USRE38723E1 - Haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells - Google Patents
Haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells Download PDFInfo
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- USRE38723E1 USRE38723E1 US08/910,090 US91009097A USRE38723E US RE38723 E1 USRE38723 E1 US RE38723E1 US 91009097 A US91009097 A US 91009097A US RE38723 E USRE38723 E US RE38723E
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- BCHIXGBGRHLSBE-UHFFFAOYSA-N CC(c(ccc(OP(O)(O)=O)c1)c1O1)=CC1=O Chemical compound CC(c(ccc(OP(O)(O)=O)c1)c1O1)=CC1=O BCHIXGBGRHLSBE-UHFFFAOYSA-N 0.000 description 1
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- CUWZHYKDMYRJKN-UHFFFAOYSA-N CCCCCCCC(=O)OC1=CC=C2C(CCl)=CC(=O)OC2=C1 Chemical compound CCCCCCCC(=O)OC1=CC=C2C(CCl)=CC(=O)OC2=C1 CUWZHYKDMYRJKN-UHFFFAOYSA-N 0.000 description 1
- ISPBECDEFRIYTA-UHFFFAOYSA-N COC1=CC=C2C(CCl)=CC(=O)OC2=C1 Chemical compound COC1=CC=C2C(CCl)=CC(=O)OC2=C1 ISPBECDEFRIYTA-UHFFFAOYSA-N 0.000 description 1
- QZQKXDVLCGEFHH-UHFFFAOYSA-P NC(=[NH2+])NC1=CC=C(C(=O)OC2=CC=C3C(=C2)OC2=C(C=CC(OC(=O)C4=CC=C(NC(N)=[NH2+])C=C4)=C2)C32OC(=O)C3=C2C=CC(CCl)=C3)C=C1.[Cl-].[Cl-] Chemical compound NC(=[NH2+])NC1=CC=C(C(=O)OC2=CC=C3C(=C2)OC2=C(C=CC(OC(=O)C4=CC=C(NC(N)=[NH2+])C=C4)=C2)C32OC(=O)C3=C2C=CC(CCl)=C3)C=C1.[Cl-].[Cl-] QZQKXDVLCGEFHH-UHFFFAOYSA-P 0.000 description 1
- VIEYMVWPECAOCY-UHFFFAOYSA-N NC1=CC=C2C(CCl)=CC(=O)OC2=C1 Chemical compound NC1=CC=C2C(CCl)=CC(=O)OC2=C1 VIEYMVWPECAOCY-UHFFFAOYSA-N 0.000 description 1
- WDDXHKZVZWXQDG-UHFFFAOYSA-N O=C1C=C(CCl)C2=CC=C(OC3OC(C(=O)O)C(O)C(O)C3O)C=C2O1 Chemical compound O=C1C=C(CCl)C2=CC=C(OC3OC(C(=O)O)C(O)C(O)C3O)C=C2O1 WDDXHKZVZWXQDG-UHFFFAOYSA-N 0.000 description 1
- NUSVNLNZXIYSRK-UHFFFAOYSA-N O=C1C=C(CCl)C2=CC=C(OC3OC(CO)C(O)C(O)C3O)C=C2O1 Chemical compound O=C1C=C(CCl)C2=CC=C(OC3OC(CO)C(O)C(O)C3O)C=C2O1 NUSVNLNZXIYSRK-UHFFFAOYSA-N 0.000 description 1
- SQAVRZOWGALISV-UHFFFAOYSA-N O=C1C=C(CCl)C2=CC=C(OP(=O)(O)O)C=C2O1 Chemical compound O=C1C=C(CCl)C2=CC=C(OP(=O)(O)O)C=C2O1 SQAVRZOWGALISV-UHFFFAOYSA-N 0.000 description 1
- BFWGUSHDLFFGPN-UHFFFAOYSA-N [H]N(C(=O)C(N)CC(C)C)C1=CC=C2C(=C1)OC1=C(C=CC(OS(C)(=O)(=O)C3=CC=C(CCl)C=C3)=C1)C21OC(=O)C2=C1C=CC=C2 Chemical compound [H]N(C(=O)C(N)CC(C)C)C1=CC=C2C(=C1)OC1=C(C=CC(OS(C)(=O)(=O)C3=CC=C(CCl)C=C3)=C1)C21OC(=O)C2=C1C=CC=C2 BFWGUSHDLFFGPN-UHFFFAOYSA-N 0.000 description 1
- QFRPCLQNNOCFNL-UHFFFAOYSA-N [H]N(C(=O)C1=CC=C(CCl)C=C1)C1=CC2=C(C=C1)C1(OC(=O)C3=C1C=CC=C3)C1=CC=C(N([H])C(=O)C(N)CC(C)C)C=C1O2 Chemical compound [H]N(C(=O)C1=CC=C(CCl)C=C1)C1=CC2=C(C=C1)C1(OC(=O)C3=C1C=CC=C3)C1=CC=C(N([H])C(=O)C(N)CC(C)C)C=C1O2 QFRPCLQNNOCFNL-UHFFFAOYSA-N 0.000 description 1
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- 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
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- 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
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
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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
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/20—Coumarin derivatives
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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
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/40—Triphenylmethane dye chromogens, e.g. fluorescein derivatives
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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
- C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
- C12Q2337/20—Coumarin derivatives
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- 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 subject invention provides fluorogenic substrates for analyzing the metabolic activity in cells by improving the retention of a detectable reporter molecule only in intact cells where a particular enzyme is present.
- improved retention results from conjugation of haloalkyl-substituted derivatives of a reporter molecule with intracellular cysteine-containing peptides, while a detectable signal is provided by unblocking the reporter molecule.
- Enzymes are also markers for the cell type since the occurrence and activity of certain enzymes is frequently characteristic of a particular cell. For instance, the activity of certain enzymes can often be used to distinguish cells of bacterial, plant or animal origin. Enzymatic activity and integrity of the cell membrane is one of the criteria for cell viability.
- Detection of the presence and activity of enzymes has been facilitated by the development of chromogenic or fluorogenic substrates that are converted by chemical action of the enzyme to a reporter molecule whose optical properties can be measured.
