US20140274798A1 - Compounds and methods relating to lysosomal storage disorders - Google Patents

Compounds and methods relating to lysosomal storage disorders Download PDF

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US20140274798A1
US20140274798A1 US14/215,885 US201414215885A US2014274798A1 US 20140274798 A1 US20140274798 A1 US 20140274798A1 US 201414215885 A US201414215885 A US 201414215885A US 2014274798 A1 US2014274798 A1 US 2014274798A1
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alkyl
alkenyl
substrate
substituent
heteroatom containing
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Alexander Cherkassky
Jason Cournoyer
Michael Gelb
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University of Washington Center for Commercialization
Revvity Health Sciences Inc
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PerkinElmer Health Sciences Inc
University of Washington Center for Commercialization
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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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present description relates to analytical reagents for detecting enzymatic activity for detecting lysosomal enzyme activity.
  • Lysosomal storage disorders are a group of inherited disorders characterized by deficiencies in specific enzymes in the body, which results in the body's inability to break down metabolic substances.
  • Fabry disease is a lysosomal storage disorder seen in one out of every 40,000 people. It is caused by a deficiency in the enzyme alpha-galactosidase which results in the body's inability to break down specific fatty substances called globotriaosylceramides.
  • Gaucher disease a lysosomal storage disorder caused by an inability to break down fatty substances or lipids called glucosylceramides (also called glucocerebrosides).
  • glucocerebrosidase an enzyme needed to break down these fatty substances. These fatty substances then accumulate in cells of the liver, spleen, and bone marrow.
  • a third example is Pompe disease, a lysosomal storage disorder caused by a deficiency in the enzyme acid alpha-glucosidase, which is needed to break down certain sugars called glycogen. When the enzyme acid alpha-glucosidase is missing, glycogen accumulates in various tissues and organs in the body.
  • Lysosomal storage disorders are, for the most part, childhood disorders although some manifest in adulthood. In most of them, patients are normal at birth and have progressive neurological deterioration beginning at some later time. The clinical phenotype depends on the type and severity of the biochemical defect. Some of these lysosomal disorders, such as Pompe disease and Krabbe disease, manifest primarily in infancy. There have been ongoing efforts in developing methods to detect such disorders before the onset of clinical symptoms so that therapeutic interventions can be initiated.
  • Tandem mass spectrometry continues to gain popularity in the clinic because this technology allows for assay of many metabolites in a single sample. For example, this technology has been implemented as a routine clinical practice for the detection of hereditary metabolic disorders in newborns using dry blood spot samples. Although lysosomal enzyme activities can be quantified using tandem mass spectrometry, published assay methods have been somewhat difficult to adapt to a clinical setting due to solubility issues and the need for incorporation of an external standard.
  • compositions and processes for detecting enzymatic reactions using detection systems such as mass spectrometry are provided. These compositions provide for improved solubility in aqueous solvent systems and/or improved reactivity with a target enzyme thereby improving assay efficiency, reproducibility, and accuracy.
  • the invention provides chemical compounds useful for assessing the level of lysosomal enzyme activity in a sample. Testing of lysosomal enzyme activity is useful, for example, when screening for metabolic disorders in newborns as well as when assessing an individual having a medical condition affecting enzyme activity or one undergoing a medical treatment such as enzyme replacement therapy, gene therapy, or bone marrow transplantation.
  • the chemical compounds described herein include substrates for target enzymes and related molecules useful as controls or standards in enzyme assays.
  • substrates suitable for detecting the activity of an enzyme illustratively an enzyme the deficiency of which leads to a lysosomal storage disorder.
  • Substrate are provided that have the general formula:
  • A is a monosaccharide or a disaccharide
  • B1 is: a C 1 -C 20 alkyl; a heteroatom containing C 1 -C 20 alkyl, C 2 -C 20 alkenyl; a heteroatom containing C 2 -C 20 alkenyl, a substituted or unsubstituted C 6 -C 20 aryl
  • B2 is: a C 2 -C 7 urethane; a C 2 -C 7 amido, a C 2 -C 7 ester, a C 2 -C 7 uriedo; a C 2 -C 7 carbamato; a C 2 -C 7 carbonyl; a C 1 -C 7 alkyl; a heteroatom containing C 1 -C 7 alkyl; a C 1 -C 7 alkyl having a substituent of N, O, or S; a C 2 -C 7 alkenyl; a heteroatom containing C 2 -C 7
  • a substituent of N, O, or S is independently optionally a hydroxyl, an amino, a thiol, ether, thioether, or secondary amine.
  • A is an aldohexose or a ketohexose, optionally linked through and ⁇ or ⁇ glycosidic bond to the remainder of the molecule.
  • A is a D-glucose or a D-galactose.
  • a B1 moiety is optionally a methylene or C 2 alkyl group.
  • a B2 moiety is optionally a C 2 -C 7 amido.
  • a B3 moiety is optionally a C 2 -C 20 alkenyl having a substituent of N, O, or S.
  • a substrate includes an A that is D-glucose or a D-galactose, A B1 that is a methylene, a B1 that is a C 2 -C 7 amido, and B3 that is a C 13 -C 20 alkenyl having a substituent of O present as a hydroxyl.
  • Formula II A is an aldohexose or ketohexose;
  • R 1 is a C 1 -C 6 alkyl or a C 2 -C 20 alkenyl; and
  • R 2 is a C 1 -C 20 alkyl, a C 1 -C 20 alkyl having a substituent of N, O, or S, a C 1 -C 20 alkenyl, or a C 1 -C 20 alkenyl having a substituent of N, O or S.
  • R 1 or R 2 could be a C 1 -C 20 alkynl optionally containing a substitution of N, O, or S, or a substituent of N, O, or S.
  • the substrate of formula II includes A as a D-glucose or D-galactose, R 1 as a C 4 -C 6 alkyl, and R 2 is a C 13 -C 20 alkyl, a C 13 -C 20 alkyl having a substituent of N, O, or S, a C 13 -C 20 alkenyl, or a C 13 -C 20 alkenyl having a substituent of N, O or S.
  • R 2 is a C 1-3 alkyl.
  • A is a monosaccharide or a disaccharide
  • B1 is: a C 1 -C 20 alkyl; a heteroatom containing C 1 -C 20 alkyl, C 2 -C 20 alkenyl; a heteroatom containing C 2 -C 20 alkenyl; and a substituted or unsubstituted C 6 -C 20 aryl
  • R 1 ′ is a substituted or unsubstituted C, or N
  • R 2 ′ is a substituted or unsubstituted C, a substituted or unsubstituted N, O, or S
  • R 3 ′ is a substituted or unsubstituted C, N, or O
  • R 4 ′ is a nullity, a substituted or unsubstituted C 1 -C 2 , O, or S
  • R 1 is: a C 1 -C 6 alkyl; a heteroatom containing C 1 -C 7 alkyl: a
  • A is optionally an aldohexose or a ketohexose, optionally linked through and ⁇ or ⁇ glycosidic bond to the remainder of the molecule.
  • A is a D-glucose or a D-galactose.
  • a substrate according to formula III is:
  • R 2 ′ is a substituted or unsubstituted C or a substituted or unsubstituted N;
  • R 2 ′′ is a H or a methyl and R 2 ′′′ is a H or a methyl; or
  • R 3 ′ is a substituted or unsubstituted C
  • R 4 ′ is a nullity, a substituted or unsubstituted C 1 -C 2 , O, or S.
