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US 2004/0073014 A1Published application
Fluorescent dyes, energy transfer couples and methods
AssigneeGuava Technologies, Inc.
InventorsRoger O'Neill, Peter V. Fisher
Life of this patent
Estimating the term…
Abstract
Fluorescent dyes, fluorescence energy transfer dye couples, multi-color dye sets, can be employed in art-recognized assays and certain novel methods, such as in proximity assays.
Claims18
A fluorescence energy transfer dye comprising Formula I:
- L1-D1-FETL-D2-L2- (Formula I)
- where:
- L1 is a link for attachment to a probe or target, for attachment to a solid support, or is absent;
- L2 is a link for attachment to a probe or target, for attachment to a solid support, or is absent;
- FETL is a fluorescence energy transfer linker;
- D1 is a donor dye represented by the formula:
- 41
- where:
- X is O or C(R*R**), where R* and R** are independently lower alkyl or —CH2—Z;
- R1 is H, CF3, perfluoropropyl, lower alkyl acid, substituted aryl, substituted heteroaryl or Z;
- R2 is H, halo, SO3−, or is taken together with R3 to form an optionally substituted fused ring having 5 to 7 atoms;
- R3 is halo, Z, or is taken together with R2 and/or R4 to form an optionally substituted fused ring having 5 to 7 atoms;
- R4 is ═O or OH, —N(R4′R4″) or ═N+(R4′R4″), or is taken together with R3 and/or R5 to form an optionally substituted fused ring having 5 to 7 atoms,
- where R4′ is H, lower alkyl or L1, and
- R4 is H, lower alkyl or CH2—Z;
- R5 is H, halo, Z, or is taken together with R4 to form an optionally substituted fused ring having 5 to 7 atoms;
- R6 is H, halo, Z, or is taken together with R7 to form an optionally substituted fused ring having 5 to 7 atoms;
- R7 is ═O or OH, —N(R7′R7″) or ═N+(R7′R7″), or is taken together with R6 and/or R8 to form an optionally substituted fused ring having 5 to 7 atoms,
- where R7′ is H, lower alkyl or L1, and
- R7 is H, lower alkyl or CH2—Z;
- R8 is halo, Z, or is taken together with R7 and/or R9 to form an optionally substituted fused ring having 5 to 7 atoms;
- R9 is H, halo, SO3−, or is taken together with R8 to form an optionally substituted fused ring having 5 to 7 atoms;
- and
- Z is a group of the formula:
- —Z*—Z1— (L, L2 or FETL), where:
- Z* is methylene, methoxy, ethoxy, aminomethyl, aminoethyl, aminopropynyl, aminobutynyl, carboxyethenyl, carboxyethynyl, optionally substituted aryl or optionally substituted heteroaryl;
- Z1 is —C(O)—, —N(Z2)—, —CH2—O—, —CH2—C(O)—, —CH2—N(Z2)—, —CH2—S—, —CH2—S(O)—, —CH2—S(O2)— or is absent;
- and
- Z2 is H, C1 to C8 optionally substituted lower alkyl, or optionally substituted aryl;
- and
- D2 is an acceptor/reporter dye represented by formula D1 or by a formula of the group:
- 42
- where:
- at least one of R21 to R36 is joined to FETL,
- n is zero, 1, 2 or 3;
- R21 and R30 are independently —CH2—Z, activated lower alkyl, or optionally substituted arlkyl;
- R22 to R29 are independently H, SO3−, or optionally substituted alkyl, or R22 and R23, R23 and R24, R24 and R25, R26 and R27. R27 and R28, and/or R28 and R29 taken together form an optionally substituted fused ring having 6 atoms;
- R33 and R32 are independently H, optionally substituted alkyl, aryl, or taken together form an optionally substituted fused ring having 6 atoms;
- R33 to R36 are independently H, SO3−, optionally substituted alkyl, aryl, or R33 and R34, R34 and R35, and/or R35 and R36 taken together form an optionally substituted fused ring having 6 atoms;
- and
- Y is —O — or —N(Y1)— where Y1 is —CH2—Z, activated lower alkyl, or optionally substituted aryl;
- provided that at least one of R3 to R8 is Z where Z* is optionally substituted aryl or optionally substituted heteroaryl, or a probe-, target- and/or support-conjugate thereof.