- Principal among the new reporter molecules have been fluorescent dyes. In many cases the high sensitivity of fluorescence detection permits measurements in living single cells with high spatial and temporal resolution that are not possible with dyes that are not fluorescent.
- a number of synthetic enzyme substrates have been used to determine the activity of enzymes in extracts of cells, in solution, or in living cells. Most of these substrates have been based on fluorescent reporter dyes such as 7-hydroxy-4-methylcoumarin ( ⁇ -methylumbelliferone), 7-amino-4-methylcoumarin, 4-methoxy-2-naphthylamine, fluorescein, resorufin, rhodamine 110 or various derivatives of ⁇ - and ⁇ -naphthol.
- fluorescent reporter dyes such as 7-hydroxy-4-methylcoumarin ( ⁇ -methylumbelliferone), 7-amino-4-methylcoumarin, 4-methoxy-2-naphthylamine, fluorescein, resorufin, rhodamine 110 or various derivatives of ⁇ - and ⁇ -naphthol.
- FDG fluorescein digalactoside
- the leakage rate of potential alternative probes for monitoring lacZ or GUS gene fusion such as the resorufin formed by hydrolysis of resorufin galactoside and ⁇ -methylumbelliferone formed by hydrolysis of ⁇ -methylumbelliferyl glucuronide tends to be even faster.
- Lipophilic derivatives of fluorescein and resorufin have been described in LIPOPHILIC FLUORESCENT GLYCOSIDASE SUBSTRATES (U.S. Pat. No. 5,208,148 to Haugland et al. (1993)) and LONG WAVELENGTH LIPOPHILIC FLUOROGENIC GLYCOSIDASE SUBSTRATES (U.S. Pat. No. 5,242,805 to Naleway et al. (1993)), where retention is enhanced by the addition of a lipophilic residue and can be used under physiological conditions.
- These substrates can be used with automated procedures but are not effective where the fluorescent label needs to be retained inside cells to which a fixative has been applied.
- Glutathione transferase is known to catalyze the reaction of a wide variety of alkylating and arylating reagents with glutathione (Mantle, et al., Glutathione S-transferases, BIOCHEM. SOC. TRANSACTIONS, 18, 175 (1990)). This is an important process for detoxification of pollutants and dangerous chemicals by cells.
- the enzyme is ubiquitous, being found in both plant and animal cells. Free glutathione levels in most normal cells are relatively high (up to >1 mM).
- Monochlorobimane is a chloroalkylated fluorescent reagent that has been used to measure glutathione levels in single cells by flow cytometry in a reaction that is catalyzed by glutathione transferase (Rice, et al., Quantitative Analysis of Cellular Glutathione by Flow Cytometry Utilizing Monochlorobimane: Some Applications to Radiation and Drug Resistance in Vitro and in Vivo, CANCER RESEARCH 46, 6105 (1986)).
- Monobromobimane is a reagent with similar utilizing however it is less selective for intracellular thiols than is monochlorobimane (Fernández-Checa, et al., The Use of Monochlorobimane to Determine Hepatic GSH Levels and Synthesis, ANALYT. BIOCHEM. 190, 212 (1990)).
- CMFDA chloromethylfluorescein diacetate
- CMEDA chloromethyleosin diacetate
- Mangel et al. has described a family of fluorogenic protease substrates having amino acids or peptides bound to the amino nitrogens of a rhodamine fluorophore (U.S. Pat. Nos. 4,557,862 (1985) and 4,649,893 (1987)). Upon cleavage of the amino acids by proteases, the rhodamine becomes fluorescent.
- the Mangel et al. compounds do not possess a haloalkyl moiety to aid in retaining the fluorescent product within cells, and typically leak from cells fairly quickly after enzyme cleavage. The Mangel et al. compounds are also generally difficult to load into cells.
- haloalkyl group is critical for retention of the reporter molecule in cells.
- chloromethyl-substituted reporter was retained in cells for several hours, while the conventional reporter leaked away quickly (Examples 15 and 16).
- the nature of the haloalkyl group itself is critical. While haloalkyl-substituted hydrolase substrates based on coumarin fluorophores have been described by Scheper et al., (U.S. Pat. No.
- FIG. 1 Diagram of two alternate pathways to formation of a detectable thioether conjugate.
- E 1 is the reaction between the intracellular thiol, which, in the case of glutathione, is catalyzed by glutathione transferase.
- E 2 is the removal of BLOCK resulting from the action of the analyte enzyme.
- This invention utilizes novel probes that simultaneously form conjugates with cysteine residues in peptides and act as substrates for an intracellular enzyme whose activity is to be detected.
- the probes allow the analysis of metabolic activity of living cells under a variety of conditions.
- the novel method for analyzing the metabolic activity of cells begins with the preparation of a biocompatible solution containing a substrate.
- a biocompatible solution is any solution that facilitates the contact between the substrate and the cells being analyzed without destroying the integrity of the cell membrane or the viability of the cell.
- the biocompatible solution may include buffering agents, a culture medium for the cells, solvents or detergents that, without materially harming the cells, assist in solubilizing the substrate or permeabilizing cell membranes, or combinations thereof.
- the substrates have the general form: XR-SPACER-REPORTER-BLOCK
- BLOCK is any group that can be removed by specific hydrolytic or oxidative action of the analyte on the bond between BLOCK and REPORTER.
- the action of the analyte removing BLOCK produces a detectable product with emission or excitation properties different from those of the substrate, i.e. as a result of action by the analyte on the substrate, REPORTER exhibits a detectable change in absorbance or, preferably, fluorescence.
- Usually action of the analyte removing BLOCK results in a shift in the absorbance of the resultant product to longer wavelengths that are not absorbed or are minimally absorbed before the removal of BLOCK.
- BLOCK is selected to be removable only by action of a specific analyte, i.e. an enzyme or chemical substance predetermined to be of analytical interest. BLOCK is only removed when the specific analyte is present.
- BLOCK is typically a monovalent moiety derived by removal of a hydroxy group from a phosphate, a sulfate, or a biologically compatible salt thereof.
