US20040191792A1 - Acridone derivatives as labels for fluorescence detection of target materials - Google Patents

Acridone derivatives as labels for fluorescence detection of target materials Download PDF

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US20040191792A1
US20040191792A1 US10/479,578 US47957803A US2004191792A1 US 20040191792 A1 US20040191792 A1 US 20040191792A1 US 47957803 A US47957803 A US 47957803A US 2004191792 A1 US2004191792 A1 US 2004191792A1
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John Smith
Richard West
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GE Healthcare UK Ltd
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Assigned to AMERSHAM BIOSCIENCES UK LIMITED reassignment AMERSHAM BIOSCIENCES UK LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SMITH, JOHN ANTHONY, WEST, RICHARD MARTIN
Publication of US20040191792A1 publication Critical patent/US20040191792A1/en
Assigned to GE HEALTHCARE UK LIMITED reassignment GE HEALTHCARE UK LIMITED CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: AMERSHAM BIOSCIENCES UK LIMITED, AMERSHAM PHARMACIA BIOTECH UK LIMITED, AMERSHAM LIFE SCIENCE LIMITED, AMERSHAM LIFE SCIENCE, BATESON
Priority to US11/943,628 priority Critical patent/US8034558B2/en
Priority to US13/233,365 priority patent/US20120015373A1/en
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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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B15/00—Acridine dyes

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  • the present invention relates to new acridone derivatives having characteristic fluorescence lifetimes that can be used as labels for attachment to and labelling of target materials.
  • the acridone derivatives of the invention may be easily distinguished, one from the other, by virtue of their fluorescence lifetimes and they may be used in multiparameter applications.
  • the invention also relates to assay methods utilising acridone derivatives and to a set of different fluorescent acridone lifetime dyes.
  • Fluorescent labels are generally stable, sensitive and a wide range of methods are now available for the labelling of biomolecules.
  • the emission spectrum of a fluorescent dye is a characteristic property of the dye, the intensity of such emission being used in the detection of materials labelled with that dye.
  • One problem with measurements of fluorescence intensity as a means of detecting and/or measuring the concentration of a fluorescent labelled biomolecule is that background fluorescence may interfere with the measurement.
  • the acridone chromophore is highly fluorescent and has been used for labelling biological molecules and subsequent detection by conventional fluorescence emission spectroscopy.
  • Faller, T. et al J.Chem.Soc.Chem.Comm., (1997), 1529-30) describe the preparation of a succinimidyl ester derivative of acridone and its use in labelling peptides for subsequent analysis by mass-spectroscopy.
  • U.S. Pat. No. 5,472,582 (Jackson) describes the use of the fluorescent label, 2-aminoacridone, for labelling and detecting carbohydrates in a mixture, following electrophoretic separation.
  • Val'kova, G. et al (Dokl. Akad. Nauk. SSR, (1978), 240(4), 884-7) have measured the fluorescence lifetimes of several acridone derivatives, however, to date, there appear to be no reports relating to the use of acridones as lifetime dyes suitable for labelling and the detection of biological materials.
  • the present invention therefore describes modifications of the acridone chromophore, to produce a range of acridone derivatives having characteristic fluorescence lifetimes and which are useful for labelling biological materials.
  • the acridone derivatives of the present invention moreover provide a valuable set of fluorescent labels having a common core structure and which are particularly useful for multiparameter analysis.
  • the absorption and emission spectra remain essentially the same, whilst the fluorescence lifetimes vary.
  • Another advantage of the present invention is that the fluorescence lifetimes of the acridone dye derivatives are generally longer than the lifetimes of other fluorescent labels, as well as naturally occurring fluorescent materials, such as proteins and polynucleotides, thereby allowing easy discrimination from background fluorescence in biological assays utilising such dyes.
  • a reagent for labelling and lifetime detection of a target material wherein said reagent is a dye of the formula (I):
  • groups R 2 and R 3 are attached to the Z 1 ring structure and groups R 4 and R 5 are attached to the Z 2 ring structure;
  • Z 1 and Z 2 independently represent the atoms necessary to complete one ring, two fused ring, or three fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur;
  • R 2 , R 3 , R 4 and R 5 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, mono- or di-nitro-substituted benzyl, amino, mono- or di-C 1 -C 4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and the group —(CH 2 —) n Y;
  • R 1 is selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20 alkyl, aralkyl, the group —E—F and the group —(CH 2 —) n Y;
  • E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group;
  • Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6.
  • the dye of formula (I) is a fluorescent dye wherein:
  • groups R 2 and R 3 are attached to the Z 1 ring structure and groups R 4 and R 5 are attached to the Z 2 ring structure, where Z 1 and Z 2 are hereinbefore defined;
  • R 2 , R 3 , R 4 and R 5 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C 1 -C 4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and the group —(CH 2 —) n Y; and
  • R 1 is selected from hydrogen, C 1 -C 20 alkyl, aralkyl, the group —E—F and the group —(CH 2 —) n Y;
  • the acridone dyes according to the first embodiment of the first aspect are particularly suitable for use as fluorescence lifetime dyes.
