulfatase.
  • extracting the enzyme product solution comprises liquid extraction with an organic solution. Extracting the enzyme product solution may comprise solid phase extraction.
  • a method may further comprise quenching the enzymatic reaction prior to extraction.
  • a method may further comprise determining the quantity of the N- acetylgalactosamine 6-sulfatase product.
  • determining the quantity of the ⁇ -acetylgalactosamine 6-sulfatase product comprises determining the ratio of the ⁇ -acetylgalactosamine 6-sulfatase product to the ⁇ -acetylgalactosamine 6-sulfatase internal standard by mass spectrometric analysis. In some embodiments, determining the quantity of the N-acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis.
  • determining the quantity of the N- acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions. In some embodiments, determining the quantity of the N- acetylgalactosamine 6-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ⁇ -acetylgalactosamine 6-sulfatase product.
  • the invention provides enzyme assays.
  • the assays are qualitative assays that determine the presence or absence of one or more particular enzymes in a sample by measuring the presence or absence of an enzyme product.
  • the assays are quantitative assays that determine the quantity of one or more particular enzymes by measuring the quantity of an enzyme product.
  • the quantity of the one or more enzymes is determined through the use of standards.
  • the standards are internal standards and the amount of enzyme is determined directly by comparing the signal from the standard to the signal associated with the enzyme product.
  • the standards are external to the sample and quantitation of the enzyme product is determined by, for example, creating a standard curve and comparing the signal associated with the enzyme product to the standard curve.
  • Qualitative assays that determine the presence or absence of an enzyme product do not require the use of standards.
  • a method for determining the presence or absence of N- acetylgalactosamine 6-sulfatase enzymatic activity in a sample comprising: (a) incubating an N- acetylgalactosamine 6-sulfatase substrate with sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 6-sulfatase product when the sample comprises N-acetylgalactosamine 6-sulfatase; and (b) determining the presence or absence of the ⁇ -acetylgalactosamine 6-sulfatase product, wherein the presence of N- acetylgalactosamine 6-sulfatase product indicates the presence of ⁇ -acetylgalactosamine 6-sulfatase enzymatic activity, and wherein the absence of ⁇ -acetylgalactosamine 6- sulfatas
  • the sample further comprises an N-acetylgalactosamine 6-sulfatase internal standard.
  • a method may further comprise extracting the enzyme product solution to provide the ⁇ -acetylgalactosamine 6-sulfatase product and the N- acetylgalactosamine 6-sulfatase internal standard when the sample comprises N- acetylgalactosamine 6-sulfatase.
  • a method may further comprise determining the quantity of the N- acetylgalactosamine 6-sulfatase product.
  • determining the quantity of the ⁇ -acetylgalactosamine 6-sulfatase product comprises determining the ratio of the ⁇ -acetylgalactosamine 6-sulfatase product to the N- acetylgalactosamine 6-sulfatase internal standard by mass spectrometric analysis. In some embodiments, determining the quantity of the ⁇ -acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis.
  • determining the quantity of the ⁇ -acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions. In some embodiments, determining the quantity of the ⁇ -acetylgalactosamine 6-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ⁇ -acetylgalactosamine 6-sulfatase product.
  • a sample may be a blood sample, such as a dried blood sample from a newborn.
  • a method may further comprise contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution.
  • a method may further comprise using the amount of ⁇ -acetylgalactosamine 6-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis IVA (Morquio Syndrome Type A).
  • a method for determining the quantity of an N- acetylgalactosamine 6-sulfatase product in a blood sample comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating (i) an N- acetylgalactosamine 6-sulfatase substrate of formula (I):
  • R j is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or substituted arylalkyl ⁇ . ⁇ ;
  • Xj is hydrogen or a counterion;
  • Y j is -OH or -NHC(0)CH3; and
  • n is an integer from 2 to 12; and (ii) an ⁇ -acetylgalactosamine 6-sulfatase internal standard, with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 6-sulfatase product when the sample comprises N- acetylgalactosamine 6-sulfatase; and (c) determining the quantity of the ⁇ -acetyl
  • the ⁇ -acetylgalactosamine 6-sulfatase internal standard may be a compound of formula (II):
  • R 2 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or; substituted arylalkyl ⁇ . ⁇ ;
  • Y 2 is - OH or -NHC(0)CH3; and
  • m is an integer from 2 to 12, wherein n ⁇ m. Any of the R 2 , Y2, and m descriptions provided herein for formula (II) are provided as well.
  • a method may further comprise extracting the enzyme product solution to provide the N- acetylgalactosamine 6-sulfatase internal standard and the ⁇ -acetylgalactosamine 6- sulfatase product when the sample comprises ⁇ -acetylgalactosamine 6-sulfatase.
  • Step (c) may comprise: (i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and (ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ⁇ -acetylgalactosamine 6-sulfatase product.
  • a blood sample may be a dried blood sample from a newborn.
  • Step (a) may be further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution.
  • a method may further comprise using the amount of ⁇ -acetylgalactosamine 6-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis IVA (Morquio Syndrome Type A).
  • R 3 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl C5 . j 2; unsubstituted arylalkyl C6 . j 4; or; substituted arylalkyl C6 . j 4;
  • X 2 is hydrogen or a counterion; and p is an integer from 2 to 12.
  • R3 may be unsubstituted alkyl cl .g.
  • R 3 may be ie/t-butyl.
  • X 2 may be a counterion.
  • the counterion may be a metal counterion or a non-metal counterion.
  • the counterion is a metal counterion, such as a sodium counterion, a potassium counterion, or a lithium counterion.
  • the metal counterion is a sodium counterion.
  • the counterion may be a non-metal counterion, such as an ammonium counterion.
  • R4 is unsubst
  • kits are also provided.
  • R 3 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or; substituted arylalkyl ⁇ . ⁇ ;
  • X2 is hydrogen or a counterion; and
  • p is an integer from 2 to 12; and with a sample for a predetermined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 4-sulfatase product when the sample comprises N-acetylgalactosamine 4-sulfatase.
  • the sample is a blood sample.
  • the sample is a dried blood sample from a newborn screening card.
  • a method may further comprise using the amount of the N- acetylgalactosamine 4-sulfatase product to determine whether the dried blood sample is from a candidate for treatment for Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome).
  • a sample may further comprise an ⁇ -acetylgalactosamine 4-sulfatase internal standard.
  • the ⁇ -acetylgalactosamine 4-sulfatase internal standard may be a compound of formula (IV):
  • R 4 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or; substituted arylalkyl ⁇ . ⁇ ; and q is an integer from 2 to 12, and wherein p ⁇ q. Any of the R 4 and q descriptions provided herein for formula (IV) are provided as well.
  • a method may further comprise extracting the enzyme product solution to provide the N- acetylgalactosamine 4-sulfatase internal standard and the N-acetylgalactosamine 4-sulfatase product when the sample comprises ⁇ -acetylgalactosamine 4-sulfatase.
  • Extracting the enzyme product solution may comprise liquid extraction with an organic solution.
  • extracting the enzyme product solution comprises solid phase extraction.
  • a method may further comprise quenching the enzymatic reaction prior to extraction.
  • a method may further comprise determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product.
  • determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product comprises determining the ratio of the ⁇ -acetylgalactosamine 4-sulfatase product to the ⁇ -acetylgalactosamine 4-sulfatase internal standard by mass spectrometric analysis. In some embodiments, determining the quantity of the N- acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis.
  • determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions. In some embodiments, determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ⁇ -acetylgalactosamine 4-sulfatase product.
  • Also provided is a method for determining the presence or absence of N- acetylgalactosamine 4-sulfatase enzymatic activity in a sample comprising: (a) incubating an N- acetylgalactosamine 4-sulfatase substrate with sample for a pre-determined time sufficient to effect enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 4-sulfatase product when the sample comprises ⁇ -acetylgalactosamine 4-sulfatase; and (b) determining the presence or absence of the ⁇ -acetylgalactosamine 4-sulfatase product, wherein the presence of N- acetylgalactosamine 4-sulfatase product indicates the presence of ⁇ -acetylgalactosamine 4-sulfatase enzymatic activity, and wherein the absence of ⁇ -acetylgalactosamine 4- sulfatas
  • the sample further comprises an N-acetylgalactosamine 4-sulfatase internal standard.
  • a method may further comprise extracting the enzyme product solution to provide the ⁇ -acetylgalactosamine 4-sulfatase product and the N- acetylgalactosamine 4-sulfatase internal standard when the sample comprises N- acetylgalactosamine 4-sulfatase.
  • a method may further comprise determining the quantity of the N-acetylgalactosamine 4-sulfatase product.
  • determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product comprises determining the ratio of the ⁇ -acetylgalactosamine 4-sulfatase product to the N- acetylgalactosamine 4-sulfatase internal standard by mass spectrometric analysis. In some embodiments, determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis.
  • determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions. In some embodiments, determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ⁇ -acetylgalactosamine 4-sulfatase product.
  • a sample may be a blood sample, such as a dried blood sample from a newborn.
  • a method may further comprise contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution.
  • a method may further comprise using the amount of ⁇ -acetylgalactosamine 4-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome).
  • a method for determining the quantity of an N- acetylgalactosamine 4-sulfatase product in a blood sample comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating (i) an N- acetylgalactosamine 4-sulfatase substrate of formula (III):
  • R 3 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl C5 . j 2; unsubstituted arylalkyl C6 . j 4; or; substituted arylalkyl C6 .
  • X 2 is hydrogen or a counterion; and p is an integer from 2 to 12; and (ii) an N- acetylgalactosamine 4-sulfatase internal standard, with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -acetylgalactosamine 4-sulfatase product when the sample comprises ⁇ -acetylgalactosamine 4-sulfatase; and (c) determining the quantity of the ⁇ -acetylgalactosamine 4-sulfatase product by tandem mass spectrometric analysis.
  • Any of the R3, X 2 , and p descriptions provided herein for formula (III) are provided as well.
  • the ⁇ -acetylgalactosamine 4-sulfatase internal standard may be a compound of formula (IV):
  • R 4 is unsubstituted alkyl cl _ 6 ; substituted alkyl cl _ 6 ; unsubstituted aryl C5 . j 2; substituted aryl ⁇ . ⁇ ; unsubstituted arylalkyl ⁇ . ⁇ ; or; substituted arylalkyl ⁇ . ⁇ ; and q is an integer from 2 to 12, and wherein p ⁇ q. Any of the R 4 and q descriptions provided herein for formula (IV) are provided as well.
  • a method may further comprise extracting the enzyme product solution to provide the ⁇ -acetylgalactosamine 4-sulfatase internal standard and the N-acetylgalactosamine 4-sulfatase product when the sample comprises N-acetylgalactosamine 4-sulfatase.
  • (c) comprises: (i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and (ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the N-acetylgalactosamine 4-sulfatase product.
  • the blood sample is a dried blood sample from a newborn.
  • (a) is further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution.
  • a method may further comprise using the amount of ⁇ -acetylgalactosamine 4-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome).
  • a method for providing a solution comprising an a-glucosidase product, an a-galactosidase product, and an a-L-iduronidase product comprising incubating an a-glucosidase substrate, an a-galactosidase substrate, and an a-L- iduronidase substrate with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an a- glucosidase product, an ⁇ -galactosidase product, and an ⁇ -L-iduronidase product when the sample comprises ⁇ -glucosidase, ⁇ -galactosidase, and ⁇ -L-iduronidase, wherein the buffer comprises an aqueous solution of acarbose.
  • the buffer has a pH range of 2-7. In some embodiments, the buffer has a pH range of 3.5-4.5. In some embodiments, the buffer has a pH of 4.4.
  • the buffer may comprise, for example, formate, acetate, citrate-phosphate, or trifluoroacetate, or a combination thereof. In some embodiments, the concentration of formate, acetate, citrate-phosphate, or trifluoroacetate ranges from 0.01-1.0 M. In some embodiments, the concentration of formate, acetate, citrate-phosphate, or trifluoroacetate, or combination thereof, is 0.1 M.
  • the buffer comprises ammonium formate, ammonium acetate, ammonium citrate-phosphate, ammonium trifluoroacetate, sodium formate, sodium acetate, sodium citrate-phosphate, or sodium trifluoroacetate, or a combination thereof.
  • the buffer comprises ammonium formate or sodium formate.
  • the buffer is a sodium buffer or an ammonium buffer.
  • the buffer may be a volatile buffer.
  • a "volatile buffer” refers to a buffer that, when concentrated to dryness by evaporation, causes the buffer components to evaporate along with the solvent.
  • a volatile buffer may comprise ammonium formate, for example. Other suitable volatile buffers are known in the art.
  • the sample is a blood sample. In some embodiments, the sample is a dried blood sample from a newborn screening card. A method may further comprise using the amount of a- glucosidase product to determine whether the dried blood sample is from a candidate for treatment for Pompe disease. A method may further comprise using the amount of a- galactosidase A product to determine whether the dried blood sample is from a candidate for treatment for Fabry disease. A method may further comprise using the amount of a- L-iduronidase product to determine whether the dried blood sample is from a candidate for treatment for mucopolysaccharidosis I (Hurler) disease (MPS IH).
  • Hurler mucopolysaccharidosis I
  • a method may further comprise using the amount of a-glucosidase product to determine whether the dried blood sample is from a candidate for treatment for Pompe disease, using the amount of a-galactosidase A product to determine whether the dried blood sample is from a candidate for treatment for Fabry disease, and using the amount of a-L-iduronidase product to determine whether the dried blood sample is from a candidate for treatment for mucopolysaccharidosis I (Hurler) disease (MPS IH).
  • a sample may further comprise an ⁇ -glucosidase internal standard, an ⁇ -galactosidase internal standard, and an a-L- iduronidase internal standard.
  • a method may further comprise extracting the enzyme product solution to provide the ⁇ -glucosidase internal standard, the a-glucosidase product, the ⁇ -galactosidase internal standard, the ⁇ -galactosidase product, the a-L- iduronidase internal standard, and the ⁇ -L-iduronidase product when the sample comprises ⁇ -glucosidase, ⁇ -galactosidase, and ⁇ -L-iduronidase.
  • extracting the enzyme product solution comprises liquid extraction with an organic solution.
  • extracting the enzyme product solution comprises solid phase extraction.
  • a method may further comprise quenching the enzymatic reaction prior to extraction.
  • the aqueous solution of acarbose has an acarbose concentration of 5-10 ⁇ .
  • a method may further comprise determining the quantity of the ⁇ -glucosidase product, the ⁇ -galactosidase product, and the ⁇ -L-iduronidase product.
  • determining the quantity of the ⁇ -glucosidase product, the a- galactosidase product, and the ⁇ -L-iduronidase product comprises determining the ratio of the ⁇ -glucosidase product, the ⁇ -galactosidase product, and the a-L-iduronidase product to the ⁇ -glucosidase internal standard, the ⁇ -galactosidase internal standard, and the ⁇ -L-iduronidase internal standard, respectively, by mass spectrometric analysis.
  • determining the quantity of the ⁇ -glucosidase product, the a- galactosidase product, and the ⁇ -L-iduronidase product comprises tandem mass spectrometric analysis. In some embodiments, determining the quantity of the a- glucosidase product, the a-galactosidase product, and the a-L-iduronidase product comprises tandem mass spectrometric analysis in which the parent ions of the products and internal standards are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, in some embodiments, determining the quantity of the ⁇ -glucosidase product, the a- galactosidase product, and the ⁇ -L-iduronidase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ⁇ -glucosidase product, the a-galactosidase product, and the
  • a method for determining the presence or absence of a- glucosidase enzymatic activity, ⁇ -galactosidase enzymatic activity, and a-L-iduronidase enzymatic activity in a sample comprising: (a) incubating an ⁇ -glucosidase substrate, an ⁇ -galactosidase substrate, an ⁇ -L-iduronidase substrate with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -glucosidase product, an ⁇ -galactosidase product, and an ⁇ -L-iduronidase product when the sample comprises ⁇ -glucosidase, ⁇ -galactosidase, and ⁇ -L-iduronidase; and (b) determining the presence or absence of the a-glucosidase product, the ⁇ -galactosidase product, and the
  • a sample may further comprise an ⁇ -glucosidase internal standard, an ⁇ -galactosidase internal standard, and an ⁇ -L-iduronidase internal standard.
  • a method may further comprise extracting the enzyme product solution to provide the a- glucosidase product, the ⁇ -glucosidase internal standard, the ⁇ -galactosidase product, the ⁇ -galactosidase internal standard, the ⁇ -L-iduronidase product, and the a-L-iduronidase internal standard when the sample comprises ⁇ -glucosidase, ⁇ -galactosidase, and a-L- iduronidase.
  • a method may further comprise determining the quantity of the a- glucosidase product, the ⁇ -galactosidase product, and the ⁇ -L-iduronidase product. Also provided is a method for determining the quantity of a a-glucosidase product, a a-galactosidase product, and a a-L-iduronidase product in a blood sample, comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating an a-glucosidase substrate, an a-glucosidase internal standard, an ⁇ -galactosidase substrate, an ⁇ -galactosidase internal standard, an a-L-iduronidase substrate, and an ⁇ -L-iduronidase internal standard with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ⁇ -
  • a method may further comprise extracting the enzyme product solution with an organic solvent to provide an organic phase comprising the ⁇ -glucosidase product, the a-glucosidase internal standard, the ⁇ -galactosidase product, the ⁇ -galactosidase internal standard, the ⁇ -L-iduronidase product, and the ⁇ -L-iduronidase internal standard when the sample comprises ⁇ -glucosidase, ⁇ -galactosidase, and ⁇ -L-iduronidase.
  • (c) comprises: (i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and (ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ⁇ -acetylgalactosamine 6-sulfatase product.
  • the blood sample is a dried blood sample from a newborn.
  • (a) is further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution.
  • a method may further comprise: (e) using the amount of ⁇ -glucosidase product to predict whether the newborn is a candidate for treatment of Pompe disease; (f) using the amount of ⁇ -galactosidase product to predict whether the newborn is a candidate for treatment of Fabry disease; or (g) using the amount of a-L- iduronidase product to predict whether the newborn is a candidate for treatment of mucopolysaccharidosis I (Hurler) disease (MPS IH).
  • a method may further comprise: (e) using the amount of ⁇ -glucosidase product to predict whether the newborn is a candidate for treatment of Pompe disease; (f) using the amount of ⁇ -galactosidase product to predict whether the newborn is a candidate for treatment of Fabry disease; and (g) using the amount of a-L-iduronidase product to predict whether the newborn is a candidate for treatment of mucopolysaccharidosis I (Hurler) disease (MPS IH).
  • any embodiment herein may recite “consisting of.”
  • the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim.
  • any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
  • any composition of the invention may be used in any method or system of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • Example 1 Preparation of Maroteaux-Lamy (MPS VI) and Morquio A (MPS rVA) Substrates and Internal Standards and Related Enzymatic Specificity Assays Structures of Maroteaux-Lamy substrate 1 and internal standard 3 and Morquio A substrate 2 and internal standard 4 are shown in FIGURE 1. Preparation of MPS ⁇ substrate 2 and internal standards 3 and 4 is shown in FIGURE 2. Preparation of MPS VI substrate 1 is shown in FIGURE 3.
  • the MPS IVA substrate 2 was synthesized from glycoside 10 by selective sulfation of the primary 6-hydroxyl group over the secondary 2, 3 and 4-hydroxyl groups as revealed by the downfield of H-6 in the sulfate relative to the non-sulfate (Sawada et al., Carbohydr. Res., 340, 1983-96 (2005)).
  • the MPS VI substrate (1) required further synthetic manipulations in order to install the sulfate at the more hindered 4-hydroxy of the sugar. Therefore, the less hindered 3- and 6-hydroxyls were selectively benzoylated to afford dibenzoate 9.
  • the glucosamine was then converted to a galactosamine by inversion of they 4-hydroxyl by formation of the triflate and displacement with sodium nitrite to afford 10. Finally, sulfation of the free hydroxyl followed by cleavage of the benzoate protecting groups gave the desired MPS VI substrate (1).
  • Te/t-butyl 5-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetamido)pentylcarbamate (6b).
  • the reaction mixture was quenched with MeOH (8 mL) and solution passed through a column of DOWEX 50WX8 (Na+ form) and eluted with methanol.
  • the eluent was concentrated under vacuum to an oil which was chromatographed over silica gel in CHCI3 : MeOH : H 2 0 (8 : 5 : 1) to yield the target compound 2 (0.051 g, 0.076 mmol) in 45% yield.
  • the compound was prepared by the analogous procedure described for the synthesis of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(2-(5-(iert-butoxy carbonyl amino)pentyl amino)-2-oxoethyl)-2-oxo-2H-chromen-7-yloxy)tetrahydro-2H-pyran- 3,4,5-triyl tri acetate.
  • glycoside B was obtained by filtration and washed 3X with DCM (20 mL) and ⁇ 3 ⁇ 40 (20 mL) to afford glycoside B as an off-white powder (513 mg, 56%).
  • the layers of the filtrate from the reaction were then saturated with citric acid and extracted with DCM (20 mL, 3X), a fitrate formed during the extraction and was isolated to afford recovered coumarin 6b (286 mg, 28% of starting coumarin recovered).
  • the layers were separated, and the organic layers were evaporated to afford recovered coumarin 6b and product.
  • the residue was taken up in MeOH and filtered.
  • the solid was galactopyranoside (180 mg, 20%: 76% total).
  • Enzymatic Assays for Specificity With the two substrates and two internal standards in hand, enzymatic activity was studied. The enzymatic activity was measured by incubating a solution of substrate and internal standard with a 2 mm diameter dried blood spot punch for 16 hr. The amount of product was quantified by tandem mass spectrometer using the internal standards. For MPS VI, the range of activity measured with 10 dried blood spots from healthy individuals was 1.6-10.5 ⁇ hr 1 (L blood) " 1 compared to 0.08 ⁇ hr 1 (L blood) -1 using a dried blood spot from an MPS VI patient.
  • this method of this Example is an assay of N- acetylgalactosamine 4- sulfatase (aryl sulfatase B) activity in dried blood spots (DBS) for the early detection of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) in newborn screening.
  • DBS dried blood spots
  • Other assays and MPS VI substrates described herein may be employed.
  • aryl sulfatase B present in DBS converted the substrate to a desulfated product which was detected by electrospray tandem mass spectrometry and quantified using a homologous internal standard. Assay and work-up procedures were optimized to be compatible with the work flow in newborn screening laboratories. Analysis of DBS from human newborns showed clear distinction of aryl sulfatase B activity from 89 healthy individuals where it ranged between 1.4-16.9 ⁇ 1/(1 ⁇ x L blood), with an average activity of 7.4 ⁇ /hr/L blood, and an MPS VI patient that had an activity of 0.12 ⁇ 1/(1 ⁇ x L blood).
  • results are also reported for the aryl sulfatase B assay in DBS from groups of normal felines and felines affected with MPS VI.
  • the presence of the umbelliferyl moiety in MPS VI substrates described herein would allow for fluorometric assays should that be desirable by laboratories who desire not to use ESI-MS/MS (see van Diggelen et al., Clin. Chim. Acta, 187: 131-40 (1990)).
  • the substrate (7-0-(2-acetamido-2-deoxy-4-0-sulfonato-P-D- galactopyranosyl) ie/t-butyl 6-(7-hydroxy-coumarin-4-acetamido)hexylcarbamate sodium salt, ASB-S) and internal standard (7-0-(2-acetamido-2-deoxy-P-D-galactopyranosyl) ie/t-butyl 5-(7-hydroxy-coumarin-4-acetamido)pentylcarbamate, ASB-IS) were synthesized as described in Example 1.
  • DBS Dried blood spots
  • IRB Washington State Institutional Review Board
  • Additional DBS from two unaffected adult donors were used to develop the assays and to characterize the enzyme kinetics.
  • DBS from a single affected anonymous donor were obtained from BioMarin Pharmaceutical Inc. (Novato, CA).
  • the MPS VI affected patient had been diagnosed previously with established clinical and biochemical procedures.
  • DBS from normal and affected felines were obtained from BioMarin Pharmaceutical Inc.
  • DBS were kept at ambient temperature during shipment ( ⁇ 10 days) and then stored at -20 °C in Ziploc® plastic bags (one bag sealed inside a second bag). Ziploc® bags were kept in a sealed plastic box containing desiccant (anhydrous CaSC ⁇ granules).
  • the sample was quenched with 100 ⁇ ⁇ of water containing 16 mg of diethylaminoethylcellulose resin (DEAE cellulose, 20 mg, Whatman, Cat. No. 4057-200), leading to a precipitate.
  • a blank assay was carried out as above but using a blank paper punch instead of a DBS.
  • Samples were submitted to liquid-liquid extraction with the addition of 400 ⁇ ⁇ ethyl acetate.
  • the solutions were mixed by aspiration with a 12-channel pipette (lOx), then centrifuged at 3000 rpm for 5 min to separate the layers. 300 ⁇ ⁇ of the top layer were transferred to a new 96-well plate (0.5 mL, Axygen Scientific, VWR International, Cat. No. 47743-982).
  • the ethyl acetate was removed under a stream of air, and the sample was reconstituted in 100 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2 % formic acid
  • the ion exchange resin was washed with methanol (2 x 0.5 mL).
  • a slurry of C-18 resin (20 mg, Aldrich, octadecyl-functionalized silica gel, Cat. No. 377635) in methanol (250 ⁇ ) was pipetted on top of the ion exchange resin in each well.
  • the 2 resin layers were washed with methanol (2 x 0.5 mL) to help it settle, then with de-ionized water (2 x 0.5 mL).
  • the sample was slowly loaded onto the column and washed with de-ionized water (2 x 0.5 mL) to remove salts.
  • ESI-MS/MS analysis was performed on a Waters Quattro Micro tandem quadrupole instrument using flow injection and selected reaction monitoring in positive ion mode. Samples were injected manually using a syringe, except for the samples using the liquid-liquid extraction protocol that were injected using an autosampler. Twenty five ⁇ L ⁇ of the 100 ⁇ ⁇ sample was injected using a Waters 2777C Sample Manager via flow injection 80/20 acetonitrile/water with 0.2% formic acid with a flow-rate of 0.1 mL/min for 1 min then 1 mL/min for 0.5 min. Data was collected during 1.5 minute of infusion, and the signal returned to the background level before the next injection.
  • the ion dissociations used for selected reaction monitoring were m/z 608.3 ⁇ m/z 508.6 and m/z 622.3 ⁇ m/z 522.6 for the internal standard and product, respectively.
  • Other ESI-MS/MS conditions were as follows: electrospray capillary voltage: 4500 V, extractor: 3 V, desolvation temperature: 350 °C, desolvation gas flow: 600 L/h, collision cell pressure: 2.23 10-3 millibar.
  • the cone voltage and ion laboratory collision energy were 15 V and 13 eV, respectively for both m/z 622.3 ⁇ m/z 522.6 and m/z 608.3 ⁇ m/z 508.6 transitions.
  • the dwell time was 100 ms with a 20 ms delay.
  • the amount of ASB product was then determined from the ion intensity ratio of the product to internal standard and converted to ASB activity ( ⁇ 1/(1 ⁇ x L of blood)) using the incubation time and blood volume in the DBS punch.
  • FIGURE 4 The strategy for the MPS VI assay using tandem mass spectrometry is outlined in FIGURE 4.
  • the enzyme in DBS is incubated with ASB-S to catalyze specific hydrolysis in the N-acetylgalactosamine-4-O-sulfate moiety yielding ASB-P.