  • A is optionally an aldohexose or a ketohexose, optionally linked through and ⁇ or ⁇ glycosidic bond to the remainder of the molecule.
  • A is a D-glucose or a D-galactose.
  • a substrate according to Formula III includes:
  • R 2 ′ is a substituted or unsubstituted C or a substituted or unsubstituted N
  • R 6 ′ is a C 1 -C 20 alkyl, a heteroatom containing C 1 -C 20 alkyl; C 1 -C 20 alkyl having a substituent of N, O, or S
  • C 4 -C 20 ether is a C 1 -C 20 ester
  • C 6 -C 20 aryl and C 6 -C 20 heterocyclic containing a heteroatom of N, O or S
  • R 6 ′ is a substituted or unsubstituted C or a substituted or unsubstituted N
  • R 6 ′ is a C 1 -C 20 alkyl, a heteroatom containing C 1 -C 20
  • R 2 ′ is a substituted or unsubstituted C or a substituted or unsubstituted N;
  • R 2 is a C 13 -C 20 alkyl, a heteroatom containing C 13 -C 20 alkyl, a C 13 -C 20 alkyl having a substituent of N, O, or S; a C 13 -C 20 alkenyl, a heteroatom containing C 13 -C 20 alkenyl; or a C 13 -C 20 alkenyl having a substituent of N, O, or S; or
  • R 2 is a C 13 -C 20 alkyl, a heteroatom containing C 13 -C 20 alkyl, a C 13 -C 20 alkyl having a substituent of N, O, or S; a C 13 -C 20 alkenyl, a heteroatom containing C 13 -C 20 alkenyl; or a C 13 -C 20 alkenyl having a substituent of N, O, or S.
  • A is optionally an aldohexose or a ketohexose, optionally linked through and ⁇ or ⁇ glycosidic bond to the remainder of the molecule.
  • A is a D-glucose or a D-galactose.
  • substrates including:
  • A is optionally an aldohexose or a ketohexose, optionally linked through and ⁇ or ⁇ glycosidic bond to the remainder of the molecule.
  • A is a D-glucose or a D-galactose.
  • any of the substrates of the invention optionally include a stable secondary prevalence isotope of an element or other label.
  • stable secondary prevalence isotope in each occurrence is selected from the group consisting of 2 H, 13 C, 15 N, 17 O, 18 O, 31 P or 34 S.
  • Such molecules optionally include the structure of formula V:
  • B1 is selected from the group consisting of: a C 1 -C 20 alkyl; a heteroatom containing C 1 -C 20 alkyl, C 2 -C 20 alkenyl; a heteroatom containing C 2 -C 20 alkenyl, a substituted or unsubstituted C 6 -C 20 aryl;
  • B2 is selected from the group consisting of: a C 2 -C 7 urethane; a C 2 -C 7 amido, a C 2 -C 7 ester, a C 2 -C 7 uriedo; a C 2 -C 7 carbamato; a C 2 -C 7 carbonyl; a C 1 -C 7 alkyl; a heteroatom containing C 1 -C 7 alkyl; a C 1 -C 7 alkyl having a substituent of N, O, or S; a C 2 -C 7 alkenyl; a heteroatom containing C 2 -C 7 alkeny
  • B1 is a methylene.
  • B2 is a C 2 -C 7 amido.
  • B3 is a C 2 -C 20 alkenyl having a substituent of N, O, or S.
  • B1 is a methylene
  • B2 is a C 2 -C 7 amido
  • B3 is a C 13 -C 20 alkenyl having a substituent of O present as a hydroxyl.
  • any of the molecules of the invention optionally include a stable secondary prevalence isotope of an element or other label.
  • stable secondary prevalence isotope in each occurrence is selected from the group consisting of 2 H, 13 C, 15 N, 17 O, 18 O, 31 P or 34 S.
  • compositions of Formulas I-VI in a method for the detection of the presence, absence, or level of an enzyme allows improved confidence in assay results from the improved solubility of the enzymes in aqueous solvent systems.
  • a method for detecting enzymatic activity includes contacting a sample containing a target enzyme with any of the substrates described herein or their equivalents, under conditions wherein the target enzyme is capable of acting on the substrate to produce an enzymatic product; and detecting the enzymatic product.
  • the said target enzyme is acid ⁇ -glucocerebrosidase and said substrate of is of formula I wherein said B2 is a C 2 -C 7 amido, and B3 is a C 2 -C 20 alkenyl having a substituent of N, O, or S.
  • the target enzyme is acid galactocerebroside ⁇ -galactosidase or acid- ⁇ -glucocerebrosidase and said wherein the B2 is a C 2 -C 7 amido, and B3 is a C 13 -C 20 having a substituent of O present as a hydroxyl.
  • a substrate for use in a method of detecting enzyme activity is the substrate of any one or more of compound 1 through 80, optionally 1 or 7, or any combination thereof.
  • a method includes adding an internal standard to the reaction solution during or following the step of contacting. Such an internal standard is optionally any standard of compositions 81-86.
  • the step of detecting is optionally by mass spectrometry, optionally by multiple reaction monitoring such as in MS/MS.
  • the step of detecting is optionally by immunoassay, HPLC, mass spectrometry, or other suitable method for detecting molecules with a molecular weight less than 1000 Daltons.
  • FIG. 1A illustrates NMR analysis of a substrate according to one embodiment
  • FIG. 1B illustrates NMR analysis of a second substrate according to one embodiment
  • FIG. 2 is an exemplary substrate structure highlighting structural moieties
  • FIG. 3 is an exemplary substrate structure highlighting structural moieties
  • FIG. 4 illustrates exemplary substrate structures and corresponding exemplary internal standard structures
  • FIG. 5 is a generic enzymatic reaction scheme using an exemplary substrate.
  • FIG. 6 is a graph of experimental results comparing the performance of substrates of the present invention (hashed) with the performance of prior art substrates (checked) in an analytical procedure for detecting lysosomal storage disorders.
  • any A element is interchangeable with any other A element
  • any B1 element is substitutable with any other B1 element
  • any B2 element is substitutable with any other B2 element
  • any B3 element is substitutable with any other B3 element
  • any R 1 element is substitutable with any other R 1 element
  • any R 2 element is substitutable with any other R 2 element
  • any R 1 element is substitutable with any other R 1 ′ element
  • any R 2 ′ element is substitutable with any other R 2 ′ element
  • any R 3 ′ element is substitutable with any other R 3 ′ element
  • any R 4 ′ element is substitutable with any other R 4 ′ element
  • any R 5 ′ element is substitutable with any other R 5′ element
  • any R 6′ element is substitutable with any other R 6 ′ element.
  • compositions provided have utility as analytical reagents for detecting hydrolase enzyme activity, such as lysosomal enzyme activities associated with lysosomal storage disorders.
  • hydrolase enzyme activity such as lysosomal enzyme activities associated with lysosomal storage disorders.
  • compositions relate to substrates that are targets for lysosomal enzymes optionally including: acid ⁇ -glucocerebrosidase (ABG), galactocerebroside ⁇ -galactosidase (GALC).
  • ABS acid ⁇ -glucocerebrosidase
  • GALC galactocerebroside ⁇ -galactosidase
  • the action of these enzymes over the substrates is used to measure the corresponding enzyme activities in a sample, and thus these substrates may be used to detect the following lysosomal storage disorders: Gaucher (ABG); and Krabbe (GALC).