- BLOCK is a monovalent moiety derived by removal of a hydroxy group from a carboxy group of an aliphatic acid (e.g. acetic or octanoic acid), an aromatic acid (e.g. p-guanidinobenzoic acid), an amino acid, a protected amino acid, or a peptide.
- BLOCK may also be a monovalent moiety derived by removal of a hydroxy group from an alcohol, a mono-, or a polysaccharide (e.g.
- hydrolytic enzymes analytes
- target groups are given in Table 1. Any of the target groups in Table 1, as well as target groups for other hydrolytic or oxidative enzymes, can be used to derive, by means known in the art, a removable BLOCK to combine with a selected REPORTER molecule.
- BLOCK is usually an aliphatic ether.
- Primary examples include 7-ethoxycoumarin, ethers of resorufin (Nakai, et al., J. BIOL. CHEM. 267, 19503 (1992)) and steroidal ethers U.S. Pat. No. 5,110,725 to Martone et al. (1992)). Because the microsomal dealkylase enzymes typically have low turnover rates in cells, it is difficult to detect the fluorescent product in single cells unless a substrate such as that described in Example 8 is used.
- REPORTER portion of the substrate is a molecule for which the emission or excitation properties change depending on the presence or absence of BLOCK.
- REPORTER is a dye that, except for being bound to BLOCK by a REPORTER-BLOCK bond, is detectable by fluorescence.
- REPORTER is a phenol or an aromatic amine and the bond linking REPORTER and BLOCK is an ester of a carboxylic, phosphoric, or sulfuric acid, or a biologically compatible salt thereof, an ether, a thioether, or an amide.
- the carboxylate ester is an ester of an aliphatic carboxylic acid having 1 to 18 carbon atoms or of an aromatic carboxylic acid having 7 to 24 carbon atoms.
- the amide bond is commonly formed by the removal of the elements of hydroxy from the carboxylic acid of an amino acid or peptide and a hydrogen atom from an amino moiety on REPORTER.
- the ether or thioether bond is formed by removal of the elements of hydroxy from an alcohol, preferably a steroidal alcohol having 30 or less
- a biologically compatible salt is not toxic as used, and does not have a substantially deleterious effect on biomolecules.
- biologically compatible anionic salts include, among others, chloride, bromide, iodide, sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate, tetrafluoroborate, tetraphenylboride, nitrate and anions of aromatic or aliphatic carboxylic acids.
- biologically compatible cationic salts include, among others, sodium, potassium, ammonium, and alkylammonium.
- Preferred salts are chloride, iodide, perchlorate, various sulfonates, sodium and potassium.
- a third component of the subject substrate is a haloalkyl moiety XR-, where X is a halogen and R is an alkyl linkage having 1-4 carbon atoms.
- X is a halogen and R is an alkyl linkage having 1-4 carbon atoms.
- the halogen is Cl or Br, and R is methylene; more preferably XR- is ClCH 2 —.
- substituents such as haloaryl, epoxides, sulfonate esters and similar functional groups that contain a group that is readily replaced in a reaction with thiols such as glutathione, that may or may not be catalyzed by glutathione transferase, are equivalent to XR- for some applications.
- XR- is attached to the aromatic system of the REPORTER moiety directly.
- SPACER is preferably a single covalent bond.
- the reactive sites XR- and BLOCK are separated from each other in their attachment to REPORTER. To provide this separation XR- is attached to REPORTER by a SPACER moiety, to yield a compound of the formula XR-SPACER-REPORTER-BLOCK.
- XR- is typically attached to REPORTER via conjugation to a heteroatom present on REPORTER, such as a fluorescein oxygen or rhodamine nitrogen, and SPACER is a covalent linkage having 0-8 carbon atoms and 1-4 heteroatoms, where the heteroatoms are O, N or S in any combination.
- a heteroatom present on REPORTER such as a fluorescein oxygen or rhodamine nitrogen
- SPACER is a covalent linkage having 0-8 carbon atoms and 1-4 heteroatoms, where the heteroatoms are O, N or S in any combination.
- SPACER is carbonyl (—(C ⁇ O)—), sulfonyl (—SO 2 —), benzoyl (—C 6 H 4 —(C ⁇ O)—), where the carbonyl is attached to the phenyl ring at the ortho, metal, or para position with respect to XR-, or benzenesulfonyl (—C 6 H 5 —SO 2 —), where the sulfonyl is attached to the phenyl ring at the ortho, meta, or para position with respect to XR-.
- REPORTER is a rhodol fluorophore
- BLOCK is optionally a mono- or polysaccharide, phosphate, sulfate, ester or ether attached to the rhodol oxygen, or an amino acid or peptide attached to the rhodol nitrogen
- XR-SPACER is optionally attached to the remaining nitrogen or oxygen, respectively (as illustrated above; rhodols type I, II and III).
- REPORTER is a rhodamine fluorophore
- both BLOCK and XR-SPACER are optionally attached to rhodamine nitrogens (as illustrated above: rhodamines type I and II).
- REPORTER is either a rhodol or rhodamine fluorophore
- XR- is optionally attached directly to the aromatic ring at the 5- or 6-position of the fluorophore, and the fluorophore is further substituted by two BLOCK groups (as illustrated above; rhodamine type III).
- Rhodamine-based substrates having only a single amino acid or peptide are smaller or less polar than bis-substituted rhodamines, and can subsequently enter cells with greater facility. Additionally, the removal of only a single BLOCK results in a fluorescent product, simplifying the kinetics of enzyme activity. Finally, less of the typically expensive amino acid BLOCK is used per fluorophore.
- Rhodol-based substrates having an XR-SPACER on either the terminal O or N atom and an enzyme-removable BLOCK on the remaining terminal O or N are also smaller and less polar than substrates based on fluorescein or rhodamine 110 . Furthermore, only a single enzymatic reaction is required to generate a fluorescent product from the substrates.
- the haloalkyl moiety (XR-) will covalently react with an intracellular thiol (Z-S-H) to form a thioether conjugate (Z-S-R).
- intracellular thiol includes glutathione and other polar or higher molecular weight thiols present in cells such as cysteine and cysteine-containing peptides and proteins. Reaction of the subject substrates with thiols other than gluthione appears to be more common with the more reactive alkylating groups (XR) such as bromomethyl and iodomethyl.