  • lifetime dye is intended to mean a dye having a measurable fluorescence lifetime, defined as the average amount of time that the dye remains in its excited state following excitation (Lackowicz, J. R., Principles of Fluorescence Spectroscopy, Kluwer Academic/Plenum Publishers, New York, (1999)).
  • the fluorescent dye has a fluorescence lifetime in the range from 2 to 30 nanoseconds, more preferably from 2 to 20 nanoseconds.
  • the dye of formula (I) is a non-fluorescent or substantially non-fluorescent dye wherein: groups R 1 , R 2 , R 3 , R 4 , R 5 , Z 1 and Z 2 are hereinbefore defined; and wherein at least one of groups R, R 2 , R 3 , R 4 and R 5 comprises at least one nitro group.
  • the at least one nitro group may be attached directly to the Z 1 and/or Z 2 ring structures.
  • a mono- or di-nitro-substituted benzyl group may be attached to the R 1 , R 2 , R 3 , R 4 or R 5 positions, which optionally may be further substituted with one or more nitro groups attached directly to the Z 1 and/or Z 2 ring structures.
  • At least one of groups R 1 , R 2 , R 3 , R 4 and R 5 in the dye of formula (I) is the group —E—F where E and F are hereinbefore defined.
  • the target bonding group F is a reactive or functional group.
  • a reactive group of a compound according to formula (I) can react under suitable conditions with a functional group of a target material; a functional group of a compound according to formula (I) can react under suitable conditions with a reactive group of the target material such that the target material becomes labelled with the compound.
  • F when F is a reactive group, it is selected from succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, acid halide, vinylsulphone, dichlorotriazine, carbodiimide, hydrazide and phosphoramidite.
  • F when F is a functional group, it is selected from hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphate and thiophosphate.
  • Z 1 and Z 2 may be selected independently from the group consisting of phenyl, pyridinyl, naphthyl, anthranyl, indenyl, fluorenyl, quinolinyl, indolyl, benzothiophenyl, benzofuranyl and benzimidazolyl moieties. Additional one, two fused, or three fused ring systems will be readily apparent to the skilled person.
  • Z 1 and Z 2 are selected from the group consisting of phenyl, pyridinyl, naphthyl, quinolinyl and indolyl moieties. Particularly preferred Z 1 and Z 2 are phenyl and naphthyl moieties.
  • At least one of the groups R 1 , R 2 , R 3 , R 4 and R 5 of the dyes of formula (I) is a water solubilising group for conferring a hydrophilic characteristic to the compound.
  • Solubilising groups for example, sulphonate, sulphonic acid and quaternary ammonium, may be attached directly to the aromatic ring structures Z 1 and/or Z 2 of the compound of formula (I).
  • solubilising groups may be attached by means of a C 1 to C 6 alkyl linker chain to said aromatic ring structures and may be selected from the group —(CH 2 —) n Y where Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6.
  • Alternative solubilising groups may be carbohydrate residues, for example, monosaccharides.
  • Examples of water solubilising constituents include C 1 -C 6 alkyl sulphonates, such as —(CH 2 ) 3 —SO 3 ⁇ and —(CH 2 ) 4 —SO 3 ⁇ .
  • one or more sulphonate or sulphonic acid groups attached directly to the aromatic ring structures of a dye of formula (I) are particularly preferred. Water solubility may be advantageous when labelling proteins.
  • Suitable spacer groups E may contain 1-60 chain atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus.
  • the spacer group may be:
  • R 1 is hydrogen, C 1 -C 4 alkyl or aryl, which may be optionally substituted with sulphonate
  • Ar is phenylene, optionally substituted with sulphonate
  • p is 1-20, preferably 1-10
  • q is 0-10
  • r is 1-10 and s is 1-5.
  • groups R 1 , R 2 , R 3 , R 4 and R 5 and the groups with which R 1 , R 2 , R 3 , R 4 and R 5 can react are provided in Table 1.
  • groups R 1 , R 2 , R 3 , R 4 and R 5 may be the functional groups of Table 1 that would react with the reactive groups of a target material: TABLE 1 Possible Reactive Substituents and Sites Reactive Therewith Reactive Groups Functional Groups succinimidyl esters primary amino, secondary amino isothiocyanates amino groups haloacetamides, maleimides sulphydryl, imidazole, hydroxyl, amine acid halides amino groups anhydrides primary amino, secondary amino, hydroxyl hydrazides, aldehydes, ketones vinylsulphones amino groups dichlorotriazines amino groups carbodiimides carboxyl groups phosphoramidites hydroxyl groups
  • Preferred reactive groups which are especially useful for labelling target materials with available amino and hydroxyl functional groups include:
  • n is 0 or an integer from 1-10.