  • a substrate has the general formula
  • A is a monosaccharide or a disaccharide
  • B1 is: a C 1 -C 20 alkyl; a heteroatom containing C 1 -C 20 alkyl, C 2 -C 20 alkenyl; a heteroatom containing C 2 -C 20 alkenyl, a substituted or unsubstituted C 6 -C 20 aryl
  • B2 is: a C 2 -C 7 urethane; a C 2 -C 7 amido, a C 2 -C 7 ester, a C 2 -C 7 uriedo; a C 2 -C 7 carbamato; a C 2 -C 7 carbonyl; a C 1 -C 7 alkyl; a heteroatom containing C 1 -C 7 alkyl; a C 1 -C 7 alkyl having a substituent of N, O, or S; a C 2 -C 7 alkenyl; a heteroatom containing C 2 -C 7
  • Specificity of the substrate for a particular lysosomal enzyme is provided in part by structural variations in the sugar moiety A such as A being a monosaccharide or a disaccharide, and in some embodiments by the particular sugar moiety employed.
  • Exemplary sugar moieties include ⁇ -D-Glucose for detecting Gaucher disease, and ⁇ -D-Galactose for detecting Krabbe disease.
  • the monosaccharide is optionally linked to the remainder of the molecule by either a ⁇ or ⁇ glycosidic bond.
  • Additional exemplary sugar moieties include but are not limited to allose, altrose, glucose, mannose, gulose, idose, galactose, talose, fructose, psicose, sorbose, and tagatose, each in either the D or L configuration.
  • B 1 is a linker moiety which functions to allow conjugation of the sugar moiety A to the remaining structure of the substrate. B 1 also functions as a spacer between the sugar moiety A and the remaining structure of the substrate so as to provide flexible access for a target enzyme.
  • the linker arm B 1 can be designed so as to control the polarity of the materials and hence their solubility characteristics. In some instances, the linker arm B 1 can have a hydrophenol structure.
  • a substrate of the general formula I can be configured to be at least partially hydrophilic in a solvent such as pure methanol or pure ethanol.
  • the fatty acid moiety in toto generally is tailored to be sufficiently hydrophilic to provide aqueous solubility to the substrate.
  • the substrates can be soluble in aqueous buffer systems; although it is to be understood that, as is known in the art, detergents or other such agents may be included in the reagent mixture to enhance the aqueous solubility of the substrate materials.
  • B 2 provides aqueous solubility to the substrates relative to long chain fatty acids of natural cerebrosides or prior substrates. It was unexpected that the short alkyl chain length of particular B 2 structures (length of C 2 -C 7 ) would allow recognition by the desired enzyme with sufficient affinity and turnover rate to be operable. In addition, the lack of saturation in the alkenyl chain according to some embodiments was similarly not expected to impart function as a substrate. Both of these elements used alone or separately allow full solubility characteristics to the substrates that was not achieved by prior methods.
  • B 2 may include one or more a nucleophilic groups for interactions with a solid support or detectable tag, such as a fluorescent tag.
  • a nucleophilic groups are optionally a nitrogen, oxygen, or sulfur nucleophilic group.
  • a nucleophilic group is an amine.
  • compositions do not need to carry a quaternary ammonium group to be used in particular detection procedures such as mass spectroscopy.
  • the relative solubility imparted by the B 2 group is sufficient to improve detection of one or more enzyme using the substrates.
  • a substrate is generally more hydrophilic without the requirement of a permanently charged moiety and requires less or no need for detergent. This results in simplified assay procedures because, like the use of chloroform, the use of detergents can require cumbersome clean up steps including the labor-intensive liquid-liquid and solid phase extractions.
  • the specificity of the substrates during the mass spectrometry analysis may also be conferred by variations in the carbon length and degree of saturation within an alkyl group of B 2 .
  • Exemplary chemical structures of B 2 include a four-carbon fatty-acyl group for detecting Gaucher disease, and a six-carbon fatty-acyl group specific for detecting Krabbe disease. It is appreciated that absolute specificity for the enzyme is not imparted by B 2 , but more appropriately by the identity of A. Different chain lengths or degree of saturation of B 2 can be used to tailor the substrates to the enzyme of interest as well as serve as a differentiator for detection in multiplex assay format.
  • a multiplex assay that combines a substrate with a B 2 including a four-carbon fatty-acyl group, and a second substrate with a B 2 having a six-carbon fatty-acyl group will allow detection and identification of which products is produced and which are not. For example, if a result indicates a four-carbon fatty-acyl group in a reaction product, this indicates the presence of ⁇ -glucerebrosidase in the material when using some embodiments of the substrates. If a result indicates a six-carbon fatty-acyl group this indicates the presence of a galactocerebroside ⁇ -galactosidase when using some embodiments of the substrates. Therefore, by combining substrates with different A groups and different product compositions, rapid identification of specific enzyme present in the sample is readily achieved.
  • a substrate is structurally terminated by a B 3 group.
  • B 3 can be structurally tailored to provide different chain lengths. Such different chain lengths are useful for distinguishing different substrates, as well as enzymatic products thereof, from each other in enzyme assays. For example, in mass spectrometry, a substrate containing a 12 carbon atom chain has a different mass-to-charge ratio than a substrate containing a 14 carbon atom chain and as such, substrates containing 12 carbon atom or 14 carbon atom chains can be distinguished. Similarly, in immunoassay formats, substrates having differing chain lengths can be distinguished using antibodies selective for particular chemical moieties. It is recognized that the same is true for any differences in chain length or other structure of B 3 in the substrates when combinations of substrates are used in multiplex or singleplex assay formats.
  • a substrate has the structure of:
  • A is a monosaccharide or a disaccharide and is linked to the remainder of the molecule via a glycosidic bond;
  • R 1 is a C 1 -C 6 alkyl or a C 2 -C 20 alkenyl, and where R 2 is a C 13 -C 20 alkyl or a C 13 -C 20 alkenyl.
  • R 1 is a C 6 alkyl or a C 4 alkyl, and R 2 is a C 1-3 alkyl.
  • A is an aldohexose or a ketohexose.
  • A is a D-glucose or D-galactose.
  • A is a D-glucose or D-galactose
  • R 1 is a C 4 alkyl, C 5 alkyl or a C 6 alkyl
  • R 2 is a C 13 -C 20 alkyl or a C 13 -C 20 alkenyl
  • A is a D-glucose or D-galactose
  • R 1 is a C 4 alkyl, C 5 alkyl or a C 6 alkyl
  • R 2 is a C 1-3 alkyl.
  • a substrate has the structure of:
  • A is a monosaccharide or a disaccharide
  • B1 is: a C 1 -C 20 alkyl; a heteroatom containing C 1 -C 20 alkyl, C 2 -C 20 alkenyl; a heteroatom containing C 2 -C 20 alkenyl; and a substituted or unsubstituted C 6 -C 20 aryl
  • R 1 ′ is a substituted or unsubstituted C, or N
  • R 2 ′ is a substituted or unsubstituted C, a substituted or unsubstituted N, O, or S
  • R 3 ′ is a substituted or unsubstituted C, N, or O
  • R 4 ′ is a nullity, a substituted or unsubstituted C 1 -C 2 , O, or S
  • R 1 is: a C 1 -C 6 alkyl; a heteroatom containing C 1 -C 7 alkyl: a
  • the activity of a particular enzyme can be assessed by its capability or rate of acting on a cognate substrate to product enzymatic products.