- thiol is glutathione
- formation of the thioether conjugate is likely to be catalyzed by the enzyme glutathione transferase, but glutathione transferase is not essential for utilization of these substrates, provided that the uncatalyzed reaction with intracellular thiols is sufficiently rapid to yield retention of at least some of the fluorescent product inside cells.
- glutathione transferase is not essential for utilization of these substrates, provided that the uncatalyzed reaction with intracellular thiols is sufficiently rapid to yield retention of at least some of the fluorescent product inside cells.
- a reaction of the chloroalkylated substrates with glutathione that is catalyzed by glutathione transferase is preferred since nonspecific reaction of protein thiols with alkylating agents is more likely to result in cytotoxicity.
- the cells being analyzed may be from any plant or animal origin that is suspected of containing the analyte, including single cell organisms such as bacteria and yeast.
- the cells may be present in an intact organism or in a medium that has been separated from the organism, such as biological fluids or cultures of essentially pure cell lines.
- the analyte may have been introduced into the cell by processes of genetic engineering familiar to one skilled in the art.
- the analyte if present, may be localized in one or more intracellular areas or organelles, which may include the inner membrane of the cell, the cytosol, microsomes, mitochondria, liposomes, or the nucleus.
- the substrate is brought into contact with the analyte enzyme under conditions where the substrate can readily enter intracellular areas which may contain the analyte of interest.
- the substrate enters intracellular areas by any technique that is suitable for transporting the substrate into the intracellular areas with minimal disruption of the viability of the cell and integrity of cell membranes. Examples of suitable processes include passive diffusion through the membrane; action of chemical agents such as detergents, enzymes or adenosine triphosphate; receptor- or transport protein-mediated uptake; pore-forming proteins; microinjection; electroporation; hypoosmotic shock (Example 12); or minimal physical disruption such as scrape loading or bombardment with solid particles coated with or in the presence of the substrate.
- it may take a few seconds to several hours for enough substrate to enter the cells to give a detectable result.
- the formation of the detectable conjugate REPORTER-SPACER-R-S-Z can occur by either of two routes (FIG. 1 ).
- Route 1 a probe whose detectable optical property is blocked, reacts with an intracellular thiol to form a blocked intermediate that is not yet detectable, at least at the optimal wavelength for detection. If the analyte is present and active, BLOCK is removed by action of the analyte.
- Conjugation of the haloalkyl moiety to glutathione or low molecular weight peptide converts the REPORTER molecule into a small peptide, REPORTER-SPACER-R-S-Z, that has the charge and approximate permeability properties of an amino acid, while having the detectability of the REPORTER molecule. Since amino acids and most peptides are generally not freely permeant to intact cell membranes, it is important for retention of the reaction product that this conjugation occurs intracellularly.
- the intracellularly thiol is glutathione, and the conjugation is catalyzed by the enzyme glutathione transferase.
- Example 14 presents evidence that the metabolic products are substrates for glutathione transferase and presents methods for determined the activity of glutathione transferase in vitro. The relative importance of the two routes to the same product depends on the concentrations and activities of the enzymes and substrates, the access of the substrates to the enzymes, the concentration of intracellular thiols, particularly glutathione, the reactivity of the haloalkyl moiety, and other factors. Significantly, in both cases formation of the detectable reporter molecule occurs only following action of the analyte.
- conjugation of the substrate to an intracellular thiol improves retention of BLOCK-REPORTER in the cell, it does not result in formation of a detectable reporter molecule unless the enzyme needed to remove the BLOCK is present and active.
- the fluorescent product is typically visible within a few minutes.
- Optimal accumulation of the thioether conjugate may be obtained within 15 minutes to 1 hour, however detectability remains for hours to days.
- the cells are prepared for measuring fluorescence or absorbance.
- the preparation required will depend on the method to be used to make such measurements.
- the preparation typically involves the transfer of cells from the biocompatible solution containing the substrate to a fresh biocompatible solution without substrate, usually a standard culture medium, and washing the cells to remove extracellular products. Then the cells may be placed in or on the appropriate container for making the measurements, such as cuvettes, culture dishes, slides, etc.
- the method using the subject substrates may also include fixing the cells using an aldehyde fixative.
- the REPORTER-thiol adduct possesses an aliphatic amine group, as in the case of a glutathione adduct, this group makes the detectable adduct capable of fixation by common aldehyde fixatives such as formaldehyde and glutaraldehyde without complete dispersion of the fluorescence (Example 11).
- aldehyde fixatives such as formaldehyde and glutaraldehyde without complete dispersion of the fluorescence (Example 11).
- the final step of the subject method for analyzing the metabolic activity of cells in making qualitative or quantitative measurements of absorbance or fluorescence of REPORTER is typically present as part of the thioether conjugate retained inside the cell. REPORTER may be blocked or unblocked, depending on whether the analyte was present in the cells.
- the measurements can be made by observation of the absorbance or fluorescence through visual inspection or by using a suitable instrument such as an absorption spectrometer, fluorometer, flow cytometer or microscope. Analysis may be of the whole organism, a tissue, a cell suspension, adherent cells or single cells.
- Example 11 can be of the REPORTER conjugate located in intact living cells (Examples 11-13), in cells that have been fixed with an aldehyde fixative in a manner so as to retain the REPORTER in the fixed cell (Example 11) or can be of the supernate that results from lysis of the cell by a lysing agent. In the latter case, detection in the supernate may be correlated with both metabolism of the BLOCK-REPORTER and the activity of the lysing agent (Example 15).
- the analysis may be by imaging or flow cytometry, where the fluorescene of REPORTER in the cell may be used to discriminate or isolate the cell based on the presence or level of activity of an analyte, using techniques well known in the art, as described by Melamed, et al., FLOW CYTOMETRY AND SORTING (2nd ed. 1990); Shapiro, PRACTICAL FLOW CYTOMETRY (1985).
- the fluorescent thioether conjugate formed by metabolic removal of BLOCK and conjugation of REPORTER to glutathione is retained inside living cells for sufficiently long to quantitatively or qualitatively measure the fluorescence or absorbance of the attached REPORTER.
- the reaction products may easily leak from the cell unless a fixing agent is present (Example 11).