  • the product is ionized by protonation in electrospray to produce the m/z 622 precursor ion, (M + H) + , which is selected by mass and subjected to collision induced dissociation (CID) forming the m/z 522 product ion.
  • CID collision induced dissociation
  • CID is steered into one predominant product ion channel by the iert-butylcarbamido ( ⁇ -BOC) group that undergoes facile elimination of isobutene and carbon dioxide.
  • the internal standard (ABS-IS) is a lower homologue of ASB-P from which it differs in the length of the diamine carbon chain which has six methylene groups in ASB-P and five methylene groups in ASB-IS.
  • CID of the ASB-IS (M + H)+ ion forms an m/z 508 fragment ion which is homologous with the m/z 522 fragment from ASB-P.
  • the ASB-P and ASB-IS structures were designed such that the m/z values of both precursor and fragment ions were distinct from those of substrates and internal standards used in ESTMS/MS assays of other lysosomal enzymes.
  • the ASB assay can be multiplexed with any other previously developed EST MS/MS assays.
  • R P /R IS The relative response in ESI-MS/MS to ASB-P and ASB-IS concentrations in the sample, R P /R IS , depends on the partition coefficients in extraction from the assay buffer, electrospray ionization efficiency, and propensity for fragmentation by elimination of isobutene and CO2.
  • This range of ASB-P concentrations corresponds to 10-500 pmol of enzymatic product formed in an assay, which is consistent with the range of enzyme activities found in DBS.
  • Assays of lysosomal enzymes are typically conducted at low pH 3.5-5.3 and under conditions achieving low substrate conversions ( ⁇ 10 ) to keep the enzyme kinetics in the initial pseudo-linear stage and also to avoid enzyme inhibition by the products.
  • Sulfatases in particular, are inhibited by free sulfate ions present in the sample or produced by enzymatic hydrolysis.
  • ASB showed no activity toward ASB-S, but full activity was recovered at lead concentrations >20 mM.
  • Lead formate and acetate showed the same effect, and so 30 mM lead(II) formate was used in all ASB assays.
  • MS/MS-based assays One of the advantages of MS/MS-based assays is the capacity for multiplexing analysis of several enzyme reactions in a single injection into the mass spectrometer. For practical purposes of work time constraints and sample throughput in newborn screening laboratories, it is also desirable to carry out the incubation of several enzymes in one common buffer. Hence, effects of pH on the enzyme activity need to be studied to find a pH range where several enzymes might have sufficient activity.
  • the ASB activity showed a three-fold increase from pH 3.4 to pH 4.0 and then the dependence flattened at pH 4-4.5.
  • the other parameters of the ASB assay were studied under the optimized conditions of pH 4 and 30 mM lead concentration.
  • the amount of ASB-generated product in DBS increased as a linear function of incubation time between 5 and 30 h. This indicated that the assay conditions were such that the enzyme kinetics was in the initial stage.
  • the incubation time was set to 16 h which is compatible with the work schedule in newborn screening laboratories.
  • Michaelis-Menten parameters were measured as shown in FIGURE 5.
  • a substrate concentration of 1 mM was used to saturate the enzyme in an effort to minimize the effect of potential competitive inhibitors present in blood.
  • the amount of product formed increased with the size of the DBS punch, with a plateau at higher blood amounts, presumably due to an increase in endogenous inhibitors.
  • a 3 mm DBS punch was used to be compatible with the protocols used in newborn screening laboratories.
  • the DBS from a previously identified MPS VI patient gave a value of 0.12 ⁇ 1/(1 ⁇ x L blood), which was very close to the activity measured with the method using solid phase extraction (Table 1).
  • Quality control DBS provided by the Centers for Disease Control and Prevention were also analyzed for ASB activity. These samples are prepared from fully and partially depleted as well as standard blood termed QC base, low, medium, and high.
  • the respective ASB activities measured in those samples by the assay were 0.20, 0.65, 3.4, and 8.8 ⁇ 1/(1 ⁇ x L blood). All values are blank subtracted using the measured activity of assay cocktail incubated without a dried blood spot. Individual values for all samples are given in Table 2.
  • Assay precision was calculated using triplicate analyses of DBS from a healthy control.
  • Thirty random newborn NBS were assayed by omitting ASB-S and only background levels of ASB-P were found in all samples showing that DBS do not contain substances that interfere with the ASB assay.
  • the new ESTMS/MS assay of aryl sulfatase B activity in dried blood spots from humans and felines unambiguously distinguishes healthy individuals from affected ones.
  • the assay uses a small amount of synthetic material (14.5 ⁇ g of substrate and 61 ng of internal standard per assay) and shows good linearity and inter-assay reproducibility.
  • the work-up procedure using solid-phase extraction of the product and internal standard generates virtually zero background but requires several liquid transfers which is acceptable for a research laboratory.
  • the work-up procedure using liquid-liquid extraction of the product and internal standard provides very low background and, due to a minimum of liquid transfer steps, is suitable for large throughput analysis such as those performed in newborn screening laboratories.
  • Example 3 Assay of MPS IV A (Morquio A Syndrome) The following Example is similar to the methodology presented in Example 2. Briefly, a highly specific enzyme activity assay for MPS IVA using ESI-MS/MS is presented that uses a novel substrate and directly measures the enzyme activity of N- acetylgalactosamine 6-sulfatase (GALNS) in rehydrated dried blood spots (DBS). Other assays and sMPS IVA substrates described herein may be employed. The assay is relatively simple to execute and may be suitable for the eventual screening of MPS IVA by newborn screening laboratories.
  • GALNS N- acetylgalactosamine 6-sulfatase
  • DBS rehydrated dried blood spots
  • GALNS-S consists of an umbelliferyl- -D-galactose with a sulfate group at the 6- position of the sugar.
  • a hydrophobic five-carbon linker with a terminal i-butylcarbamate was incorporated at the 4-position of the umbelliferyl unit to facilitate the post-assay purification and increase the sensitivity of the ESTMS/MS signal (due to a dominant fragmentation pathway involving cleavage of the i-butylcarbamate) (FIGURE 7).
  • GALNS-IS The internal standard
  • ESI-MS/MS enables separate detection and quantification of GALNS-P and GALNS-IS by their fragment ions, after collision induced dissociation of their parent ions (FIGURE 7A).
  • the presence of the umbelliferyl moiety in GALNS-S would allow for fluorometric assay of GALNS should that be desirable by laboratories who desire not to use ESI- MS/MS (see van Diggelen et al., Clin. Chim. Acta, 187: 131-40 (1990)).
  • a single 2-mm diameter DBS punch (containing approximately 1.6 ⁇ ⁇ of blood) was obtained with a leather punch and was placed in a 0.6 mL Eppendorf tube.
  • a slurry of CI 8 silica gel (20 mg, Aldrich, octadecyl-functionalized silica gel cat. #377635) in methanol (300 ⁇ ) was pipetted on top of the ion exchange resin in each well.
  • the dual layer was sequentially washed with methanol (2 x 0.5 mL) and then with de-ionized water (2 x 1 mL).
  • the quenched sample was centrifuged for 5 min, and the supernatant was loaded onto the column and washed with de-ionized water (2 x 1 mL) to remove salts.
  • GALNS-P and GALNS-IS were eluted with methanol (2 x 0.5 mL) into a deep well plate (Neptune, cat. # 2405). The methanol was removed in a Speed- Vac, and the residue was reconstituted in 30 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2% formic acid.
  • the assay sample was quenched by adding a suspension of 64 mg DEAE-cellulose (DE52, Whatman Cat. 4057-200, pre-swollen) in 250 ⁇ ⁇ water. Ethyl acetate (500 ⁇ ) was added to extract GALNS-P and GALNS-IS. After mixing on a vortexer (-15 sec) the sample was centrifuged for 5 min. The ethyl acetate portion (300 ⁇ ) was transferred to a new Eppendorf tube, solvent removed with a stream of N2, and the residue reconstituted in 30 ⁇ L ⁇ of 80/20 acetonitrile/water with 0.2% formic acid.
  • DEAE-cellulose DE52, Whatman Cat. 4057-200, pre-swollen
  • the post-assay purification is used because the buffer salts present in high concentrations will interfere with the electrospray ionization, and the unreacted sulfated substrate can dissociate in the source of the mass spectrometer forming GALNS-P ions, and thus giving rise to false positive GALNS activity.
  • the buffer salts are removed with water whilst the hydrophobic GALNS-S, GALNS-IS and GALNS-P are retained on the C18 resin.
  • the anionic GALNS-S is retained on the DEAE-cellulose, and GALNS-P and GALNS-IS pass through.
  • GALNS-S is captured on the DEAE-cellulose, and GALNS-IS and GALNS-P are extracted into ethyl acetate.
  • ESI-MS/MS analysis was carried out on a Waters Quattro Micro tandem quadrupole instrument operating in positive-ion, multiple-reaction-monitoring mode.
  • MassLynx 4.1 Data acquisition was carried out with MassLynx 4.1 software with the following settings: capillary voltage, 3500 V; cone voltage, 80 V; extractor, 2 V; RF, 0.0 V; source temperature, 80 °C; desolvation temperature, 250 °C; cone gas flow, 30 L/h; desolvation gas flow, 550 L/h; collision gas flow, 0.20 mL/min; LM 1 resolution, 15; HM 1 resolution, 15; ion energy 1, 0.2; MS/MS mode entrance, 15; MS/MS collision energy, 30 eV (Gal-6S-P) and 30 eV (Gal-6S-IS); MS/MS mode exit, 15; LM 2 resolution, 15.0; HM 2 resolution, 15.0; ion energy 2, 2.0; Multiplier, 650; collision cell pressure, ⁇ 10 ⁇ 4 mbar; collision gas, argon. Multiple-reaction-monitoring mode was used for m/z 589.2 — > 489.1 and 603.2
  • the sample (10 ⁇ ⁇ of the 30 ⁇ ⁇ sample in 80/20 acetonitrile/ water with 0.2% formic acid) was injected into the mass spectrometer with a flow-rate of 0.1 mL/min. Data was collected during 1 minute of infusion, and after 1 min, the MS/MS signal has returned to the background level. The amount of product was calculated from the ion abundance ratio of product to internal standard, minus that from a minus DBS blank control, multiplied by the amount of added internal standard. Enzymatic activity was calculated from the amount of product divided by the incubation time and the volume of blood (1.6 ⁇ ⁇ of blood in a 2 mm DBS punch).
  • the parent ions for GALNS-P and GALNS-IS were each isolated by mass and subjected to collision-induced dissociation.
  • the amount of GALNS-P was calculated by comparing the ion peak intensity of GALNS-P to that of GALNS-IS.
  • the signal generated from the minus blood blank assay was -1 % of that seen with the complete assay.
  • Assay optimization depicted maximum GALNS activity at pH 4.0 in formate buffer. Sulfate and phosphate ions are competitive inhibitors of sulfatases. Therefore, lead(II) formate (30 mM) was used to suppress these inhibitors.
  • the amount of GALNS- P increases approximately linearly with reaction time from 0-30 h. The incubation time of 16 h was chosen in order to allow for overnight incubation to simplify work schedules of laboratories.
  • the amount of GALNS-P decreases when the size of the DBS punch is increased from 2 to 4 mm presumably due to the presence of endogenous inhibitors in the DBS. Therefore, a 2 mm DBS punch was chosen for the assays.
  • a control assay containing all assay components except GALNS-S was performed with 30 different DBS, and the signal was -1% of that measured with the complete assay. This rules out any ESI-MS/MS signal coming from sources other than GALNS action.
  • Enzyme stability studies showed that GALNS is stable in DBS maintained at -20 °C for at least 1 yr, but about 50% of the activity is lost at 37 °C over 3 days.
  • a tandem mass spectrometry assay is provided in this Example in which the enzymatic activities of three lysosomal enzymes, cc-glucosidase (GAA), cc-galactosidase A (GLA), and ⁇ -L-iduronidase (IDUA), were quantified in dried blood spots using a single assay buffer with minimal workup.
  • GAA cc-glucosidase
  • GLA cc-galactosidase A
  • IDUA ⁇ -L-iduronidase
  • GLA-IS GLA internal standard
  • GLA-S GLA substrate
  • GAA-IS GAA internal standard
  • GAA-S GAA substrate
  • DBS institutional review board
  • Fabry, Pompe, and MPS IH were obtained from Genzyme Corp. or from the inventors' clinical program as anonymous samples, in compliance with IRB requirements.
  • the Fabry samples were from affected males only; the Pompe samples were from both infantile and late-onset clinical forms; and the MPS IH samples were from patients with early childhood presentations (Hurler).
  • DBS were obtained as anonymous samples from the Washington state newborn screening laboratory. DBS were obtained from birthing centers and kept at ambient temperature during shipment ( ⁇ 10 days).
  • DBS For assays carried out in the Washington state newborn screening laboratory, DBS were used after all routine testing was performed (i.e., leftover DBS) and were up to ⁇ 6 months old and kept at ambient laboratory temperature. Triplex assay. The following experimental details were carried out in the Washington state newborn screening laboratory. Experimental details for assays carried out during the optimization phase of the project at the University of Washington are presented in Example 5.
  • the final assay cocktail contains 0.48 mM IDUA-S, 3.1 ⁇ IDUA-IS, 0.2 mM GAA-S, 2.0 ⁇ GAA-IS, 0.6 mM GLA-S, 1.2 ⁇ GLA-IS, and 8 ⁇ acarbose.
  • Excess assay buffer can be stored at 4 °C and is used the following day without loss of activity.
  • the other four vials of dried and mixed reagents were stored at -20 °C and reconstituted on the day of the assay.
  • Each 96-deep well plate also contained 6 wells with a blank filter paper punch and 2 wells of each of the CDC quality control dried blood spot samples (base, low, medium and high, obtained from Drs. H. Zhou and V. De Jesus at the CDC in Atlanta, stored at -20 °C in a Ziploc® plastic bag).
  • the blanks and QC samples are on the first and last columns of the plate. From top to bottom, there are two blanks, then QC base, QC low, QC medium, QC high, an adult DBS and then another blank. QC samples were manually punched with a l/8th inch whole punch.
  • Assay cocktail (30 ⁇ ) was added to each well in the 96-deep well plate using a Rainin Liquidator 96-tip pipette. The plate was sealed with plate sealing film (VWR, Cat. No. 14230-062) and incubated at 37 °C for 16 h (overnight) with orbital shaking at 150 rpm.
  • the plate was returned to the Rainin Liquidator 96-tip pipette, and 200 ⁇ ⁇ of the top layer (ethyl acetate) was transferred to a new 96-shallow well plate (0.5 mL, Axygen Scientific, VWR International, Cat. No. 47743-982).
  • the ethyl acetate was evaporated using a stream of air (SPE Dry 96 Dual Argonaut sample concentrator system, Biotage) with a flow rate of 40-80 PSI of air and heating ⁇ 35 °C (typically ⁇ 30 min).
  • the residue in the wells was resuspended in mass spectrometry mobile phase (100 ⁇ , 80% acetonitrile: 20% water with 0.2% formic acid) using the Rainin Liquidator 96-tip pipette. After addition of the mobile phase, the plate was mixed for a few minutes in an orbital shaker. The plate was covered with aluminum foil (not sealing foil since acetonitrile can dissolve the glue) and placed in the auto-injector tray for mass spectrometric analysis.
  • ESI-MS/MS ESI-MS/MS analysis was performed on a Waters Acquity TQD
  • Ultra Performance tandem quadrupole mass spectrometer using positive mode multiple reaction monitoring mode and flow injection Ten microliters of the 150 ⁇ ⁇ sample was injected for each analysis at a flow rate of 0.1 mL/min using a Waters 2777C Sample Manager via flow injection with 80/20 acetonitrile/water with 0.2% formic acid for 1.10 min then 0.5 mL/min for 0.4 min (most of the sample elutes during the 1.10 min phase, and the faster flow phase is used to reduce the time of sample clearance in preparation for the next injection).
  • MassLynx 4.1 software was used to record all ion signals. Data was collected during 1.5 minute of infusion, and the signal returned to the background level before the next injection.
  • the mass transitions used for multiple reaction monitoring are IDUA-IS: m/z 377.2 ⁇ m/z 277.1, IDUA-P: m/z 391.2 ⁇ m/z 291.2, GLA-P: m/z 484.2 ⁇ m/z 384.3, GLA-IS: m/z 489.4 ⁇ m/z 389.2, GAA-P: m/z 498.4 ⁇ m/z 398.4, GAA-IS: m/z 503.4 ⁇ m/z 403.4.
  • Other ESI-MS/MS parameters are given in Tables 4A and 4B. All transitions were conducted with the following settings: dwell time, 0.1 s; delay, 0.02 s.
  • Table 4A ESI-MS/MS parameters for newborn screening studies.
  • Table 4B ESI-MS/MS parameters for newborn screening studies.
  • the amount of product was calculated from the ion abundance ratio of the product to the internal standard for the sample minus that of the blank (filter paper only punch, average of 6 blanks was used), multiplied by the amount of added internal standard and divided by the response factor ratio of the product to internal standard.
  • the response factor was determined from a calibration curve obtained from standards containing ratios of product and internal standard from 0.0-5.0 for GAA, GLA (obtained from Drs. H. Zhou and V. De Jesus at the CDC) and IDUA (obtained from Drs. K. Zhang and J. Keutzer at Genzyme Corporation).
  • the enzyme activity in units of ⁇ - ⁇ 1 - ⁇ of blood) -1 was calculated from the measured amount of product assuming the 3 mm DBS punch contains 3.2 ⁇ L of blood.
  • the enzyme activity of IDUA was low in sodium citrate and sodium citrate-phosphate buffers at all pH values, so sodium formate was chosen as the preferred buffer salt.
  • GalNAc N- acetylgalactosamine
  • Blank assays were carried out with the assay cocktail incubated with a filter paper punch that does not contain blood.
  • the GLA enzyme activity in five punches obtained from male patients with Fabry disease were 0.33 - 0.63 ⁇ /h/L of blood with GalNAc and 0.44 - 0.58 without the GalNAc added.
  • the measured activity is from a- galactosidase.
  • Substrate concentration should be high enough to provide enzyme activities with a significant difference between the normal newborn blood with low activity and affected patient blood. Also, the use of substrate concentrations well above the K M minimizes effects of competitive inhibitors that may be present in blood. On the other hand, higher substrate concentrations increase the load on the mass spectrometer and lead to a higher background signal and to higher reagent costs.
  • the assays are conducted at a substrate concentration lower than the apparent K m of the enzymes, so the activity of the enzymes should double when the substrate concentration is doubled. This would be true if DBS samples do not contain competitive inhibitors or if the amount of competitive inhibitors is similar in all samples.
  • the solid-phase extraction step has been eliminated, so there is the potential for non- volatile buffer salts that are extracted into ethyl acetate to build up in the mass spectrometer source and also to suppress the electrospray ionization process (although most of the salts will remain in the aqueous phase).
  • volatile buffers components were evaluated.
  • a concern with eliminating the solid-phase extraction step was that using only the liquid-liquid extraction with ethyl acetate would lead to higher levels of enzyme substrates in the mass spectrometer infusion solvent. Because substrates may fragment to give products in the source of the mass spectrometer, one would expect higher product levels in control reactions.
  • a mixture of the internal standards and substrates for GAA, GLA, and IDUA were prepared in sodium formate buffer and purified without incubation by both liquid-liquid extraction and liquid-liquid extraction plus solid-phase extraction on silica gel.
  • the samples purified by solid-phase extraction had a lower background signal for the products of all three enzymes than those with just the liquid-liquid extraction workup, but the background signal of the samples purified without solid-phase extraction is very small compared to the signal observed from enzyme activity in DBS punches.
  • the amount of in- source fragmentation of the substrate to product was kept to a minimum by carefully tuning the mass spectrometric parameters (cone voltage and collision energy, Tables 4A and 4B) while still maintaining a high signal for the product ion.
  • the DBS punch was placed directly into the well of a 96-deep well plate followed by addition of the assay cocktail. This eliminates the step in which the DBS punch is extracted with buffer prior to assay initiation.
  • the use of the deep well plate allows the liquid-liquid extraction to be carried out in the same well as the incubation. After incubation, quench buffer and ethyl acetate are added directly to the assay well. After extraction, the top ethyl acetate layer is transferred to a 96-shallow well plate, and solvent is removed with a stream of air.
  • the residue is taken up in the mobile phase for infusion into the mass spectrometer.
  • This plate is used directly in the autosampler.
  • the new method uses two 96-well plates and five boxes of pipette tips to assay the activity of three enzymes as opposed to the seven 96-well plates, a 96-well filter plate and eighteen boxes of pipette tips for the same three enzymes in the previous method (Li et al., Clin. Chem. , 50: 1785-1796 (2004)).
  • Blank assays were carried out with the assay cocktail incubated with a filter paper punch that does not contain blood.
  • FIGURE 8 A summary of an exemplary screening protocol using the optimized triplex assay described in the previous section is shown in FIGURE 8.
  • the entire protocol fits within a 48 hr period including data analysis.
  • the analysis of 4 plates containing 320 newborn DBS samples, 40 QC DBS samples and 24 blanks in this 48 hr period requires the items listed in Table 7.
  • Equipment required is a DBS punch machine, a manually operated 96- tip pipette, an incubator for 96-well plates, a centrifuge to spin 96-well plates, a simple solvent evaporation system for 96-well plates, an autosampler, and a ESTMS/MS instrument. Robotics are not used.
  • the table does not include buffer salts, ethyl acetate and 96-well plate sealers, but the cost of these are insignificant.
  • FIGURES 9A, 9B, and 9C show the distribution of GLA, GAA, and IDUA activities, respectively, for 5,990 DBS submitted to the triplex assay.
  • the activity of GLA and GAA is similar to the activity previously documented using citrate-phosphate or acetate buffer.
  • the mean activity of IDUA is approximately 50% less than prior determinations (pH 3.4) but still clearly separates affected patients from unaffected newborns using formate buffer at pH 4.4.
  • QC samples for inclusion in the DBS enzyme assay were obtained from the Center for Disease Control (CDC). These are artificially prepared blood samples that represent the range of enzyme activity expected from newborn blood spots submitted for screening. They are expected to approximate 5%, 50%, and 90% of the normal range. The inclusion of two QC samples for each activity level in each 96- well plate serves as a reliable indicator of assay integrity.
  • Table 8 summarizes the enzyme activity data obtained for 80 punches of the low, medium and high QC samples from the CDC [8 punches per day (distributed 2 per 96-well plate in 4 plates) over a 10- day period] .
  • the coefficient of variation percentages are generally higher for the low QC samples as expected since the medium enzyme activity in these samples is -10% of those for the high QC samples.
  • the coefficient of variation percentages are in the range 7.9-13.5%. These values include all variation from the assay procedure and from punching the DBS at different spots, since multiple punches were taken from each DBS.
  • the first number in each box is the mean enzyme activity followed by the covariance in percent (in parenthesis).
  • the triplex assay has the added benefit of having an internal quality control.
  • DBS punches that are not fully saturated with blood.
  • these samples appear to come from affected patients.
  • such a DBS would have low activity in all three enzymes, flagging an error in the testing procedure.
  • Fabry disease is an X-linked disease
  • females will have a wide spectrum of cc-galactosidase activity due to random X-chromosome inactivation.
  • the assay reported here, or any enzyme activity assay will primarily detect hemizygous males with absent, or low, cc-galactosidase activity.
  • the enzyme activities of three separate enzymes can be efficiently determined in a single incubation of a DBS with the appropriate enzyme substrates and internal standards.
  • the new assay reliably distinguishes blood from affected patients with Fabry, Pompe, and MPS IH from unaffected newborns.
  • the assay has an internal control for integrity that minimizes the occurrence of false positives.
  • the present study also demonstrates that a single buffer will not be required for each lysosomal enzyme to be assayed, and the pre-ESTMS/MS sampling handling steps have been simplified to the level that they fit well into the high throughput regiment of a newborn screening laboratory.
  • the new procedure is simplified from published procedures as it requires fewer manipulations and resources and has been designed to be compatible with the typical schedule of a newborn screening laboratory.
  • Table 9B ESTMS/MS arameters for o timization studies.
  • Assay cocktails preparation Assay components were added to a vial as a solution in methanol: GAA-S (20 ⁇ . of 10 mM), GAA-IS (5 ⁇ . of 1 mM), GLA-S (100 ⁇ . of 10 mM), GLA-IS (5 ⁇ . of 1 mM), IDUA-S (50 ⁇ . of 10 mM), IDUA-IS (5 ⁇ . of 1 mM).
  • Additives were added as a solution in water: Acarbose (8 ⁇ ⁇ of 1 mM) and saccharic acid 1,4 lactone (50 ⁇ ⁇ of 1 mM).
  • the solvent was evaporated using a centrifugal concentrator (SpeedVac), the residue was taken up in 1 mL of the appropriate buffer solution, and the vial was vortex mixed until the residue dissolved.
  • the final assay cocktail contains 0.5 mM IDUA-S, 5 ⁇ IDUA-IS, 0.2 mM GAA-S, 5 ⁇ GAA-IS, 1.0 mM GLA-S, 5 ⁇ GLA-IS, 8 ⁇ acarbose, and 50 ⁇ saccharic acid 1,4-lactone.
  • Buffer Screen For each buffer composition, a single 2-mm diameter DBS punch (containing approximately 1.6 ⁇ ⁇ of blood) was obtained with a leather punch and was placed in a 1.5 mL Eppendorf tube. To this tube was added assay cocktail (20 ⁇ ) prepared as described above and containing 0.5 mM IDUA-S, 5 ⁇ IDUA-IS, 0.2 mM GAA-S, 5 ⁇ GAA-IS, 1.0 mM GLA-S, 5 ⁇ GLA-IS, 8 ⁇ acarbose, and 50 ⁇ saccharic acid 1,4-lactone. Similarly, a blank containing all the assay components except the DBS punch was also prepared.
  • the samples were incubated at 37 °C for 16 hrs in a thermostated air shaker operating at 250 rpm.
  • the layers were mixed by aspirating with a pipette (5 times), and then the tubes were centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 ⁇ ) was transferred to a new centrifuge tube and dried down under a stream of nitrogen.
  • the residue was resuspended in 100 ⁇ ⁇ ethyl acetate: methanol (19: 1) and applied to a plug of silica gel (-100 mg in a 1 mL pipette tip containing a cotton plug. The plug was eluted 4 times with 400 ⁇ ⁇ ethyl acetate: methanol (19: 1).
  • Solid-phase extraction was carried out using a vacuum manifold (Millipore Inc, MAVM0960R) connected to a water aspirator. The solvent was evaporated with a stream of nitrogen, and the residue was reconstituted in 100 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 ⁇ ⁇ aliquot was used for the mass spectrometric analysis.
  • a 20 ⁇ ⁇ aliquot of assay cocktail was purified as described in the buffer screen experiments using the liquid-liquid extraction and the solid phase extraction. The residue was reconstituted in 100 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 ⁇ ⁇ aliquot was used for the mass spectrometric analysis.
  • a single 3-mm diameter DBS punch (containing approximately 3.2 ⁇ ⁇ of blood) was obtained with a leather punch and was placed in a single well of a 96 deep-well plate.
  • the assays contained the following concentrations of detergent: no detergent, 1.6 g/L sodium taurocholate, 5.6 g/L sodium taurocholate, 9.6 g/L sodium taurocholate, 2 g/L CHAPS, 4 g/L CHAPS, and 6 g/L CHAPS.