  • action of a target enzyme results in generation of two products: A (and/or A-H, since, as is shown in FIG. 5 , the sugar group typically leave with an —OH group on the first carbon) and HO-fatty acid portion (B) since the B portion, as is shown in FIG. 5 , also typically leaves in a hydroxylated form.
  • A and/or A-H, since, as is shown in FIG. 5 , the sugar group typically leave with an —OH group on the first carbon
  • B HO-fatty acid portion
  • the activity of the target enzyme can be determined.
  • a known amount of an internal standard corresponding to the non-A portion of Formula I-III as is described in more detail below and is optionally labeled, can be included in the sample.
  • an exemplary sugar moiety is ⁇ -D-glucose and an exemplary fatty acid portion (e.g. B 1 (—B 2 )(—B 3 )) includes a B of methylene linked to a C7-amido and a substituent containing alkenyl of 1-20 carbons in length.
  • an exemplary sugar moiety is ⁇ -D-galactose and an exemplary fatty acid portion includes a B 1 of methylene linked to a C 5 -amido and a substituent containing alkenyl of 1-20 carbons in length.
  • a substrate can be tailored for assaying a variety of enzymes, in particular, enzymes associated with a disease state or birth defect, or one otherwise useful for medical purposes. Such tailoring is possible because a variety of monosaccharide and disaccharide groups can be present at A of the general formula I. Even for a newly identified target enzyme, once its specificity for monosaccharide and/or disaccharide groups is determined using routine methods, a substrate can be readily prepared using guidance provided herein.
  • Non-limiting examples of enzymes which can be assayed using a substrate as described herein include acid ⁇ -glucocerebrosidase, galactocerebroside ⁇ -galactosidase, and acid sphingomyelinase.
  • This system provides for optional multiplex assays where two or more lysosomal enzymes are analyzed in the same sample or sample receptacle using structurally similar yet enzyme specific substrates.
  • the present invention provides compounds that function as experimental controls or standards useful for assessing the amount of enzymatic product in a sample or sample receptacle.
  • an internal standard corresponding to a particular substrate is structurally identical to its enzymatic product (i.e. fatty acid portion), except that the internal standard differs in mass-to-charge (m/z) ratio.
  • the internal standards as provided include modified forms of enzymatic products, for example, stable isotope-labeled analogs of enzymatic products in which one or more atoms are replaced by corresponding atomic isotopes so as to create a differentially detectable mass difference with the corresponding enzymatic product.
  • the resulting spectrum reveals a spatial separation of the internal standard and enzymatic product, each represented by its own peak.
  • the known amount of internal standard is reflected by peak magnitude at its known m/z ratio.
  • the amount of enzymatic product can be assessed by comparison of peak magnitude at its known m/z, relative to the peak magnitude of the internal standard.
  • An example of isotopic labeling to produce an internal standard is the replacement of 1 H on an acyl group of B2 or B3 (or both) with 2 H (i.e. deuterium, D).
  • a “heavier” internal standard molecule with the substituted 2 H has a different m/z from the enzymatic product, as detected on a mass spectrum.
  • an internal standard is labeled with deuterium to cause a mass change of 3 to 9 Daltons from the corresponding cleaved product
  • the substrates are labeled with a detectable tag or a heavy atom label.
  • a detectable tag may be found interacting with a B 2 group, a B 3 group, or both.
  • Many fluorescent probes are recognized in the art as useful for labeling reactive amines.
  • some embodiments include a B2 group or a B3 group that terminates in a nucleophilic group suitable for interacting with a label or a substrate surface if desired.
  • An illustrative nucleophilic group includes a nitrogen or oxygen nucleophile.
  • a particularly sensitive target for specific labeling of biomolecules is a terminal amine group.
  • a particular embodiment of the substrates includes a B2 or B3 that possesses this active terminal amino group.
  • detectable tags suitable for labeling the substrates include fluorophores such as isothiocyanates, dansyl and other sulfonyl chlorides, 7-nitrobenz-2-oxa-1,3-diazole derivatives, fluorescamine, and the like.
  • a substrate can be used in a variety of physical formats, for example, in solution as well as linked or immobilized to solid supports.
  • a solid support can be composed of a natural or synthetic material, an organic or inorganic material, such as a polymer, resin, metal or glass, and combinations thereof.
  • a suitable solid support can have a variety of physical formats, which can include for example, a membrane; column; a hollow, solid, semi-solid, pore or cavity-containing particle such as a bead; a gel; a fiber, including a fiber optic material; a matrix and sample receptacle.
  • sample receptacles include sample wells, tubes, capillaries, vials and any other vessel, groove or indentation capable of holding a sample.
  • a sample receptacle can be contained on a multi-sample platform, such as a microplate, slide, microfluidics device, and the like.
  • a multi-sample platform such as a microplate, slide, microfluidics device, and the like.
  • suitable particles are known in the art and illustratively include Luminex®-type encoded particles, encoded fiber optic particles, magnetic particles, and glass particles. Covalent interaction of a substrate and/or enzymatic cleavage product thereof with a solid support is useful for retaining the substrate and/or product during washing procedures performed in some assay formats, thus, producing a robust and accurate signal of enzymatic activity.
  • High binding solid supports are surfaces having exposed moieties that are chemically active or otherwise capable of covalent or high affinity binding to a substrate or internal standard.
  • Corning Life Sciences produces high-binding microwell plates that are irradiated to break the benzene ring and produce exposed carboxylic acids. These carboxylic acids are amenable to nucleophilic attack such as by the terminal amino group on the lysine derivative component of an embodiment substrate. This reaction is rapid and produces a tight interaction between the substrate/product and the high-binding surface.
  • compositions provided can be performed in a multiplexed format such that a plurality of samples are assayed simultaneously.
  • An illustrative multiplexed format involves using physically and/or chemically coded particles.
  • Use of coded particles in multiplexed formats has been described, for example, in U.S. Pat. No. 6,649,414 and U.S. Pat. No. 6,939,720. Because the codes allow particles to be distinguished from each other, a plurality of distinct particles can be present in a single reaction mixture, allowing a plurality of different samples or different enzymes to be assayed simultaneously. Codes on particles can correspond, for example, to sample origins, particular enzymes to be assayed, particular substrates present, and the like, depending on the experimental goal of the user.
  • a sample useful in the processes provided contains or is suspected of containing one or more target enzymes.
  • Target enzymes can be contained in samples obtained from an individual, as well as from laboratory materials, such as cell lines, and synthetic protein sources.
  • Exemplary sample sources illustratively include: tissue homogenates; cell culture lysates; and biological fluids including urine, blood in liquid or dry form, tears, saliva, and cerebrospinal fluid.
  • a sample can be further fractionated, if desired, to a fraction containing particular cell types.
  • a blood sample can be fractionated into serum or into fractions containing particular types of blood cells such as red blood cells or white blood cells (leukocytes).
  • a sample can be a combination of samples from a subject such as a combination of a tissue and fluid sample, and the like.
  • the sample is blood, which can be, for example, whole blood or a blood fraction thereof, or reconstituted from a dry blood sample.