- the change in fluorescence intensity that results from loss of the detectable product in therefore a useful means of detecting cell death, especially when caused by cytotoxic agents.
- Metabolism of the substrate to a polar tracer by cells that are positive for the enzymes that remove BLOCK is the basis of a test for cytotoxicity of extrinsic reagents (Example 15) or extrinsic conditions such as heat.
- extrinsic reagents Example 15
- extrinsic conditions such as heat.
- glucosidase is an enzyme that is commonly found in many cells, making the subject glucosidase substrates also useful for this application.
- substrates such as the galactosides, glucuronides and phosphates are preferred since these enzymes are not as widely distributed, particularly in mammalian cells.
- the subject substrates XR-SPACER-REPORTER-BLOCK may be derived from a variety of fluorophores (Examples 1-10) with a range of spectral properties from the near ultraviolet to about 650 nm and that the BLOCK group may be any of several groups that can be removed by action of a specific analyte (Examples 1-10).
- Methods for synthesis of the REPORTER-BLOCK conjugates are similar or identical to those of the structurally similar molecules that do not contain the chloroalkyl groups.
- haloalkylated REPORTERS are typically synthesized using the same or similar chemistry as is used to prepare non-haloalkylated REPORTERS by the substitution of a haloalkyl moiety on one of the reactants (Examples 2 and 6).
- haloalkylated REPORTERS can be prepared by the conversion of other residues present on REPORTER, such as carboxylic acids, to haloalkyl by methods well known in the art (Example 1).
- the subject substrates are advantageous for measurement of the enzymatic activity in single cells in that they are permeable to the intracellular organelle containing the enzyme (Examples 11-13 and 15), they are converted to a detectable reporter molecule by specific action of the enzyme (Examples 11-13 and 15), the reporter molecule is retained under physiological conditions for the period required for its detection (Example 11-13 and 15), a direct relationship between the enzymatic activity and the amount of dye retained in the cell can be established (Example 13), and the reporter molecule and the substrate are substantially non-toxic to the cells (Example 11-13). In addition, while not essential for utility, the reporter molecule is still detectable flowing fixation of the cell (Example 11).
- This reaction mixture is stirred for another 30 minutes at ⁇ 5° C., allowed to reach +5° C., and sodium borohydride (5.8 g, 150 mmoles) is added portionwise, followed by dropwise addition of methanol (25 mL) to the stirred mixture at 5° C. over 30 minutes.
- the reaction mixture is allowed to come to room temperature and acidified to pH 2 by dropwise addition of 6M aqueous HCl.
- the precipitated salts are filtered and rinsed with tetrahydrofuran (2 ⁇ 50 mL). The filtrate is evaporated to yield an orange solid, which is added to a solution of acetic anhydride (100 mL) containing pyridine (50 mL).
- 5-Acetoxymethylfluorescein (4.9 g, 10 mmoles) is dissolved with heating and stirring in acetic acid (200 mL). Concentrated hydrochloric acid (200 mL) is added gradually. The mixture is heated to reflux for 15 hours and the solvents are evaporated. Concentrated hydrochloric acid (400 mL) is again added to the residue and heating to reflux with stirring is continued for another 15 hours. The reaction mixture is cooled to room temperature, crushed ice is added (500 mL) and the pH is increased to about 2-3 by gradual addition of aqueous 1M sodium hydroxide solution. The resulting orange solid is filtered and washed with water.
- the mixture is allowed to stir as described above for 96 hours, after which it is filtered through a pad of diatomaceous earth.
- the precipitate is washed with chloroform (5 ⁇ 15 mL).
- the combined filtrates are extracted sequentially with 1M aqueous hydrochloric acid solution (1 ⁇ 75 mL), saturated aqueous sodium bicarbonate solution (1 ⁇ 75 mL), 0.1M aqueous sodium thiosulfate solution (1 ⁇ 75 mL) then water (1 ⁇ 75 mL).
- the combined organic layers are dried over anhydrous sodium sulfate, filtered, evaporated and dried in vacuo to a bright yellow foam (620 mg).
- CMFDG 5-chloromethylfluorescein di- ⁇ -D-galactopyranoside
- a suspension of 5-chloromethylfluorescein di- ⁇ -D-galactopyranoside, octaacetate (60 mg, 58 ⁇ moles) in anhydrous methanol (15 mL) is cooled to 0° C. in an ice bath while under an atmosphere of dry nitrogen gas.
- a solution of freshly prepared sodium methoxide in methanol (20 ⁇ L, 0.75M solution) is added, and this mixture is allowed to stir at 0° C. for 2.15 hours.
- the reaction is neutralized with washed, dry Amberlite IRC50 (H + ) resin (pH 4), filtered and evaporated to approximately 25 mL total volume.
- the product is crystallized by addition of anhydrous diethyl ether (150 mL) and stored at ⁇ 12° C.
- the colorless crystalline product is isolated by filtration through a Nylon 66 membrane filter (0.45 pore size), washed with fresh diethyl ether and dried in vacuo to an off-white powder (1st crop, 15 mg, 37% ).
- Silica gel TLC analysis of this material shows one major product with an R f of 0.48 (15:1 chloroform:methanol).
- 2.0 g of this material is purified on a 4 cm ⁇ 15 cm column of 35-70 ⁇ m silica gel. Elution is carded out by a stepwise gradient starting with chloroform (500 mL) and finished with 9:1 chloroform:methanol (500 mL). Ten 100 mL fractions are collected and monitored by silica gel TLC (15:10 chloroform:methanol). Fractions containing the pure product are combined, evaporated and dried (1.1 g, 55% recovery, 40% yield).
- the precipitate is washed with chloroform (5 ⁇ 10 mL), and the combined organic filtrates are extracted with 1M aqueous HCl (1 ⁇ 75 mL), saturated aqueous sodium carbonate solution (1 ⁇ 75 mL), 1M sodium thiosulfate (1 ⁇ 75 mL), and water (1 ⁇ 75 mL).
- the organic layer is dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to a pale yellow oil (400 mg).
- the oil is applied to a column of silica gel G (100 g) and eluted by gradient elution using 0-10% ethyl acetate in chloroform.