  • Aryl is an aromatic substituent containing one or two fused aromatic rings containing 6 to 10 carbon atoms, for example phenyl or naphthyl, the aryl being optionally and independently substituted by one or more substituents, for example halogen, hydroxyl, straight or branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl and C 1 -C 6 alkoxy, for example methoxy, ethoxy, propoxy and n-butoxy.
  • Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ring system containing at least one and no more than 3 heteroatoms which may be selected from N, O, and S and is optionally and independently substituted by one or more substituents, for example halogen, hydroxyl, straight or branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl and C 1 -C 6 alkoxy, for example methoxy, ethoxy, propoxy and n-butoxy.
  • substituents for example halogen, hydroxyl, straight or branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl and C 1 -C 6 alkoxy, for example methoxy, ethoxy, propoxy and n-butoxy.
  • Aralkyl is a C 1 to C 6 alkyl group substituted by an aryl or heteroaryl group.
  • Halogen and halo groups are selected from fluorine, chlorine, bromine and iodine.
  • Exemplary dyes according to the first embodiment of the first aspect are as follows:
  • the dyes of the present invention may be used to label and thereby impart fluorescent properties to a variety of target biological materials.
  • a method for labelling a target biological material comprising:
  • groups R 2 and R 3 are attached to the Z 1 ring structure and groups R 4 and R 5 are attached to the Z 2 ring structure, where Z 1 and Z 2 are hereinbefore defined;
  • R 2 , R 3 , R 4 and R 1 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C 1 -C 4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and the group —(CH 2 —) n Y;
  • R 1 is selected from hydrogen, C 1 -C 20 alkyl, aralkyl, the group —E—F and the group —(CH 2 —) n Y;
  • the fluorescent dyes of the present invention wherein at least one of the groups R 1 to R 5 contains a charge, for example, quaternary amino, may be used to bind non-covalently to charged biological molecules such as, for example, DNA and RNA.
  • fluorescent dyes of the present invention wherein at least one of the groups R 1 to R 5 is an uncharged group, for example, a long chain alkyl or an aryl group, may be used to bind to uncharged biological molecules such as, for example, biological lipids, as well as to intact cell membranes, membrane fragments and cells.
  • the fluorescent dyes may be used to covalently label a target biological material.
  • the target bonding group may be a reactive group for reacting with a functional group of the target material.
  • the target bonding group may be a functional group for reacting with a reactive group on the target biological material.
  • the method comprises incubating the target material with an amount of the dye according to the invention under conditions to form a covalent linkage between the target and the dye.
  • the target may be incubated with an amount of a compound according to the present invention having at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 that includes a reactive or functional group as hereinbefore defined that can covalently bind with the functional or reactive group of the target biological material.
  • Suitable biological materials include, but are not limited to the group consisting of antibody, lipid, protein, peptide, carbohydrate, nucleotides which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, and oxy or deoxy polynucleic acids which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, microbial materials, drugs, hormones, cells, cell membranes and toxins.
  • the fluorescent dyes according to the invention having a target bonding group in at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 may be used in an assay method for determining the presence or the amount of an analyte in a sample.
  • a method for the assay of an analyte in a sample which method comprises:
  • groups R 2 and R 3 are attached to the Z 1 ring structure and groups R 4 and R 5 are attached to the Z 2 ring structure, where Z 1 and Z 2 are hereinbefore defined;
  • At least one of groups R 1 , R 2 , R 3 , R 4 and R 5 is the group —E—F where E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group;
  • said remaining groups R 2 , R 3 , R 4 and R 5 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C 1 -C 4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH 2 —) n Y; and, when group R 1 is not said group —E—F, it is selected from hydrogen, C 1 -C 20 alkyl, aralkyl and the group —(CH 2 —) n Y;
  • step ii) may be performed by measurement of the fluorescence intensity or fluorescence lifetime of the sample, preferably the fluorescence lifetime.
  • the assay method is a direct assay for the measurement of an analyte in a sample.
  • a known or putative inhibitor compound may be included in the assay mix.
  • the assay may be a competitive assay wherein a sample containing an analyte competes with a fluorescent tracer for a limited number of binding sites on a binding partner that is capable of specifically binding the analyte and the tracer.
  • the tracer is a labelled analyte or a labelled analyte analogue, in which the label is a fluorescent dye of formula (I).
  • Increasing amounts (or concentrations) of the analyte in the sample will reduce the amount of the fluorescent labelled analyte or fluorescent labelled analyte analogue that is bound to the specific binding partner.
  • the fluorescence signal is measured and the concentration of analyte may be obtained by interpolation from a standard curve.
  • the binding assay may employ a two-step format, wherein a first component (which may be optionally coupled to an insoluble support) is bound to a second component to form a specific binding complex, which is bound in turn to a third component.
  • the third component is capable of specifically binding to either the second component, or to the specific binding complex.
  • Either of the second or the third component may be labelled with a fluorescent dye according to the present invention. Examples include “sandwich” assays, in which one component of a specific binding pair, such as a first antibody, is coated onto a surface, such as the wells of a multiwell plate.