39 more elements
An compound represented by the formula:
- 47
- where:
- X is O or C(R*R**), where R* and R** are independently lower alkyl or —CH2—Z;
- R1 is H, CF3, perfluoropropyl, lower alkyl acid, substituted aryl, substituted heteroaryl or Z;
- R2 is H, halo, SO3−, or is taken together with R3 to form an optionally substituted fused ring having 5 to 7 atoms;
- R3 is halo, Z, or is taken together with R2 and/or R4 to form an optionally substituted fused ring having 5 to 7 atoms;
- R4 is ═O or OH, —N(R4′R4′) or ═N+(R4′R4′), or is taken together with R3 and/or R5 to form an optionally substituted fused ring having 5 to 7 atoms,
- where R4′ is H, lower alkyl or L1, and
- R4 is H, lower alkyl or CH2—Z;
- R5 is H, halo, Z, or is taken together with R4 to form an optionally substituted fused ring having 5 to 7 atoms;
- R6 is H, halo, Z, or is taken together with R7 to form an optionally substituted fused ring having 5 to 7 atoms;
- R7 is ═O or OH, —N(R7′R7″) or ═N+(R7′R7″), or is taken together with R6 and/or R8 to form an optionally substituted fused ring having 5 to 7 atoms,
- where R7′ is H, lower alkyl or L1, and
- R7′ is H, lower alkyl or CH2—Z;
- R8 is halo, Z, or is taken together with R7 and/or R9 to form an optionally substituted fused ring having 5 to 7 atoms;
- R9 is H, halo, SO3−, or is taken together with R3 to form an optionally substituted fused ring having 5 to 7 atoms;
- and
- Z is a group of the formula:
- —Z*—Z1— (L, L2 or FETL), where:
- Z* is methylene, methoxy, ethoxy, aminomethyl, aminoethyl, aminopropynyl, aminobutynyl, carboxyethenyl, carboxyethynyl, optionally substituted aryl or optionally substituted heteroaryl;
- Z1 is —C(O)—, —N(Z2)—, —CH2—O—, —CH2—C(O)—, —CH2—N (Z2), —CH2—S—, —CH2—S(O)—, —CH2—S(O2)— or is absent;
- and
- Z2 is H, C1 to C8 optionally substituted lower alkyl, or optionally substituted aryl;
- L1 being a link for attachment to a probe or target, for attachment to a solid support, or is absent;
- L2 being a link for attachment to a probe or target, for attachment to a solid support, or is absent;
- and
- FETL being a fluorescence energy transfer linker. provided that at least one of R3 to R8 is Z, where Z* is optionally substituted aryl or optionally substituted heteroaryl, or a FETL—, probe—, target- and/or support-conjugate thereof.
24 more elements
A fluorescence energy transfer dye represented by Formula II:
- Probe-L1-(D1 or D2)-FETL-(low affinity false target) (Formula II)
- where:
- Probe is a polynucleotide, antibody, triglyceride, low density lipoprotein or lectin;
- L1 is a link for attachment to a the Probe;
- FETL is a fluorescence energy transfer linker comprising a symmetric, rigid or sterically hindered, divalent moiety joined to D1 and D2 via an amine, carbonyl, activated carboxylic acid ester, disulfide, thiol or thiol ester;
- low affinity false target is an analyte for the Probe disposed on FETL to deactivate, block or otherwise prevent coupling of FETL to a corresponding D2 or D1 until said low affinity false target is displaced by a higher affinity true target;
- D1 is a donor dye represented by the formula:
- 48
- where:
- X is O or C(R*R**), where R* and R** are independently lower alkyl or —CH2—Z;
- R1 is H, CF3, perfluoropropyl, lower alkyl acid, substituted aryl, substituted heteroaryl or Z;
- R2 is H, halo, SO3−, or is taken together with R3 to form an optionally substituted fused ring having 5 to 7 atoms;
- R3 is halo, Z, or is taken together with R2 and/or R4 to form an optionally substituted fused ring having 5 to 7 atoms;
- R4 is ═O or OH, —N(R4′R4″) or ═N (R4′R4), or is taken together with R3 and/or R5 to form an optionally substituted fused ring having 5 to 7 atoms,
- where R4′ is H, lower alkyl or L1, and
- R4 is H, lower alkyl or CH2—Z;
- R5 is H, halo, Z, or is taken together with R4 to form an optionally substituted fused ring having 5 to 7 atoms;