  • the layers were mixed by aspirating with a pipette (5 times), and the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 ⁇ ) was transferred to a new 96 shallow-well plate, the solvent was evaporated with a stream of N 2 , and the residue was reconstituted in 100 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 ⁇ ⁇ aliquot was used for the mass spectrometric analysis.
  • a separate assay cocktail containing the same concentrations as well as 96 mM GalNAc (TCI American, A1245) was also prepared.
  • the layers were mixed by aspirating with a pipette (5 times), and then the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 ⁇ ) was transferred to a new 96 shallow-well plate, the solvent was evaporated with a stream of nitrogen gas and the residue was reconstituted in 150 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 ⁇ ⁇ aliquot was used for the mass spectrometric analysis.
  • the layers were mixed by aspirating with a pipette (5 times), and then the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 ⁇ ) was transferred to a new 96 shallow-well plate, the solvent was evaporated with a stream of N 2 , and the residue was reconstituted in 150 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 ⁇ ⁇ aliquot was used for the mass spectrometric analysis.
  • Test of Sodium versus Ammonium Formate Buffers Two separate assay cocktails were prepared as described above and contained 0.5 mM IDUA-S, 6.7 ⁇ IDUA-IS, 0.4 mM GAA-S, 6.7 ⁇ GAA-IS, 1.0 mM GLA-S, 6.7 ⁇ GLA-IS, and 8 ⁇ acarbose.
  • the ammonium formate buffer was prepared as described above, but the pH of the buffer was adjusted with ammonium hydroxide instead of sodium hydroxide.
  • the layers were mixed by aspirating with a pipette (5 times), and then the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 ⁇ ) was transferred to a new 96 shallow-well plate, the solvent was evaporated with a stream of N 2 , and the residue was reconstituted in 150 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 ⁇ ⁇ aliquot was used for the mass spectrometric analysis.
  • a single 3-mm diameter DBS punch (containing approximately 3.2 ⁇ _, of blood) was obtained from each DBS source with a leather punch and was placed in a single well of a 96 deep-well plate. To this well was added 30 ⁇ ⁇ of assay cocktail in sodium formate buffer.
  • top layer ethyl acetate, 250 ⁇
  • solvent was evaporated with a stream of N 2
  • residue was reconstituted in 150 ⁇ ⁇ of 80/20 acetonitrile/water with 0.2 % formic acid.
  • a 20 ⁇ ⁇ aliquot was used for the mass spectrometric analysis.
  • Average activity and standard deviation is based on assays in triplicate.
  • the first number in each box is the mean enzyme activity followed by the covariance in percent (in parenthesis).

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Abstract

Provided herein are substrates, internal standards, enzymatic assays, and screening methods pertaining to the analysis of various lysosomal storage diseases. In particular, mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome), mucopolysaccharidosis IVA (Morquio A Syndrome), Fabry Syndrome, Pompe Syndrome, and mucopolysaccharidosis I (Hurler) (MPS IH) Syndrome may be analyzed, particularly with respect to the screening of patients.

Description

MASS SPECTROMETRIC COMPOSITIONS AND METHODS FOR LYSOSOMAL
STORAGE DISEASE SCREENING
CROSS-REFERENCES TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.
61/377,069 filed August 25, 2010, and U.S. Provisional Application No. 61/380,633 filed September 7, 2010, each of which is incorporated herein by reference in its entirety.
STATEMENT OF GOVERNMENT LICENSE RIGHTS
This invention was made with Government support under Grant Nos. R01DK067859 and HHSN26700603429 awarded by the National Institutes of Health. The Government has certain rights in the invention.
BACKGROUND
The mucopolysaccharidoses (MPS I to VII) are a group of metabolic diseases caused by a deficiency of one of the lysosomal enzymes degrading the glycosaminoglycans heparan, dermatan, keratan, or chondroitin sulfate (Neufeld, E. F.; Muenzer, J. The Mucopolysaccharidoses. In: Scriver, C; Beaudet, A.; Sly, W.; eds. The Metabolic and Molecular Bases of Inherited Disease. New York, NY: McGraw Hill; 2001; 3421-3452). The pertinent enzymes consist of five sulfatases, four exoglycosidases, and one non-hydrolytic acetyl-N-transferase. These syndromes result in non-degraded or partially degraded glycosaminoglycans amassing in the lysosome resulting in irreversible multisystemic organ damage.
Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome) is caused by deficiency of /V-acetylgalactosamine 4-sulfatase (also called aryl sulfatase B, ASB, EC 3.1.56.12), which is one of several enzymes involved in step-wise degradation of chondroitin and dermatan sulfate. ASB deficiency is inherited as an autosomal recessive trait. The affected children may appear normal at birth but within 1-3 years develop skeletal abnormalities causing short stature and joint stiffness, as well as other symptoms such as corneal clouding. Early detection of MPS VI seems prudent to maximize the potential benefit of treatment, and thus there is the need to develop tests that are appropriate for early diagnosis. Likewise, there is a need for developing a fast, inexpensive, and reliable diagnostic procedure that uses dried blood spots (DBS) as a sample source, such as those submitted to newborn screening laboratories. Morquio Syndrome Type A, or mucopolysaccharidosis IVA (MPS IVA), is an autosomal recessive disorder due to deficiency of lysosomal ^-acetylgalactosamine 6- sulfatase (GALNS) (EC 3.1.6.4) activity. The enzyme hydrolyzes the sulfate ester bond of galactose 6-sulphate present in keratin sulfate and of ^-acetylgalactosamine 6-sulfate present in chondroitin C. Early detection of MPS rVA seems prudent to maximize the potential benefit of treatment, and thus there is the need to develop tests that are appropriate for early diagnosis, e.g., for use with DBS submitted to newborn screening laboratories.
Other lysosomal storage diseases for which early recognition and treatment has been shown to be efficacious include Pompe, Fabry, and mucopolysaccharidosis I (Hurler) (MPS IH) diseases. Recently, treatments have become available for some of the MPS syndromes; however, optimal benefits from these treatments would require commencement of treatment prior to the onset of the irreversible symptoms.
SUMMARY
Provided herein are substrates, internal standards, enzymatic assays, and screening methods pertaining to the analysis of various lysosomal storage diseases in patients, such as newborns.
Accordingly, provided herein is a compound of formula (I):
wherein: Rj is unsubsti
Figure imgf000003_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or substituted arylalkylC6.j4; Xj is hydrogen or a counterion; Yj is -OH or -NHC(0)CH3; and n is an integer from 2 to 12. Also provided is a compound of formula (II):
wherein: R2 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; Y2 is - OH or -NHC(0)CH3; and m is an integer from 2 to 12. Further provided is a method for providing an ^-acetylgalactosamine 6-sulfatase product, comprising incubating an N- acetylgalactosamine 6-sulfatase substrate of formula (I):
Figure imgf000004_0002
wherein: Rj is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or substituted arylalkyl^.^; Xj is hydrogen or a counterion; Yj is -OH or -NHC(0)CH3; and n is an integer from 2 to 12; with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 6-sulfatase product when the sample comprises ^-acetylgalactosamine 6-sulfatase. A further method provided is a method for determining the presence or absence of ^-acetylgalactosamine 6-sulfatase enzymatic activity in a sample, comprising: (a) incubating an N- acetylgalactosamine 6-sulfatase substrate with sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 6-sulfatase product when the sample comprises N- acetylgalactosamine 6-sulfatase; and (b) determining the presence or absence of the N- acetylgalactosamine 6-sulfatase product, wherein the presence of N-acetylgalactosamine 6-sulfatase product indicates the presence of N-acetylgalactosamine 6-sulfatase enzymatic activity, and wherein the absence of N-acetylgalactosamine 6-sulfatase product indicates the absence of N-acetylgalactosamine 6-sulfatase enzyme activity. Further provided is a method for determining the quantity of an N-acetylgalactosamine 6-sulfatase product in a blood sample, comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating (i) an ^-acetylgalactosamine 6-sulfatase substrate of formula (I):
wherein: Rj is unsubsti
Figure imgf000005_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or substituted arylalkylC6.j4; Xj is hydrogen or a counterion; Yj is -OH or -NHC(0)CH3; and n is an integer from 2 to 12; and (ii) an ^-acetylgalactosamine 6-sulfatase internal standard, with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 6-sulfatase product when the sample comprises ^-acetylgalactosamine 6-sulfatase; and (c) determining the quantity of the N-acetylgalactosamine 6-sulfatase product by tandem mass spectrometric analysis.
Further provided is a compound of formula (III):
wherein: R3 is unsubst
Figure imgf000005_0002
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4; X2 is hydrogen or a counterion; and p is an integer from 2 to 12. Another compound provided is a compound of formula (IV):
Figure imgf000006_0001
wherein: R4 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; and q is an integer from 2 to 12. Also provided is a method for providing an N- acetylgalatosamine 4-sulfatase product, comprising incubating an N-acetylgalactosamine 4-sulfatase substrate of formula (III):
wherein: R3 is unsubst
Figure imgf000006_0002
substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; X2 is hydrogen or a counterion; and p is an integer from 2 to 12; and with a sample for a predetermined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 4-sulfatase product when the sample comprises ^-acetylgalactosamine 4-sulfatase. Further provided is a method for determining the presence or absence of N-acetylgalactosamine 4-sulfatase enzymatic activity in a sample, comprising: (a) incubating an ^-acetylgalactosamine 4-sulfatase substrate with sample for a pre-determined time sufficient to effect enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 4-sulfatase product when the sample comprises ^-acetylgalactosamine 4-sulfatase; and (b) determining the presence or absence of the ^-acetylgalactosamine 4-sulfatase product, wherein the presence of N-acetylgalactosamine 4-sulfatase product indicates the presence of N-acetylgalactosamine 4-sulfatase enzymatic activity, and wherein the absence of N- acetylgalactosamine 4-sulfatase product indicates the absence of ^-acetylgalactosamine 4-sulfatase enzymatic activity. Further provided is a method for determining the quantity of an ^-acetylgalactosamine 4-sulfatase product in a blood sample, comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating (i) an ^-acetylgalactosamine 4-sulfatase substrate of formula (III):
Figure imgf000007_0001
wherein: R3 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^: X2 is hydrogen or a counterion; and p is an integer from 2 to 12; and (ii) an N- acetylgalactosamine 4-sulfatase internal standard, with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 4-sulfatase product when the sample comprises N-acetylgalactosamine 4-sulfatase; and (c) determining the quantity of the N- acetylgalactosamine 4-sulfatase product by tandem mass spectrometric analysis.
A further embodiment provides a method for providing a solution comprising an a-glucosidase product, an a-galactosidase product, and an a-L-iduronidase product, comprising incubating an a-glucosidase substrate, an a-galactosidase substrate, and an a- L-iduronidase substrate with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an a- glucosidase product, an α-galactosidase product, and an a-L-iduronidase product when the sample comprises α-glucosidase, α-galactosidase, and α-L-iduronidase, wherein the buffer comprises an aqueous solution of acarbose. Also provided is A method for determining the presence or absence of α-glucosidase enzymatic activity, a-galactosidase enzymatic activity, and α-L-iduronidase enzymatic activity in a sample, comprising: (a) incubating an α-glucosidase substrate, an α-galactosidase substrate, an a-L-iduronidase substrate with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an α-glucosidase product, an α-galactosidase product, and an α-L-iduronidase product when the sample comprises a- glucosidase, α-galactosidase, and α-L-iduronidase; and (b) determining the presence or absence of the a-glucosidase product, the α-galactosidase product, and the a-L- iduronidase product, wherein the presence of a-glucosidase product indicates the presence of α-glucosidase enzymatic activity, wherein the presence of a-galactosidase product indicates the presence of α-galactosidase enzymatic activity, and wherein the presence of α-L-iduronidase product indicates the presence of α-L-iduronidase activity, and wherein the absence of α-glucosidase product indicates the absence of a-glucosidase enzymatic activity, wherein the absence of α-galactosidase product indicates the absence of α-galactosidase enzymatic activity, and wherein the absence of a-L-iduronidase product indicates the absence of α-L-iduronidase activity. Also provided is a method for determining the quantity of a α-glucosidase product, a α-galactosidase product, and a a-L- iduronidase product in a blood sample, comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating an α-glucosidase substrate, an a- glucosidase internal standard, an α-galactosidase substrate, an α-galactosidase internal standard, an α-L-iduronidase substrate, and an α-L-iduronidase internal standard with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an α-glucosidase product, an a- galactosidase product, and an α-L-iduronidase product when the sample comprises a- glucosidase, α-galactosidase, and α-L-iduronidase, wherein the buffer comprises an aqueous solution of acarbose; and (c) determining the quantity of the a-glucosidase product, the α-galactosidase product, and the α-L-iduronidase product by tandem mass spectrometric analysis.
DESCRIPTION OF THE DRAWINGS
The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.
FIGURE 1 : Structures of Maroteaux-Lamy substrate 1 and internal standard 3 and Morquio A substrate 2 and internal standard 4.
FIGURE 2: Preparation of MPS IVA substrate 2 and internal standards 3 and 4.
Reagents and conditions: (a) N-Boc-l, 6-hexanediamine, N-(3-dimethylamino- propy^-N!-ethylcarbodiimide hydrochloride, DMF, CH2CI2, overnight, 52%; (b) N-Boc-1, 5-pentanediamine, N-Q-diemthylaminopropyl^N!-ethylcarbodiimide hydrochloride, DMF, CH2CI2, overnight, 62%; (c) glycosyl chloride (7a, 7b), peracetylated glycosyl bromide (9), tetrabutylammonium hydrogen sulfate, aq. NaOH (1 M), DCM, 30-60 min; (d) sodium methoxide, MeOH, CHC13, 4 h, 8 (from 6a and 7a) : 53% over two steps; 3 (from 6b and 7b) : 50% over two steps; 10 (from 6b and 9) : 28% over two steps; 4 (from 6a and 9) : 35% over two steps; (e) sulfur trioxide-pyridine complex, pyridine; 5 h; DOWEX 50WX8 (Na+ form), 45%.
FIGURE 3: Preparation of MPS VI substrate 1. Reagents and conditions: (a) benzoyl chloride (2.1 equiv), pyridine, 0°C, 3 h, 58%; (b) triflic anhydride, pyridine, DCM, -20° C, 3 h; (c) sodium nitrite, DMF, overnight, 51% over two steps; (d) sulfur trioxide-pyridine complex, pyridine, 3 h; DOWEX 50WX8 (Na+ form), 50%; (e) sodium methoxide, MeOH, 5 d, 80%.
FIGURE 4: Structures of aryl sulfatase B (ASB-S), aryl sulfatase B product (ASB-P), and aryl sulfatase B internal standard (ASB-IS) for the ASB reaction. The structures of the daughter ions from ASB-P and ASB-IS after collision-induced dissociation (CID) in the mass spectrometer are also shown. ASB-P and ASB-IS were quantified by electrospray ionization-tandem mass spectrometry (ESTMS/MS) in positive-ion multiple-reaction-monitoring mode ((M + H)+).
FIGURE 5: Amount of aryl sulfatase B (ASB)-generated product measured in dried blood spots (DBS) as a function of the concentration of substrate. Reactions were carried out at 37 °C for 16 h using the standard assay described herein using solid phase extraction method. Error bars are shown for triplicate analyses. The solid line shows the regression fit of the data to the Michaelis-Menten equation.
FIGURE 6: Distribution of ASB activities in DBS from humans. Black bars show data for 89 unaffected newborns obtained with the liquid-liquid extraction method. White bars show data for 10 unaffected newborns obtained with the solid phase extraction method. The hashed bar is data for 1 MPS VI patient. ASB activity values for each sample are given in Tables 1 and 2.
FIGURES 7 A and 7B: FIGURE 7 A: Structures of N- acetylgalactosamine 6- sulfatase substrate (GALNS-S), N-acetylgalactosamine 6-sulfatase product (GALNS-P), and ^-acetylgalactosamine 6-sulfatase internal standard (GALNS-IS) for the GALNS reaction. The structures of the daughter ions from GALNS-P and GALNS-IS after collision-induced dissociation (CID) in the mass spectrometer are also shown. GALNS-P and GALNS-IS were quantified by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in positive-ion multiple-reaction-monitoring mode as the sodiated species (M + Na)+. FIGURE 7B: GALNS activities were measured in DBS by the standard assay described herein with ethyl acetate extraction workup.
FIGURE 8: Typical work-flow of the triplex assay run in a newborn screening laboratory. Assay results are obtained within 48 h of the beginning of the procedure.
FIGURES 9A, 9B, and 9C: Activity distribution of cc-galactosidase A (GLA) (FIGURE 9A), cc-glucosidase (GAA) (FIGURE 9B), and a-L-iduronidase (IDUA) (FIGURE 9C) from 5,990 anonymous newborn blood spots. Assays were performed in the Washington State Newborn Screening laboratory using the triplex assay.
DETAILED DESCRIPTION
Provided herein are substrates, internal standards, enzymatic assays, and screening methods pertaining to the analysis of various lysosomal storage diseases. In particular, mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome), mucopolysaccharidosis IVA (Morquio A Syndrome), Fabry Syndrome, Pompe Syndrome, and mucopolysaccharidosis I (Hurler) (MPS IH) Syndrome may be analyzed, particularly with respect to the screening of newborns.
Substrates that can be used to assay N-acetylgalactosamine 4-sulfatase, the enzyme deficient in Maroteaux-Lamy Syndrome, and galactose 6-sulfatase, the enzyme deficient in Morquio A syndrome, are provided herein. The substrates are readily prepared, specific for the enzymes, and tagged for detection in the newborn screening laboratory. Natural substrates for the sulfatases are oligosaccharides containing a sulfate on the terminal sugar. While these oligosaccharides would be selective substrates for the enzymes, they are difficult to prepare on the scale needed for worldwide newborn screening (-10 g/year). In contrast, aryl sulfatases are readily available but show low specificity between the sulfatase enzymes. Substrate design was based on the terminal sugar of the natural substrates, i.e., ^-acetylgalactosamine 4-sulfate and galactose 6-sulfate. The anomeric carbon of these sugars were coupled to an umberferryl moiety, which could be used for fluorescence assays in laboratories lacking tandem mass spectrometers. A carbon chain with a fragmentable N-iert-butyl carbamate was attached to direct the fragmentation of the parent ion in the mass spectrometer along a single reaction pathway, which increases the sensitivity of the tandem mass spectrometry assay. Other carbamates may be employed, as described herein. The linker chain lengths were chosen such that the mass of the products and internal standards allow for the assays to be multiplexed in the mass spectrometry analysis. See Example 1 for exemplary preparations of MPS VI and MPS IVA substrates and internal standards.
Two separate enzyme assays have been developed that directly measure the enzyme activity of aryl sulfatase B for detection of MPS VI and the enzyme activity of N- acetylgalactosamine 6-sulfatase for detection of MPS rVA. In general, each assay is highly specific and can be used, for example, with the substrates described herein to directly measure the relevant enzyme in a sample. Mass spectrometric methods may be employed in these assays, such as electrospray ionization-tandem mass spectrometry (ESTMS/MS), which offers the capability of assaying products of several enzymes by a single infusion into the mass spectrometer for simultaneous sampling, and may provide more accurate data than fluorometric assays that are presently used. See Example 2 for an exemplary assay regarding MPS VI and Example 3 for an exemplary assay regarding MPS IVA.
A triplex assay to detect Fabry, Pompe, and MPS IH lysosomal storage diseases is also provided. These disorders were selected because each may be difficult to recognize clinically, and recent studies have shown enzyme replacement therapy or bone marrow transplantation to improve the natural history of the conditions. The assay for the three related lysosomal enzymes (cc-glucosidase, cc-galactosidase A, and cc-L-iduronidase) can be performed using a minimal quantity of disposable supplies and equipment in a timely manner. The procedure is adaptable to a newborn screening laboratory. By assaying three enzymes at once, problematic samples due, for example, to insufficient blood in the assay are readily flagged for reanalysis. Example 4 presents an exemplary triplex assay.
Accordingly, provided herein is a compound of formula (I):
Figure imgf000011_0001
(I)
wherein: Rj is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or substituted arylalkyl^.^; Xj is hydrogen or a counterion; Yj is -OH or -NHC(0)CH3; and n is an integer from 2 to 12. Rj may be unsubstituted alkylcl_6. Rj may be ie/t-butyl. Xj may be a counterion. The counterion may be a metal counterion or a non-metal counterion. In some embodiments, the counterion is a metal counterion, such as a sodium counterion, a potassium counterion, or a lithium counterion. In some embodiments, the metal counterion is a sodium counterion. The counterion may be a non-metal counterion, such as an ammonium counterion. In some embodiments, n = 2-6. In some embodiments, n = 5.
Also provided is a compound of formula (II):
wherein: R2 is unsubst
Figure imgf000012_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4; Y2 is - OH or -NHC(0)CH3; and m is an integer from 2 to 12. R2 may be unsubstituted alkyl^j. 5. R2 may be ie/t-butyl. In some embodiments, m = 2-6. In some embodiments, m = 6.
Kits are also provided, such as a kit comprising a compound of formula (I) and a compound of formula (II), wherein n≠ m. In some embodiments, n = 5 and m = 6.
Further provided is a method for providing an ^-acetylgalactosamine 6-sulfatase product, comprising incubating an ^-acetylgalactosamine 6-sulfatase substrate of formula (I):
Figure imgf000012_0002
wherein: Rj is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5_i2; substituted aryl^.^; unsubstituted arylalkyl^.^; or substituted arylalkyl^.^; Xj is hydrogen or a counterion; Yj is -OH or -NHC(0)CH3; and n is an integer from 2 to 12; with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an N-acetylgalactosamine 6-sulfatase product when the sample comprises N-acetylgalactosamine 6-sulfatase. Any of the Rj, Xj, Yj, and n descriptions provided herein for formula (I) are provided as well. In some embodiments, the sample is a blood sample, such as a dried blood sample from a newborn screening card. A method may further comprise using the amount of the N- acetylgalactosamine 6-sulfatase product to determine whether the dried blood sample is from a candidate for treatment for Mucopolysaccharidosis IVA (Morquio Syndrome Type A). A sample may further comprise an ^-acetylgalactosamine 6-sulfatase internal standard. In some embodiments, the ^-acetylgalactosamine 6-sulfatase internal standard is a compound of formul
Figure imgf000013_0001
wherein: R2 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; Y2 is - OH or -NHC(0)CH3; and m is an integer from 2 to 12, wherein n≠ m. Any of the R2, Y2, and m descriptions provided herein for formula (II) are provided as well. A method may further comprise extracting the enzyme product solution to provide the N- acetylgalactosamine 6-sulfatase internal standard and the ^-acetylgalactosamine 6- sulfatase product when the sample comprises ^-acetylgalactosamine 6-sulfatase. In some embodiments, extracting the enzyme product solution comprises liquid extraction with an organic solution. Extracting the enzyme product solution may comprise solid phase extraction. A method may further comprise quenching the enzymatic reaction prior to extraction. A method may further comprise determining the quantity of the N- acetylgalactosamine 6-sulfatase product. In some embodiments, determining the quantity of the ^-acetylgalactosamine 6-sulfatase product comprises determining the ratio of the ^-acetylgalactosamine 6-sulfatase product to the ^-acetylgalactosamine 6-sulfatase internal standard by mass spectrometric analysis. In some embodiments, determining the quantity of the N-acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis. In some embodiments, determining the quantity of the N- acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions. In some embodiments, determining the quantity of the N- acetylgalactosamine 6-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 6-sulfatase product.
In certain aspects, the invention provides enzyme assays. In certain embodiments, the assays are qualitative assays that determine the presence or absence of one or more particular enzymes in a sample by measuring the presence or absence of an enzyme product. In other embodiments, the assays are quantitative assays that determine the quantity of one or more particular enzymes by measuring the quantity of an enzyme product. In representative quantitative assays, the quantity of the one or more enzymes is determined through the use of standards. In certain embodiments, the standards are internal standards and the amount of enzyme is determined directly by comparing the signal from the standard to the signal associated with the enzyme product. In other embodiments, the standards are external to the sample and quantitation of the enzyme product is determined by, for example, creating a standard curve and comparing the signal associated with the enzyme product to the standard curve. Qualitative assays that determine the presence or absence of an enzyme product do not require the use of standards.
Further provided is a method for determining the presence or absence of N- acetylgalactosamine 6-sulfatase enzymatic activity in a sample, comprising: (a) incubating an N- acetylgalactosamine 6-sulfatase substrate with sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 6-sulfatase product when the sample comprises N-acetylgalactosamine 6-sulfatase; and (b) determining the presence or absence of the ^-acetylgalactosamine 6-sulfatase product, wherein the presence of N- acetylgalactosamine 6-sulfatase product indicates the presence of ^-acetylgalactosamine 6-sulfatase enzymatic activity, and wherein the absence of ^-acetylgalactosamine 6- sulfatase product indicates the absence of ^-acetylgalactosamine 6-sulfatase enzyme activity. In some embodiments, the sample further comprises an N-acetylgalactosamine 6-sulfatase internal standard. A method may further comprise extracting the enzyme product solution to provide the ^-acetylgalactosamine 6-sulfatase product and the N- acetylgalactosamine 6-sulfatase internal standard when the sample comprises N- acetylgalactosamine 6-sulfatase. A method may further comprise determining the quantity of the N- acetylgalactosamine 6-sulfatase product. In some embodiments, determining the quantity of the ^-acetylgalactosamine 6-sulfatase product comprises determining the ratio of the ^-acetylgalactosamine 6-sulfatase product to the N- acetylgalactosamine 6-sulfatase internal standard by mass spectrometric analysis. In some embodiments, determining the quantity of the ^-acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis. In some embodiments, determining the quantity of the ^-acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions. In some embodiments, determining the quantity of the ^-acetylgalactosamine 6-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 6-sulfatase product. A sample may be a blood sample, such as a dried blood sample from a newborn. A method may further comprise contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution. A method may further comprise using the amount of ^-acetylgalactosamine 6-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis IVA (Morquio Syndrome Type A).
Further provided is a method for determining the quantity of an N- acetylgalactosamine 6-sulfatase product in a blood sample, comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating (i) an N- acetylgalactosamine 6-sulfatase substrate of formula (I):
Figure imgf000015_0001
(I)
wherein: Rj is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or substituted arylalkyl^.^; Xj is hydrogen or a counterion; Yj is -OH or -NHC(0)CH3; and n is an integer from 2 to 12; and (ii) an ^-acetylgalactosamine 6-sulfatase internal standard, with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 6-sulfatase product when the sample comprises N- acetylgalactosamine 6-sulfatase; and (c) determining the quantity of the ^-acetylgalactosamine 6-sulfatase product by tandem mass spectrometric analysis. Any of the Rj, Xj, Yj, and n descriptions provided herein for formula (I) are provided as well. The ^-acetylgalactosamine 6-sulfatase internal standard may be a compound of formula (II):
Figure imgf000016_0001
wherein: R2 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; Y2 is - OH or -NHC(0)CH3; and m is an integer from 2 to 12, wherein n≠ m. Any of the R2, Y2, and m descriptions provided herein for formula (II) are provided as well. A method may further comprise extracting the enzyme product solution to provide the N- acetylgalactosamine 6-sulfatase internal standard and the ^-acetylgalactosamine 6- sulfatase product when the sample comprises ^-acetylgalactosamine 6-sulfatase. Step (c) may comprise: (i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and (ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 6-sulfatase product. A blood sample may be a dried blood sample from a newborn. Step (a) may be further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution. A method may further comprise using the amount of ^-acetylgalactosamine 6-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis IVA (Morquio Syndrome Type A).