  • Such methods include the use of appropriate buffers and/or inhibitors, including nuclease, protease and phosphatase inhibitors, which preserve or minimize changes in the molecules in the sample.
  • Such inhibitors include, for example, chelators such as ethylenediamine tetraacetic acid (EDTA), ethylene glycol bis(P-aminoethyl ether)N,N,N1,N1-tetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain and the like, and phosphatase inhibitors such as phosphate, sodium fluoride, vanadate and the like.
  • chelators such as ethylenediamine tetraacetic acid (EDTA), ethylene glycol bis(P-aminoethyl ether)N,N,N1,N1-tetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain and the like, and phosphatase inhibitors such as phosphat
  • Appropriate buffers and conditions for isolating molecules are well known to those skilled in the art and can be varied depending, for example, on the type of molecule in the sample to be characterized (see, for example, Ausubel et al. Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999); Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press (1988); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); Tietz Textbook of Clinical Chemistry, 3rd ed. Burtis and Ashwood, eds. W.B. Saunders, Philadelphia, (1999)).
  • a sample also can be processed to eliminate or minimize the presence of interfering substances.
  • Samples in the form of a dry blood spot are commonly used when screening blood from newborns and child patients.
  • blood is collected and retained on filter paper.
  • the dried blood is eluted from the filter paper into an aqueous solution, which generally contains a buffer such as phosphate buffered saline and a protease inhibitor.
  • protease inhibitor conditions include for example, one or more of the following: AEBSF hydrochloride in a final concentration of 50 to 400 ⁇ g/ml, EDTA disodium dehydrate in a final concentration of 0.2 to 25 mg/ml, leupeptin hemisulfate in a final concentration of 0.5 to 1 ⁇ g/ml, and pepstatin A in a final concentration of 0.5 to 1 ⁇ g/ml.
  • protease inhibitor conditions include for example, one or more of the following: AEBSF hydrochloride in a final concentration of 50 to 400 ⁇ g/ml, EDTA disodium dehydrate in a final concentration of 0.2 to 25 mg/ml, leupeptin hemisulfate in a final concentration of 0.5 to 1 ⁇ g/ml, and pepstatin A in a final concentration of 0.5 to 1 ⁇ g/ml.
  • Protease inhibitor cocktails known commonly used in the art may be used.
  • a single extraction of a dry sample avoids the need to obtain several sample punches from the same sample, or to collect aliquots of other sample sources and accordingly reduces variation caused by inhomogeneous distribution of blood on the filter paper and errors in sample transfer.
  • extraction efficiency may vary with the different enzymes being analyzed.
  • the target enzymes may have different levels of activities when contained in different assay solutions.
  • a composition of a universal assay solution is optionally chosen such that each enzyme to be tested is active.
  • the dried blood spot or a punch derived from it is placed directly into an assay buffer including one or more substrates and optionally internal standards or and allowed to incubate for a time sufficient to allow enzymes present in the sample to convert the substrate(s) to product(s) prior to detection by one or more detection methods.
  • the substrates and products provided can be used in a variety of assay formats.
  • the substrate can be detected in an assay when it is desired to observe substrate consumption during an enzymatic assay, while the product can be detected in the assay when it is desired to observe its formation during an enzymatic assay.
  • Both substrate and product can be detected when it is desired to observe the enzymatic reaction from both perspectives, for example, to confirm that the amount of product produced correlates with the amount of substrate consumed.
  • the amount of substrate or product can be detected using established tandem mass spectrometry procedures.
  • An exemplary enzyme assay employing mass spectrometry can be performed as follows. A sample is incubated with a substrate for a time period that allows formation of an enzymatic product. During the incubation period, the substrate is cleaved by a target enzyme present in a blood sample to form a respective product. The reaction is then quenched by adding a reagent that precipitates protein components. Exemplary reagents include alcohol, acetonitrile and dilute trifluoro acetic acid. A portion of the incubation mixture is then transferred to a new assay vessel.
  • a dilution reagent such as methanol, acetonitrile, water-methanol mixtures or water-acetonitrile can be added to dilute the transferred portion.
  • the sample so diluted reduces the amount of endogenous competing material so as to relatively increase the sensitivity of the tandem mass spectrometry analysis.
  • Other types of reagents are selected by those skilled in the art to be compatible with analyses by mass spectrometry of many varieties.
  • the diluted sample is directly injected into the tandem mass spectrometer either manually or automatically with the aid of autosamplers and liquid handlers. If desired, the sample can be derivatized prior to analysis.
  • Reagents are selected to be non-hostile to the MS/MS system. For example, suitable solvents lack detergents and corrosive agents, such as chloroform. Pure ethanol and pure methanol are often used simply because they easily vaporized upon mechanical drying processes.
  • the tandem mass spectrometer can be set to simultaneously detect the added substrate, the corresponding resulting enzymatic product and the corresponding internal standards. Such detection is accomplished by means of parent ion scans, precursor ion scans or multiple reaction monitoring scans.
  • the amount of substrate consumed or product formed during an enzymatic assay also can be detected using antibodies and other target-specific binding molecules.
  • an antibody can be used to detect the substrate, product or both.
  • Antibodies useful in such methods can be specific, such that they recognize individual substrates, or non-specific, such that they recognize many or all substrates.
  • a substrate or product optionally includes a label such as biotin or avidin to allow specific detection.
  • the antibody is illustratively produced in animals including mouse, rat, rabbit, horse, donkey, or other suitable animal used for the production of antibodies.
  • a detectable tag such as a fluorescent tag.
  • detection can be performed by using a secondary antibody that is specific for the species IgG of the primary antibody is labeled illustratively with a fluorescent marker such as rhodamine.
  • a fluorescent marker such as rhodamine.
  • other antibody detection systems are similarly operable in the instant invention such as horseradish peroxidase labeled antibodies, or alkaline phosphatase labeled antibodies.
  • samples containing target enzymes are contacted with substrates linked to particles in an assay solution.
  • each particle is linked to a particular substrate, and there are multiple particles representing each substrate.
  • the target enzymes act on the substrates to produce products (A) and the fatty acid containing portion of the molecule (B).
  • the product remains bound to the particle, while the A product is released into solution.
  • Antibodies that recognize specific products are then contacted with the assay solution.
  • the antibodies will bind to the products, if produced during the enzymatic assay, to produce particles having bound antibodies.
  • antibodies having different product specificities can have different detectable moieties, such as different fluorescent tags.
  • antibodies that recognize substrate can be used to detect substrate remaining on the beads after incubation with enzymes. In this situation, either product would remain attached to the bead, if an enzymatic reaction occurred. In either case the selected substrate specific antibody would not significantly cross-react with product attached to the bead.
  • samples containing target enzymes are contacted with substrates linked to encoded particles in an assay solution.
  • the encoded particles have a feature, such as a bar code or optical profile, which allows them to be distinguished from each other.
  • encoded particles can have different bar codes corresponding to different target enzyme substrates.
  • the target enzymes act on the substrates to produce products.
  • the fatty acid product would remain bound to the particle, while the A product would be released into solution, or visa versa.
  • Antibodies that recognize specific products are then contacted with the assay solution. Because the encoding of the particle indicates which substrate is attached to the particle, antibodies need not be specific for particular products, and thus one type of antibody can be used to detect products derived from multiple different substrates.
  • Such non-specific antibodies will bind to the products, if produced during the enzymatic assay, to produce particles having bound antibodies.