- a suspension of 4-chloromethylcoumarin 7-(2,3,4-tri-O-acetyl ⁇ -D-glucopyranosiduronate, methyl ester (560 mg, 1.06 mmole) in anhydrous methanol (50 mL) is cooled to 0° C. in an ice-bath under an atmosphere of dry nitrogen gas.
- a solution of freshly prepared sodium methoxide is added in two portions (2 ⁇ 500 ⁇ L, 1.37M solution) over a period of one hour. This mixture is allowed to stir as above for 2 hours.
- the product residue is purified by chromatography (5 ⁇ 17 cm column of 35-70 ⁇ m silica gel G), and eluted with chloroform. Fractions containing product are combined, evaporated and dried in-vacuo. The non-fluorescent residue is collected after crystallization from methanol:diethyl ether (1:5).
- L-leucine-rhodamine 110-p-chloromethylbenzenesulfonamide is prepared by reacting the rhodamine 110 with p-chloromethylbenzenesulfonyl chloride in place of p-chloromethylbenzoyl chloride.
- Similar substrates having a carbonyl spacer or sulfonyl spacer are prepared using chloroacetyl chloride and chloromethylsulfonyl chloride, respectively, in the presence of a suitable base.
- the protected substrate is deprotected using a procedure exactly analogous to that described in Example 9.
- N-(p-Chloromethylbenzoyl)-rhodol To a stirred cold solution of rhodol (1.0 g, 3.0 mmol) and triethylamine (0.37 g, 3.6 mmoles) in 15 mL dry DMF in an ice bath is added p-chloromethylbenzoic acid chloride (0.57 g, 3.0 mmol) in 5 mL dry DMF dropwise over 1 ⁇ 2 hr. The solution is stirred in the ice bath for 2 hours, and then warmed to room temperature and stirred an additional 24 hrs. The solvent is removed at room temperature under high vacuum. The crude product is purified using column chromatography. The product is characterized using 1 H NMR spectroscopy.
- N-(p-Chloromethylbenzenesulfonyl-rhodol This product is prepared exactly analogously to N-(p-chloromethylbenzoyl)rhodol (above) except using p-chloromethylbenzenesulfonyl chloride in place of p-chloromethylbenzoyl chloride.
- Similar reporter molecules including the glycosides and phosphates, are prepared by treating the desired XR-rhodol intermediate (as prepared above) with the desired BLOCK precursors, for example those described for fluorescein in Examples 1 and 5.
- NIH/3T3 cells lacZ negative
- CRE BAG 2 cells 3T3 cells transformed with a retrovirus containing lacZ gene
- Both cell lines can be obtained from American Type Culture Collection Co., Rockville, Md.
- the cells are grown in a humidified atmosphere of 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 50 ⁇ g/mL gentamicin, 300 ⁇ g/mL L-glutamine and 10 mM HEPES pH 7.4.
- a working medium containing 100 ⁇ M Cl-MUG.
- This medium is filter-sterilized by passing through an AcrodiscTM filter (0.45 ⁇ m pore size).
- a working medium containing MUG is prepared in the same way.
- This substrate shows no cytotoxicity to either type of cell.
- Cells incubated in 100 ⁇ M Cl-MUG working medium for 24 hours look morphologically normal and have the same population doubling time as the cells incubated in substrate-free medium.
- Cells preincubated in the working medium for 6 hours can be subcultured and incubated in fresh medium resulting in the formation of a second generation of cells that is normal. Fluorescence can be detected in the cells for at least 24 hours following washing of the cells with fresh medium.
- the cells grown on coverslips and incubated with either Cl-MUG or MUG are immersed in 3.7% paraformaldehyde in 80 mM Pipes buffer, pH 6.5 for 4 minutes to allow the paraformaldehyde to penetrate the cells.
- the cells are then transferred to 3.7% paraformaldehyde in PBS. After 6 minutes, the cells are washed three times with PBS buffer for ten minutes.
- the coverslips are put on the slides and sealed.
- the fixed cells are examined for their fluorescence under a Zeiss microscope equipped with a Hoechst filter set.
- the cells stained with Cl-MUG are still brightly fluorescent while those stained with MUG are barely fluorescent. This indicates that the fluorescent products, enzymatically cleaved from Cl-MUG, react with intracellular thiols and form cell-impermeant fluorescent adducts, which improves the dye retention after cell fixation.
- Example 11 The same cell lines as used in Example 11 are used for these experiments.
- CMFDG 5-Chloromethylfluorescein di- ⁇ -D-galactoside
- Example 1 is dissolved in DMSO to get 20 mM stock solution.
- a 40 mM stock solution offluorescein di- ⁇ -D-galaetoside (FDG) (Molecular Probes) is also made in DMSO as a control.
- the stock solutions are kept sealed in brown reagent bottles and stored at ⁇ 20° C.
- FCS-PBS Solution and Staining Solution
- the CMFDG substrate Because the CMFDG substrate generates a product that is so well retained by cells (Examples 11 and 12), it can be used for quantitative measurements of lacZ expression.
- Product accumulation can be quantitated by the following procedure: 1 mL of the reaction mixture is extracted with 2 mL of ethyl acetate and the pH is adjusted to about 4 (3.5-4.3) with 1M HCl ( ⁇ 20 ⁇ L). The aqueous layer is separated using a modified transfer pipette as a miniature separatory funnel and the absorbance or fluorescence is determined as above.
- CMFDG 5-Chloromethylfluorescein di- ⁇ -D-galactopyranoside
- Cl-MUG 7-( ⁇ -D-galactopyranosyloxy)-4-chloromethylcoumarin
- NIH/3T3 cells lacZ negative
- CRE BAG 2 cells 3T3 cells transformed with a retrovirus containing latZ gene
- Both cell lines can be obtained from American Type Culture Collection Co., Rockville, Md.
- the cells are grown in a humidified atmosphere of 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 50 ⁇ g/mL gentamicin, 300 ⁇ g/mL L-glutamine and 10 mM HEPES pH7.4.
- the experiment is most conveniently performed in a CytoFluorTM 2300 fluorescence plate reader (Millipore) but can be performed qualitatively on a microscope slide or quantitatively in a flow cytometery, or in any system that permits washing of the cells following addition of the cytotoxic agent.