Also provided is a compound of formula (III):
Figure imgf000017_0001
wherein: R3 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4; X2 is hydrogen or a counterion; and p is an integer from 2 to 12. R3 may be unsubstituted alkylcl.g. R3 may be ie/t-butyl. X2 may be a counterion. The counterion may be a metal counterion or a non-metal counterion. In some embodiments, the counterion is a metal counterion, such as a sodium counterion, a potassium counterion, or a lithium counterion. In some embodiments, the metal counterion is a sodium counterion. The counterion may be a non-metal counterion, such as an ammonium counterion. In some embodiments, p = 2-6. In some embodiments, p = 6.
Also provided is a compound of formula (IV):
wherein: R4 is unsubst
Figure imgf000017_0002
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4; and q is an integer from 2 to 12. R4 may be unsubstituted alkylci. . R4 may be iert-butyl. In some embodiments, q = 2-6. In some embodiments, q = 5.
As noted, kits are also provided. In one embodiment, a kit comprises a compound of formula (III) and a compound of formula (IV), wherein p≠ q. In some embodiments, p = 6 and q = 5. Further provided is a method for providing an N-acetylgalatosamine 4-sulfatase product, comprising incubating an ^-acetylgalactosamine 4-sulfatase substrate of formula
(HI):
Figure imgf000018_0001
wherein: R3 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; X2 is hydrogen or a counterion; and p is an integer from 2 to 12; and with a sample for a predetermined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 4-sulfatase product when the sample comprises N-acetylgalactosamine 4-sulfatase. Any of the R3, X2, and p descriptions provided herein for formula (III) are provided as well. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a dried blood sample from a newborn screening card. A method may further comprise using the amount of the N- acetylgalactosamine 4-sulfatase product to determine whether the dried blood sample is from a candidate for treatment for Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome). A sample may further comprise an ^-acetylgalactosamine 4-sulfatase internal standard. The ^-acetylgalactosamine 4-sulfatase internal standard may be a compound of formula (IV):
Figure imgf000018_0002
wherein: R4 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; and q is an integer from 2 to 12, and wherein p≠ q. Any of the R4 and q descriptions provided herein for formula (IV) are provided as well. A method may further comprise extracting the enzyme product solution to provide the N- acetylgalactosamine 4-sulfatase internal standard and the N-acetylgalactosamine 4-sulfatase product when the sample comprises ^-acetylgalactosamine 4-sulfatase. Extracting the enzyme product solution may comprise liquid extraction with an organic solution. In some embodiments, extracting the enzyme product solution comprises solid phase extraction. A method may further comprise quenching the enzymatic reaction prior to extraction. A method may further comprise determining the quantity of the ^-acetylgalactosamine 4-sulfatase product. In some embodiments, determining the quantity of the ^-acetylgalactosamine 4-sulfatase product comprises determining the ratio of the ^-acetylgalactosamine 4-sulfatase product to the ^-acetylgalactosamine 4-sulfatase internal standard by mass spectrometric analysis. In some embodiments, determining the quantity of the N- acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis. In some embodiments, determining the quantity of the ^-acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions. In some embodiments, determining the quantity of the ^-acetylgalactosamine 4-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 4-sulfatase product.
Also provided is a method for determining the presence or absence of N- acetylgalactosamine 4-sulfatase enzymatic activity in a sample, comprising: (a) incubating an N- acetylgalactosamine 4-sulfatase substrate with sample for a pre-determined time sufficient to effect enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 4-sulfatase product when the sample comprises ^-acetylgalactosamine 4-sulfatase; and (b) determining the presence or absence of the ^-acetylgalactosamine 4-sulfatase product, wherein the presence of N- acetylgalactosamine 4-sulfatase product indicates the presence of ^-acetylgalactosamine 4-sulfatase enzymatic activity, and wherein the absence of ^-acetylgalactosamine 4- sulfatase product indicates the absence of ^-acetylgalactosamine 4-sulfatase enzymatic activity. In some embodiments, the sample further comprises an N-acetylgalactosamine 4-sulfatase internal standard. A method may further comprise extracting the enzyme product solution to provide the ^-acetylgalactosamine 4-sulfatase product and the N- acetylgalactosamine 4-sulfatase internal standard when the sample comprises N- acetylgalactosamine 4-sulfatase. A method may further comprise determining the quantity of the N-acetylgalactosamine 4-sulfatase product. In some embodiments, determining the quantity of the ^-acetylgalactosamine 4-sulfatase product comprises determining the ratio of the ^-acetylgalactosamine 4-sulfatase product to the N- acetylgalactosamine 4-sulfatase internal standard by mass spectrometric analysis. In some embodiments, determining the quantity of the ^-acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis. In some embodiments, determining the quantity of the ^-acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions. In some embodiments, determining the quantity of the ^-acetylgalactosamine 4-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 4-sulfatase product. A sample may be a blood sample, such as a dried blood sample from a newborn. A method may further comprise contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution. A method may further comprise using the amount of ^-acetylgalactosamine 4-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome).
Further provided is a method for determining the quantity of an N- acetylgalactosamine 4-sulfatase product in a blood sample, comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating (i) an N- acetylgalactosamine 4-sulfatase substrate of formula (III):
Figure imgf000021_0001
wherein: R3 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4: X2 is hydrogen or a counterion; and p is an integer from 2 to 12; and (ii) an N- acetylgalactosamine 4-sulfatase internal standard, with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 4-sulfatase product when the sample comprises ^-acetylgalactosamine 4-sulfatase; and (c) determining the quantity of the ^-acetylgalactosamine 4-sulfatase product by tandem mass spectrometric analysis. Any of the R3, X2, and p descriptions provided herein for formula (III) are provided as well. The ^-acetylgalactosamine 4-sulfatase internal standard may be a compound of formula (IV):
Figure imgf000021_0002
wherein: R4 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; and q is an integer from 2 to 12, and wherein p≠ q. Any of the R4 and q descriptions provided herein for formula (IV) are provided as well. A method may further comprise extracting the enzyme product solution to provide the ^-acetylgalactosamine 4-sulfatase internal standard and the N-acetylgalactosamine 4-sulfatase product when the sample comprises N-acetylgalactosamine 4-sulfatase. In some embodiments, (c) comprises: (i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and (ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the N-acetylgalactosamine 4-sulfatase product. In some embodiments, the blood sample is a dried blood sample from a newborn. In some embodiments, (a) is further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution. A method may further comprise using the amount of ^-acetylgalactosamine 4-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome).
Further provided is a method for providing a solution comprising an a-glucosidase product, an a-galactosidase product, and an a-L-iduronidase product, comprising incubating an a-glucosidase substrate, an a-galactosidase substrate, and an a-L- iduronidase substrate with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an a- glucosidase product, an α-galactosidase product, and an α-L-iduronidase product when the sample comprises α-glucosidase, α-galactosidase, and α-L-iduronidase, wherein the buffer comprises an aqueous solution of acarbose. In some embodiments, the buffer has a pH range of 2-7. In some embodiments, the buffer has a pH range of 3.5-4.5. In some embodiments, the buffer has a pH of 4.4. The buffer may comprise, for example, formate, acetate, citrate-phosphate, or trifluoroacetate, or a combination thereof. In some embodiments, the concentration of formate, acetate, citrate-phosphate, or trifluoroacetate ranges from 0.01-1.0 M. In some embodiments, the concentration of formate, acetate, citrate-phosphate, or trifluoroacetate, or combination thereof, is 0.1 M. In some embodiments, the buffer comprises ammonium formate, ammonium acetate, ammonium citrate-phosphate, ammonium trifluoroacetate, sodium formate, sodium acetate, sodium citrate-phosphate, or sodium trifluoroacetate, or a combination thereof. In some embodiments, the buffer comprises ammonium formate or sodium formate. In some embodiments, the buffer is a sodium buffer or an ammonium buffer. The buffer may be a volatile buffer. As used herein, a "volatile buffer" refers to a buffer that, when concentrated to dryness by evaporation, causes the buffer components to evaporate along with the solvent. A volatile buffer may comprise ammonium formate, for example. Other suitable volatile buffers are known in the art. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a dried blood sample from a newborn screening card. A method may further comprise using the amount of a- glucosidase product to determine whether the dried blood sample is from a candidate for treatment for Pompe disease. A method may further comprise using the amount of a- galactosidase A product to determine whether the dried blood sample is from a candidate for treatment for Fabry disease. A method may further comprise using the amount of a- L-iduronidase product to determine whether the dried blood sample is from a candidate for treatment for mucopolysaccharidosis I (Hurler) disease (MPS IH). A method may further comprise using the amount of a-glucosidase product to determine whether the dried blood sample is from a candidate for treatment for Pompe disease, using the amount of a-galactosidase A product to determine whether the dried blood sample is from a candidate for treatment for Fabry disease, and using the amount of a-L-iduronidase product to determine whether the dried blood sample is from a candidate for treatment for mucopolysaccharidosis I (Hurler) disease (MPS IH). A sample may further comprise an α-glucosidase internal standard, an α-galactosidase internal standard, and an a-L- iduronidase internal standard. A method may further comprise extracting the enzyme product solution to provide the α-glucosidase internal standard, the a-glucosidase product, the α-galactosidase internal standard, the α-galactosidase product, the a-L- iduronidase internal standard, and the α-L-iduronidase product when the sample comprises α-glucosidase, α-galactosidase, and α-L-iduronidase. In some embodiments, extracting the enzyme product solution comprises liquid extraction with an organic solution. In some embodiments, extracting the enzyme product solution comprises solid phase extraction. A method may further comprise quenching the enzymatic reaction prior to extraction. In some embodiments, the aqueous solution of acarbose has an acarbose concentration of 5-10 μΜ. A method may further comprise determining the quantity of the α-glucosidase product, the α-galactosidase product, and the α-L-iduronidase product. In some embodiments, determining the quantity of the α-glucosidase product, the a- galactosidase product, and the α-L-iduronidase product comprises determining the ratio of the α-glucosidase product, the α-galactosidase product, and the a-L-iduronidase product to the α-glucosidase internal standard, the α-galactosidase internal standard, and the α-L-iduronidase internal standard, respectively, by mass spectrometric analysis. In some embodiments, determining the quantity of the α-glucosidase product, the a- galactosidase product, and the α-L-iduronidase product comprises tandem mass spectrometric analysis. In some embodiments, determining the quantity of the a- glucosidase product, the a-galactosidase product, and the a-L-iduronidase product comprises tandem mass spectrometric analysis in which the parent ions of the products and internal standards are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, in some embodiments, determining the quantity of the α-glucosidase product, the a- galactosidase product, and the α-L-iduronidase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the α-glucosidase product, the a-galactosidase product, and the a-L- iduronidase product.
Further provided is a method for determining the presence or absence of a- glucosidase enzymatic activity, α-galactosidase enzymatic activity, and a-L-iduronidase enzymatic activity in a sample, comprising: (a) incubating an α-glucosidase substrate, an α-galactosidase substrate, an α-L-iduronidase substrate with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an α-glucosidase product, an α-galactosidase product, and an α-L-iduronidase product when the sample comprises α-glucosidase, α-galactosidase, and α-L-iduronidase; and (b) determining the presence or absence of the a-glucosidase product, the α-galactosidase product, and the α-L-iduronidase product, wherein the presence of α-glucosidase product indicates the presence of α-glucosidase enzymatic activity, wherein the presence of α-galactosidase product indicates the presence of a- galactosidase enzymatic activity, and wherein the presence of α-L-iduronidase product indicates the presence of α-L-iduronidase activity, and wherein the absence of a- glucosidase product indicates the absence of α-glucosidase enzymatic activity, wherein the absence of α-galactosidase product indicates the absence of α-galactosidase enzymatic activity, and wherein the absence of α-L-iduronidase product indicates the absence of a- L-iduronidase activity. A sample may further comprise an α-glucosidase internal standard, an α-galactosidase internal standard, and an α-L-iduronidase internal standard. A method may further comprise extracting the enzyme product solution to provide the a- glucosidase product, the α-glucosidase internal standard, the α-galactosidase product, the α-galactosidase internal standard, the α-L-iduronidase product, and the a-L-iduronidase internal standard when the sample comprises α-glucosidase, α-galactosidase, and a-L- iduronidase. A method may further comprise determining the quantity of the a- glucosidase product, the α-galactosidase product, and the α-L-iduronidase product. Also provided is a method for determining the quantity of a a-glucosidase product, a a-galactosidase product, and a a-L-iduronidase product in a blood sample, comprising: (a) contacting a blood sample with a first buffer solution to provide a solution; (b) incubating an a-glucosidase substrate, an a-glucosidase internal standard, an α-galactosidase substrate, an α-galactosidase internal standard, an a-L-iduronidase substrate, and an α-L-iduronidase internal standard with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an α-glucosidase product, an α-galactosidase product, and an α-L-iduronidase product when the sample comprises α-glucosidase, α-galactosidase, and α-L-iduronidase, wherein the buffer comprises an aqueous solution of acarbose; and (c) determining the quantity of the α-glucosidase product, the α-galactosidase product, and the α-L-iduronidase product by tandem mass spectrometric analysis. A method may further comprise extracting the enzyme product solution with an organic solvent to provide an organic phase comprising the α-glucosidase product, the a-glucosidase internal standard, the α-galactosidase product, the α-galactosidase internal standard, the α-L-iduronidase product, and the α-L-iduronidase internal standard when the sample comprises α-glucosidase, α-galactosidase, and α-L-iduronidase. In some embodiments, (c) comprises: (i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and (ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 6-sulfatase product. In some embodiments, the blood sample is a dried blood sample from a newborn. In some embodiments, (a) is further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution. A method may further comprise: (e) using the amount of α-glucosidase product to predict whether the newborn is a candidate for treatment of Pompe disease; (f) using the amount of α-galactosidase product to predict whether the newborn is a candidate for treatment of Fabry disease; or (g) using the amount of a-L- iduronidase product to predict whether the newborn is a candidate for treatment of mucopolysaccharidosis I (Hurler) disease (MPS IH). A method may further comprise: (e) using the amount of α-glucosidase product to predict whether the newborn is a candidate for treatment of Pompe disease; (f) using the amount of α-galactosidase product to predict whether the newborn is a candidate for treatment of Fabry disease; and (g) using the amount of a-L-iduronidase product to predict whether the newborn is a candidate for treatment of mucopolysaccharidosis I (Hurler) disease (MPS IH).
The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. It is specifically contemplated that any listing of items using the term "or" means that any of those listed items may also be specifically excluded from the related embodiment.
Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In any embodiment discussed in the context of a numerical value used in conjunction with the term "about," it is specifically contemplated that the term about can be omitted.
As used herein, "a" or "an" may mean one or more, unless clearly indicated otherwise.
The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.
As an alternative to or in addition to "comprising," any embodiment herein may recite "consisting of." The transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim.
It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method or system of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
EXAMPLES
Example 1: Preparation of Maroteaux-Lamy (MPS VI) and Morquio A (MPS rVA) Substrates and Internal Standards and Related Enzymatic Specificity Assays Structures of Maroteaux-Lamy substrate 1 and internal standard 3 and Morquio A substrate 2 and internal standard 4 are shown in FIGURE 1. Preparation of MPS ΓΥΑ substrate 2 and internal standards 3 and 4 is shown in FIGURE 2. Preparation of MPS VI substrate 1 is shown in FIGURE 3.
Overview. The synthesis of both the compounds (Schemes 1 and 2 above) started with the amide coupling of 7-hydroxycoumarinyl-4-acetic acid with either mono-Boc protected 1,6-hexanediamine or 1,5-pentanediamine. The resulting substituted coumarins were glycosylated with two different glycosyl halide donors under phase transfer catalysis to afford the β-glycosides (Carriere et al., J. Mol. Cat. A: Chemical, 154, 9-22 (2000)). The acetate esters were then deprotected to afford glycosides 8, 3, 4, and 10. Glycosides 3 and 4 may be used as internal standards for the enzymatic assays and were used without further elaboration. The MPS IVA substrate 2 was synthesized from glycoside 10 by selective sulfation of the primary 6-hydroxyl group over the secondary 2, 3 and 4-hydroxyl groups as revealed by the downfield of H-6 in the sulfate relative to the non-sulfate (Sawada et al., Carbohydr. Res., 340, 1983-96 (2005)).
The MPS VI substrate (1) required further synthetic manipulations in order to install the sulfate at the more hindered 4-hydroxy of the sugar. Therefore, the less hindered 3- and 6-hydroxyls were selectively benzoylated to afford dibenzoate 9. The glucosamine was then converted to a galactosamine by inversion of they 4-hydroxyl by formation of the triflate and displacement with sodium nitrite to afford 10. Finally, sulfation of the free hydroxyl followed by cleavage of the benzoate protecting groups gave the desired MPS VI substrate (1).
Synthetic Details. Unless otherwise indicated, all anhydrous solvents were commercially obtained and stored under nitrogen. Reactions were performed under an atmosphere of dry nitrogen in oven dried glassware and were monitored for completeness by thin layer chromatography (TLC) using silica gel 60 F-254 (0.25 mm) plates with detection under UV light. !HNMR spectra were recorded on dilute solutions in CDCI3,
CD3OD or DMSO-d at 300 MHz. Chemical shifts are reported in parts per million (δ) downfield from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Electrospray ionization mass spectra were acquired on a Bruker Esquire LC00066. Flash chromatography was carried out with silica gel (40-63 micron). Preparative reverse phase HPLC was performed on an automated Varian Prep star system using a gradient of 20% MeOH to 100% MeOH (with 0.1% trifluoroacetic acid) at 12 mL/min over 30 min using a YMC S5 ODS column (20x100 mm, Waters Inc.).
Synthesis of Reagents for MPS IVA Assay: To a stirred ice-cooled solution of 33% hydrobromic acid (50 mL) in acetic acid under nitrogen was added β-D-galactose pentaacetate (4.68 g, 12 mmol) and stirring continued for 15 minutes. Then the reaction mixture was brought to room temperature and stirred for 2 hrs. The mixture was diluted with toluene and rotary evaporated to a residue which was then diluted with 250 mL of ethyl acetate followed by successive washing with 250 mL of cold saturated NaHCC^ and 150 mL of cold brine solutions.
The organic layer was dried over MgSC^, filtered and concentrated under vacuum to yield the crude bromide derivative which was used as such for the next step.
Te/t-butyl 5-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetamido)pentylcarbamate (6b). To a stirred ice-cooled mixture of 7 -hydroxycoumarin-4- acetic acid (2.20 g, 10 mmol) and N-Boc- l,5-diaminopentane (2.22 g, 11 mmol) in DMF (2 mL) and anhydrous dichloromethane (20 mL) was added l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.43 g, 18 mmol) and the stirring was continued overnight at room temperature. TLC in CHC13 : MeOH : AcOH : H20 (96 : 12 : 4: 1) and detection with UV light showed the complete consumption of the acid. The reaction mixture was diluted with 10 mL CH2C12 and 20 mL acetone and washed successively with water (20 mL, pH 8), 1 M HC1 : H20 (10 mL : 20 mL, pH 1), 5% NaHC03 : water (10 mL : 10 mL, pH 9) and finally with water (20 mL, pH 7). The aqueous layer was back extracted with DCM and dried over MgSC^, filtered and rotary evaporated to a solid residue which was successively washed with acetone to yield the desired product (2.0 g, 4.95 mmol) in 50% yield. ¾ NMR (300 MHz, DMSO-d6) δ 8.15 (brs, 1H), 7.59 (d, J = 11.9 Hz, 1H), 6.80-
6.73 (m, 2H), 6.11 (s, 1H), 3.62 (s, 2H), 3.04 (m, 2H), 2.86 (m, 2H), 1.37 (s, 13H), 1.21 (m, 2H).
(2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(4-(2-(5-(ieri-butoxy carbonyl amino) pentyl amino)-2-oxoethyl)-2-oxo-2H-chromen-7-yloxy)tetrahydro-2H-pyran-3,4,5-triyl tri acetate. 7-Hydroxycoumarin-4-acetamide 6b (1.70 g, 4.20 mmol), acetobromogalactose 9 (0.844 g, 2.10 mmol) and tetrabutyl ammonium hydrogen sulfate (0.709 g, 2.10 mmol) were stirred in equimolar ratio of CH2C12 (4.5 mL) and 1 M NaOH
(4.5 mL) at room temperature for 50 minutes. The reaction mixture was diluted with ethyl acetate (30 mL) and successively washed the organic phase with 30 mL of 1 M NaOH (10 mL x 3), 60 mL of water (30 mL x 2) and finally with 15 mL of saturated brine solution. The organic phase was dried over MgSC^, filtered and rotary evaporated to a yellow semisolid. Column chromatography over silica gel in CHCI3 : EtOAc : (CH3)2CHOH (91 : 6 : 3) afforded the desired product (1.60 g, 2.18 mmol) in 52% yield. !H NMR (300 MHz, DMSO-d6) δ 8.17 (brs, 1H), 7.72 (d, J = 8.8 Hz, 1H ), 7.06 (d, J =
2.2 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 6.74 (brs, 1H), 6.31 (s, 1H), 5.65 (d, J = 6.7 Hz,
1H), 5.37 (s, 1H), 5.25 (d, J = 5.7 Hz, 2H), 4.50 (t, J = 6.4 Hz, 1H), 4.11 (d, J = 6.2 Hz, 2H), 3.68 (s, 2H), 3.03 (t, J = 5.8 Hz, 2H), 2.87 (t, J = 6.3 Hz, 2H), 2.15 (s, 3H), 2.04 (s,
3H), 2.02 (s, 3H), 1.95 (s, 3H), 1.36 (s, 13H), 1.23-1.20 (m, 2H).
Γέτί-butyl 5-(2-(2-oxo-7-((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yloxy)-2H-chromen-4-yl)acetamido)pentylcarbamate (10). To an ice-cooled stirred solution of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(2-(5-(ie/t- butoxy carbonyl amino) pentyl amino)-2-oxoethyl)-2-oxo-2H-chromen-7- yloxy)tetrahydro-2H-pyran-3,4,5-triyl tri acetate (1.46 g, 2.0 mmol) in anhydrous methanol (10 mL) was added a solution of 0.5 M sodium methoxide in methanol (66.7 μί, 1.0 mmol) dropwise and then stirring continued at 0 °C for 1.5 hrs. The reaction mixture was neutralized with Amberlite® IR- 120 (H+), filtered and concentrated to a solid residue. Column chromatography of the latter over silica gel in CH2CI2 : MeOH (9 :
1) afforded the desired compound (0.60 g, 1.06 mmol) in 53% yield. ¾ NMR (300 MHz, DMSO-d6) δ 8.17 (brs, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.05-7.01 (m, 2H), 6.74 (brs, 1H),
6.26 (s, 1H), 5.23 (d, J = 5.1 Hz, 1H), 4.98 (d, J = 7.5 Hz, 1H), 4.89 (d, J = 5.5 Hz, 1H), 4.68 (s, 1H), 4.54 (d, J = 4.3 Hz, 1H), 3.67 (s, 2H), 3.51 (s, 2H), 3.04 (t, J = 6.3 Hz, 2H), 2.88 (t, J = 6.6 Hz, 2H), 1.36 (s, 13H), 1.23 (brs, 2H).
Sodium ((2R,3R,4S,5R,6S)-6-(4-(2-(5-(ieri-butoxycarbonylamino)pentylamino)- 2-oxoethyl)-2-oxo-2H-chromen-7-yloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- yl)methyl sulfate (2). A mixture of compound 10 (0.096 g, 0.17 mmol) and SO3 - pyridine complex (0.056 g, 0.35 mmol) in dry pyridine (5 mL) was stirred at 40 °C for 5 hrs. The reaction mixture was quenched with MeOH (8 mL) and solution passed through a column of DOWEX 50WX8 (Na+ form) and eluted with methanol. The eluent was concentrated under vacuum to an oil which was chromatographed over silica gel in CHCI3 : MeOH : H20 (8 : 5 : 1) to yield the target compound 2 (0.051 g, 0.076 mmol) in 45% yield. ¾ NMR (300 MHz, DMSO-d6) 5 7.55 (d, J = 8.4 Hz, 1H), 7.01-6.91 (m, 2H), 6.13 (s, 1H), 4.52 (d, J = 5.1 Hz, 1H), 4.06 (d, J = 1.5 Hz, 1H), 3.93 (d, J = 5.1 Hz, 1H), 3.83 (d, J = 3.6 Hz, 1H), 3.68 (d, J = 8.4 Hz, 1H), 3.60 (d, J = 6.8 Hz, 2H), 3.51 (s, 2H), 3.06 (t, J = 6.4 Hz, 2H), 2.86 (t, J = 6.3 Hz, 2H), 1.29- 1.27 (m, 13H), 1.18 (m, 2H). ESI-MS m/z 645.4 (M - H)-. Internal Standard Synthesis:
Synthesis of iert-butyl 6-(7-hydroxy-coumarin-4-acetamido)hexylcarbamate (6a). Coumarin 5 (3.86 g, 17.5 mmol) and N-Boc- l,6-hexanediamine (4.64 g, 21.6 mmol, 1.2 equiv) were dissolved in DCM (35 mL) and DMF (3.5 mL) and cooled in an ice bath. N- (3-Dimethylaminopropyl)-A^-ethylcarbodiimide hydrochloride (6.00 g, 31.3 mmol, 1.8 equiv) were added and the solution was allowed to warm to rt. After 16 h, the solution was diluted with DCM (10 mL) and acetone (20 mL) and washed with Η20 (20 mL), aq.
HC1 (0.33 M, 30 mL), aq. NaHC03 (2.5%, 20 mL), and H20 (20 mL). Each of the aqueous layers were extracted a second time with a mixture of DCM (40 mL) and acetone (10 mL). The combined organic layers were evaporated followed by evaporation with acetone to afford a yellow solid. This material was recrystallized in from acetone to yield coumarin 6a as a white crystalline solid (2.79 g, 38%). Recrystallization of the mother liquor afforded additional product as a white crystalline solid (1.00 g, 14%, 52% total) Rf 0.33 (5% MeOH in CH2C12); ¾ NMR (500 MHz, DMSO-d6): δ 10.58 (1H, br s), 8.19 (1H, t, J= 5.4 Hz), 7.60 (1H, d, J= 8.7 Hz), 6.78 (1H, dd, J= 8.7, 2.5 Hz), 6.74 (1H, br s), 6.72 (1H, d, J= 2.5 Hz), 6.15 (1H, s), 3.62 (2H, s), 3.04 (2H, td, J= 6.4, 6.0 Hz), 2.87 (2H, ddd, J= 6.8, 6.4, 6.2 Hz), 1.36 (9H, s), 1.42-1.28 (4H, m), 1.25- 1.18 (4H, m); ESI- MS: m/z 419.3 (M + H)+.