  • Particles having bound antibodies are then distinguished from those without antibodies, for example, by detecting a tag on the antibodies or physical behavior of the particles.
  • the different products contained on the antibody-bound particles can be determined based on the encoding of each particle.
  • antibodies directed to particular substrates are generated. Following quenching of an enzymatic reaction, the reaction solution is transferred to a high-binding microtiter plate whereby the reactive B 2 moiety (for example) covalently attaches to the plate via a terminal amino group. The enzyme and assay solution components are removed by washing. The specific primarily antibody is then incubated in each assay well followed by subsequent washing to remove unbound antibody. A secondary antibody is optionally used for detection and quantitation. The more product formed per unit time of initial reaction the greater the activity of the measured enzyme.
  • an antibody specific for the fatty acid subgroup is optionally used as a capture antibody on the surface of the microtiter plate in a standard sandwich ELISA assay.
  • a primary antibody with a unique epitope on the product such as one directed to the fatty acid moiety (or the fatty acid moiety is modified with a specific binding pair member such as biotin) is used for detection.
  • a labeled secondary antibody is optionally used for detection as described above.
  • an exemplary antibody reacts with the ⁇ -D-glucose A group bound to the B 1 moiety.
  • the substrate may be attached to a solid support using an amino terminated fatty acid moiety.
  • the substrate is provided in solution, the reaction is transferred to a sample receptacle, in which following quenching of an exemplary enzymatic reaction, the reaction solution is transferred to a high-binding microtiter plate whereby the reactive fatty acid moiety covalently attaches to the receptacle via a terminal amino group.
  • a capture antibody specific for an alternate epitope on the product/substrate is employed. The unreacted enzyme and buffer components are removed by washing. The antibody specific to the A-B 1 moiety is then incubated in each assay well for detection and quantitation of remaining substrate. The greater the substrate remaining after the initial enzyme reaction, the lower the activity of the enzyme.
  • the antibody is illustratively unlabeled and produced in animals including mouse, rat, rabbit, horse, donkey, or other suitable animal used for the production of antibodies.
  • a secondary antibody that is specific for the species IgG of the primary antibody is labeled illustratively with a fluorescent marker such as rhodamine and subsequently used for detection of remaining substrate. It is appreciated in the art that other antibody detection systems are similarly operable in the instant invention such as horseradish peroxidase labeled antibodies, or alkaline phosphatase labeled antibodies.
  • monoclonal mouse antibody specific for the exemplary the ⁇ -D-glucose A group bound to the B 1 moiety and/or to a portion of a fatty acid moiety is itself labeled illustratively by a fluorescent marker.
  • multiple lysosomal enzymes are optionally simultaneously analyzed for activity toward a variety of specific substrates.
  • An illustrative example includes a two enzyme system wherein two substrate are employed, one specific for GALC and another specific for ABG. Each substrate is simultaneously added to the reaction with the biological sample. As each substrate optionally contains an amine terminated fatty acid group, both will similarly bind to the high-binding microtiter plate.
  • Each antibody is illustratively labeled with a different fluorophore such as rhodamine or cyanine. As such the binding of each antibody is detected and quantitated without interference from the other, and the amount of each enzyme activity is detectable in the same well of the microtiter plate from the same sample.
  • a different fluorophore such as rhodamine or cyanine.
  • an assay for target enzymes is performed by first obtaining a sample illustratively including serum, plasma, whole blood, urea, saliva, other biological fluids or tissue lysates, recombinant or native purified enzyme in solution, or chemically or functionally modified enzyme in biological fluid or liquid medium.
  • a portion of the filter paper sample is then excised and deposited in a non-binding assay tube or micro titer plate well to which an assay solution is added.
  • the assay solution comprises aqueous buffers, a substrate, a standard, as well as protease inhibitors.
  • the sample mixture is then incubated for a determined period of time in the range of 30 minutes to 20 hours at a particular temperature ranging from 30 to 41° C.
  • a stopping solution is illustratively 0.4 M glycine/NaOH pH 10.4 added at 6 ⁇ reaction volume.
  • a stopping solution is illustratively 0.4 M glycine/NaOH pH 10.4 added at 6 ⁇ reaction volume.
  • the amount of product formation is determined by transferring a known volume of sample to a high-binding assay tube or microtiter plate and incubated for 5 minutes to 2 hours. The unbound material is removed by washing. Detection of the intact substrates or products is illustratively performed using a coupled peroxidase enzyme approach.
  • the level of released glucose or galactose product is measured in real time by a coupled enzyme approach.
  • a non-limiting example involves the release of glucose from a substrate specific for ⁇ -glucocereborsidase in diagnosis of Gaudier disease.
  • glucose is reacted with glucose oxidase producing glucolactone and releasing hydrogen peroxide.
  • the released hydrogen peroxide is detected by reaction with peroxidase to produce a fluorescent molecule that is measured on a standard fluorometer.
  • suitable peroxidases are horseradish peroxidase or any other peroxidase known in the art.
  • the hydrogen peroxide released by glucose oxidase interacts with a detector substrate molecule.
  • a detector molecule suitable for use with the substrates includes Amplex Red that is oxidized in to produce the fluorescent product resorufin.
  • Amplex Red and kits for detecting free glucose are available from Invitrogen Corp.
  • the increase in red fluorescent product is detected on a fluorometer set with an excitation wavelength at 571 and an emission wavelength at 585 with the band pass set at 5 nm. The greater amount of glycosidase activity the more rapidly the red fluorescent product is produced.
  • multiple substrates for different lysosomal enzymes are generated with unique fatty acid structure(s). This prevents product inhibition of one enzyme that is particularly important should the catalytic activity of one enzyme toward one substrate be much greater than the catalytic activity of the other enzyme for its corresponding substrate. This is additionally important in conditions where a single mutant glycosidase is being screened in a panel of substrates for 6 or more lysosomal enzymes.
  • the product formed by the other lysosomal enzymes may inhibit the function of the lower activity enzyme such that its activity is not accurately measured.
  • the specificity of the substrate and the product for each enzyme is appreciated to be optionally distinct.
  • the substrates may differ not only in the type of sugar moiety which confers enzyme specificity, but also in the length of the any component of the fatty acid portion. This is particularly important with the use of MS/MS as a detection tool since the differentiated substrate molecules having corresponding differentiated mass index correspond to various enzymes being examined.
  • the approach described for assaying enzymes using the described substrate and standard compounds can be expanded to assay a plurality of enzymes simultaneously in a single reaction, obviating the need for multiple assays to assist in confirming diagnoses of medical disorders.
  • the methods can also be used to measure several enzymes simultaneously when evaluating the rate of chemical flux through a specific biochemical pathway or for monitoring biochemical signaling pathways. Because of the high sensitivity of mass spectrometry detection employable using the compounds described herein, which can require only sub-microgram quantities of the substrate reagents per assay, the synthesis of several hundred substrate reagents on a low-gram scale becomes practical and economical.
  • two, three, four, five, six, or more lysosomal enzymes are simultaneously measured for activity by the use of substrates as provided.
  • the substrates can be labeled with the same fluorophore, but possess significant mass or charge characteristics that differentiate one from the other.
  • the amount of product produced following an enzymatic cleavage reaction is detected by reversed phase high performance liquid chromatography (HPLC). Reactions are quenched by the addition of alcohol, acetonitrile or dilute trifluoro acetic acid. A portion of the incubation mixture is transferred to a new assay vessel to which is added a neat solution such as methanol, acetonitrile, water-methanol mixtures or water-acetonitrile.