- 200 ⁇ L of NIH 3T3 cells are transferred to several wells of a sterilized 96 well plate and permitted to recover and attach for 1 hour.
- the cells are loaded with 10 ⁇ M CMFDG for 30 minutes at 37° C.
- the brightly fluorescent cells are washed with fresh medium then reconstituted to 200 ⁇ L total volume. Fluorescence of each well is determined using a 485 nm excitation filter, a 530 nm emission filter and sensitivity setting 1.
- saponin as a lysing agent in concentrations varying from 0 to 0.004%. Following an incubation of approximately 15 minutes the wells are washed with fresh medium, reconstituted to 200 ⁇ L with fresh medium and remeasured as above. Loss of fluorescence from the sample is correlated with the loss of integrity of the membrane and thus the cytotoxicity of the reagent. Using the above conditions it is found that 50% of the signal is lost when the cells are incubated with about 0.001% saponin while greater than 90% signal loss is observed using a 0.02% solution.
- the CRE BAG 2 cells are loaded with Cl-MUG as described in Example 11.
- the assay of viability of the lacZ transfected cells is conducted essentially as described immediately above for the assay of NIH 3T3 cells using CMFDG.
- the results of the saponin cytotoxicity measurement using the two reagents are comparable for the two reagents.
- NIH/3T3 cells are prepared as described in Example 15.
- Staining solutions of bis-(L-leucine)-rhodamine 110 and L-leucine-rhodamine 110-p-chloromethylbenzamide are prepared that are 25 ⁇ M in concentration.
- Equivalent NIH/3T3 cells are incubated with the same amount of peptidase substrate for thirty (30) minutes at 37° C. in Modified Tyrode's buffered saline (135 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 10 mM NaHEPES, 5.6 mM glucose, pH 7.5, plus 0.1% bovine serum albumin).
- the cells stained with the bis-L-leucine substrate exhibit substantial leakage of the rhodamine 110 enzyme reaction product, while the cells stained with the chloromethyl-substituted Rhodamine 110 substrate show significantly less leakage.
- the cells are washed and placed in fresh media for several hours.
- the cells stained with bis-(L-leucine)-rhodamine 110 show no fluorescent signal upon illumination, while the L-leucine-Rhodamine 110-p-chloromethylbenzamide stained cells show a bright, well-defined staining pattern when viewed using a fluorescein long-pass filter set.
- CRE-BAG2 cells are prepared as described in Example 15.
- Staining solutions of 4-methylcoumarin ⁇ -D-galactopyranoside, 4-chloromethylcoumarin ⁇ -D-galactopyranoside and 4-trifluoromethylcoumarin ⁇ -D-galactopyranoside, respectively, are prepared that are 10 ⁇ M in concentration.
- Each galactopyranoside substrate is loaded into CRE-BAG2 cells expressing ⁇ -galactosidase using brief hypotonic shock for less than five minutes. Cells are then washed free of extracellular substrate and incubated for three hours in fresh culture medium at 37° C. Each cell sample is then illuminated at 360 ⁇ 20 nm, and the fluorescence is observed at >400 nm.
- Fluorescence intensity data is acquired using a cooled CCD camera and subsequently stored in digital format.
- the cell samples incubated with the methylcoumarin and trifluoromethylcoumarin substrates show low fluorescent intensity, while the cells incubated with the galactosidase substrate having a choromethyl substituent are brightly fluorescent.
Abstract
XR-SPACER-REPORTER-BLOCK
-
- wherein -BLOCK is a group selected to be removable by action of a specific analyte, to give REPORTER spectral properties different from those of the substrate,
- -REPORTER- is a molecule that, when no longer bound to BLOCK by a BLOCK-REPORTER bond, has spectral properties different from those of the substrate,
- -SPACER- is a covalent linkage, and
- XR- is a haloalkyl moiety that can covalently react with an intracellular thiol (Z-S-H) to form a thioether conjugate (Z-S-R).
After the substrate enters the cells, the analyte removes BLOCK to make REPORTER detectable by the change in spectral properties, and the haloalkyl XR reacts with the intracellular thiol to form the thioether conjugate inside the cells, which is well-retained in the cells.
Description
XR-SPACER-REPORTER-BLOCK
TABLE 1 |
REPRESENTATIVE ENZYMES |
E.C. NO. | ENZYME | TARGET GROUP |
3.2.1.20 | α-Glucosidase | α-D-Glucose |
3.2.1.21 | β-Glucosidase | β-D-Glucose |
3.2.1.22 | α-Galactosidase | α-D-Galactose |
3.2.1.23 | β-Galactosidase | β-D-Galactose |
3.2.1.24 | α-Mannosidase | α-D-Mannose |
3.2.1.25 | β-Mannosidase | β-D-Mannose |
3.2.1.30 | N-Acetyl-β-D-Glucos- | β-D-N-Acetyl-Glucosamine |
amidase | ||
3.2.1.31 | β-Glucuronidase | β-D-Glucoronic Acid |
3.2.1.38 | β-D-Fucosidase | β-D-Fucose |
3.2.1.51 | α-L-Fucosidase | α-L-Fucose |
3.2.1-- | β-L-Furosidase | β-L-Fucose |
3.2.1.76 | L-Iduronidase | α-L-Iduronic Acid |
3.2.1.4 | Cellulase | β-D-Cellobiose |
3.2.1-- | α-Arabinopyranosidase | α-L-Arabinopyranose |
3.2.1.37 | β-Xylosidase | β-D-Xylose |
3.2.1.18 | α-N-Acetyl- | α-D-N-Acetyl-neuraminic acid |
neuraminidase | (Sialic acid) | |
3.1.1-- | guanidinobenzoatase | aryl esters of p-guanidinobenzoate |
3.1.3.1 | alkaline phosphatase | aryl or alkyl phosphate monoesters |
3.1.3.2 | acid phosphatase | aryl or alkyl phosphate monoesters |
3.1.6.1 | aryl sulfatase | aryl sulfate monoesters |
3.3.3.41 | 4-nitrophenylphosphatase | aryl phosphates |
3.4.11.l | leucine amino peptidase | leucine residues at α-carboxyl |
carbon atoms, or a lower alcohol having 6 or less carbon atoms, and a hydrogen atom from a phenolic or thiophenolic moiety on REPORTER. Alternatively the ether or thioether bond is formed by removal of hydroxy from a mono- or polysaccharide and a hydrogen atom from a phenolic or thiophenolic moiety on REPORTER.