(2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(4-(2-(6-(ieri-butoxy carbonylamino) hexyl amino)-2-oxoethyl)-2-oxo-2H-chromen-7-yloxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate. The compound was prepared by the analogous procedure described for the synthesis of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(2-(5-(iert-butoxy carbonyl amino)pentyl amino)-2-oxoethyl)-2-oxo-2H-chromen-7-yloxy)tetrahydro-2H-pyran- 3,4,5-triyl tri acetate. 7-hydroxycoumarin-4-acetamide 6a (1.50 g, 3.59 mmol) and acetobromogalactose (0.72 g, 1.80 mmol) were reacted with tetrabutyl ammonium hydrogen sulfate (1.22 g, 3.60 mmol) to yield the product (1.40 g, 1.87 mmol) in 52% yield. ¾ NMR (300 MHz, CDC13) δ 7.97 (s, 1H), 7.66 (d, J = 9.5 Hz, 1H), 6.94 (s, 1H),
6.30 (s, 1H), 6.10 (brs, 1H), 5.49 (d, J = 7.7 Hz, 1H), 5.16-5.12 (m, 2H), 4.59 (brs, 1H), 4.18-4.12 (m, 3H), 3.66 (s, 2H), 3.22 (dd, J = 6.5, 5.7 Hz, 2H), 3.08 (q, J = 6.4 Hz, 2H), 2.18 (s, 3H), 2.11 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 1.55 (s, 6H), 1.43 (s, 9H), 1.27 (brs, 2H).
Γέτί-butyl 6-(2-(2-oxo-7-((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yloxy)-2H-chromen-4-yl)acetamido)hexylcarbamate (4). The compound was prepared by the analogous procedure described for the synthesis of compound 10. To an ice-cooled stirred solution of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)- 6-(4-(2-(6-(ieri-butoxycarbonylamino)hexylamino)-2-oxoethyl)-2-oxo-2H-chromen-7- yloxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (20 mg, 0.027 mmol) in anhydrous methanol (3 mL) was added a solution of 0.5 M sodium methoxide in methanol (1.66 μί, 0.025 mmol) dropwise to yield the product (10 mg, 0.017 mmol) in 65% yield. !H NMR (300 MHz, CD3OD) 5 7.72 (d, J = 9.3 Hz, 1H), 7.12-7.11 (m, 2H), 6.52 (brs, 1H), 6.29
(s, 1H), 5.01 (d, J = 7.7 Hz, 1H), 4.57 (s, 1H), 3.94-3.82 (m, 2H), 3.78-3.75 (m, 3H), 3.63 (s, 2H), 3.21 (t, J = 6.8 Hz, 2H), 3.02 (t, J = 6.0 Hz, 2H), 1.52 (m, 2H), 1.44 (s, 11H), 1.32 (m, 4H). ESI-MS: m/z 581.3 (M + H)+.
Synthesis of Reagents for MPS VI assay:
Synthesis of ie/t-butyl 5-(7-hydroxy-coumarin-4-acetamido)pentylcarbamate (6b). Coumarin 5 (1.10 g, 5.0 mmol) and N-Boc-l,5-pentanediamine (1.12 g, 5.5 mmol, 1.1 equiv) were dissolved in DCM (10 mL) and DMF (1 mL) and cooled in an ice bath. N- (3-Dimethylaminopropyl)-A^-ethylcarbodiimide hydrochloride (1.73 g, 9.0 mmol, 1.8 equiv) were added and the solution was allowed to warm to rt. After 16 h, the solution was diluted with DCM (5 mL) and acetone (10 mL) and washed with ¾0 (5 mL), aq. HC1 (0.33 M, 5 mL), aq. NaHC03 (2.5%, 5 mL), and H20 (5 mL). Each of the aqueous layers were extracted a second time with a mixture of DCM (20 mL) and acetone (5 mL). The combined organic layers were evaporated followed by evaporation with acetone to afford a yellow solid. This material was recrystallized from acetone to yield coumarin 6b as a white crystalline solid (1.25 g, 62%). Rf0.33 (5% MeOH in CH2C12); ¾ NMR (300 MHz, DMSO-d6): δ 10.59 (1H, br s), 8.18 (1H, t, J= 5.4 Hz), 7.59 (1H, d, J= 8.8 Hz),
6.78 (1H, d, J= 9.8 Hz), 6.75 (1H, dd, J= 5.7, 5.4 Hz), 6.72 (1H, br s), 6.15 (1H, s), 3.62 (2H, s), 3.03 (1H, app q, J= 6.3 Hz), 2.87 (1H, app q, J= 6.3 Hz), 1.36 (9H, s), 1.42- 1.30
(4H, m), 1.24- 1.17 (2H, m); ESI-MS: m/z 405.2 (M + H)+.
Synthesis of 7-O-(2-acetamido-3,4,6-tri-0-acetyl-2-deoxy- -D-glucopyranosyl) ie/t-butyl 6-(7-hydroxy-coumarin-4-acetamido)hexylcarbamate (A). A solution of 2 acetamido-3,4,6-tri-O-acetyl-2-deoxy-P-D-glycopyranosyl chloride (7a, 1.10 g, 3.0 mmol), coumarin 6a (2.51 g, 6.0 mmol, 2 equiv) and tetrabutyl ammonium hydrogen sulfate (1.02 g, 3.0 mmol, 1 equiv) in DCM (10 mL) was vigorously stirred and aqueous
NaOH was added (1 M, 10 mL). The solution turned bright yellow and solid began precipitating. After 45 min, no glycosyl chloride remained by TLC (H2SO4 stain). The precipitate was filtered and washed 3X with DCM (20 mL) and H20 (20 mL). The precipitate was recrystallized from 1-butanol to afford glycoside A as an off-white powder (1.058 g, 47%). The layers of the filtrate from the reaction were then saturated with citric acid and extracted with DCM (20 mL, 3X). The layers were separated, and the organic layers were evaporated to afford recovered coumarin 6a which was recrystallized from acetone (1.26 g, 50% of starting coumarin recovered). Ry0.19 (5% MeOH in DCM);
!H NMR (500 MHz, DMSO-d6): δ 8.16 (1H, t, J= 6.1 Hz), 8.11 (1H, d, J= 9.2 Hz) 7.70
(1H, d, J= 8.8 Hz), 7.12 (1H, d, J= 2.6 Hz), 6.98 (1H, dd, J= 8.8, 2.6 Hz), 6.75 (1H, t, J= 5.3 Hz), 6.30 (1H, s), 5.50 (1H, d, J= 8.4 Hz), 5.22 (1H, dd, J= 10.3, 9.5 Hz), 4.93 (1H, dd, J= 9.9, 9.5 Hz), 4.25 (1H, ddd, J= 5.3, 2.4 Hz), 4.19 (1H, dd, J= 11.9, 5.3 Hz), 4.10-
4.01 (2H, m), 3.68 (2H, s), 3.04 (1H, ddd, J= 7.0, 6.8, 6.1 Hz), 2.88 (1H, dd, J= 6.8, 6.6,
6.2 Hz), 2.10 (3H, s), 2.10 (3H, s), 1.95 (3H, s), 1.77 (3H, s), 1.36 (9H, s), 1.43- 1.29 (4H, m), 1.25-1.18 (4H, m); ESI-MS: m/z Π0.2 (M + Na)+.
Synthesis of 7-O-(2-acetamido-2-deoxy- -D-glucopyranosyl) iert-butyl 6-(7- hydroxycoumarin-4-acetamido)hexylcarbamate (8). Sodium methoxide in methanol (0.5 M, 0.23 mL) was added to a solution of triacetyl glycoside A (871 mg, 1.16 mmol) in methanol (24 mL) and chloroform (12 mL). After 4 h, no starting material remained by TLC, and DOWEX 50WX8 (H+ form) was added until solution was neutral by litmus paper. The solution was filtered, rinsing with methanol and the combined organic layers were evaporated to afford the trialcohol 8 as a white solid (687 mg, 95%). Ry0.32 (20% MeOH in DCM); ¾ NMR (500 MHz, DMSO-d6): δ 8.16 (1H, dd, J= 5.8, 5.4 Hz), 7.81
(1H, d, J= 8.8 Hz), 7.68 (1H, d, J= 8.1 Hz), 7.00 (1H, d, J= 2.5 Hz), 6.93 (1H, dd, J= 8.8, 2.5 Hz), 6.73 (1H, t, J=5.2 Hz), 6.27 (1H, s), 5.13 (2H, dd, J= 6.9, 1.3 Hz), 5.10 (1H, d, J= 5.4 Hz), 4.62 (1H, t, J= 5.7 Hz), 3.76-3.68 (2H, m), 3.67 (2H, s) 3.52-3.38 (3H, m), 3.19 (1H, ddd, J= 9.1, 5.7, 3.5 Hz), 3.04 (1H, ddd, J= 6.9, 6.6, 6.0 Hz), 2.88 (1H, td, J= 6.6, 6.3 Hz), 1.81 (3H, s), 1.36 (9H, s), 1.42-1.31 (4H, m), 1.27- 1.17 (4H, m); ESI-MS: m/z 622.3 (M + H)+.
Synthesis of 7-O-(2-acetamido-3,4,6-tri-0-acetyl-2-deoxy- -D-galactopyranosyl) ie/t-butyl 5-(7-hydroxy-coumarin-4-acetamido)pentylcarbamate (B). A solution of 2- acetamido-3,4,5-tri-O-acetyl-2-deoxy-P-D-galactopyranosyl chloride (7b, 460 mg, 1.26 mmol), coumarin 6b (1.02 g, 2.5 mmol, 2 equiv) and tetrabutyl ammonium hydrogen sulfate (427 g, 1.26 mmol, 1 equiv) in DCM (4.2 mL) was vigorously stirred and aqueous NaOH was added (1 M, 4.2 mL). The solution turned bright yellow and solid began precipitating. After 1 h 15 min, no glycosyl chloride remained by TLC (H2SO4 stain). The precipitate was filtered and washed 3X with DCM (20 mL) and Ι¾0 (20 mL) to afford glycoside B as an off-white powder (513 mg, 56%). The layers of the filtrate from the reaction were then saturated with citric acid and extracted with DCM (20 mL, 3X), a fitrate formed during the extraction and was isolated to afford recovered coumarin 6b (286 mg, 28% of starting coumarin recovered). The layers were separated, and the organic layers were evaporated to afford recovered coumarin 6b and product. The residue was taken up in MeOH and filtered. The solid was galactopyranoside (180 mg, 20%: 76% total). R^O.19 (5% MeOH in DCM); ¾ NMR (500 MHz, DMSO-d6): δ 8.18 (1H, dd, J= 5.5, 5.1 Hz), 8.00 (1H, d, J= 9.2 Hz) 7.71 (1H, d, J= 9.0 Hz), 7.10 (1H, d, J=
2.2 Hz), 6.96 (1H, dd, J= 9.0, 2.2 Hz), 6.76 (1H, dd, J= 5.5, 4.5 Hz), 6.29 (1H, s), 5.39 (1H, d, J= 8.4 Hz), 5.32 (1H, d, J= 3.3 Hz), 5.10 (1H, dd, J= 11.2, 3.3 Hz), 4.43 (1H, dd, J= 6.6, 5.9 Hz), 4.22 (1H, ddd, J= 10.8, 9.2, 9.0 Hz), 4.15-4.04 (2H, m), 3.68 (2H, s),
3.03 (1H, ddd, J= 6.8, 6.6, 6.1 Hz), 2.87 (1H, dd, J= 6.6, 6.4, 6.2 Hz), 2.14 (3H, s), 2.02 (3H, s), 1.93 (3H, s), 1.79 (3H, s), 1.36 (9H, s), 1.42- 1.31 (4H, m), 1.31-1.15 (4H, m);
ESI-MS: m/z 734.3 (M + H)+.
Synthesis of 7-O-(2-acetamido-2-deoxy- -D-galactopyranosyl) ie/t-butyl 5-(7- hydroxy-coumarin-4-acetamido)pentylcarbamate (3). Sodium methoxide in methanol (0.5 M, 0.14 mL) was added to a solution of triacetyl glycoside B (508 mg, 0.69 mmol) in methanol (14 mL) and chloroform (7 mL). After 4 h, no starting material remained by TLC, and DOWEX 50WX8 (H+ form) was added until solution was neutral by litmus paper. The solution was filtered, rinsing with methanol (-400 mL) and the combined organic layers were evaporated to afford the trialcohol 3 as a white solid (351 mg, 83%). Rf 0.32 (20% MeOH in DCM); ¾ NMR (500 MHz, DMSO-d6): δ 8.18 (1H, t, J= 5.7 Hz), 7.76 (1H, d, J= 9.2 Hz), 7.68 (1H, d, J= 9.0 Hz), 7.01 (1H, d, J= 2.6 Hz), 6.94 (1H, dd, J= 9.0, 2.6 Hz), 6.77 (1H, t, J= 5.7 Hz), 6.27 (1H, s), 5.10 (1H, d, J= 8.3 Hz), 4.79 (1H, d, J= 6.4 Hz), 4.72-4.68 (2H, m) 4.01 (1H, ddd, J= 10.1, 9.2, 8.6 Hz), 3.73 (1H, t, J= 3.9 Hz) 3.66 (2H, s), 3.66-3.62 (1H, m), 3.61-3.49 (3H, m), 3.03 (2H, ddd, J= 6.8, 6.1, 5.7 Hz), 2.87 (2H, ddd, J= 6.8, 6.6, 5.7 Hz) 1.80 (3H, s), 1.36 (9H, s), 1.42- 1.31 (4H, m), 1.26- 1.18 (2H, m); ESI-MS: m/z 630.2 (M + Na)+.
Synthesis of 7-O-(2-acetamido-3,6-di-0-benzoyl-2-deoxy- -D-glucopyranosyl) ie/t-butyl 6-(7-hydroxy-coumarin-4-acetamido)hexylcarbamate (9). Triol 8 (425 mg, 0.68 mmol) was taken up in pyridine (6.8 mL) and cooled in an ice bath. Added benzoyl chloride (79 μί, 0.68 mmol) and solution was stirred for 1 h until most of the solid had dissolved. More benzoyl chloride (95 μί, 0.82 mmol) was added and the solution was stirred for an additional 2 h. Methanol (10 mL) was then added and the solvent was evaporated. The residue was purified by column chromatography (2.5% MeOH in DCM - 5% MeOH in DCM) to afford dibenzoate 9 as a white solid (334 mg, 59%). Ry 0.22 (5% MeOH in DCM); ¾ NMR (500 MHz, DMSO-d6): δ 8.20-8.15 (2H, m), 8.03 (2H, d,
J= 6.9 Hz) 7.99 (2H, d, J= 7.3 Hz), 7.70-7.64 (3H, m), 7.60-7.53 (4H, m), 7.22 (1H, d, J= 2.2 Hz), 7.00 (1H, dd, J= 8.8, 2.5 Hz), 6.75 (1H, dd, J= 5.7, 5.4 Hz), 6.33 (1H, s), 5.89 (1H, d, J= 6.0 Hz), 5.55 (1H, d, J= 8.5 Hz), 5.31 (1H, dd, J= 10.1, 9.5 Hz), 4.69 (1H, d, J= 10.4 Hz), 4.38 (1H, dd, J= 12.0, 6.9 Hz), 4.27-4.19 (2H, m), 3.78 (1H, ddd, J= 9.5, 6.0, 5.7 Hz), 3.68 (1H, d, J= 16.7 Hz), 3.67 (1H, d, J= 16.7 Hz), 3.05 (1H, td, J= 6.6, 6.3 Hz), 2.89 (1H, td, J= 6.6, 6.3 Hz), 1.68 (3H, s), 1.37 (9H, s), 1.42- 1.30 (4H, m), 1.26-1.19 (4H, m); ESI-MS: m/z 852.3 (M + Na)+.
Synthesis of 7-O-(2-acetamido-3,6-di-0-benzoyl-2-deoxy- -D-galactopyranosyl) ie/t-butyl 6-(7-hydroxy-coumarin-4-acetamido)hexylcarbamate (10). Alcohol 9 (334 mg, 0.40 mmol) was taken up in pyridine (2.6 mL) and DCM (8 mL) and cooled in an ice bath. Added triflic anhydride (85
Figure imgf000034_0001
0.5 mmol) and the solid dissolved and the solution turned orange. After 1 h additional triflic anhydride was added (85
Figure imgf000034_0002
0.5 mmol). After an additional 2 h, the reaction poured over aq. HC1 (0.1 M) and diluted with CHCI3. The layers were separated and the organic layer was washed with water, aq. NaHC03 (sat.), and water. The solvent was then evaporated to afford an orange oil that was used in the next reaction without further purification. The triflate was taken up in DMF (0.4 mL) and sodium nitrite (222 mg, 3.2 mmol) was added. The solution was stirred overnight, then poured over water and diluted with chloroform. The layers were separated and the aqueous layer was extracted with chloroform. The combined organic layers were washed with aq. HC1 (0.1 M) and brine, dried (MgSO^ and evaporated. The residue was recrystallized from MeOH to afford galactopyranoside 10 as a white crystalline solid (167 mg, 50%). Rf O.22 (5% MeOH in DCM); ¾ NMR (500 MHz, DMSO-d6): δ 8.15 (1H, dd, J= 5.7, 5.4 Hz), 8.03-7.97 (5H, m), 7.70-7.63 (3H, m), 7.59-7.53 (4H, m), 7.20 (1H, d, J= 2.5 Hz), 6.99 (1H, dd, J= 8.8, 2.5 Hz), 6.73 (1H, t, J=5.0 Hz), 6.31 (1H, s), 5.59 (1H, d, J= 6.0 Hz), 5.45 (1H, d, J= 8.5 Hz), 5.07 (1H, dd, J= 11.0, 3.2 Hz), 4.60 (1H, dd, J= 19.9, 9.0 Hz), 4.48 (1H, d, J= 7.3 Hz), 4.44 (1H, d, J= 8.2 Hz), 4.42 (1H, d, J= 8.2 Hz), 4.22 (1H, dd, J= 5.7, 3.2 Hz), 3.68 (1H, d, J= 15.3 Hz), 3.65 (1H, d, J= 15.3 Hz), 3.04 (1H, ddd, J= 6.9, 6.6, 6.0 Hz), 2.88 (1H, td, J= 6.6, 6.3 Hz), 1.74 (3H, s), 1.36 (9H, s),
1.41- 1.30 (4H, m), 1.25- 1.18 (4H, m); ESI-MS: m/z 852.3 (M + Na)+.
Synthesis of 7-0-(2-acetamido-3,6-di-O-benzoyl-2-deoxy-4-0-sulfonato- -D- galactopyranosyl) ie/t-butyl 6-(7-hydroxy-coumarin-4-acetamido)hexylcarbamate sodium salt (C). Alcohol 10 (87 mg, 0.11 mmol) was taken up in pyridine (2.6 mL) and S03-pyridine (33 mg, 0.21 mmol) was added. After 3 h, MeOH (1 mL) was added and the solution was applied to a plug of DOWEX 50WX8 (Na+ form) and rinsed with MeOH (20 mL). The eluent was collected and evaporated. The residue was purified by column chromatography (10% MeOH in DCM - 20% MeOH in DCM) to afford sulfate C as a white powder (55 mg, 56%). Rf 0.71 (20% MeOH in DCM); ¾ NMR (500 MHz,
DMSO-d6): δ 8.17 (1H, t, J= 5.5 Hz), 8.04-7.94 (5H, m), 7.65-7.59 (3H, m), 7.56 (2H, dd, J= 7.9, 7.5 Hz), 7.49 (2H, dd, J= 7.7, 7.5 Hz), 7.16 (1H, d, J= 2.4 Hz), 6.97 (1H, dd, J= 8.6, 2.4 Hz), 6.76 (1H, t, J=6.0 Hz), 6.30 (1H, s), 5.45 (1H, d, J= 8.4 Hz), 5.13 (1H, dd, J= 11.2, 3.3 Hz), 4.79 (1H, d, J= 3.2 Hz), 4.59 (1H, dd, J= 11.7, 3.1 Hz), 4.52-4.37 (3H, m), 3.67 (1H, d, J= 15.2 Hz), 3.63 (1H, d, J= 15.2 Hz), 3.04 (1H, ddd, J= 6.8, 6.6, 6.0 Hz), 2.87 (1H, dt, J= 7.0, 6.2 Hz), 1.74 (3H, s), 1.36 (9H, s), 1.40-1.31 (4H, m), 1.28- 1.18 (4H, m); ESI-MS: m/z 908.9 (M - H)-.
Synthesis of 7-O-(2-acetamido-2-deoxy-4-0-sulfonato- -D-galactopyranosyl) ie/t-butyl 6-(7-hydroxy-coumarin-4-acetamido)hexylcarbamate sodium salt (1). Compound C (105 mg, 0.11 mmol) was taken up in MeOH (4 mL) and sodium methoxide (0.22 mL, 0.5 M, 0.11 mmol) was added. The reaction was stirred and monitored by MS (EST). After 5 h, all dibenzoate (908 m/z) and monobenzoate (804 m/z) were converted to diol (700 m/z). The reaction was quenched by adding Amberlite® IRC-50 (H+ form) until the solution was neutral by litmus paper. The solution was filtered and solvent evaporated to afford a yellow solid. Purification by column chromatography (silica gel, CHCl3:MeOH with 5% H20, 0% to 30%) afforded the deprotected galactopyranoside 1 as a white solid (62 mg, 77%). Ry O. lO (CHC^iMeOH with 5% H20, 3: 1); JH NMR (500 MHz, DMSO-d6): δ 8.16 (1H, dd, J= 5.8, 5.5 Hz),
7.81 (1H, d, J= 9.2 Hz), 7.68 (1H, d, J= 8.9 Hz), 7.01 (1H, d, J= 2.4 Hz), 6.93 (1H, dd, J= 9.2, 2.4 Hz), 6.76 (1H, t, J=5.8 Hz), 6.27 (1H, s), 5.10 (1H, d, J= 8.5 Hz), 4.82 (1H, d, J= 6.7 Hz), 4.53 (1H, dd, J= 7.6, 5.2 Hz), 4.51 (1H, d, J= 3.1 Hz), 3.93 (1H, ddd, J= 10.7, 8.9, 8.5 Hz), 3.86 (1H, dd, J= 7.0, 5.8 Hz), 3.74 (1H, ddd, J= 10.7, 6.7, 3.1 Hz), 3.66 (2H, br s), 3.56 (1H, ddd, J= 11.3, 5.8, 5.2 Hz) 3.49 (1H, ddd, J= 11.3, 7.3, 7.0 Hz) 3.04 (1H, app dt, J= 7.0, 6.7 Hz), 2.88 (1H, app td, J= 7.0, 6.1 Hz), 1.79 (3H, s), 1.36 (9H, s), 1.41- 1.30 (4H, m), 1.25- 1.18 (4H, m); ESI-MS: m/z 700.8 (M - H)-.
Enzymatic Assays for Specificity. With the two substrates and two internal standards in hand, enzymatic activity was studied. The enzymatic activity was measured by incubating a solution of substrate and internal standard with a 2 mm diameter dried blood spot punch for 16 hr. The amount of product was quantified by tandem mass spectrometer using the internal standards. For MPS VI, the range of activity measured with 10 dried blood spots from healthy individuals was 1.6-10.5 μιηοΐ hr1 (L blood)" 1 compared to 0.08 μιηοΐ hr 1 (L blood)-1 using a dried blood spot from an MPS VI patient. For MPV IVA, the values were 0.21-0.35 μιηοΐ hr1 (L blood)" 1 for 30 dried blood spots from healthy patients and 0.00039-0.00043 μιηοΐ hr1 (L blood)- 1 from 6 patients with MPS IVA. These results serve to illustrate that the substrates are highly specific for the respective enzymes.
Example 2: Assay of MPS VI (Maroteaux-Lamy Syndrome)
In general, this method of this Example is an assay of N- acetylgalactosamine 4- sulfatase (aryl sulfatase B) activity in dried blood spots (DBS) for the early detection of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) in newborn screening. Other assays and MPS VI substrates described herein may be employed. A synthetic substrate, N-acetylgalactosamine-4-sulfate moiety glycosidically linked to a hydrophobic residue and furnished with a iert-butyloxycarbamido group as a marker for specific mass spectrometric fragmentation, was employed. Incubation with aryl sulfatase B present in DBS converted the substrate to a desulfated product which was detected by electrospray tandem mass spectrometry and quantified using a homologous internal standard. Assay and work-up procedures were optimized to be compatible with the work flow in newborn screening laboratories. Analysis of DBS from human newborns showed clear distinction of aryl sulfatase B activity from 89 healthy individuals where it ranged between 1.4-16.9 μιηο1/(1ι x L blood), with an average activity of 7.4 μιηοΙ/hr/L blood, and an MPS VI patient that had an activity of 0.12 μιηο1/(1ι x L blood). Results are also reported for the aryl sulfatase B assay in DBS from groups of normal felines and felines affected with MPS VI. In addition, the presence of the umbelliferyl moiety in MPS VI substrates described herein would allow for fluorometric assays should that be desirable by laboratories who desire not to use ESI-MS/MS (see van Diggelen et al., Clin. Chim. Acta, 187: 131-40 (1990)).
Materials. The substrate (7-0-(2-acetamido-2-deoxy-4-0-sulfonato-P-D- galactopyranosyl) ie/t-butyl 6-(7-hydroxy-coumarin-4-acetamido)hexylcarbamate sodium salt, ASB-S) and internal standard (7-0-(2-acetamido-2-deoxy-P-D-galactopyranosyl) ie/t-butyl 5-(7-hydroxy-coumarin-4-acetamido)pentylcarbamate, ASB-IS) were synthesized as described in Example 1. Dried blood spots (DBS) from anonymous newborns were obtained from the Washington State Newborn Screening Laboratory with approval of the Washington State Institutional Review Board (IRB). Additional DBS from two unaffected adult donors were used to develop the assays and to characterize the enzyme kinetics. DBS from a single affected anonymous donor were obtained from BioMarin Pharmaceutical Inc. (Novato, CA). The MPS VI affected patient had been diagnosed previously with established clinical and biochemical procedures. DBS from normal and affected felines were obtained from BioMarin Pharmaceutical Inc. DBS were kept at ambient temperature during shipment (<10 days) and then stored at -20 °C in Ziploc® plastic bags (one bag sealed inside a second bag). Ziploc® bags were kept in a sealed plastic box containing desiccant (anhydrous CaSC^ granules).
Standard Assay Using Liquid-Liquid Extraction. A 3-mm punch from a dried blood spot was placed in a single well of a 96-deep well plate (1 mL, Costar, Fisher Scientific, Cat. No. 09-761-116) containing 20 μΐ^ of assay cocktail (100 mM sodium formate, pH 4.0, 30 mM lead (II) acetate, 1 mM ASB substrate (ASB-S) and 5 μΜ ASB internal standard (ASB-IS)). The plate was centrifuged briefly to bring all components to the well bottom. The plate was sealed and incubated at 37 °C for 16 hours in a thermostated air shaker. The sample was quenched with 100 μΐ^ of water containing 16 mg of diethylaminoethylcellulose resin (DEAE cellulose, 20 mg, Whatman, Cat. No. 4057-200), leading to a precipitate. A blank assay was carried out as above but using a blank paper punch instead of a DBS. Samples were submitted to liquid-liquid extraction with the addition of 400 μΐ^ ethyl acetate. The solutions were mixed by aspiration with a 12-channel pipette (lOx), then centrifuged at 3000 rpm for 5 min to separate the layers. 300 μΐ^ of the top layer were transferred to a new 96-well plate (0.5 mL, Axygen Scientific, VWR International, Cat. No. 47743-982). The ethyl acetate was removed under a stream of air, and the sample was reconstituted in 100 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid.