  • a fluorescent labelled second antibody is added to the assay mix, so as to bind with the antigen-first antibody complex.
  • the fluorescence signal is measured and the concentration of antigen may be obtained by interpolation from a standard curve.
  • analyte-specific binding partner pairs include, but are not restricted to, antibodies/antigens, lectins/glycoproteins, biotin/streptavidin, hormone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood that any molecules which possess a specific binding affinity for each other may be employed, so that the fluorescent dyes of the present invention may be used for labelling one component of a specific binding pair, which in turn may be used in the detection of binding to the other component.
  • the fluorescent dyes according to first embodiment of the first aspect may be used in applications that include detecting and distinguishing between various components in a mixture.
  • the present invention provides a set of two or more different fluorescent dyes according to the invention, each dye of said set of dyes having the formula (I):
  • groups R 2 and R 3 are attached to the Z 1 ring structure and groups R 4 and R 5 are attached to the Z 2 ring structure, where Z 1 and Z 2 are hereinbefore defined;
  • R 2 , R 3 , R 4 and R 5 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C 1 -C 4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and the group —(CH 2 —) n Y;
  • R 1 is selected from hydrogen, C 1 -C 20 alkyl, aralkyl, the group —E—F and the group —(CH 2 —) n Y;
  • E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group;
  • Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6;
  • each dye of said set has a distinguishably different fluorescence lifetime compared with the lifetimes of the remaining dyes of the set.
  • each dye of the set of dyes at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 is the group —E—F where E and F are hereinbefore defined.
  • the set of fluorescent dyes according to the invention will comprise four different dyes, each dye of the set having a different fluorescence lifetime.
  • each of the fluorescent dyes in the set has a fluorescence lifetime in the range from 2 to 30 nanoseconds. More preferably the fluorescent dyes in the set will have fluorescence lifetimes in the range from 2 to 20 nanoseconds.
  • the difference in the lifetimes of the fluorescent emission of two such dyes is preferably at least 15% of the value of the shorter lifetime dye.
  • the set of dyes may be used in a detection method wherein different fluorescent dyes of the set of dyes are covalently bonded to a plurality of different primary components, each primary component being specific for a different secondary component, in order to identify each of a plurality of secondary components in a mixture of secondary components.
  • the method comprises covalently binding different dyes of a set of fluorescent dyes according to the fourth aspect of the invention to different primary components in a multicomponent mixture wherein each dye of the set has a different fluorescence lifetime, compared with the fluorescence lifetimes of the remaining dyes of the set; adding the dye-labelled primary components to a preparation containing secondary components under conditions to enable binding of at least a portion of each of said dye-labelled primary components to its respective secondary component; and determining the presence or the amount of the bound secondary component by measuring the fluorescence lifetime of each of the labelled primary component-secondary component complexes.
  • any unreacted primary components may be removed or separated from the preparation by, for example washing, to prevent interference with the analysis.
  • a single wavelength of excitation can be used to excite fluorescence from two or more materials in a mixture, where each fluoresces having a different characteristic fluorescent lifetime.
  • the set of fluorescent dyes according to the present invention may be used in any system in which the creation of a fluorescent primary component is possible.
  • an appropriately reactive fluorescent dye according to the invention can be conjugated to a DNA or RNA fragment and the resultant conjugate then caused to bind to a complementary target strand of DNA or RNA.
  • Other examples of primary component-secondary component complexes which may be detected include antibodies/antigens and biotin/streptavidin.
  • the set of dyes according to the present invention may also be advantageously used in fluorescent DNA sequencing based upon fluorescence lifetime discrimination of the DNA fragments.
  • each one of a set of dyes may be coupled to a primer.
  • Various primers are available, such as primers from pUC/M13, ⁇ gt10, ⁇ gt11 and the like (see Sambrook et al, Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press 1989).
  • DNA sequences are cloned into an appropriate vector having a primer sequence joined to the DNA fragment to be sequenced. After hybridisation to the DNA template, polymerase enzyme-directed synthesis of a complementary strand occurs.
  • DNA sequencing may also be performed using dideoxynucleotide terminators covalently labelled with the fluorescent dyes according to the present invention.
  • non-fluorescent or substantially non-fluorescent dyes according to the second embodiment of the first aspect may be used as the substrate for an enzyme and which upon reaction with the enzyme, yields a fluorescent product.
  • one or more nitro groups on an organic molecule may be reduced to a hydroxylamine (—NHOH) group which may subsequently be converted to an amine (—NH 2 ) group.
  • groups R 1 , R 2 , R 3 , R 4 , R 5 , Z 1 and Z 2 are hereinbefore defined and wherein at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 comprises at least one nitro group;
  • the fluorescence lifetime of the fluorescent product of the reduction is in the range from 2 to 30 nanoseconds.
  • reduction is by means of nitroreductase.