  • the reaction products and unreacted substrate are separated on a 5 ⁇ m particle size C 18 HPLC column and detected by a fluorescent detector or set of detectors. The amount of product is calculated based on a standard curve generated using increasing amounts of the relevant product.
  • each product is resolvable, for example, on an HPLC column and can be quantified in a single assay.
  • each substrate is labeled with a different fluorophore that has different or the same excitation or emission properties.
  • Detection may be by a family of fluorescent detectors that can simultaneously quantify individual products from each other and their corresponding labeled substrate. Other methods of detection are similarly suitable and are known in the art.
  • FIG. 2 depicts an exemplary substrate structure for detecting lysosomal storage disorders.
  • the structure is composed of a sugar (A) in the form of a glucose or a galactose where galactose is illustrated and an aliphatic group B.
  • Group B includes a linker arm (B 1 ) in the form of a methylene, a B 2 subgroup of a C 5 -amido, and a B 3 subgroup in the form of an alkenyl with carbon length in the range of 10 to 30 and a substituent of O.
  • B 1 linker arm
  • FIG. 3 depicts an exemplary substrate structure for detecting lysosomal storage disorders.
  • the structure is composed of a sugar (A) in the form of a glucose or a galactose where galactose is illustrated and an aliphatic group B.
  • Group B includes a linker arm (B 1 ) in the form of a methylene, a B 2 subgroup of a C 7 amido, and a B 3 subgroup in the form of an alkenyl with carbon length in the range of 10 to 30 and a substituent of O.
  • B 1 linker arm
  • FIG. 5 demonstrates a generic enzymatic reaction using an exemplary substrate.
  • the substrate Upon specific affinity binding and enzymatic reaction, the substrate is cleaved into two groups, a sugar moiety A and an aliphatic group B.
  • the group B is optionally composed of amide and long-chain alkyl or alkenyl moieties. Both groups are optionally then analyzed by MS/MS.
  • An internal standard is also concurrently subject to the MS/MS analysis.
  • the internal standard is optionally an isotopically labeled analog of B with deuterium to replace hydrogen atom(s) on a methyl group or other portion of the molecule.
  • B1 is: a C 1 -C 20 alkyl; a heteroatom containing C 1 -C 20 alkyl, C 2 -C 20 alkenyl; a heteroatom containing C 2 -C 20 alkenyl, a substituted or unsubstituted C 6 -C 20 aryl;
  • B2 is: a C 2 -C 7 urethane; a C 2 -C 7 amido, a C 2 -C 7 ester, a C 2 -C 7 uriedo; a C 2 -C 7 carbamato; a C 2 -C 7 carbonyl; a C 1 -C 7 alkyl; a heteroatom containing C 1 -C 7 alkyl; a C 1 -C 7 alkyl having a substituent of N, O, or S; a C 2 -C 7 alkenyl; a heteroatom containing C 2 -C 7 alkenyl; a C 2 -C 7 alkenyl
  • the structure B 1 -B2-B 3 has the structure of:
  • R 1 is a C 1 -C 6 alkyl or a C 2 -C 20 alkenyl
  • R 2 is a C 1 -C 20 alkyl; a C 1 -C 20 alkenyl; a C 1 -C 20 alkyl having a substituent of N, O, or S, a C 1 -C 20 alkenyl, or a C 1 -C 20 alkenyl having a substituent of N, O or S
  • R 2 is as defined for Formula II.
  • R 1 is a C 6 alkyl or a C 4 alkyl
  • R 2 is a C 1-3 alkyl.
  • R 1 is a C 4 alkyl, C 5 alkyl or a C 6 alkyl
  • R 2 is a C 13 -C 20 alkyl or a C 13 -C 20 alkenyl.
  • R 1 is a C 4 alkyl or a C 6 alkyl
  • R 2 is a C 1-3 alkyl.
  • the compositions of Formula V optionally include one or more stable secondary prevalence isotopes that are optionally 2 H, 13 C, 15 N, 17 O, 18 O, 31 P, 34 S, or combinations thereof.
  • x is a value between 2 and 4, and where R 2 is a C 1 -C 20 alkyl, a C 1 -C 20 alkenyl, or a C 1 -C 20 alkynl, any of which may be substituted with an N, O, or S, or include a substituent of N, O, or S.
  • x is 2 or 4 and R 2 is a C 1-3 alkyl.
  • the heavy isotope is optionally present on the ⁇ -carbon in the amido group, on any position on the hydroxyl containing aliphatic group, or any combination thereof.
  • the compound of Formula V or VI is also functional as an antagonist, an analytical control, or for clinical treatment of disease such as hypothyroidism, diabetes, and HIV.
  • the substrates of Formulas I-III are optionally synthesized with a non-hydrolyzable link between A and B 1 . This produces suicide substrates that maintain high specificity for their target lysosomal enzyme. These molecules serve as more specific and potent inhibitors of enzyme function.
  • All reagents including the substrates, enzymatic products, and internal standards can be optionally purified by reverse-phase HPLC and characterized by ESI-MS, either n an online HPLC-MS assay or offline by collection of the appropriate fractions.
  • ⁇ -D-glucose modified sphingosine (D-glucosyl- ⁇ 1-1′-D-erythro-sphingosine, Avanti Polar Lipids, Alabaster, Ala.) is dissolved in 1 mL of dry DMF (Aldrich). 1.5 eq. of pentanoyl-NHS ester (B) was added (from a 50 mg/mL stock solution in DMF, stored ⁇ 20° C.). The reaction is completed in 2 minutes as measured by TLC. The reaction product was subjected to centrifugation in a Speed-Vac overnight out heating.
  • A D-glucosyl- ⁇ 1-1′-D-erythro-sphingosine, Avanti Polar Lipids, Alabaster, Ala.
  • the resulting Compound 1 is analyzed by NMR to confirm identity. Results are demonstrated in FIG. 1A .
  • the observed molecular weight of the resulting compound is 545.3948.
  • the above process is similarly used to synthesize other compounds by substituting the pentanoyl-NHS ester with those of varying alkyl chain lengths.
  • the reaction product is subjected to centrifugation in a Speed-Vac overnight without heating.
  • the dried reside was dissolved in 1.4 mL of DMF and 0.2 mL portions were injected onto the HPLC column as above.
  • Product fractions were pooled from all of the HPLC runs, and solvent was removed in a centrifugal concentrator (Speed-Vac, oil pump vacuum, room temperature) to give the desired product in 81% yield.
  • Compound synthesis is confirmed by NMR as illustrated in FIG. 1B .
  • Example NHS esters used are: pentanoyl-NHS ester, heptanoyl-NHS ester, pentanoyl-NHS ester (each with or without H to D substitution). Internal standards are purified as above by HPLC and are obtained in yields of 20-90%.
  • the resulting exemplary Compound 81 is analyzed by NMR to confirm synthesis with a resulting molecular weight of 424.
  • the process of Scheme II is similarly used to synthesize other labeled internal standards that include a H to D substitution in either the sphingosine or the NHS ester precursor. Additional exemplary compounds according to Formula V synthesized are presented in Table 2.
  • the substrates of Compounds 1 and 7 are used to detect the presence of acid- ⁇ -glucocerebrosidase (ABG) and galactocerebroside- ⁇ -galactosidase (GALC) respectively.