Standard Assay Using Solid Phase Extraction. A 2-mm punch from a dried blood spot was placed in a 0.5 mL polypropylene tube (Eppendorf) containing 20 μΐ^ of assay cocktail (100 mM sodium formate, pH 4.0, 30 mM lead (II) acetate, 1 mM substrate
(ASB-S) and 5 μΜ internal standard (ASB-IS). The solution was vortexed briefly then centrifuged briefly to bring all components to the tube bottom. The capped tube was incubated at 37 °C for 16 hours in a thermostated air shaker. The sample was quenched with 100 μΐ^ of 25 mM Na2HP04, leading to a precipitate. A blank assay was carried out as above but using a blank paper punch. Samples were submitted to solid phase extraction using a vacuum manifold (Millipore Inc, MAVM0960R) system connected to an aspirator. DEAE cellulose resin (20 mg) in acetic acid (250 μί) was pipetted into each well of a 96 well filter plate (Innovative microplate, Cat. No. F20005). The ion exchange resin was washed with methanol (2 x 0.5 mL). A slurry of C-18 resin (20 mg, Aldrich, octadecyl-functionalized silica gel, Cat. No. 377635) in methanol (250 μί) was pipetted on top of the ion exchange resin in each well. The 2 resin layers were washed with methanol (2 x 0.5 mL) to help it settle, then with de-ionized water (2 x 0.5 mL). The sample was slowly loaded onto the column and washed with de-ionized water (2 x 0.5 mL) to remove salts. Finally the product and internal standard were eluted with methanol (2 x 0.5 mL) into a deep well plate (Neptune, Cat. No. 2405). The methanol was removed in a centrifugal concentrator (Speed- Vac), and the sample was reconstituted in 30 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid.
Mass Spectrometry. ESI-MS/MS analysis was performed on a Waters Quattro Micro tandem quadrupole instrument using flow injection and selected reaction monitoring in positive ion mode. Samples were injected manually using a syringe, except for the samples using the liquid-liquid extraction protocol that were injected using an autosampler. Twenty five μL· of the 100 μΐ^ sample was injected using a Waters 2777C Sample Manager via flow injection 80/20 acetonitrile/water with 0.2% formic acid with a flow-rate of 0.1 mL/min for 1 min then 1 mL/min for 0.5 min. Data was collected during 1.5 minute of infusion, and the signal returned to the background level before the next injection. The ion dissociations used for selected reaction monitoring were m/z 608.3→ m/z 508.6 and m/z 622.3→ m/z 522.6 for the internal standard and product, respectively. Other ESI-MS/MS conditions were as follows: electrospray capillary voltage: 4500 V, extractor: 3 V, desolvation temperature: 350 °C, desolvation gas flow: 600 L/h, collision cell pressure: 2.23 10-3 millibar. The cone voltage and ion laboratory collision energy were 15 V and 13 eV, respectively for both m/z 622.3→ m/z 522.6 and m/z 608.3→ m/z 508.6 transitions. The dwell time was 100 ms with a 20 ms delay. The amount of ASB product was then determined from the ion intensity ratio of the product to internal standard and converted to ASB activity (μιηο1/(1ι x L of blood)) using the incubation time and blood volume in the DBS punch.
Results and Discussion. The strategy for the MPS VI assay using tandem mass spectrometry is outlined in FIGURE 4. The enzyme in DBS is incubated with ASB-S to catalyze specific hydrolysis in the N-acetylgalactosamine-4-O-sulfate moiety yielding ASB-P. The product is ionized by protonation in electrospray to produce the m/z 622 precursor ion, (M + H)+, which is selected by mass and subjected to collision induced dissociation (CID) forming the m/z 522 product ion. CID is steered into one predominant product ion channel by the iert-butylcarbamido (ί-BOC) group that undergoes facile elimination of isobutene and carbon dioxide. The internal standard (ABS-IS) is a lower homologue of ASB-P from which it differs in the length of the diamine carbon chain which has six methylene groups in ASB-P and five methylene groups in ASB-IS. CID of the ASB-IS (M + H)+ ion forms an m/z 508 fragment ion which is homologous with the m/z 522 fragment from ASB-P. The ASB-P and ASB-IS structures were designed such that the m/z values of both precursor and fragment ions were distinct from those of substrates and internal standards used in ESTMS/MS assays of other lysosomal enzymes. Thus, the ASB assay can be multiplexed with any other previously developed EST MS/MS assays.
The relative response in ESI-MS/MS to ASB-P and ASB-IS concentrations in the sample, RP/RIS, depends on the partition coefficients in extraction from the assay buffer, electrospray ionization efficiency, and propensity for fragmentation by elimination of isobutene and CO2. The RP/RIS ratio was established as 2.09 from a calibration curve which showed excellent linearity (r2 = 0.994) for ASB-P/ASB-IS concentration ratios ranging from 0.1 to 5.0. This range of ASB-P concentrations corresponds to 10-500 pmol of enzymatic product formed in an assay, which is consistent with the range of enzyme activities found in DBS.
The assay conditions and work up procedures were thoroughly investigated and optimized. Assays of lysosomal enzymes are typically conducted at low pH 3.5-5.3 and under conditions achieving low substrate conversions (<10 ) to keep the enzyme kinetics in the initial pseudo-linear stage and also to avoid enzyme inhibition by the products. Sulfatases, in particular, are inhibited by free sulfate ions present in the sample or produced by enzymatic hydrolysis. Inorganic sulfate is sequestered as insoluble lead(II) sulfate (pKsp = 6.20) by in-situ precipitation with soluble lead salts, such as lead formate or acetate. In the absence of a lead salt, ASB showed no activity toward ASB-S, but full activity was recovered at lead concentrations >20 mM. Lead formate and acetate showed the same effect, and so 30 mM lead(II) formate was used in all ASB assays.
One of the advantages of MS/MS-based assays is the capacity for multiplexing analysis of several enzyme reactions in a single injection into the mass spectrometer. For practical purposes of work time constraints and sample throughput in newborn screening laboratories, it is also desirable to carry out the incubation of several enzymes in one common buffer. Hence, effects of pH on the enzyme activity need to be studied to find a pH range where several enzymes might have sufficient activity. The ASB activity showed a three-fold increase from pH 3.4 to pH 4.0 and then the dependence flattened at pH 4-4.5. The other parameters of the ASB assay were studied under the optimized conditions of pH 4 and 30 mM lead concentration. The amount of ASB-generated product in DBS increased as a linear function of incubation time between 5 and 30 h. This indicated that the assay conditions were such that the enzyme kinetics was in the initial stage. The incubation time was set to 16 h which is compatible with the work schedule in newborn screening laboratories.
Michaelis-Menten parameters were measured as shown in FIGURE 5. The amount of product formed increased in a hyperbolic fashion versus substrate concentration measured between 0.1 mM and 1 mM to give KM = 0.45 mM after non- linear regression fit. A substrate concentration of 1 mM was used to saturate the enzyme in an effort to minimize the effect of potential competitive inhibitors present in blood. The amount of product formed increased with the size of the DBS punch, with a plateau at higher blood amounts, presumably due to an increase in endogenous inhibitors. A 3 mm DBS punch was used to be compatible with the protocols used in newborn screening laboratories.
Post-incubation purification was necessary due to the high concentration of buffer salts, which would interfere with electrospray ionization. In addition, it was found that the sulfated substrate (ASB-S) undergoes fragmentation upon electrospray and ion transfer to the vacuum system that resulted in dissociative desulfation forming ABS-P ions. Although this dissociation is a minor process, it gains importance due to the large excess of ABS-S in the assay sample and produces a large background signal in MS/MS. Both the buffer salts and the sulfated substrate could be easily removed with a single liquid-liquid extraction step using ethyl acetate and water containing an anion exchange resin. The inorganic buffer salts partitioned into the aqueous layer and the anionic sulfate substrate was retained on the anion exchange resin.
Two procedures were developed to isolate the hydrophobic ASB-P and ASB-IS. Early investigation of the assay utilized a solid-phase extraction procedure using C18 silica gel and DEAE cellulose resin in an acetate form. The results obtained with solid phase extraction are listed in Table 1, and the distribution of activities is shown by the blue bars in FIGURE 6. The measurements showed negligibly low blanks and a 2.2- 12.9 μιηο1/(1ι x L of blood) range of enzyme activities for healthy newborns. The affected patient showed a substantially lower activity (0.11 μιηο1/(1ι x L blood) that was clearly separated from those of the healthy individuals.
Table 1. ASB Activities for Individual DBS in Humans Using Solid Phase Extraction Method.
Sample Enzyme Activity in μι ο1/(1ιουΓ x L of blood) Blank" (n = 3) 0.00
MPS VI patient 0.11
Newborns 1- 10 2.2, 12.9, 4.6, 4.5, 14.5, 11.3, 3.8, 3.8, 3.3, 10.5
'Blanks used a blank paper punch instead of a blood spot.
The sample work up using solid-phase extraction requires multiple liquid transfers which may be cumbersome in a newborn screening laboratory setup. Therefore, an alternative procedure was developed using liquid-liquid extraction to ethyl acetate. This procedure is more expedient as it involves fewer liquid transfer steps and thus facilitates a high-throughput execution of the assay. Using the optimized assay conditions and the liquid-liquid extraction protocol, ASB activities in DBS from 89 unaffected newborns and 1 MPS VI patient were analyzed. The unaffected newborns displayed an activity range of 1.4-16.9 μιηο1/(1ι x L blood), with an average activity of 7.4 μιηοΙ/hr/L blood (Table 2). The distribution of activities is shown as black bars in FIGURE 6. The DBS from a previously identified MPS VI patient gave a value of 0.12 μιηο1/(1ι x L blood), which was very close to the activity measured with the method using solid phase extraction (Table 1). Quality control DBS provided by the Centers for Disease Control and Prevention were also analyzed for ASB activity. These samples are prepared from fully and partially depleted as well as standard blood termed QC base, low, medium, and high. The respective ASB activities measured in those samples by the assay were 0.20, 0.65, 3.4, and 8.8 μιηο1/(1ι x L blood). All values are blank subtracted using the measured activity of assay cocktail incubated without a dried blood spot. Individual values for all samples are given in Table 2.
Table 2. ASB Activities for Individual DBS in Humans using Liquid-Liquid Extraction Method.
Sample Enzyme Activity in μι ο1/(1ιουΓ x L of blood)
Blank 0.17 ± 0.08
CDC QC Base 0.20 ± 0.20 (n = 3)
CDC QC Low 0.65 ± 0.26 (n = 3)
CDC QC Medium 3.4 ± 0.35 (n = 3)
CDC QC High 8.8 ± 2.0 (n = 3)
MPS VI Patient 0.12
Random newborns 1- 12 6.2, 7.1, 7.6, 16.9, 16.2, 3.2, 2.8, 5.6, 10.7, 3.9, 15.5, 4.8,
Random newborns 13-24 3.6, 9.8, 13.8, 11.0, 5.2, 4.2, 3.3, 11.7, 8.7, 5.8, 8.7, 4.9,
Random newborns 25-36 7.7, 3.3, 5.5, 10.0, 6.2, 2.1, 5.6, 13.2, 8.5, 6.8, 2.7, 4.2,
Random newborns 37-48 3.6, 4.6, 7.2, 4.5, 6.1, 5.4, 2.0, 8.1, 8.6, 9.9, 8.8, 5.3,
Random newborns 49-60 1.4, 8.3, 8.1, 13.0, 8.5, 6.5, 6.1, 11.8, 12.7, 13.1, 5.7, 15.0,
Random newborns 61-72 3.9, 15.8, 11.9, 6.6, 6.8, 11.2, 3.0, 12.4, 2.8, 8.7, 6.9, 7.6,
Random newborns 73-84 5.5, 4.3, 4.3, 10.5, 7.6, 7.3, 3.1, 16.5, 4.7, 3.7, 10.7, 4.1,
Random newborns 85-89 3.6, 4.2, 5.4, 5.8, 7.9
Assay precision was calculated using triplicate analyses of DBS from a healthy control. The within-assay coefficient of variation (CV) was 1.1% (n = 3) while the inter- assay CV was 4.5% (n = 8 punches of the same DBS, avoiding the DBS perimeter). Thirty random newborn NBS were assayed by omitting ASB-S and only background levels of ASB-P were found in all samples showing that DBS do not contain substances that interfere with the ASB assay.
Because cats have been used as an animal model in MPS VI studies (Harmatz, et al.. J. Pediatrics, 144, 574-580 (2004); Harmatz et al. Mol. Gen. Metabol, 94, 469-475 (2008)), a comparison was made for animal samples by measuring the ASB activity with the solid-phase extraction method in feline DBS using three normal felines, three MPS VI carrier felines, and three MPS VI affected felines. The normal felines displayed an activity range of 160-360 μιηο1/(1ι x L blood). The carrier felines displayed values of 35- 156 μιηο1/(1ι x L blood) and the MPS VI affected felines displayed values of 1-5 μιηο1/(1ι x L blood). Individual values for all samples are given in Table 3.
Table 3. ASB Activities for Individual DBS in Felines using Solid Phase Extraction Method.
Enzyme Activity in μι ο1/(1ιουΓ x L of blood)
Blank Healthy Kitten MPS VI Carriers MPS VI Affected n = 3 n = 3 n = 3 n = 3
0.00 170 36 6
0.00 358 73 4
0.00 160 156 1
The new ESTMS/MS assay of aryl sulfatase B activity in dried blood spots from humans and felines unambiguously distinguishes healthy individuals from affected ones. The assay uses a small amount of synthetic material (14.5 μg of substrate and 61 ng of internal standard per assay) and shows good linearity and inter-assay reproducibility. The work-up procedure using solid-phase extraction of the product and internal standard generates virtually zero background but requires several liquid transfers which is acceptable for a research laboratory. The work-up procedure using liquid-liquid extraction of the product and internal standard provides very low background and, due to a minimum of liquid transfer steps, is suitable for large throughput analysis such as those performed in newborn screening laboratories. Example 3: Assay of MPS IV A (Morquio A Syndrome) The following Example is similar to the methodology presented in Example 2. Briefly, a highly specific enzyme activity assay for MPS IVA using ESI-MS/MS is presented that uses a novel substrate and directly measures the enzyme activity of N- acetylgalactosamine 6-sulfatase (GALNS) in rehydrated dried blood spots (DBS). Other assays and sMPS IVA substrates described herein may be employed. The assay is relatively simple to execute and may be suitable for the eventual screening of MPS IVA by newborn screening laboratories.
All experiments were conducted in compliance with institutional review board guidelines. In samples obtained from affected patients, MPS rVA had been previously diagnosed with established clinical and biochemical procedures. DBS were kept at ambient temperature during shipment (<10 days) and then stored at -20 °C in Ziploc® plastic bags (one bag sealed inside a second bag). Ziploc® bags were kept in a sealed plastic box containing desiccant (anhydrous CaSC^ granules). Preparations of a GALNS substrate (GALNS-S) and an internal standard (GALNS-IS) are discussed in Example 1.
GALNS-S consists of an umbelliferyl- -D-galactose with a sulfate group at the 6- position of the sugar. A hydrophobic five-carbon linker with a terminal i-butylcarbamate was incorporated at the 4-position of the umbelliferyl unit to facilitate the post-assay purification and increase the sensitivity of the ESTMS/MS signal (due to a dominant fragmentation pathway involving cleavage of the i-butylcarbamate) (FIGURE 7).
Incubation of substrate with DBS leads to enzymatic release of the sulfate group to produce the product GALNS-P (FIGURE 7A). The internal standard (GALNS-IS) is a homologue of GALNS-P possessing one additional methylene unit in the linker chain. ESI-MS/MS enables separate detection and quantification of GALNS-P and GALNS-IS by their fragment ions, after collision induced dissociation of their parent ions (FIGURE 7A). The presence of the umbelliferyl moiety in GALNS-S would allow for fluorometric assay of GALNS should that be desirable by laboratories who desire not to use ESI- MS/MS (see van Diggelen et al., Clin. Chim. Acta, 187: 131-40 (1990)).
A single 2-mm diameter DBS punch (containing approximately 1.6 μΐ^ of blood) was obtained with a leather punch and was placed in a 0.6 mL Eppendorf tube. A 20 μΐ^ aliquot of assay cocktail containing 100 mM sodium formate buffer (pH 4.0), 30 mM lead(II) formate, 1 mM GALNS-S and 0.5 μΜ GALNS-IS (stock solutions of both in methanol stored at -20 °C, methanol removed before adding buffer) was added to the tube. The mixture was vortexed (-15 sec) and centrifuged for 5 minutes. Similarly, a blank containing all the assay components except the DBS punch was also prepared. The samples were incubated at 37 °C for 16 hr in a thermostated air shaker. The reaction was quenched by adding 100 μΐ^ of 25 mM Na2HPC"4 (pH 7.85) (product analysis before and after a 4 hr post-quench incubation showed that the quench stopped the reaction). Solid- phase extraction was carried out to purify the product and the internal standard using a vacuum manifold (Waters, Cat. WAT200606) connected to a water aspirator. DEAE- cellulose resin (20 mg) in acetic acid (300 μί) was pipetted into each well of a 96 well filter plate (Innovative microplate, cat. #F20005) and was washed with methanol (2 x 0.5 mL). A slurry of CI 8 silica gel (20 mg, Aldrich, octadecyl-functionalized silica gel cat. #377635) in methanol (300 μί) was pipetted on top of the ion exchange resin in each well. The dual layer was sequentially washed with methanol (2 x 0.5 mL) and then with de-ionized water (2 x 1 mL). The quenched sample was centrifuged for 5 min, and the supernatant was loaded onto the column and washed with de-ionized water (2 x 1 mL) to remove salts. GALNS-P and GALNS-IS were eluted with methanol (2 x 0.5 mL) into a deep well plate (Neptune, cat. # 2405). The methanol was removed in a Speed- Vac, and the residue was reconstituted in 30 μΐ^ of 80/20 acetonitrile/water with 0.2% formic acid.
For the liquid extraction recovery of GALNS-P and GALNS-IS, the assay sample was quenched by adding a suspension of 64 mg DEAE-cellulose (DE52, Whatman Cat. 4057-200, pre-swollen) in 250 μΐ^ water. Ethyl acetate (500 μί) was added to extract GALNS-P and GALNS-IS. After mixing on a vortexer (-15 sec) the sample was centrifuged for 5 min. The ethyl acetate portion (300 μί) was transferred to a new Eppendorf tube, solvent removed with a stream of N2, and the residue reconstituted in 30 μL· of 80/20 acetonitrile/water with 0.2% formic acid.
The post-assay purification is used because the buffer salts present in high concentrations will interfere with the electrospray ionization, and the unreacted sulfated substrate can dissociate in the source of the mass spectrometer forming GALNS-P ions, and thus giving rise to false positive GALNS activity. In the solid-phase extraction, the buffer salts are removed with water whilst the hydrophobic GALNS-S, GALNS-IS and GALNS-P are retained on the C18 resin. The anionic GALNS-S is retained on the DEAE-cellulose, and GALNS-P and GALNS-IS pass through. In the liquid-phase extraction, GALNS-S is captured on the DEAE-cellulose, and GALNS-IS and GALNS-P are extracted into ethyl acetate. ESI-MS/MS analysis was carried out on a Waters Quattro Micro tandem quadrupole instrument operating in positive-ion, multiple-reaction-monitoring mode. Data acquisition was carried out with MassLynx 4.1 software with the following settings: capillary voltage, 3500 V; cone voltage, 80 V; extractor, 2 V; RF, 0.0 V; source temperature, 80 °C; desolvation temperature, 250 °C; cone gas flow, 30 L/h; desolvation gas flow, 550 L/h; collision gas flow, 0.20 mL/min; LM 1 resolution, 15; HM 1 resolution, 15; ion energy 1, 0.2; MS/MS mode entrance, 15; MS/MS collision energy, 30 eV (Gal-6S-P) and 30 eV (Gal-6S-IS); MS/MS mode exit, 15; LM 2 resolution, 15.0; HM 2 resolution, 15.0; ion energy 2, 2.0; Multiplier, 650; collision cell pressure, < 10~4 mbar; collision gas, argon. Multiple-reaction-monitoring mode was used for m/z 589.2 — > 489.1 and 603.2— > 503.1 transitions with the following settings: dwell time, 0.1 s; delay, 0.02 s.
The sample (10 μΐ^ of the 30 μΐ^ sample in 80/20 acetonitrile/ water with 0.2% formic acid) was injected into the mass spectrometer with a flow-rate of 0.1 mL/min. Data was collected during 1 minute of infusion, and after 1 min, the MS/MS signal has returned to the background level. The amount of product was calculated from the ion abundance ratio of product to internal standard, minus that from a minus DBS blank control, multiplied by the amount of added internal standard. Enzymatic activity was calculated from the amount of product divided by the incubation time and the volume of blood (1.6 μΐ^ of blood in a 2 mm DBS punch).
The parent ions for GALNS-P and GALNS-IS (m/z 589.2 and 603.2, respectively) were each isolated by mass and subjected to collision-induced dissociation. The daughter ions of m/z 489.1 and 503.1 derived from GALNS-P and GALNS-IS, respectively, were quantified. The amount of GALNS-P was calculated by comparing the ion peak intensity of GALNS-P to that of GALNS-IS. The signal generated from the minus blood blank assay was -1 % of that seen with the complete assay.
Assay optimization depicted maximum GALNS activity at pH 4.0 in formate buffer. Sulfate and phosphate ions are competitive inhibitors of sulfatases. Therefore, lead(II) formate (30 mM) was used to suppress these inhibitors. The amount of GALNS- P increases approximately linearly with reaction time from 0-30 h. The incubation time of 16 h was chosen in order to allow for overnight incubation to simplify work schedules of laboratories. The amount of GALNS-P decreases when the size of the DBS punch is increased from 2 to 4 mm presumably due to the presence of endogenous inhibitors in the DBS. Therefore, a 2 mm DBS punch was chosen for the assays. The ESTMS/MS response to GALNS-P is 1.10 times that of GALNS-IS, and a linear standard curve was observed. Assay imprecision was calculated by replicate analyses of the DBS from a healthy control and the intra- and inter-assay CV was 6.4% (n = 3) and 8.3% (n = 10), respectively. A control assay containing all assay components except GALNS-S was performed with 30 different DBS, and the signal was -1% of that measured with the complete assay. This rules out any ESI-MS/MS signal coming from sources other than GALNS action. Enzyme stability studies showed that GALNS is stable in DBS maintained at -20 °C for at least 1 yr, but about 50% of the activity is lost at 37 °C over 3 days.
Using the GALNS assay with liquid-phase extraction, enzyme activity in DBSs from 9 MPS IVA patients (range, 0.0019-0.018; mean, 0.0096 μπιοΙ/h/L blood) was well below the range of activity in blood samples obtained from 30 healthy newborns (range, 0.109-0.550; mean, 0.279 μπιοΙ/h/L blood) (FIGURE 7B). The relative amounts of GALNS-P and GALNS-IS that extract into ethyl acetate was determined by ESI-MS/MS analysis after extraction of buffer containing equal moles of analytes. As expected, the less hydrophobic GALNS-P is extracted less than GALNS-IS. The combined factor is 1.81 for relative extraction and ionization efficiency, and the ESI-MS/MS signal for GALNS-P was multiplied by this factor to obtain the specific enzymatic activities reported.
Evaluation of enzyme activities of 60 additional DBS from healthy newborns along with the DBS from 9 MPS IVA patients was also performed using the solid-phase extraction workup method. The values are in the same range as those for the liquid extraction method. The latter is preferred in newborn screening laboratories as it is easier and faster to execute.
The foregoing demonstrates that a high-throughput enzyme assay based on tandem mass spectroscopy has been developed for the diagnosis of MPS IVA that may be used, for example, for newborn screening. The assay uses a substrate that can be easily synthesized on a large scale. The assay is expected to have the additional advantage of being performed in a multiplex fashion in tandem mass spectrometry with other lysosomal enzyme assays. Example 4: A Tandem Mass Spectrometry Triplex Assay for the detection of Fabry,
Pompe, and Mucopolysaccharidosis IH
A tandem mass spectrometry assay is provided in this Example in which the enzymatic activities of three lysosomal enzymes, cc-glucosidase (GAA), cc-galactosidase A (GLA), and α-L-iduronidase (IDUA), were quantified in dried blood spots using a single assay buffer with minimal workup. Data from 5,990 anonymous newborn dried blood spots shows an approximate bell-shaped distribution of enzymatic activities (average values of 19.0, 11.5, and 3.5 μιηοΐ h"1 (L blood)"1 for cc-glucosidase, cc- galactosidase A, and α-L-iduronidase, respectively, with minus-blood blank values of 0.13, 0.24, and 0.45 μιηοΐ h"1 (L blood)"1, respectively). The method demonstrates that a triplex assay in a single buffer and with minimal supplies and labor can be adapted to a high throughput screening method (e.g., in a newborn screening laboratory) for the analysis of Pompe, Fabry, and mucopolysaccharidosis IH diseases.
Materials and Methods.
Materials. GLA internal standard (GLA-IS), GLA substrate (GLA-S), GAA internal standard (GAA-IS), and GAA substrate (GAA-S) are from the Centers for Disease Control and Prevention, Atlanta. IDUA-IS and IDUA-S are from Genzyme Corp., Cambridge, Massachusetts (for structures, see Li et al., Clin. Chem., 50: 1785-1796 (2004) and Blanchard et al., Clin. Chem. 54:2067-2070 (2008)). The present studies used reagents synthesized by Genzyme Corp. in Liestal, Switzerland and approved for use as Analyte Specific Reagents by the FDA.
All experiments were conducted in compliance with institutional review board (IRB) guidelines. DBS from patients diagnosed with Fabry, Pompe, and MPS IH were obtained from Genzyme Corp. or from the inventors' clinical program as anonymous samples, in compliance with IRB requirements. The Fabry samples were from affected males only; the Pompe samples were from both infantile and late-onset clinical forms; and the MPS IH samples were from patients with early childhood presentations (Hurler). For assay development, DBS were obtained as anonymous samples from the Washington state newborn screening laboratory. DBS were obtained from birthing centers and kept at ambient temperature during shipment (<10 days). For assays carried out in the Washington state newborn screening laboratory, DBS were used after all routine testing was performed (i.e., leftover DBS) and were up to ~6 months old and kept at ambient laboratory temperature. Triplex assay. The following experimental details were carried out in the Washington state newborn screening laboratory. Experimental details for assays carried out during the optimization phase of the project at the University of Washington are presented in Example 5.
Preparation of assay buffer: Ammonium formate (1.24 g, >99.0 , Sigma-
Aldrich, cat. no. 09735) was added to water (200 mL, DI Nanopure, Barnstead, CAP type 1) and stirred to dissolve the powder. Formic acid (0.2 mL, 97%, Alfa Aesar, Cat. No. A 13285) was added, and the pH was adjusted to 4.4 with ammonium hydroxide or formic acid (not hydrochloric acid or sodium hydroxide). The solution was brought up to 250 mL with water, sterile filtered and stored at 2-8 °C for up to 6 months in a glass bottle. The final formate concentration is 0.1 mol/L (pH = 4.4).
Preparation of quench buffer: Ammonium acetate (3.45 g, >99.0%, Sigma- Aldrich, Cat. No. 09689) was added to water (400 mL) and stirred to dissolve the powder. Acetic acid (0.3 mL, glacial, JT Baker, Cat. No. 9508-02) was added, and the pH was adjusted to 5.5 with ammonium hydroxide or acetic acid (not hydrochloric acid or sodium hydroxide). The solution was brought up to 500 mL with water, sterile filtered and stored at 2-8 °C for up to 6 months in a glass bottle. The final acetate concentration is 0.1 mol/L (pH = 5.5).