  • This can be achieved by enzymatic conversion of a nitro group in a compound of formula (I) to a —NHOH or —NH 2 group by the action of the nitroreductase.
  • the fluorescence emission from the product of the nitroreductase reaction may typically have a lifetime in the range 2 to 30 nanoseconds.
  • the fluorescence lifetime characteristics of the reaction product can be altered to suit the application by means of additional substitutents, whilst retaining the nitro group(s) that are involved in the reaction with nitroreductase.
  • fluorescent reporters compatible for use with other fluors in multiplex systems can be provided.
  • a method for detecting nitroreductase enzyme activity in a composition comprising:
  • groups R 1 , R 2 , R 3 , R 4 , R 5 , Z 1 and Z 2 are hereinbefore defined and wherein at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 comprises at least one nitro group;
  • the measurement of step ii) may be of the fluorescence intensity and/or fluorescence lifetime of the dye.
  • the composition comprises a cell or cell extract.
  • a cell or cell extract any type of cell can be used, i.e. prokaryotic or eukaryotic (including bacterial, mammalian and plant cells).
  • a cell extract can be prepared from a cell, using standard methods known to those skilled in the art (Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press 1989), prior to measuring fluorescence.
  • Typical conditions for nitroreductase activity comprise incubation of the composition in a suitable medium and the dye at approximately 37° C. in the presence of NADH and FMN.
  • an assay method comprising:
  • groups R 1 , R 2 1 R 3 1 R 4 , R 5 , Z 1 and Z 2 are hereinbefore defined and wherein at least one of groups R 1 , R 2 , R 3 , R 4 and R 1 comprises at least one nitro group;
  • said specific binding pair is selected from the group consisting of antibodies/antigens, lectins/glycoproteins, biotin/streptavidin, hormone/receptor, enzyme/substrate, DNA/DNA, DNA/RNA and DNA/binding protein.
  • an in vitro assay method for the detection of antibody binding may be configured as follows.
  • An antibody specific for an antigen of interest may be labelled by covalently linking it to an enzymatically active nitroreductase.
  • the labelled antibody can then be introduced into the test sample containing the antigen under conditions suitable for binding. After washing to remove any unbound antibody, the amount of bound antibody is detected by incubating the sample with a substrate comprising a compound of formula (I) having at least one nitro group under conditions for nitroreductase activity and measuring an increase in fluorescence.
  • the amount of fluorescence detected will be proportional to the amount of nitroreductase-labelled antibody that has bound to the antigen.
  • either of the pair of target and probe nucleic acid is immobilised by attachment to a membrane or surface.
  • the second member of the pair is labelled with nitroreductase and incubated under hybridising conditions with the immobilised nucleic acid. Unbound, labelled nucleic acid is washed off and the amount of bound, labelled nucleic acid is measured by incubating the membrane or surface with a compound of formula (I) having at least one nitro group under conditions suitable for nitroreductase activity. The amount of increase in fluorescence gives a measure of the amount of bound labelled DNA.
  • Coupling may be achieved by direct means, for example by use of a suitable bifunctional crosslinking agent (e.g. N-[ ⁇ -maleimidopropionic acid]hydrazine, Pierce) to covalently link the enzyme and binding partner.
  • a suitable bifunctional crosslinking agent e.g. N-[ ⁇ -maleimidopropionic acid]hydrazine, Pierce
  • coupling may be achieved by indirect means, for example by separately biotinylating the enzyme and the binding partner using a chemically reactive biotin derivative, (e.g. N-hydroxysuccinimido-biotin, Pierce) and subsequently coupling the molecules through a streptavidin bridging molecule.
  • Cell based assays are increasingly attractive over in vitro biochemical assays for use in high throughput screening (HTS). This is because cell based assays require minimal manipulation and the readouts can be examined in a biological context that more faithfully mimics the normal physiological situation.
  • Such in vivo assays require an ability to measure a cellular process and a means to measure its output. For example, a change in the pattern of transcription of a number of genes can be induced by cellular signals triggered, for example, by the interaction of an agonist with its cell surface receptor or by internal cellular events such as DNA damage. The induced changes in transcription can be identified by fusing a reporter gene to a promoter region which is known to be responsive to the specific activation signal.
  • an assay method which comprises:
  • groups R 1 , R 2 , R 3 , R 4 , R 5 , Z 1 and Z 2 are hereinbefore defined and wherein at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 comprises at least one nitro group;
  • the assay method is conducted in the presence of a test agent whose effect on gene expression is to be determined.
  • reporter gene is chosen to allow the product of the gene to be measurable in the presence of other is cellular proteins and is introduced into the cell under the control of a chosen regulatory sequence which is responsive to changes in gene expression in the host cell.
  • Typical regulatory sequences include those responsive to hormones, second messengers and other cellular control and signalling factors.
  • agonist binding to seven transmembrane receptors is known to modulate promoter elements including the cAMP responsive element, NF-AT, SRE and AP1; MAP kinase activation leads to modulation of SRE leading to Fos and Jun transcription; DNA damage leads to activation of transcription of DNA repair enzymes and the tumour suppressor gene p53.
