Cyanine dye labelling reagents with meso-substitution

A method for labelling a target biological material comprising:

• i) adding to a liquid containing said target material a fluorescent dye of formula (1):
• wherein:
• groups R3 and R4 are attached to the Z1 ring structure and groups R5 and R6 are attached to the Z2 ring structure, and n=1, 2 or 3;
• Z1 and Z2 independently represent the atoms necessary to complete one ring, or two 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;
• X and Y are the same or different and are selected from oxygen, sulphur, —CH═CH— and the group:
• at least one of groups R1, R2, R3, R4, R5, R6 (and R8 and R9 if present) is the group -E-F where E is a spacer group having a chain from 1–20 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group;
• one of groups R7 is selected from —CN and —CF3;
• remaining groups R7 are hydrogen or two of R7 together with the group:
• form a hydrocarbon ring system which may optionally contain a heteroatom selected from —O—, —S—, or >NH and n is hereinbefore defined;
• when any of groups R3, R4, R5 and R6 is not said group -E-F, said remaining groups R3, R4, R5 and R6 are independently selected from hydrogen, halogen, hydroxyl, amino, mono- or di-C1–C4 alkyl-substituted amino, sulphydryl, carboxyl, C1–C6 alkoxy, C1–C6 alkyl, aryl, heteroaryl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH2)k—W;
• when any of groups R8 and R9 is not said group -E-F, said remaining groups R8 or R9 are independently selected from C1–C4 alkyl and —(CH2)k—W;
• when any of groups R1 and R2 is not said group -E-F, said remaining groups R1 or R2 are selected from hydrogen, C1–C6 alkyl, benzyl and —(CH2)k—W;
• wherein W is selected from sulphonate, sulphonic acid, quaternary ammonium and carboxyl;
• and k is an integer from 1 to 6;
• and
• ii) incubating said fluorescent dye with said target material under conditions suitable for binding to and thereby labelling said material.

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

• 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 (1):
• wherein:
• groups R3 and R4 are attached to the Z1 ring structure and groups R5 and R6 are attached to the Z2 ring structure, and n=1, 2 or 3;
• Z1 and Z2 independently represent the atoms necessary to complete one ring, or two 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;
• X and Y are the same or different and are selected from oxygen, sulphur, —CH═CH— and the group:
• at least one of groups R1, R2, R3, R4, R5, R6 (and R8 and R9 if present) is the group -E-F where E is a spacer group having a chain from 1–20 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group;
• one of groups R7 is selected from —CN and —CF3;
• remaining groups R7 are hydrogen or two of R7 together with the group:
• form a hydrocarbon ring system which may optionally contain a heteroatom selected from —O—, —S—, or >NH and n is hereinbefore defined;
• when any of groups R3, R4, R5 and R6 is not said group -E-F, said remaining groups R3, R4, R5 and R6 are independently selected from hydrogen, halogen, hydroxyl, amino, mono- or di-C1–C4 alkyl-substituted amino, sulphydryl, carboxyl, C1–C6 alkoxy, C1–C6 alkyl, aryl, heteroaryl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH2)k—W;
• when any of groups R8 and R9 is not said group -E-F, said remaining groups R8 or R9 are independently selected from C1–C4 alkyl and —(CH2)k—W;
• when any of groups R1 and R2 is not said group -E-F, said remaining groups R1 or R2 are selected from hydrogen, C1–C6 alkyl, benzyl and —(CH2)k—W;
• wherein W is selected from sulphonate, sulphonic acid, quaternary ammonium and carboxyl;
• and k is an integer from 1 to 6;
• 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.

A set of two or more different fluorescent dyes comprising at least one dye of formula (1):

• wherein:
• groups R3 and R4 are attached to the Z1 ring structure and groups R5 and R6 are attached to the Z2 ring structure, and n=1, 2 or 3;
• Z1 and Z2 independently represent the atoms necessary to complete one ring, or two 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;
• X and Y are the same or different and are selected from oxygen, sulphur, —CH═CH— and the group:
• at least one of groups R1, R2, R3, R4, R5, R6 (and R8 and R9 if present) is the group -E-F where E is a spacer group having a chain from 1–20 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group;
• one of groups R7 is selected from —CN and —CF3;
• remaining groups R7 are hydrogen or two of R7 together with the group:
• form a hydrocarbon ring system which may optionally contain a heteroatom selected from —O—, —S—, or >NH and n is hereinbefore defined;
• when any of groups R3, R4, R5 and R6 is not said group -E-F, said remaining groups R3, R4, R5 and R6 are independently selected from hydrogen, halogen, hydroxyl, amino, mono- or di-C1–C4 alkyl-substituted amino, sulphydryl, carboxyl, C1–C6 alkoxy, C1–C6 alkyl, aryl, heteroaryl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group —(CH2)k—W;
• when any of groups R8 and R9 is not said group -E-F, said remaining groups R8 or R9 are independently selected from C1–C4 alkyl and —(CH2)k—W;
• when any of groups R1 and R2 is not said group -E-F, said remaining groups R1 or R2 are selected from hydrogen, C1–C6 alkyl, benzyl and —(CH2)k—W;
• wherein W is selected from sulphonate, sulphonic acid, quaternary ammonium and carboxyl;
• and k is an integer from 1 to 6;
• and
• wherein each dye of said set has a distinguishably different fluorescence emission wavelength compared with the emission wavelengths of the remaining dyes of the set.