Preparation of assay cocktail: The IDUA and GAA substrate/internal standard vials from Genzyme and CDC were dissolved in methanol (6 mL). The GLA substrate/internal standard vials from CDC were dissolved in methanol (10 mL). Portions of the contents of the vials were then transferred to a new vial (1 mL each of IDUA, GAA, and GLA solutions). This was repeated four times to generate five vials total of the mixture of IDUA, GAA, and GLA substrates and internal standards. The methanol was evaporated with a stream of N2 with slight heating (<40 °C). The solution of remaining GLA substrate/internal standard vial was further distributed into four additional vials (1 mL each). The methanol was removed as above, and the vials were stored at -20 °C for later use by addition of IDUA and GAA vial reagents as above.
On the day of the assay, the residue in a vial was taken up in assay buffer (9.9 mL, 0.1 M ammonium formate, pH = 4.4), and acarbose (Sigma Aldrich, A8980-1G) was added (0.1 mL, 0.8 mM in water, stock solution stored at -20 °C). The solution was stirred with a magnetic stir bar until all the residue was dissolved (slight heating, <50 °C, should be used to ensure that all material is dissolved). The final assay cocktail contains 0.48 mM IDUA-S, 3.1 μΜ IDUA-IS, 0.2 mM GAA-S, 2.0 μΜ GAA-IS, 0.6 mM GLA-S, 1.2 μΜ GLA-IS, and 8 μΜ acarbose. Excess assay buffer can be stored at 4 °C and is used the following day without loss of activity. The other four vials of dried and mixed reagents were stored at -20 °C and reconstituted on the day of the assay.
Assay setup and incubation: Three dried blood spot (DBS) punches (1/8 inch,
3.175 mm) punched with a blood spot puncher (BSD Technologies International, BSD 600 Duet) were taken from each newborn sample. Punching near the edge of the blood region was avoided. One punch was placed in a single well of a 96-deep well plate (1 mL, Costar, Fisher Scientific, Cat. No. 09-761-116) and used for the triplex assay. The two extra punches were placed in a separate 96- well plate, sealed with aluminum foil plate sealing mats (VWR, cat no. 14230-062), and stored at room temperature in a gasketed plastic storage container. These extra punches are to be used for confirmatory testing, if necessary. Each 96-deep well plate also contained 6 wells with a blank filter paper punch and 2 wells of each of the CDC quality control dried blood spot samples (base, low, medium and high, obtained from Drs. H. Zhou and V. De Jesus at the CDC in Atlanta, stored at -20 °C in a Ziploc® plastic bag). The blanks and QC samples are on the first and last columns of the plate. From top to bottom, there are two blanks, then QC base, QC low, QC medium, QC high, an adult DBS and then another blank. QC samples were manually punched with a l/8th inch whole punch. Assay cocktail (30 μί) was added to each well in the 96-deep well plate using a Rainin Liquidator 96-tip pipette. The plate was sealed with plate sealing film (VWR, Cat. No. 14230-062) and incubated at 37 °C for 16 h (overnight) with orbital shaking at 150 rpm.
Assay workup: After 16 h, the assay was quenched with ammonium acetate buffer (100 μί, 0.1 M, pH = 5.5) transferred directly into the 96-deep well plate using a Rainin Liquidator 96-tip pipette. Ethyl acetate (400 μί) was added, and the wells were mixed by aspirating five times using the Rainin Liquidator 96-tip pipette. The plate was covered with foil and centrifuged for 5 min at 3000 rpm to separate the layers. The plate was returned to the Rainin Liquidator 96-tip pipette, and 200 μΐ^ of the top layer (ethyl acetate) was transferred to a new 96-shallow well plate (0.5 mL, Axygen Scientific, VWR International, Cat. No. 47743-982). The ethyl acetate was evaporated using a stream of air (SPE Dry 96 Dual Argonaut sample concentrator system, Biotage) with a flow rate of 40-80 PSI of air and heating <35 °C (typically <30 min). The residue in the wells was resuspended in mass spectrometry mobile phase (100 μί, 80% acetonitrile: 20% water with 0.2% formic acid) using the Rainin Liquidator 96-tip pipette. After addition of the mobile phase, the plate was mixed for a few minutes in an orbital shaker. The plate was covered with aluminum foil (not sealing foil since acetonitrile can dissolve the glue) and placed in the auto-injector tray for mass spectrometric analysis.
ESI-MS/MS: ESI-MS/MS analysis was performed on a Waters Acquity TQD
Ultra Performance tandem quadrupole mass spectrometer using positive mode multiple reaction monitoring mode and flow injection. Ten microliters of the 150 μΐ^ sample was injected for each analysis at a flow rate of 0.1 mL/min using a Waters 2777C Sample Manager via flow injection with 80/20 acetonitrile/water with 0.2% formic acid for 1.10 min then 0.5 mL/min for 0.4 min (most of the sample elutes during the 1.10 min phase, and the faster flow phase is used to reduce the time of sample clearance in preparation for the next injection). MassLynx 4.1 software was used to record all ion signals. Data was collected during 1.5 minute of infusion, and the signal returned to the background level before the next injection. The mass transitions used for multiple reaction monitoring are IDUA-IS: m/z 377.2→ m/z 277.1, IDUA-P: m/z 391.2→ m/z 291.2, GLA-P: m/z 484.2→ m/z 384.3, GLA-IS: m/z 489.4→ m/z 389.2, GAA-P: m/z 498.4→ m/z 398.4, GAA-IS: m/z 503.4→ m/z 403.4. Other ESI-MS/MS parameters are given in Tables 4A and 4B. All transitions were conducted with the following settings: dwell time, 0.1 s; delay, 0.02 s.
Table 4A: ESI-MS/MS parameters for newborn screening studies.
Figure imgf000051_0001
MS/MS Mode Entrance 1
MS/MS mode Exit 0.5
LM 2 Resolution 15
HM 2 Resolution 15
Ion Energy 2 0.4
Multiplier 650
Collision Cell Pressure 2.76 e-3 mbar
Collision Gas Argon
Table 4B: ESI-MS/MS parameters for newborn screening studies.
Figure imgf000052_0001
The amount of product was calculated from the ion abundance ratio of the product to the internal standard for the sample minus that of the blank (filter paper only punch, average of 6 blanks was used), multiplied by the amount of added internal standard and divided by the response factor ratio of the product to internal standard. The response factor was determined from a calibration curve obtained from standards containing ratios of product and internal standard from 0.0-5.0 for GAA, GLA (obtained from Drs. H. Zhou and V. De Jesus at the CDC) and IDUA (obtained from Drs. K. Zhang and J. Keutzer at Genzyme Corporation). The enzyme activity in units of μιηοΐ-ΐτ1 -^ of blood)-1 was calculated from the measured amount of product assuming the 3 mm DBS punch contains 3.2 μL of blood.
Results and Discussion.
Assay development prior to studies in the newborn screening laboratory. The goal of the first set of studies was to carefully examine the original buffer conditions reported previously for each assay (Fabry, Pompe, and MPS IH) to determine the buffer components that best help maintaining high enzymatic activity. The original buffer systems for the individual assays ranged from pH = 3.4 - 4.6 and used formate, citrate- phosphate and acetate buffering salts. Assays were conducted in sodium formate, sodium citrate, and sodium citrate-phosphate buffers at pH = 3.6, 4.0, and 4.4 on a single DBS using GLA-S, GAA-S, and IDUA-S and GLA-IS, GAA-IS, and IDUA-IS. The enzyme activity of IDUA was low in sodium citrate and sodium citrate-phosphate buffers at all pH values, so sodium formate was chosen as the preferred buffer salt. The enzyme activity of GLA was significantly higher at higher pH, and the minus DBS blanks for all the assays were lower at higher pH, so pH = 4.4 was used for further optimizations.
The Pompe and Fabry enzyme activity assays described previously used CHAPS and sodium taurocholate, respectively (Li et al., Clin. Chem., 50: 1785-1796 (2004)). Sodium taurocholate significantly reduced the activity of all three enzymes at all concentrations tested. Likewise, CHAPS reduced the activity of all three enzymes at 4-6 g/L CHAPS, while at 2 g/L CHAPS, the enzyme activities were similar to the activities without detergent. In an effort to keep the assay mixtures as simple as possible, subsequent assays were conducted without detergent.
In the original GLA enzyme assay, GalNAc (N- acetylgalactosamine) was added as an inhibitor for N-acetyl-galactoseaminidase because the GLA substrate could potentially be a substrate for this enzyme resulting in product formation in Fabry patients with low a-galactosidase activity. The activity of GLA in normal DBS punches and in DBS punches from Fabry patients was determined using the triplex assay conditions with and without GalNAc added. GLA enzyme activity was similar in normal DBS punches with and without the GalNAc added (Table 5).
Table 5. Effect of GalNAc on GLA activity in DBS
GLA activity (μιηοΙ/h/L of blood)
No GalNAc 96 mM GalNAc
Blank1'2 0.26 + 0.08 0.26 + 0.01
QC Base 0.53 + 0.05 0.61 + 0.05
QC Low 1.2 + 0.3 0.99 + 0.06
QC Medium 4.6 + 0.5 4.6 + 0.2
QC High 8.9 + 0.6 8.6
Fabry Patient #1 0.54 0.63
Fabry Patient #2 0.58 0.54 Fabry Patient #3 0.50 0.42
Fabry Patient #4 0.44 0.31
Fabry Patient #5 051 0.33
Values reported are the average + standard deviation for triplicate runs of different punches from the same DBS.
Blank assays were carried out with the assay cocktail incubated with a filter paper punch that does not contain blood.
The GLA enzyme activity in five punches obtained from male patients with Fabry disease were 0.33 - 0.63 μιηοΙ/h/L of blood with GalNAc and 0.44 - 0.58 without the GalNAc added. Thus there is minimal conversion of the GLA substrate to product by /V-acetyl- galactoseaminidase under these conditions, and the measured activity is from a- galactosidase.
Substrate concentration should be high enough to provide enzyme activities with a significant difference between the normal newborn blood with low activity and affected patient blood. Also, the use of substrate concentrations well above the KM minimizes effects of competitive inhibitors that may be present in blood. On the other hand, higher substrate concentrations increase the load on the mass spectrometer and lead to a higher background signal and to higher reagent costs. The assays are conducted at a substrate concentration lower than the apparent Km of the enzymes, so the activity of the enzymes should double when the substrate concentration is doubled. This would be true if DBS samples do not contain competitive inhibitors or if the amount of competitive inhibitors is similar in all samples. Thus, the variation in GLA, GAA, and IDUA activity was measured when the concentration of the three substrates were varied 2-fold, and this analysis was carried out with 30 DBS from different individuals. The enzyme activity approximately doubles for all three enzymes when the substrate concentration doubles. In addition, the same 30 DBS were incubated in the assay buffer without the three enzyme substrates. Analysis of these samples did not detect an ion for the enzymatic product. The data show that differential competitive inhibition is not a problem and that DBS do not contain substances that give rise to false product signals in the ESI-MS/MS analysis (i.e., all product ESI-MS/MS signal comes from enzymatic conversion of added substrates).
As described herein, the solid-phase extraction step has been eliminated, so there is the potential for non- volatile buffer salts that are extracted into ethyl acetate to build up in the mass spectrometer source and also to suppress the electrospray ionization process (although most of the salts will remain in the aqueous phase). Thus, volatile buffers components were evaluated. The enzyme activities for GAA, GLA, and IDUA were compared in sodium formate and ammonium formate buffers at pH = 4.4. IDUA enzyme activity was slightly higher in ammonium formate than in sodium formate, and GAA and GLA enzyme activities were comparable for the two buffers. Therefore, the volatile buffer ammonium formate was used as the buffer for the triplex assay.
A concern with eliminating the solid-phase extraction step was that using only the liquid-liquid extraction with ethyl acetate would lead to higher levels of enzyme substrates in the mass spectrometer infusion solvent. Because substrates may fragment to give products in the source of the mass spectrometer, one would expect higher product levels in control reactions. A mixture of the internal standards and substrates for GAA, GLA, and IDUA were prepared in sodium formate buffer and purified without incubation by both liquid-liquid extraction and liquid-liquid extraction plus solid-phase extraction on silica gel. The samples purified by solid-phase extraction had a lower background signal for the products of all three enzymes than those with just the liquid-liquid extraction workup, but the background signal of the samples purified without solid-phase extraction is very small compared to the signal observed from enzyme activity in DBS punches. The amount of in- source fragmentation of the substrate to product was kept to a minimum by carefully tuning the mass spectrometric parameters (cone voltage and collision energy, Tables 4A and 4B) while still maintaining a high signal for the product ion.
Other actions were taken to minimize the number of liquid transfer steps required, thereby reducing the number of plates and pipette tips used, as well as reducing the overall assay time and the potential for error. The DBS punch was placed directly into the well of a 96-deep well plate followed by addition of the assay cocktail. This eliminates the step in which the DBS punch is extracted with buffer prior to assay initiation. The use of the deep well plate allows the liquid-liquid extraction to be carried out in the same well as the incubation. After incubation, quench buffer and ethyl acetate are added directly to the assay well. After extraction, the top ethyl acetate layer is transferred to a 96-shallow well plate, and solvent is removed with a stream of air. The residue is taken up in the mobile phase for infusion into the mass spectrometer. This plate is used directly in the autosampler. In total, the new method uses two 96-well plates and five boxes of pipette tips to assay the activity of three enzymes as opposed to the seven 96-well plates, a 96-well filter plate and eighteen boxes of pipette tips for the same three enzymes in the previous method (Li et al., Clin. Chem. , 50: 1785-1796 (2004)).
Using the optimized assay conditions, patient blood from 11 Pompe patients, 8 MPS IH patients, and 5 male Fabry patients were tested, along with quality control DBS from the CDC (Table 6).
Table 6. Enzyme activities in DBS.
Enzyme Activity (μιηοΙ/h/L of blood)
MPS IH: Fabry:
IDUA GLA Pompe: GAA
CDC QC Blank1'2 0.45 + 0.031 0.24 + 0.02 0.13 + 0.01
CDC QC Base1 0.28 + 0.02 0.78 + 0.10 0.28 + 0.10
CDC QC Low1 0.74 + 0.02 1.5 + 0.1 1.4 + 0.1
CDC QC Medium1 5.4 + 0.6 8.4 + 0.6 9.8 + 2.6
CDC QC High1 9.4 + 1.0 16.1 + 1.0 14.7 + 0.5
Pompe Patient 1 7.63 9.52 0.S3
Pompe Patient 2 3.35 6.92 0.46
Pompe Patient 3 6.04 8.77 0.7 1
Pompe Patient 4 4.05 6.47 0.46
Pompe Patient 5 6.74 7.07 0.66
Pompe Patient 6 4.66 8.15 0.56
Pompe Patient 7 2.47 6.15 0.41
Pompe Patient 8 3.91 6.60 0.6S
Pompe Patient 9 2.64 2.80 0. 1 7
Pompe Patient 10 1.53 4.09 0.47
Pompe Patient 11 4.75 3.71 0.36
MPS IH Patient 1 0.48 5.10 14.13
MPS IH Patient 2 0.5S 5.33 20.14
MPS IH Patient 3 0.30 6.61 9.69
MPS IH Patient 4 0.28 5.17 7.04
MPS IH Patient 5 0.2l> 3.52 5.83
MPS IH Patient 6 0.74 7.35 11.31
MPS IH Patient 7 0.35 2.10 2.77 MPS IH Patient 8 || 0.34 5.01 8.99
Fabry Patient 1 4.35 O. 8.29
Fabry Patient 2 3.28 0.52 8.31
Fabry Patient 3 5.38 O.SO 12.83
Fabry Patient 4 2.83 0.52 6.69
Fabry Patient 5 2.99 O.iiO 5.58
Values reported are the average + standard deviation for triplicate runs of different punches from the same DBS.
Blank assays were carried out with the assay cocktail incubated with a filter paper punch that does not contain blood.
Blood collected from affected patients had low activity of the enzyme corresponding to the disease but normal activity for the other two enzymes assayed. For example, Fabry patients had low GLA enzyme activity but normal activity for GAA and IDUA. The activity of the affected enzyme of each of the patient blood samples screened is below the QC low sample which represents approximately 5% of the mean activity measured in a population screen.
A summary of an exemplary screening protocol using the optimized triplex assay described in the previous section is shown in FIGURE 8. The entire protocol fits within a 48 hr period including data analysis. The analysis of 4 plates containing 320 newborn DBS samples, 40 QC DBS samples and 24 blanks in this 48 hr period requires the items listed in Table 7. Equipment required is a DBS punch machine, a manually operated 96- tip pipette, an incubator for 96-well plates, a centrifuge to spin 96-well plates, a simple solvent evaporation system for 96-well plates, an autosampler, and a ESTMS/MS instrument. Robotics are not used.
Table 7. Items required for triplex enzyme assay of four 96-well plates in a 48 hr period.1
Item Required amount
GAA-S/GAA-IS 1.5 mg/0.012 mg
GLA-S/GLA-IS 4.5 mg/0.007 mg
IDUA-S/IDUA-IS 3.3 mg/0.014 mg
acarbose 0.060 mg 96-well, deep-well plates 4
96-well, shallow- well plates 4
large volume pipet tips (Liquidator pipette tips) 8 boxes of 96 tips
200 μΐ^ pipette tips 16 tips
Technician 1
Data analyst 0.5 - 1
The table does not include buffer salts, ethyl acetate and 96-well plate sealers, but the cost of these are insignificant.
FIGURES 9A, 9B, and 9C show the distribution of GLA, GAA, and IDUA activities, respectively, for 5,990 DBS submitted to the triplex assay. The activity of GLA and GAA is similar to the activity previously documented using citrate-phosphate or acetate buffer. The mean activity of IDUA is approximately 50% less than prior determinations (pH 3.4) but still clearly separates affected patients from unaffected newborns using formate buffer at pH 4.4.
Quality control (QC) samples for inclusion in the DBS enzyme assay were obtained from the Center for Disease Control (CDC). These are artificially prepared blood samples that represent the range of enzyme activity expected from newborn blood spots submitted for screening. They are expected to approximate 5%, 50%, and 90% of the normal range. The inclusion of two QC samples for each activity level in each 96- well plate serves as a reliable indicator of assay integrity. Table 8 summarizes the enzyme activity data obtained for 80 punches of the low, medium and high QC samples from the CDC [8 punches per day (distributed 2 per 96-well plate in 4 plates) over a 10- day period] . The coefficient of variation percentages are generally higher for the low QC samples as expected since the medium enzyme activity in these samples is -10% of those for the high QC samples. For the high QC samples, the coefficient of variation percentages are in the range 7.9-13.5%. These values include all variation from the assay procedure and from punching the DBS at different spots, since multiple punches were taken from each DBS.
Table 8. Assay results for multiple analysis of the quality control samples.1
Enzyme Activity in μιηοΙ/h/L of blood
(%CV)
DBS Pompe Fabry MPS IH CDC QC Low 1.1 (18.6) 1.2 (25.5) 0.8 (12.7)
CDC QC Medium 7.8 (17.8) 6.9 (8.9) 3.7 (13.6)
CDC QC High 11.5 (13.5) 13.2 (7.7) 6.5 (10.0)
The first number in each box is the mean enzyme activity followed by the covariance in percent (in parenthesis).
The triplex assay has the added benefit of having an internal quality control. In the newborn screening laboratory setting, it is possible to obtain DBS punches that are not fully saturated with blood. When tested for a single enzyme, these samples appear to come from affected patients. In the triplex, such a DBS would have low activity in all three enzymes, flagging an error in the testing procedure.
Since Fabry disease is an X-linked disease, females will have a wide spectrum of cc-galactosidase activity due to random X-chromosome inactivation. Thus, the assay reported here, or any enzyme activity assay, will primarily detect hemizygous males with absent, or low, cc-galactosidase activity.
In conclusion, the enzyme activities of three separate enzymes can be efficiently determined in a single incubation of a DBS with the appropriate enzyme substrates and internal standards. The new assay reliably distinguishes blood from affected patients with Fabry, Pompe, and MPS IH from unaffected newborns. In addition, since three enzyme activities are measured simultaneously, the assay has an internal control for integrity that minimizes the occurrence of false positives. The present study also demonstrates that a single buffer will not be required for each lysosomal enzyme to be assayed, and the pre-ESTMS/MS sampling handling steps have been simplified to the level that they fit well into the high throughput regiment of a newborn screening laboratory. Thus, the new procedure is simplified from published procedures as it requires fewer manipulations and resources and has been designed to be compatible with the typical schedule of a newborn screening laboratory.
Example 5: Experimental Details for Triplex Assays Carried Out during the
Optimization Phase (see Example 4 as well)
A Waters Quattro Micro tandem triple quadrupole instrument was used for all sample analysis in positive ion mode. MassLynx 4.1 software was used to record all ion signals. The ESTMS/MS settings were as follows: Table 9A: ESTMS/MS parameters for optimization studies.
Figure imgf000060_0001
Table 9B: ESTMS/MS arameters for o timization studies.
Figure imgf000060_0002
All transitions were conducted with the following settings: dwell time, 0.1 s; delay, 0.02 s. Ten μΐ^ of the 150 μΐ^ sample was injected using a Waters 2777C Sample Manager via flow injection with 80/20 acetonitrile/water with 0.2% formic acid with a flow-rate of 0.1 mL/min for 1 min then 1 mL/min for 0.5 min. Data was collected during 1.5 minute of infusion, and the signal returned to the background level before the next injection.
Experimental details used for triplex assay optimization.
Assay buffer preparation. For formate and citrate buffers, 1 mmol of formic acid or citric were dissolved in water, and the pH was adjusted with sodium hydroxide (1 M) to the desired pH (pH = 3.6, 4.0, and 4.4). Water was added to adjust the buffer volume to 10 mL. For the citrate-phosphate buffer, 1 mmol of NaH2P04 monohydrate and 0.5 mmol of citric acid were dissolved in water, and the pH was adjusted with sodium hydroxide (1 M) to the desired pH (pH = 3.6, 4.0, and 4.4). Water was added to adjust the buffer volume to 10 mL.
Assay cocktails preparation. Assay components were added to a vial as a solution in methanol: GAA-S (20 μΐ. of 10 mM), GAA-IS (5 μΐ. of 1 mM), GLA-S (100 μΐ. of 10 mM), GLA-IS (5 μΐ. of 1 mM), IDUA-S (50 μΐ. of 10 mM), IDUA-IS (5 μΐ. of 1 mM). Additives were added as a solution in water: Acarbose (8 μΐ^ of 1 mM) and saccharic acid 1,4 lactone (50 μΐ^ of 1 mM). The solvent was evaporated using a centrifugal concentrator (SpeedVac), the residue was taken up in 1 mL of the appropriate buffer solution, and the vial was vortex mixed until the residue dissolved. The final assay cocktail contains 0.5 mM IDUA-S, 5 μΜ IDUA-IS, 0.2 mM GAA-S, 5 μΜ GAA-IS, 1.0 mM GLA-S, 5 μΜ GLA-IS, 8 μΜ acarbose, and 50 μΜ saccharic acid 1,4-lactone.
In the following studies, every sample is run in triplicate except patients.
Buffer Screen. For each buffer composition, a single 2-mm diameter DBS punch (containing approximately 1.6 μΐ^ of blood) was obtained with a leather punch and was placed in a 1.5 mL Eppendorf tube. To this tube was added assay cocktail (20 μί) prepared as described above and containing 0.5 mM IDUA-S, 5 μΜ IDUA-IS, 0.2 mM GAA-S, 5 μΜ GAA-IS, 1.0 mM GLA-S, 5 μΜ GLA-IS, 8 μΜ acarbose, and 50 μΜ saccharic acid 1,4-lactone. Similarly, a blank containing all the assay components except the DBS punch was also prepared. The samples were incubated at 37 °C for 16 hrs in a thermostated air shaker operating at 250 rpm. The assay was quenched with 100 μΐ^ of 1: 1 methanol: ethyl acetate followed by the addition of 100 μΐ^ sodium acetate (0.1 M, pH = 5.5), 300 μΐ^ water and 400 μΐ^ ethyl acetate. The layers were mixed by aspirating with a pipette (5 times), and then the tubes were centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 μί) was transferred to a new centrifuge tube and dried down under a stream of nitrogen. The residue was resuspended in 100 μΐ^ ethyl acetate: methanol (19: 1) and applied to a plug of silica gel (-100 mg in a 1 mL pipette tip containing a cotton plug. The plug was eluted 4 times with 400 μΐ^ ethyl acetate: methanol (19: 1). Solid-phase extraction was carried out using a vacuum manifold (Millipore Inc, MAVM0960R) connected to a water aspirator. The solvent was evaporated with a stream of nitrogen, and the residue was reconstituted in 100 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 μΐ^ aliquot was used for the mass spectrometric analysis.
Post-Assay Purification. Assay cocktail was prepared as described above with the following concentrations: 0.5 mM IDUA-S, 5 μΜ IDUA-IS, 0.2 mM GAA-S, 5 μΜ GAA-IS, 0.5 mM GLA-S, 5 μΜ GLA-IS, 8 μΜ acarbose, and 50 μΜ saccharic acid 1,4- lactone in 0.1 M sodium formate buffer at pH = 3.6. A 20 μΐ^ aliquot of assay cocktail was purified as described in the buffer screen experiments using the liquid-liquid extraction and the solid phase extraction. The residue was reconstituted in 100 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 μΐ^ aliquot was used for the mass spectrometric analysis.
A different 20 μΐ^ aliquot of assay cocktail was purified using just the liquid- liquid extraction described above. After the ethyl acetate layer was dried down, the residue was reconstituted in 100 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 μΐ^ aliquot was used for the mass spectrometric analysis.
Detergent Concentrations. A single 3-mm diameter DBS punch (containing approximately 3.2 μϊ^ of blood) was obtained with a leather punch and was placed in a single well of a 96 deep-well plate. Into this well was added assay cocktail (30 μί) which was prepared as described above and containing 0.5 mM IDUA-S, 6.7 μΜ IDUA-IS, 0.4 mM GAA-S, 6.7 μΜ GAA-IS, 1.0 mM GLA-S, 6.7 μΜ GLA-IS, and 8 μΜ acarbose in 0.1 M sodium formate buffer at pH = 4.4. In addition, the assays contained the following concentrations of detergent: no detergent, 1.6 g/L sodium taurocholate, 5.6 g/L sodium taurocholate, 9.6 g/L sodium taurocholate, 2 g/L CHAPS, 4 g/L CHAPS, and 6 g/L CHAPS. A blank containing all the assay components except the DBS punch was also prepared for each detergent concentration. The samples were incubated at 37 °C for 16 hrs in a thermostated air shaker. The assay was quenched with 100 μΐ^ sodium acetate (0.1 M, pH = 5.5) and 400 μΐ^ ethyl acetate. The layers were mixed by aspirating with a pipette (5 times), and the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 μί) was transferred to a new 96 shallow-well plate, the solvent was evaporated with a stream of N2, and the residue was reconstituted in 100 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 μΐ^ aliquot was used for the mass spectrometric analysis.