  • promoter elements including the cAMP responsive element, NF-AT, SRE and AP1;
  • MAP kinase activation leads to modulation of SRE leading to Fos and Jun transcription;
  • DNA damage leads to activation of transcription of DNA repair enzymes and the tumour suppressor gene p53.
  • the nitroreductase gene may be isolated by well known methods, for example by amplification from a cDNA library by use of the polymerase chain reaction (PCR) (Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press (1989) pp 14.5-14.20). Once isolated, the nitroreductase gene may be inserted into a vector suitable for use with mammalian promoters (Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press (1989) pp 16.56-16.57) in conjunction with and under the control of the gene regulatory sequence under study.
  • PCR polymerase chain reaction
  • the vector containing the nitroreductase reporter and associated regulatory sequences may then be introduced into the host cell by transfection using well known techniques, for example by use of DEAE-Dextran or Calcium Phosphate (Molecular Cloning, A Laboratory Manual 2 nd Edition, Cold Spring Harbour Laboratory Press (1989) pp 16.30-16.46). Other suitable techniques will be well known to those skilled in the art.
  • at least one of groups R 1 , R 2 , R 3 , R 4 or R 5 comprises a cell membrane permeabilising group.
  • Membrane permeant compounds can be generated by masking hydrophilic groups to provide more hydrophobic compounds. The masking groups can be designed to be cleaved from the substrate within the cell to generate the derived substrate intracellularly.
  • Suitable cell membrane permeabilising groups may be selected from acetoxymethyl ester which is readily cleaved by endogenous mammalian intracellular esterases (Jansen, A. B. A. and Russell, T. J., J.Chem.Soc., (1965), 2127-2132 and Daehne W. et al. J.Med.Chem., (1970) 13, 697-612) and pivaloyl ester (Madhu et al., J. Ocul.Pharmacol.Ther., (1998), 14(5), 389-399) although other suitable groups will be recognised by those skilled in the art.
  • cells transfected with the nitroreductase reporter are incubated with the test agent, followed by addition of a dye of formula (I) wherein at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 in said dye comprises at least one nitro group, said compound being made cell permeant.
  • a dye of formula (I) wherein at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 in said dye comprises at least one nitro group, said compound being made cell permeant.
  • the fluorescence from the cells is measured at an emission wavelength appropriate for the chosen dye. Measurement of fluorescence may be readily achieved by use of a range of detection instruments including fluorescence microscopes (e.g.
  • LSM 410 Zeiss
  • microplate readers e.g. CytoFluor 4000, Perkin Elmer
  • CCD imaging systems e.g. LEADseekerTM, Amersham Pharmacia Biotech
  • Flow Cytometers e.g. FACScalibur, Becton Dickinson
  • the measured fluorescence is compared with fluorescence from control cells not exposed to the test agent and the effects, if any, of the test agent on gene expression modulated through the regulatory sequence, is determined by the detection of the characteristic fluorescence in the test cells.
  • a cell extract can be prepared using conventional methods.
  • Suitable means for expressing a nitroreductase enzyme include an expression plasmid or other expression construct. Methods for preparing such expression constructs are well known to those skilled in the art.
  • groups R 2 and R 3 are attached to the Z 1 ring structure and groups R 4 and R 5 are attached to the Z 2 ring structure;
  • Z 1 and Z 2 independently represent the atoms necessary to complete one ring, two fused ring, or three fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur;
  • At least one of groups R 1 , R 2 , R 3 , R 4 and R 5 is the group —E—F where E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group; and,
  • said remaining groups R 2 , R 3 , R 4 and R 5 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, amino, mono- or di-C 1 -C 4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH 2 —) n Y; and,
  • group R 1 is not said group —E—F, it is selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20 alkyl, aralkyl and the group —(CH 2 —) n Y;
  • E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group;
  • Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6; provided that at least one of groups R 1 , R 2 , R 3 , R 4 and R 5 is a water solubilising group.
  • the target bonding group F comprises a reactive group for reacting with a functional group on a target material, or a functional group for reacting with a reactive group on a target material.
  • Preferred reactive groups may be selected from carboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone, dichlorotriazine and phosphoramidite.
  • Preferred functional groups may be selected from hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphate and thiophosphate.
  • the spacer group E is selected from:
  • R 1 is hydrogen, C 1 -C 4 alkyl or aryl, which may be optionally substituted with sulphonate
  • Ar is phenylene, optionally substituted with sulphonate
  • p is 1-20, preferably 1-10
  • q is 0-10
  • r is 1-10 and s is 1-5.
  • the dyes of formula (I) may be prepared from the corresponding diphenylamine-2-carboxylic acid according to published methods (see Albert, A. and Ritchie, B., Org. Syntheses , (1942), 22, 5; also U.S. Pat. No. 3,021,334).
