Quinacridone derivatives as labelling reagents for flurescence detection of bilogical materials

A method for labelling a target biological material the method comprising:

• i) adding to a liquid containing said target biological material a dye of formula:
• 26
• wherein:
• groups R3 and R4 are attached to the Z1 ring structure and groups R5 and R6 are attached to the Z2 ring structure, where Z1 and Z2 are hereinbefore defined;
• R3, R4, R5, R6, R7 and R8 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C1-C4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C1-C6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C1-C20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and the group —(CH2—)nY;
• R1 and R2 are independently selected from hydrogen, C1-C20 alkyl, aralkyl, the group —E—F and the group —(CH2—)nY;
• where E, F, Y and n are hereinbefore defined;
• and
• ii) incubating said dye with said target biological material under conditions suitable for labelling said target.

A method for the assay of an analyte in a sample which method comprises:

• i) contacting the analyte with a specific binding partner for said analyte under conditions suitable to cause the binding of at least a portion of said analyte to said specific binding partner to form a complex and wherein one of said analyte and said specific binding partner is labelled with a fluorescent dye of formula:
• 27
• wherein:
• groups R3 and R4 are attached to atoms of the Z1 ring structure and groups R5 and R6 are attached to atoms of the Z2 ring structure, where Z1 and Z2 are hereinbefore defined;
• at least one of groups R1, R2, R3, R4, R5, R6, R7 and R8 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;
• when any of said groups R3, R4, R5, R6, R7 and R8 is not said group —E—F, said remaining groups R3, R4, R5, R6, R7 and R8 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C1-C4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C1-C6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C1-C20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH2—)nY;
• and,
• when any of groups R1 and R2 is not said group —E—F, said remaining groups R1 and R2 are independently selected from hydrogen, C1-C20 alkyl, aralkyl and the group —(CH2—)nY;
• wherein Y and n are hereinbefore defined;
• ii) measuring the emitted fluorescence of the labelled complex;
• and
• iii) correlating the emitted fluorescence with the presence or the amount of said analyte in said sample.

An assay method for the determination of an enzyme in a sample, the method comprising:

• i) providing a substrate for the enzyme wherein the substrate is labelled with a fluorescent dye of formula:
• 28
• wherein:
• groups R3 and R4 are attached to atoms of the Z1 ring structure and groups R5 and R6 are attached to atoms of the Z2 ring structure, where Z1 and Z2 are hereinbefore defined;
• at least one of groups R1, R2, R3, R4, R5, R6, R7 and R8 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;
• when any of said groups R3, R4, R5, R6, R7 and R8 is not said group —E—F, said remaining groups R3, R4, R5, R6, R7 and R8 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C1-C4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C1-C6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C1-C20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH2—)nY;
• and,
• when any of groups R1 and R2 is not said group —E—F, said remaining groups R1 and R2 are independently selected from hydrogen, C1-C20 alkyl, aralkyl and the group —(CH2—)nY;
• wherein Y and n are hereinbefore defined;
• ii) combining the labelled substrate with the enzyme under conditions suitable for initiating the enzymatic reaction;
• and
• iii) measuring the fluorescence polarisation of the sample to determine the extent of reaction.

A set of two or more different fluorescent dyes, each dye of said set of dyes having the formula:

• 29
• wherein:
• groups R3 and R4 are attached to atoms of the Z1 ring structure and groups R5 and R6 are attached to atoms of the Z2 ring structure, where Z1 and Z2 are hereinbefore defined;
• R3, R4, R5, R6, R7 and R8 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C1-C4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C1-C6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C1-C20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and the group —(CH2—)nY;
• R1 and R2 are independently selected from hydrogen, C1-C20 alkyl, aralkyl, the group —E—F and the group —(CH2—)nY;
• 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;
• wherein each dye of said set has a distinguishably different fluorescence lifetime compared with the lifetimes of the remaining dyes of the set.

A method of increasing the fluorescence of a non-fluorescent or substantially non-fluorescent dye of formula:

• 30
• wherein:
• groups R1, R2, R3, R4, R5, R6, R7, R8, Z1 and Z2 are hereinbefore defined;
• and wherein at least one of groups R1, R2, R3, R4, R5, R6, R7 and R8 comprises at least one nitro group;
• characterised by the reduction of said at least one nitro group to —NHOH or —NH2

A method for detecting nitroreductase enzyme activity in a composition comprising:

• i) mixing said composition under conditions to promote nitroreductase activity with a dye of formula:
• 31
• wherein:
• groups R1, R2, R3, R4, R5, R6, R7, R8, Z1 and Z2 are hereinbefore defined and wherein at least one of groups R1, R2, R3, R4, R5, R6, R7 and R8 comprises at least one nitro group;
• and
• ii) measuring an increase in fluorescence wherein said increase is a measure of the amount of nitroreductase activity.

An assay method comprising:

• i) binding one component of a specific binding pair to a surface;
• ii) adding a second component of the specific binding pair under conditions to promote binding between the components, said second component being labelled with a nitroreductase enzyme;
• iii) adding a dye of formula:
• 32
• wherein:
• groups R1, R2, R3, R4, R5, R6, R7, R8, Z1 and Z2 are hereinbefore defined and wherein at least one of groups R1, R2, R3, R4, R5, R6, R7 and R8 comprises at least one nitro group;
• and
• iv) detecting binding of the second component to the first component by measuring an increase in fluorescence as a measure of bound nitroreductase activity.

An assay method which comprises:

• i) contacting a host cell which has been transfected with a nucleic acid molecule comprising expression control sequences operably linked to a sequence encoding a nitroreductase with a dye of formula:
• 33
• wherein:
• groups R1, R2, R3, R4, R5, R6, R7, R8, Z1 and Z2 are hereinbefore defined and wherein at least one of groups R1, R2, R3, R4, R5, R6, R7 and R8 comprises at least one nitro group;
• and
• ii) measuring an increase in fluorescence as a measure of nitroreductase gene expression.

A dye of formula:

• 34
• wherein:
• groups R3 and R4 are attached to atoms of the Z1 ring structure and groups R5 and R6 are attached to atoms of the Z2 ring structure;
• Z1 and Z2 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 R1, R2, R3, R4, R5, R6, R7 and R8 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,
• when any of said groups R3, R4, R5, R6, R7 and R8 is not said group —E—F, said remaining groups R3, R4, R5, R6, R7 and R8 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, amino, mono- or di-C1-C4 alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C1-C6 alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C1-C20 alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH2—)nY;
• and,
• when any of groups R1 and R2 is not said group —E—F, said remaining groups R1 and R2 are independently selected from hydrogen, mono- or di-nitro-substituted benzyl, C1-C20 alkyl, aralkyl and the group —(CH2—)nY;
• 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 R3, R4, R5, R6, R7 and R8 is selected from sulphonate, sulphate, quaternary ammonium and the group —(CH2—)nY;
• and/or at least one of groups R1 and R2 is the group —(CH2—)nY, where Y and n are hereinbefore defined.