Test for /V-acetyl galactosamine (GalNAc) addition. Assay cocktail was prepared as described above and contained 0.5 mM IDUA-S, 6.7 μΜ IDUA-IS, 0.4 mM GAA-S, 6.7 μΜ GAA-IS, 1.0 mM GLA-S, 6.7 μΜ GLA-IS, and 8 μΜ acarbose in 0.1 M sodium formate buffer at pH = 4.4. A separate assay cocktail containing the same concentrations as well as 96 mM GalNAc (TCI American, A1245) was also prepared. A single 3-mm diameter DBS punch (containing approximately 3.2 μΙ_, of blood) was obtained from each DBS source with a leather punch and was placed in a single well of a 96 deep-well plate. To this well was added 30 μΐ^ of assay cocktail without GalNAc. In a separate well, a 3-mm diameter DBS punch was incubated with 30 μΐ^ of assay cocktail containing GalNAc. Blank samples containing all the assay components except the DBS punches were also prepared. The samples were incubated at 37 °C for 16 hrs in a thermostated air shaker. The assay was quenched with 100 μΐ^ sodium acetate (0.1 M, pH = 5.5) and 400 μΐ^ ethyl acetate. The layers were mixed by aspirating with a pipette (5 times), and then the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 μί) was transferred to a new 96 shallow-well plate, the solvent was evaporated with a stream of nitrogen gas and the residue was reconstituted in 150 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 μΐ^ aliquot was used for the mass spectrometric analysis.
Substrate Concentration Tests
Three assay cocktails were prepared containing the following concentrations: Table 10. Assay cocktail components.
Figure imgf000063_0001
GLA-IS 6.7 μΜ 6.7 μΜ 6.7 μΜ
IDUA-S 0 0.25 ηιΜ 0.5 ηιΜ
IDUA-IS 6.7 μΜ 6.7 μΜ 6.7 μΜ
A single 3-mm diameter DBS punch (containing approximately 3.2 μΙ_, of blood) was obtained from each DBS source with a leather punch and was placed in a single well of a 96 deep-well plate. To this well was added 30 μΐ^ of assay cocktail with no substrate. In two additional wells, a 3-mm diameter DBS punch was incubated with 30 μΐ^ of assay cocktail with IX substrate and 2X substrate, respectively. Blank samples containing all the assay components except the DBS punch were also prepared. The samples were incubated at 37 °C for 16 hrs in a thermostated air shaker. The assay was quenched with 100 μΐ^ sodium acetate (0.1 M, pH = 5.5) and 400 μΐ^ ethyl acetate. The layers were mixed by aspirating with a pipette (5 times), and then the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 μί) was transferred to a new 96 shallow-well plate, the solvent was evaporated with a stream of N2, and the residue was reconstituted in 150 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 μΐ^ aliquot was used for the mass spectrometric analysis.
Test of Sodium versus Ammonium Formate Buffers. Two separate assay cocktails were prepared as described above and contained 0.5 mM IDUA-S, 6.7 μΜ IDUA-IS, 0.4 mM GAA-S, 6.7 μΜ GAA-IS, 1.0 mM GLA-S, 6.7 μΜ GLA-IS, and 8 μΜ acarbose. One assay cocktail was prepared in 0.1 M sodium formate buffer at pH = 4.4. A separate assay cocktail was prepared in 0.1 M ammonium formate at pH = 4.4. The ammonium formate buffer was prepared as described above, but the pH of the buffer was adjusted with ammonium hydroxide instead of sodium hydroxide.
A single 3-mm diameter DBS punch (containing approximately 3.2 μΙ_, of blood) was obtained from each DBS source with a leather punch and was placed in a single well of a 96 deep-well plate. To this well was added 30 μΐ^ of assay cocktail in sodium formate buffer. In a separate well, a 3-mm diameter DBS punch was incubated with 30 μΐ^ of assay cocktail in ammonium formate. Blank samples containing all the assay components except the DBS punch were also prepared. The samples were incubated at 37 °C for 16 hrs in a thermostated air shaker. The assay was quenched with 100 μΐ^ sodium acetate (0.1 M, pH = 5.5) and 400 μΐ^ ethyl acetate. The layers were mixed by aspirating with a pipette (5 times), and then the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 μί) was transferred to a new 96 shallow-well plate, the solvent was evaporated with a stream of N2, and the residue was reconstituted in 150 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 μΐ^ aliquot was used for the mass spectrometric analysis.
Test of DBS samples from Pompe Patients, Fabry Patients, and MPS IH Patients. Assay cocktail was prepared as described above and contained 0.5 mM IDUA-S, 6.7 μΜ IDUA-IS, 0.4 mM GAA-S, 6.7 μΜ GAA-IS, 1.0 mM GLA-S, 6.7 μΜ GLA-IS, and 8 μΜ acarbose in 0.1 M ammonium formate at pH = 4.4. A single 3-mm diameter DBS punch (containing approximately 3.2 μΙ_, of blood) was obtained from each DBS source with a leather punch and was placed in a single well of a 96 deep-well plate. To this well was added 30 μΐ^ of assay cocktail in sodium formate buffer. In a separate well, a 3-mm diameter DBS punch was incubated with 30 μΐ^ of assay cocktail in ammonium formate. Blank samples containing all the assay components except the DBS punch were also prepared. The samples were incubated at 37 °C for 16 hrs in a thermostated air shaker. The assay was quenched with 100 μΐ^ sodium acetate (0.1 M, pH = 5.5) and 400 μΐ^ ethyl acetate. The layers were mixed by aspirating with a pipette (5 times), and then the plate was centrifuged to separate the layers. A portion of the top layer (ethyl acetate, 250 μί) was transferred to a new 96 shallow- well plate, the solvent was evaporated with a stream of N2, and the residue was reconstituted in 150 μΐ^ of 80/20 acetonitrile/water with 0.2 % formic acid. A 20 μΐ^ aliquot was used for the mass spectrometric analysis.
Figure imgf000065_0001
Average Activity1 Average Blank μιηοΙ/h/L of blood μιηοΙ/h/L of blood
Pompe 2.41 + 0.40 0.16 + 0.01
5.6 g/L Sodium Taurocholate
MPS-IH 3.14 + 0.54 0.17 + 0.07
Fabry 6.46 + 0.49 1.15 + 0.07
Pompe 3.55 + 0.46 0.17 + 0.02
1.6 g/L Sodium Taurocholate
MPS-IH 4.05 + 0.16 0.15 + 0.05
Fabry 6.36 + 1.16 1.74 + 0.06
Pompe 5.42 + 1.10 0.32 + 0.03
6 g/L CHAPS
MPS-IH 1.86 + 0.10 0.07 + 0.04 Fabry 3.37 + 0.18 0.72 + 0.04 Pompe 4.87 + 0.29 0.14 + 0.02
4 g/L CHAPS
MPS-IH 2.26 + 0.27 0.10 + 0.03 Fabry 4.51 + 0.28 0.91 + 0.03 Pompe 6.16 + 0.56 0.19 + 0.00
2 g/L CHAPS
MPS-IH 5.19 + 0.16 0.09 + 0.03 Fabry 13.26 + 2.32 2.46 + 0.06 Pompe 17.72 + 3.17 0.53 +0.05
Average activity and standard deviation is based on assays in triplicate.
Blank assays were carried out with the assay cocktail incubated with paper punch that does not contain blood. Effect of solid-phase extraction on the amount of background product
Figure imgf000067_0001
These blank assays contained all components but were not subject to incubation prior to workup.
The observed activity in the non-incubated blank was compared to the average activity observed in triplicate assays conducted in 0.1 M sodium formate buffer, pH = 4.4 that were incubated to obtain the percentages listed in the Table.
Table 13. Assay results for multiple analysis of the quality control samples.
Enzyme Activity in μιηοΙ/h/L of blood
(%CV)
DBS Pompe Fabry MPS IH
CDC QC Low 1.1 (18.6) 1.2 (25.5) 0.8 (12.7)
CDC QC Medium 7.8 (17.8) 6.9 (8.9) 3.7 (13.6)
CDC QC High 11.5 (13.5) 13.2 (7.7) 6.5 (10.0)
The first number in each box is the mean enzyme activity followed by the covariance in percent (in parenthesis).
While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A compound of formula (I):
wherein:
Rj is unsubstituted
Figure imgf000068_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or substituted arylalkylC6.j4;
Xj is hydrogen or a counterion;
Yj is -OH or -NHC(0)CH3; and
n is an integer from 2 to 12.
2. The compound of Claim 1, wherein Rj is unsubstituted alkylcl_6.
3. The compound of Claim 1, wherein Rj is iert-butyl.
4. The compound of Claim 1, wherein Xj is a counterion.
5. The compound as in any of Claims 1-4, wherein n = 2-6.
6. A compound of formula (II):
Figure imgf000068_0002
wherein:
R2 is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or; substituted arylalkyl^.^; Y2 is -OH or -NHC(0)CH3; and
m is an integer from 2 to 12.
7. The compound of Claim 6, wherein R2 is unsubstituted alkylcl_6.
8. The compound of Claim 6, wherein R2 is iert-butyl.
9. The compound of Claim 6, wherein m = 2-6.
10. A kit comprising a compound of Claim 1 and a compound of Claim 6, wherein n≠ m.
11. A method for providing an ^-acetylgalactosamine 6-sulfatase product, comprising incubating an N- acetylgalactosamine 6-sulfatase substrate of formula (I):
Figure imgf000069_0001
wherein:
Rj is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or substituted arylalkyl^.^;
Xj is hydrogen or a counterion;
Yj is -OH or -NHC(0)CH3; and
n is an integer from 2 to 12;
with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an N-acetylgalactosamine 6-sulfatase product when the sample comprises N-acetylgalactosamine 6-sulfatase.
12. The method of Claim 11, wherein the sample is a blood sample.
13. The method of Claim 11, wherein the sample is a dried blood sample from a newborn screening card.
14. The method as in any of Claims 11-13, further comprising using the amount of the ^-acetylgalactosamine 6-sulfatase product to determine whether the dried blood sample is from a candidate for treatment for Mucopolysaccharidosis IVA (Morquio Syndrome Type A).
15. The method of Claim 14, wherein the sample further comprises an N- acetylgalactosamine 6-sulfatase internal standard.
16. The method of Claim 15, wherein the ^-acetylgalactosamine 6-sulfatase internal standard is a compound of formula (II):
wherein:
R > is unsubstituted
Figure imgf000070_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4;
Y2 is -OH or -NHC(0)CH3; and
m is an integer from 2 to 12,
wherein n≠ m.
17. The method as in Claims 15 or 16, further comprising extracting the enzyme product solution to provide the N-acetylgalactosamine 6-sulfatase internal standard and the N-acetylgalactosamine 6-sulfatase product when the sample comprises ^-acetylgalactosamine 6-sulfatase.
18. The method of Claim 17, further comprising quenching the enzymatic reaction prior to extraction.
19. The method of Claims 11 or 15, further comprising determining the quantity of the ^-acetylgalactosamine 6-sulfatase product.
20. The method of Claim 19, wherein determining the quantity of the N- acetylgalactosamine 6-sulfatase product comprises determining the ratio of the N- acetylgalactosamine 6-sulfatase product to the ^-acetylgalactosamine 6-sulfatase internal standard by mass spectrometric analysis.
21. The method of Claim 19, wherein determining the quantity of the N- acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis.
22. The method of Claim 19, wherein determining the quantity of the N- acetylgalactosamine 6-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions.
23. The method of Claim 22, wherein determining the quantity of the N- acetylgalactosamine 6-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 6-sulfatase product.
24. A method for determining the presence or absence of N- acetylgalactosamine 6-sulfatase enzymatic activity in a sample, comprising:
(a) incubating an N- acetylgalactosamine 6-sulfatase substrate with sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 6-sulfatase product when the sample comprises ^-acetylgalactosamine 6-sulfatase; and
(b) determining the presence or absence of the ^-acetylgalactosamine 6- sulfatase product, wherein the presence of ^-acetylgalactosamine 6-sulfatase product indicates the presence of ^-acetylgalactosamine 6-sulfatase enzymatic activity, and wherein the absence of ^-acetylgalactosamine 6-sulfatase product indicates the absence of ^-acetylgalactosamine 6-sulfatase enzyme activity.
25. The method of Claim 24, wherein the sample further comprises an N- acetylgalactosamine 6-sulfatase internal standard.
26. The method of Claim 25, further comprising extracting the enzyme product solution to provide the ^-acetylgalactosamine 6-sulfatase product and the N- acetylgalactosamine 6-sulfatase internal standard when the sample comprises N- acetylgalactosamine 6-sulfatase.
27. The method as in any of Claims 24-26, further comprising determining the quantity of the ^-acetylgalactosamine 6-sulfatase product.
28. A method for determining the quantity of an ^-acetylgalactosamine 6- sulfatase product in a blood sample, comprising:
(a) contacting a blood sample with a first buffer solution to provide a solution;
(b) incubating
(i) an N-acetylgalactosamine 6-sulfatase substrate of formula (I):
Figure imgf000072_0001
wherein:
Rj is unsubstituted alkylcl_6; substituted alkylcl_6; unsubstituted arylC5.j2; substituted aryl^.^; unsubstituted arylalkyl^.^; or substituted arylalkyl^.^;
Xj is hydrogen or a counterion;
Yj is -OH or -NHC(0)CH3; and
n is an integer from 2 to 12; and
(ii) an N-acetylgalactosamine 6-sulfatase internal standard,
with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an N- acetylgalactosamine 6-sulfatase product when the sample comprises N- acetylgalactosamine 6-sulfatase; and
(c) determining the quantity of the ^-acetylgalactosamine 6-sulfatase product by tandem mass spectro metric analysis.
29. The method of Claim 28, wherein the ^-acetylgalactosamine 6-sulfatase internal standard is a compound of formula (II):
Figure imgf000073_0001
wherein:
R > is unsubstituted
Figure imgf000073_0002
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4;
Y2 is -OH or -NHC(0)CH3; and
m is an integer from 2 to 12, wherein n≠ m.
30. The method of Claims 28 or 29, further comprising extracting the enzyme product solution to provide the ^-acetylgalactosamine 6-sulfatase internal standard and the ^-acetylgalactosamine 6-sulfatase product when the sample comprises N- acetylgalactosamine 6-sulfatase.
31. The method of Claims 28 or 29, wherein (c) comprises:
(i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and
(ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 6- sulfatase product.
32. The method of Claim 28, wherein the blood sample is a dried blood sample from a newborn.
33. The method of Claim 32, wherein (a) is further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution.
34. The method of Claims 32 or 33, further comprising using the amount of N- acetylgalactosamine 6-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis IVA (Morquio Syndrome Type A).
35. A compound of formula (III):
wherein:
R3 is unsubstituted
Figure imgf000074_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4;
X2 is hydrogen or a counterion; and
p is an integer from 2 to 12.
36. The compound of Claim 35, wherein R3 is unsubstituted alkylci. .
37. The compound of Claim 35, wherein R3 is iert-butyl.
38. The compound of Claim 35, wherein X2 is a counterion.
39. The compound as in any of Claims 35-38, wherein p = 2-6.
40. A compound of formula (IV):
wherein:
R4 is unsubstituted
Figure imgf000074_0002
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4; and q is an integer from 2 to 12.
41. The compound of Claim 40, wherein R4 is unsubstituted alkylcl_6.
42. The compound of Claim 40, wherein R4 is iert-butyl.
43. The compound of Claim 40, wherein q = 2-6.
44. A kit comprising a compound of Claim 35 and a compound of Claim 40, wherein p≠ q.
45. A method for providing an N-acetylgalatosamine 4-sulfatase product, comprising incubating an N- acetylgalactosamine 4-sulfatase substrate of formula (III):
wherein:
R3 is unsubstituted
Figure imgf000075_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4;
X2 is hydrogen or a counterion; and
p is an integer from 2 to 12;
with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an N-acetylgalactosamine 4-sulfatase product when the sample comprises ^-acetylgalactosamine 4-sulfatase.
46. The method of Claim 45, wherein the sample is a blood sample.
47. The method of Claim 45, wherein the sample is a dried blood sample from a newborn screening card.
48. The method as in any of Claims 45-47, further comprising using the amount of the ^-acetylgalactosamine 4-sulfatase product to determine whether the dried blood sample is from a candidate for treatment for Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome).
49. The method of Claim 48, wherein the sample further comprises an N- acetylgalactosamine 4-sulfatase internal standard.
50. The method of Claims 49, wherein the ^-acetylgalactosamine 4-sulfatase internal standard is a compound of formula (IV):
wherein:
R4 is unsubstituted
Figure imgf000076_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4; and q is an integer from 2 to 12, and wherein p≠ q.
51. The method as in Claims 49 or 50, further comprising extracting the enzyme product solution to provide the N-acetylgalactosamine 4-sulfatase internal standard and the N-acetylgalactosamine 4-sulfatase product when the sample comprises ^-acetylgalactosamine 4-sulfatase.
52. The method of Claim 51, further comprising quenching the enzymatic reaction prior to extraction.
53. The method of Claims 45 or 49, further comprising determining the quantity of the ^-acetylgalactosamine 4-sulfatase product.
54. The method of Claim 53, wherein determining the quantity of the N- acetylgalactosamine 4-sulfatase product comprises determining the ratio of the N- acetylgalactosamine 4-sulfatase product to the ^-acetylgalactosamine 4-sulfatase internal standard by mass spectrometric analysis.
55. The method of Claim 53, wherein determining the quantity of the N- acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis.
56. The method of Claim 53, wherein determining the quantity of the N- acetylgalactosamine 4-sulfatase product comprises tandem mass spectrometric analysis in which the parent ions of the product and internal standard are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions.
57. The method of Claim 56, wherein determining the quantity of the N- acetylgalactosamine 4-sulfatase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 4-sulfatase product.
58. A method for determining the presence or absence of N- acetylgalactosamine 4-sulfatase enzymatic activity in a sample, comprising:
(a) incubating an N- acetylgalactosamine 4-sulfatase substrate with sample for a pre-determined time sufficient to effect enzymatic reaction to provide an enzyme product solution comprising an ^-acetylgalactosamine 4-sulfatase product when the sample comprises ^-acetylgalactosamine 4-sulfatase; and
(b) determining the presence or absence of the ^-acetylgalactosamine 4- sulfatase product, wherein the presence of ^-acetylgalactosamine 4-sulfatase product indicates the presence of ^-acetylgalactosamine 4-sulfatase enzymatic activity, and wherein the absence of ^-acetylgalactosamine 4-sulfatase product indicates the absence of ^-acetylgalactosamine 4-sulfatase enzymatic activity.
59. The method of Claim 58, wherein the sample further comprises an N- acetylgalactosamine 4-sulfatase internal standard.
60. The method of Claim 59, further comprising extracting the enzyme product solution to provide the ^-acetylgalactosamine 4-sulfatase product and the N- acetylgalactosamine 4-sulfatase internal standard when the sample comprises N- acetylgalactosamine 4-sulfatase.
61. The method as in any of Claims 58-60, further comprising determining the quantity of the ^-acetylgalactosamine 4-sulfatase product.
62. A method for determining the quantity of an ^-acetylgalactosamine 4- sulfatase product in a blood sample, comprising:
(a) contacting a blood sample with a first buffer solution to provide a solution;
(b) incubating
(i) an N-acetylgalactosamine 4-sulfatase substrate of formula (III):
wherein:
R3 is unsubstituted
Figure imgf000078_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4:
X2 is hydrogen or a counterion; and
p is an integer from 2 to 12; and
(ii) an N-acetylgalactosamine 4-sulfatase internal standard,
with the first buffer solution for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an N- acetylgalactosamine 4-sulfatase product when the sample comprises N- acetylgalactosamine 4-sulfatase; and
(c) determining the quantity of the N-acetylgalactosamine 4-sulfatase product by tandem mass spectrometric analysis.
63. The method of Claim 62, wherein the ^-acetylgalactosamine 4-sulfatase internal standard is a compound of formula (IV): wherein:
R4 is unsubstituted
Figure imgf000079_0001
substituted arylC5.j2; unsubstituted arylalkylC6.j4; or; substituted arylalkylC6.j4; and q is an integer from 2 to 12, and wherein p≠ q.
64. The method of Claims 62 or 63, further comprising extracting the enzyme product solution to provide the ^-acetylgalactosamine 4-sulfatase internal standard and the ^-acetylgalactosamine 4-sulfatase product when the sample comprises N- acetylgalactosamine 4-sulfatase.
65. The method of Claims 62 or 63, wherein (c) comprises:
(i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and
(ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 4- sulfatase product.
66. The method of Claim 62, wherein the blood sample is a dried blood sample from a newborn.
67. The method of Claim 66, wherein (a) is further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution.
68. The method of Claims 66 or 67, further comprising using the amount of N- acetylgalactosamine 4-sulfatase product to predict whether the newborn is a candidate for treatment of Mucopolysaccharidosis VI (Maroteaux-Lamy Syndrome).
69. A method for providing a solution comprising an a-glucosidase product, an a-galactosidase product, and an a-L-iduronidase product, comprising incubating an a- glucosidase substrate, an α-galactosidase substrate, and an α-L-iduronidase substrate with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an a-glucosidase product, an a- galactosidase product, and an α-L-iduronidase product when the sample comprises a- glucosidase, α-galactosidase, and a-L-iduronidase,
wherein the buffer comprises an aqueous solution of acarbose.
70. The method of Claim 69, wherein the buffer has a pH range of 2-7.
71. The method of Claim 69, wherein the buffer comprises formate, acetate, citrate-phosphate, or trifluoroacetate, or a combination thereof.
72. The method of Claim 71, wherein the buffer comprises ammonium formate, ammonium acetate, ammonium citrate-phosphate, ammonium trifluoroacetate, sodium formate, sodium acetate, sodium citrate-phosphate, or sodium trifluoroacetate, or a combination thereof.
73. The method of Claim 69, wherein the buffer is a volatile buffer.
74. The method of Claim 69, wherein the sample is a blood sample.
75. The method of Claim 69, wherein the sample is a dried blood sample from a newborn screening card.
76. The method as in Claims 69-75, further comprising using the amount of a- glucosidase product to determine whether the dried blood sample is from a candidate for treatment for Pompe disease.
77. The method as in Claims 69-75, further comprising using the amount of a- galactosidase A product to determine whether the dried blood sample is from a candidate for treatment for Fabry disease.
78. The method as in Claims 69-75, further comprising using the amount of a- L-iduronidase product to determine whether the dried blood sample is from a candidate for treatment for mucopolysaccharidosis I (Hurler) disease (MPS IH).
79. The method of Claim 69, wherein the sample further comprises an a- glucosidase internal standard, an α-galactosidase internal standard, and an a-L- iduronidase internal standard.
80. The method of Claim 79, further comprising extracting the enzyme product solution to provide the α-glucosidase internal standard, the a-glucosidase product, the α-galactosidase internal standard, the α-galactosidase product, the a-L- iduronidase internal standard, and the α-L-iduronidase product when the sample comprises α-glucosidase, α-galactosidase, and a-L-iduronidase.
81. The method of Claim 80, further comprising quenching the enzymatic reaction prior to extraction.
82. The method of Claims 69 or 79, further comprising determining the quantity of the α-glucosidase product, the α-galactosidase product, and the a-L- iduronidase product.
83. The method of Claim 82, wherein determining the quantity of the a- glucosidase product, the α-galactosidase product, and the α-L-iduronidase product comprises determining the ratio of the α-glucosidase product, the α-galactosidase product, and the α-L-iduronidase product to the α-glucosidase internal standard, the a- galactosidase internal standard, and the α-L-iduronidase internal standard, respectively, by mass spectrometric analysis.
84. The method of Claim 82, wherein determining the quantity of the a- glucosidase product, the α-galactosidase product, and the α-L-iduronidase product comprises tandem mass spectrometric analysis.
85. The method of Claim 82, wherein determining the quantity of the a- glucosidase product, the α-galactosidase product, and the α-L-iduronidase product comprises tandem mass spectrometric analysis in which the parent ions of the products and internal standards are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions.
86. The method of Claim 85, wherein determining the quantity of the a- glucosidase product, the α-galactosidase product, and the a-L-iduronidase product comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the a-glucosidase product, the a- galactosidase product, and the α-L-iduronidase product.
87. A method for determining the presence or absence of a-glucosidase enzymatic activity, α-galactosidase enzymatic activity, and α-L-iduronidase enzymatic activity in a sample, comprising:
(a) incubating an α-glucosidase substrate, an α-galactosidase substrate, an a- L-iduronidase substrate with a sample for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an a-glucosidase product, an α-galactosidase product, and an α-L-iduronidase product when the sample comprises α-glucosidase, α-galactosidase, and α-L-iduronidase; and
(b) determining the presence or absence of the α-glucosidase product, the a- galactosidase product, and the α-L-iduronidase product, wherein the presence of a- glucosidase product indicates the presence of α-glucosidase enzymatic activity, wherein the presence of α-galactosidase product indicates the presence of a-galactosidase enzymatic activity, and wherein the presence of α-L-iduronidase product indicates the presence of α-L-iduronidase activity, and
wherein the absence of α-glucosidase product indicates the absence of a- glucosidase enzymatic activity, wherein the absence of α-galactosidase product indicates the absence of α-galactosidase enzymatic activity, and wherein the absence of a-L- iduronidase product indicates the absence of α-L-iduronidase activity.
88. The method of Claim 87, wherein the sample further comprises an a- glucosidase internal standard, an α-galactosidase internal standard, and an a-L- iduronidase internal standard.
89. The method of Claim 88, further comprising extracting the enzyme product solution to provide the α-glucosidase product, the α-glucosidase internal standard, the a-galactosidase product, the a-galactosidase internal standard, the a-L- iduronidase product, and the a-L-iduronidase internal standard when the sample comprises a-glucosidase, α-galactosidase, and a-L-iduronidase.
90. The method as in any of Claims 87-89, further comprising determining the quantity of the α-glucosidase product, the α-galactosidase product, and the a-L- iduronidase product.
91. A method for determining the quantity of a α-glucosidase product, a a- galactosidase product, and a α-L-iduronidase product in a blood sample, comprising:
(a) contacting a blood sample with a first buffer solution to provide a solution;
(b) incubating an α-glucosidase substrate, an α-glucosidase internal standard, an α-galactosidase substrate, an α-galactosidase internal standard, an a-L-iduronidase substrate, and an α-L-iduronidase internal standard with a sample in a buffer for a pre-determined time sufficient to effect an enzymatic reaction to provide an enzyme product solution comprising an α-glucosidase product, an α-galactosidase product, and an α-L-iduronidase product when the sample comprises α-glucosidase, α-galactosidase, and a-L-iduronidase,
wherein the buffer comprises an aqueous solution of acarbose; and
(c) determining the quantity of the α-glucosidase product, the a-galactosidase product, and the α-L-iduronidase product by tandem mass spectrometric analysis.
92. The method of Claim 91, further comprising extracting the enzyme product solution with an organic solvent to provide an organic phase comprising the a- glucosidase product, the α-glucosidase internal standard, the α-galactosidase product, the α-galactosidase internal standard, the α-L-iduronidase product, and the a-L-iduronidase internal standard when the sample comprises α-glucosidase, α-galactosidase, and a-L- iduronidase.
93. The method of Claim 91, wherein (c) comprises:
(i) generating, isolating, and subjecting the parent ions of the products and the internal standards to collision-induced dissociation to provide product fragment ions and internal standard fragment ions, and (ii) comparing the ion peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the ^-acetylgalactosamine 6- sulfatase product.
94. The method of Claim 91, wherein the blood sample is a dried blood sample from a newborn.
95. The method of Claim 94, wherein (a) is further defined as contacting a dried blood sample from a newborn screening card with a first buffer solution to provide a solution.
96. The method of Claims 94 or 95, further comprising:
(e) using the amount of a-glucosidase product to predict whether the newborn is a candidate for treatment of Pompe disease;
(f) using the amount of a-galactosidase product to predict whether the newborn is a candidate for treatment of Fabry disease; or
(g) using the amount of a-L-iduronidase product to predict whether the newborn is a candidate for treatment of mucopolysaccharidosis I (Hurler) disease (MPS IH).
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