  • the diphenylamine-2-carboxylic acid may be heated in the presence of an acidic dehydrating agent such as phosphorus oxychloride or concentrated sulfuric acid.
  • the diphenylamine-2-carboxylic acid derivatives may be prepared by reaction of a 2-halobenzoic acid with a suitable primary aminobenzene (having at least one aryl ring position unsubstituted ortho- to the amino group), which reaction may be performed in the presence of catalytic copper metal/salt (see Ullmann, F., Chem. Ber., (1903), 36, 2382; also British Pat. 649197).
  • the 2-halobenzoic acid is heated with the aminobenzene, in the presence of a base such as an alkali metal carbonate, in a solvent such as 1-butanol or 1-pentanol.
  • a catalytic amount of copper metal powder or a copper salt such as anhydrous copper acetate is also usually included, although sometimes this is not required.
  • dyes of the present invention may be useful as intermediates for conversion to other dyes by methods well known to those skilled in the art.
  • the dyes of the present invention may be synthesized by the methods disclosed herein. Derivatives of the compounds having a particular utility are prepared either by selecting appropriate precursors or by modifying the resultant compounds by known methods to include functional groups at a variety of positions.
  • the dyes of the present invention may be modified to include certain reactive groups for preparing a dye according to the present invention, or charged or polar groups may be added to enhance the solubility of the compound in polar or nonpolar solvents or materials.
  • an ester may be converted to a carboxylic acid or may be converted to an amido derivative.
  • Groups R 1 to R 5 may be chosen so that the dyes of the present invention have different lifetime characteristics, thereby providing a number of related dyes which can be used in multiparameter analyses wherein the presence and quantity of different compounds in a single sample may be differentiated based on the wavelengths and lifetimes of a number of detected fluorescence emissions.
  • the dyes of the present invention may be made soluble in aqueous, other polar, or non-polar media containing the material to be labelled by appropriate selection of R-groups.
  • FIG. 1 shows the absorbance spectra (1A) and the emission spectra (1B) of four acridone dyes according to the present invention
  • FIG. 2 shows the fluorescence lifetime decay plot of four acridone dyes according to the present invention, as follows:
  • FIG. 4 is a lifetime decay plot following immunoprecipitation with anti-BSA antibody as described in Example 16;
  • FIG. 5 illustrates fluorescence lifetime detection in capillary electrophoresis of four acridone dye-labelled DNA fragments as described in Example 17;
  • FIG. 6 shows the lifetime detection of a mixture of two different acridone dye-labelled BSA conjugates and co-electrophoresed in SDS PAGE as described in Example 19.
  • CyTM is a trademark of Amersham Biosciences UK Limited.
  • the reaction mixture was then poured into 0.25M aqueous HCl (200 ml) and the precipitated product collected by vacuum filtration, washing with more dilute HCl and water.
  • the still-damp solid was recrystallized from ethanol/water and dried under vacuum over phosphorus pentoxide to give 6-(9-oxo-9H-acridin-4-carboxamido)hexanoic acid (1.97 g, 62%).
  • ⁇ max (EtOH) 408, 390, 256 nm.
  • 6-(2-Amino-9-oxo-9H-acridin-10-yl)hexanoic acid 370 mg: 1.14 mmol was dissolved in anhydrous pyridine (10 ml) and acetic anhydride (100 ⁇ l) followed by diisopropylethylamine (350 ⁇ l). The mixture was stirred for 3 hours. The solution was evaporated to dryness under vacuum and the gummy residue dissolved in dichloromethane. The solution was washed with 1.0M hydrochloric acid and then brine. The organic phase was dried with anhydrous magnesium sulphate, filtered and the solvent removed by rotary evaporation to leave a sticky solid. Trituration with ether gave a solid which was dried under vacuum to give 360 mg (86%) of 6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoic acid.
  • O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate (2.0 g; 6.0 mmol) was dissolved in conc. sulphuric acid (10ml) and the solution heated to 120° C. for 20 hrs. The mixture was allowed to cool and added to ⁇ 50 gm of crushed ice. The precipitate was collected by centrifugation, washed with 3.0M hydrochloric acid and dried under vacuum and over phosphorous pentoxide to give 2.1 g (90%) of 6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid.
  • N-Butoxycarbonyl-2-(aminoethyl)maleimide (200 mg; 0.83 mmol) was stirred under nitrogen with 4M hydrochloric acid in dioxan (4 ml). A white precipitate started to form after a few minutes. Stirring was continued for 40 minutes and then the solvent was removed by rotary evaporation. The resultant white solid was dried under vacuum to give 180 mg (100%) of N-(aminoethyl)maleimide hydrochloride. ⁇ H (200 MHz, CD 3 OD) 1.38(2H, s), 3.14(2H, t), 3.81(2H, t), 6.90(2H, s).
  • N-(4-Fluorophenyl)anthranilic acid (0.70 gm; 3 mmol) and phosphorous oxychloride (3 ml) were stirred together and heated to 115° C. for 3.5 hours, then allowed to cool.