  • Blood is obtained by venipuncture from consenting adult humans and blotted on filter paper.
  • a disk of 3 mm diameter is punched from the areas of dried blood into a well of a 96-well microtiter plate.
  • the blood disk is then incubated directly with an assay solution containing substrates at a final concentration of 500 ⁇ mol/L and corresponding internal standards at a final concentration of 5 ⁇ mol/L.
  • a final concentration of 0.5 mol/L sodium acetate buffer with sodium taurocholate is also added.
  • the assay mixture containing the blood disk is incubated for 15 to 24 hours at 37° Celsius with orbital shaking (150 rpm) in a thermostatic air shaker. After the incubation period, an aliquot of pure methanol is added to each tube or well to terminate the enzymatic reaction. Before going into the mass spectrometer, the incubated reaction mixture is diluted with pure methanol.
  • the electrospray source is operated in positive mode, and the ions are detected in parent-ion scan mode. The amount of enzymatic product is calculated from the ion abundance ratio of the product to the internal standard minus that of a blank.
  • FIG. 6 illustrates the unexpectedly improved results using compound 1 as the ABG substrate relative to previously used substrates illustrating a 2-fold improvement in product detection over a similar compound substituting the C 5 amido for a C 19 amido.
  • Samples positive for GALC demonstrate a positive ion with molecular weight 264.27.
  • the corresponding internal standard produces a positive ion with molecular weight 271.31.
  • the substrates of Compounds 1 and 7 are used simultaneously to detect the presence of acid- ⁇ -glucocerebrosidase (ABG) and galactocerebroside- ⁇ -galactosidase (GALC) respectively.
  • Blood is obtained by venipuncture from consenting adult humans and blotted on filter paper.
  • a disk of 3 mm diameter is punched from the areas of dried blood into a well of a 96-well microtiter plate.
  • the blood disk is then incubated directly with an assay solution containing substrates at a final concentration of 100 ⁇ mol/L and corresponding internal standards at a final concentration of 1 ⁇ mol/L.
  • a final concentration of 0.5 mol/L sodium acetate with sodium taurocholate buffer is also added (30 ⁇ l final assay volume).
  • the assay mixture containing the blood disk is incubated for 15 to 24 hours at 37° Celsius with orbital shaking (150 rpm) in a thermostatic air shaker. After the incubation period, a 100 ⁇ l aliquot of 50:50 methanol/ethyl acetate is added to each tube or well to terminate the enzymatic reaction.
  • the reaction is then supplemented with 400 ⁇ l HPLC grade ethyl acetate and 200 ⁇ l water. The reaction is centrifuged and the resulting top layer of the liquid is transferred to a new assay plate and evaporated under nitrogen.
  • An analysis buffer of 83% acetonitrile/17% water with 0.1% formic acid is added to the assay plate and the samples subjected to analysis by MS/MS to detect enzymatic products and internal standards.
  • the electrospray source is operated in positive mode, and the ions are detected in parent-ion scan mode.
  • the amount of enzymatic product is calculated from the ion abundance ratio of the product to the internal standard minus that of a blank. Detection of both ABG and GALC is achieved.
  • the product of the reaction with the substrates of Formula I is quantified by immunoassay.
  • Blood spotted on filter paper is reconstituted in buffer to liberate the active components.
  • One or an array of substrates is added to the reaction chamber and the reaction allowed to proceed overnight ( ⁇ 14 hours).
  • the reaction is quenched by the addition of 6 ⁇ volume glycine/NaOH pH 10.4.
  • a sample of each reaction is added to the wells of a high-binding irradiated microtiter plate and incubated overnight to allow sufficient binding of the reaction product to the wells of the plate.
  • a standard curve of product in similar buffer/sample is also added to the plate to serve as a basis for quantitation.
  • the wells are washed twice with phosphate buffered saline (PBS) by the use of a squirt bottle, plate washer, or any other automated or non-automated plate washing system. Any additional sites for protein binding are subsequently blocked by the addition of a blocking agent illustratively including 3% bovine serum albumin in PBS or any other synthetic or natural blocking agent known in the art.
  • the blocking agent is incubated for two hours at room temperature.
  • the wells are washed 3 ⁇ with PBS.
  • the primary antibody(s) is then added to the wells to recognize and bind the remaining substrate, or the product.
  • the antibody(s) is incubated in the wells for at least 2 hours.
  • the plate is washed four times to remove unbound antibody. If the primary antibody is labeled the plate is used for detection. Optionally, a labeled secondary antibody is placed in the plate wells and allowed to incubate for an additional 2 hours followed by washing 4 times and detection by the appropriate method such as by a fluorescent or optical plate reader.
  • the product of the reaction with the substrates of Formula III is quantified by immunoassay.
  • Blood spotted on filter paper is reconstituted in buffer to liberate the active components.
  • One or an array of substrates immobilized to encoded particles is added to the reaction chamber, preferably a microplate well, and the reaction allowed to proceed overnight ( ⁇ 14 hours).
  • a standard curve of enzyme in similar buffer/sample is also added to separate sets of encoded particles to serve as a basis for quantitation.
  • the reaction is quenched by the addition of 6 ⁇ volume glycine/NaOH pH 10.4.
  • the primary antibody(s) is then added to the wells to recognize and bind the remaining substrate, or the product.
  • the antibody(s) is incubated for at least 30 minutes. If the primary antibody is labeled the assay is ready for detection.
  • a labeled secondary antibody is placed in the plate wells and allowed to incubate for an additional 30 minutes. Detection is accomplished a flow cytometer.
  • an active terminal amino group on the B 2 of an exemplary embodiment of Formula I or the R 1 or R 2 group of Formula II or III is amenable to numerous labeling procedures.
  • the terminal amine is specifically labeled with fluoroisothiocyanate (FITC).
  • FITC fluoroisothiocyanate
  • Derivitization of the substrate is performed by addition of a FITC molecule to the terminal amine of the B 2 group. Similar modification may be performed on amine groups that are not on the terminal carbon, if present.
  • the purified/lyophilized substrate is resuspended in 0.1 M sodium bicarbonate buffer, pH 9.0 at a concentration of 5 mg/ml. Immediately prior to reaction with substrate, dissolve 5 mg of FITC dye in 0.5 ml of DMSO in the dark.
  • the labeled substrate is used in a reaction for the detection of glucocerebrosidase activity and the detection of Gaucher's disease.
  • a disk of 3 mm diameter is punched from the areas of dried blood on a filter paper into a micro-centrifuge tube or a well of a 96-well microtiter plate.
  • the blood disk is then incubated directly with an assay solution containing labeled substrate at a final concentration of 5 ⁇ mol/L and internal standards at a final concentration of 0.1 ⁇ mol/L.
  • the assay mixture containing the blood disk is incubated for 15 to 24 hours at 37° C. with orbital shaking (150 rpm) in a thermostatic air shaker.
  • Fluorescence intensity is continuously monitored using a fluorescence detector (model L-7480; Hitachi, Naperville, Ill.) at a medium gain sensitivity.
  • the amount of labeled product in the sample is determined by comparing the area of the peak to that of an external standard comprised of labeled product at a known concentration.
  • the concentration of product in the reaction is readily determined and the activity of glucocerebrosidase determined by dividing the moles product/per unit of reaction time.

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