- R6 is H, halo, Z, or is taken together with R7 to form an optionally substituted fused ring having 5 to 7 atoms;
- R7 is ═O or OH, —N(R7′ R7) or ═N+(R7′R7″), or is taken together with R6 and/or R8 to form an optionally substituted fused ring having 5 to 7 atoms,
- where R7′ is H, lower alkyl or L1, and
- R7′ is H, lower alkyl or CH2—Z;
- R8 is halo, Z, or is taken together with R7 and/or R9 to form an optionally substituted fused ring having 5 to 7 atoms;
- R9 is H, halo, SO3−, or is taken together with R8 to form an optionally substituted fused ring having 5 to 7 atoms;
- and
- Z is a group of the formula:
- —Z*Z1 (L1, L2 or FETL), where:
- Z* is methylene, methoxy, ethoxy, aminomethyl, aminoethyl, aminopropynyl, aminobutynyl, carboxyethenyl, carboxyethynyl, optionally substituted aryl or optionally substituted heteroaryl;
- Z1 is —C(O)—, —N(Z2), —CH2—O—, —CH2—C(O)—, —CH2—N(Z2)_, —CH2—S—, —CH2—S(O)—, —CH2—S(O2)— or is absent;
- and
- Z2 is H, C1 to C8 optionally substituted lower alkyl, or optionally substituted aryl;
- and
- D2 is an acceptor/reporter dye represented by formula D1 or by a formula of the group:
- 49
- where:
- at least one of R21 to R36 is joined to FETL,
- n is zero, 1, 2 or 3;
- R2′ and R30 are independently —CH2—Z, activated lower alkyl, or optionally substituted aryl;
- R22 to R29 are independently H, SO3−, or optionally substituted alkyl, or R22 and R23, R23 and R24, R24 and R25, R26 and R27, R27 and R28, and/or R28 and R29 taken together form an optionally substituted fused ring having 6 atoms;
- and
- R3 and R32 are independently H, optionally substituted alkyl, aryl, or taken together form an optionally substituted fused ring having 6 atoms;
- R33 to R36 are independently H, SO3−, optionally substituted alkyl, aryl, or R33 and R34, R34 and R35, and/or R35 and R36 taken together form an optionally substituted fused ring having 6 atoms;
- and
- Y is —O — or —N(Y1)— where Y1 is —CH2—Z, activated lower alkyl, or optionally substituted arlkyl.
40 more elements
An assay requiring no pre-analysis purification for removal of unbound dye, comprising either the steps of:
- (a) in a suitable assay vessel, contacting a substance to be tested and a Probe1-L1-D1 conjugate that is site-specific for a first epitope on a target;
- (b) introducing a Probe2-L2-D2 conjugate to the vessel, where said Probe2 is site-specific for bound Probe 1 or for a second epitope spatially proximate to said first epitope;
- (c) causing D1 to absorb energy;
- and
- (d) measuring the level of D2 emission, or the steps of:
- (e) contacting a substance to be tested and a target site specific Probe-L1-(D1 or D2)-FETL-(low affinity false target) conjugate in a suitable assay vessel under conditions suitable for preferential binding of the Probe to the target site, as opposed to the low affinity false target;
- (f) introducing a D2 or D1 fluorescence energy transfer dye having an activated site for coupling to the FETL into the vessel, under conditions suitable for coupling to FETL where the low affinity false target is not bound to the Probe;
- (g) causing D1 to absorb energy;
- and
- (h) measuring the level of D2 emission.
7 more elements
A proximity assay comprising the steps of:
- (a) contacting a substance to be tested and a target-site-specific donor dye in a suitable assay vessle;
- (b) introducing a target-site-specific reporter dye into the vessel, where said reporter dye's target is either spatially proximate to said donor dye target or specific for a given target to be tested for spatial proximity to said donor dye target, and said reporter dye's energy absorption spectra overlaps the emission spectra of said donor dye;
- (c) causing the donor dye to absorb energy;
- and
- (d) measuring the level of reporter dye emission.
2 more elements
Dependencies are inferred from the claim text.