  • the reaction mixture was placed on ice and small pieces of ice added, a vigorous reaction occurred with the evolution of hydrogen chloride.
  • water (15 ml) was added and the mixture was boiled for 2 hours. On cooling, a solid precipitated out. This was filtered off and washed with water until the filtrate was colourless. The precipitate was further washed with cold methanol then diethyl ether and dried under vacuum to give 383 mg (59%) of 2-fluoroacridone.
  • O-Ethyl-6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoate (367 mg; 1.0 mmol) was dissolved in ethanol (10 ml) and 1.0M sodium hydroxide solution (2.0 ml) added and the mixture heated to 90° C. for 1 hour. The mixture was cooled and water (20 ml) added. The mixture was cooled on ice and acidified with conc. hydrochloric acid when a yellow oil separated. The oil slowly crystallised to a bright yellow solid.
  • FIG. 2 is a plot showing the fluorescence lifetimes of certain acridone dyes according to the invention.
  • M13 DNA primers were labelled using standard techniques with each of four acridone dyes according to the present invention, i.e:
  • succinimidyl ester of each dye was conjugated to an amine-modified M13 forward sequencing primer in 0.1M carbonate pH 9.3/DMF (final composition 2:1). Purification by HPLC on a C18 column used a triethylammonium bicarbonate/MeCN solvent system.
  • Real-time fluorescence lifetime detection was achieved by interfacing a commercial multiharmonic Fourier transform (MHF) fluorescence lifetime instrument (Model 4850MHF, Spectronics Instruments, Rochester, N.Y.) to a Beckman P/ACE 5000 CE system (Li, L. et al, J. Chromatogr. B, (1997), 695, 85-92).
  • MHF multiharmonic Fourier transform
  • the excitation source was a continuous wave violet diode laser that supplied 25-30 nW at 405 nm.
  • the laser beam was focused onto the detection window of the capillary using either a 45 mm focusing lens or a 6.3 ⁇ microscope objective with a focal length of 22 mm.
  • the emission signal was collected by a 40 ⁇ microscope objective.
  • Emission was selected through a 435 nm long pass filter.
  • a cross-correlation frequency of 9.4 Hz was used in the lifetime measurements, resulting in 9.4 phase and modulation measurements per second.
  • Ten successive measurements were then averaged prior to data analysis to yield approximately one lifetime measurement per second. Scattered light from the capillary provided the lifetime reference.
  • Electrophoresis was performed using a MultiPhor II flat bed electrophoresis system with ExcelGel SDS buffer strips (anode strip: 0.45 mol/Tris/acetate pH 6.6, 4 g/L SDS and 0.05 g/L Orange G; cathode strip: 0.08 mol/L Tris, 0.80 mol/L Tricine and 6.0 g/L SDS pH 7.1).
  • Duplicate samples were applied to the surface of a pre-formed Excel 8-18 SDS PAGE gradient gel (Amersham Biosciences) using a paper sample application strip placed at locations corresponding with 96-well microplate centres required for scanning the gel.
  • Molecular weight markers were run in separate lanes, so that part of the gel could be stained using Coomassie Blue to check the integrity of the samples, monitor molecular weight and to orientate the gel for lifetime scanning. Electrophoresis was initiated at constant current to a maximum voltage of 500V for 85 minutes with the flat bed temperature maintained at 15° C. Prior to scanning, the gel was fixed in aqueous solution of 25% methanol, 5% acetic acid v/v for at least 30 minutes. Following lifetime scanning, the gel was stained for 10-20 minutes in 0.1% Coomassie Blue G-250 in aqueous solution of 25% methanol, 5% acetic acid v/v and de-stained in aqueous solution of 25% methanol, 5% acetic acid v/v.
  • FIG. 6 shows the 4 ns dye-labelled BSA and the 1 4 ns dye-labelled BSA (mixed in a ratio of 2:1) and co-electrophoresed in the same gel lane. Two peaks are resolved at 4 ns and 14 ns, both corresponding to the position of BSA relative to molecular weight markers. The two labelled BSA species are co-located but distinguishable by lifetime discriminated intensity. Both BSA species are resolved by the gel to the same location in the gel as indicated by reference to molecular weight markers and post electrophoresis staining (66 kD).

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US20080139788A1 (en) 2008-06-12
US20120015373A1 (en) 2012-01-19
US8034558B2 (en) 2011-10-11
GB0326889D0 (en) 2003-12-24
JP2005500406A (ja) 2005-01-06
GB2391870B (en) 2005-12-14
CA2449201A1 (en) 2002-12-12
WO2002099424A3 (en) 2003-12-04
WO2002099424A2 (en) 2002-12-12
JP4583027B2 (ja) 2010-11-17
GB2391870A (en) 2004-02-18
CA2754169A1 (en) 2002-12-12
EP1392776A2 (en) 2004-03-03

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