TABLE 2 |
REPRESENTATIVE REPORTER MOLECULES |
Reporter Molecules | Sample Substrate |
6-aminobenzphenalenones |
|
7-aminocoumarin |
|
6-aminophenalenone |
|
anthracene |
|
fluorescein |
|
6-hydroxybenzphenalenones |
|
7-hydroxycoumarin |
|
6-hydroxyphenalenone |
|
4-methoxy-2-naphthyl amine |
|
resorufin |
|
rhodamine, type I |
|
rhodamine, type II |
|
rhodamine, type III |
|
rhodol, type I |
|
rhodol, type II |
|
rhodol, type III |
|
acridinone |
|
Preparation of 7-(2,3,4,6-tetra-O-acetyl β-D-galactopyranosyloxy)-4-chloromethylcoumarin:
Preparation of 4-chloromethyl-7-coumarinyl-(2,3,4-tri-O-acetyl β-D-glucopyranosiduronide, methyl ester):
-
- (A) FCS-PBS Solution: Phosphate buffered saline (GIBCO), 4% fetal calf serum, 10 mM HEPES, pH 7.2
- (B) Staining Solution: CMFDG: 200 μL of stock solution is diluted with distilled water to 4 mL to get a 1 mM staining solution. FDG: 400 μL of stock solution is diluted to 4 mL with distilled water to get a 4 mM staining solution.
4. Staining and Examination of Cells:
- 1. A series of lacZ gene containing plasmids, each with a modified yeast promoter are used. When transferred into the appropriate strains of Saccharomyces cerevisiae, each plasmid confers a different level of lacZ gene expression. Yeast strains EG123 and HR125-5d, transformed with these plasmids, are grown in special minimal medium for plasmid maintenance and, after reaching the appropriate level of growth, are assayed for lacZ gene expression as measured by β-galactosidase activity.
- 2. The β-galactosidase activity of one aliquot of these transformed yeast cells is measured using o-nitrophenyl-β-D-galactopyranoside (ONPG).
- 3. Another aliquot of yeast cells is loaded with the fluorogenic substrate CMFDG as indicated in Example 12. Following washing to remove any dye that has leaked from the cells, the fluorescence intensity of the remaining cells as measured in a CytoFluor 2300 fluorescence plate reader (Millipore) can be measured over time. Each lacZ-positive yeast strain shows a linear increase in fluorescence when plotted over time. The amount of increase over time can be calculated from the slope of the plotted increase using linear regression. Each yeast strain accumulates fluorescent product at a characteristic rate, as reflected in the differences in the slopes of the various lines.
- 4. The slopes of these lines can be used to calculate the β-galactosidase activity of each yeast strain. The obtained values are compared with the assays using ONPG substrate to confirm that the fluorescence intensity as measured over time correlates well with the level of lacZ expression (enzyme activity) in these transformed yeast cells. Intensity measurements in single cells by flow cytometry using FDG have been extensively described by Nolan et al. [Nolan G. P., Fiering, S., Nicolas, J. F., Herzenberg, L. A., PROC. NATL. ACAD. SCI. USA 85, 2603 (1988] that can be improved by use of CMFDG, which is shown in Example 12 to give a product that is better retained than that formed from FDG and to be useable under physiological conditions of incubation. Certain products such as 4-chloromethylumbelliferyl β-D-galactoside (Cl-MUG) can be loaded into living cells without requiring hypotonic shock (see Example 11).
- 1) Reagents: The stock reagents required are: 1) 5-Chloromethylfluorescein (CMF) substrate (Example 1) 10 mg/mL in DMF, diluted 1:10 in reaction buffer to 1 mg/mL; 2). Reaction buffer 100 mM potassium phosphate, 15% glycerol pH 6.8; 3). Glutathione (GSH) 155 mM in 20 mM TRIS, 2 mM DTT, 15% glycerol pH 7.8; 4). Glutathione Transferase from Equine Liver (Sigma #G 6511) 1.0 mg/mL in reaction buffer.
- 2) Assay. The reaction mixture consists of 0.7 mL reaction buffer, 0.1 mL GSH, 0.1 mL substrate and 0.1 mL enzyme. The background is measured and subtracted by using 0.1 mL of the reaction buffer instead of the enzyme. Enzymatic activity can be detected at less than 5 minutes at room temperature. Product formation can be detected by separation of the polar product from the less polar substrate by a suitable means such as thin layer chromatography or high performance liquid chromatography. For instance, product accumulation can be detected by thin layer chromatography using A) 15:85 methanol:chloroform or B) 2:5:15 NH4OH:isopropyl alcohol:methanol. In system A the product does not move from the origin whereas the substrate has an intermediate mobility relative to the solvent front. In system B the product moves from the origin, with an Rf identical to that of an authentic standard for the glutathione adduct of chloromethyl fluorescein prepared by a nonenzyme catalyzed reaction, whereas the substrate moves with the solvent front. For quantitative measurement the products are scraped from the plate, dissolved in 1% aqueous ammonium hydroxide and the absorbance at 492 nm determined using an extinction coefficient of 75,000 cm−1M−1 for fluorescein. Alternatively the fluorescence of the product is determined using a fluorometer versus a fluorescein standard with excitation at 492 and emission measured at 515 nm.
Claims (25)
XR-SPACER-REPORTER-BLOCK
XR-SPACER-REPORTER-BLOCK
XR-SPACER-REPORTER-BLOCK
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US08/026,633 US5362628A (en) | 1991-08-23 | 1993-03-05 | Fluorescent haloalkyl derivatives of reporter molecules well retained in cells |
US08/336,285 US5576424A (en) | 1991-08-23 | 1994-11-08 | Haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells |
US08/910,090 USRE38723E1 (en) | 1991-08-23 | 1997-08-12 | Haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells |
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