Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
  • C07D263/52—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings condensed with carbocyclic rings or ring systems
  • C07D263/54—Benzoxazoles; Hydrogenated benzoxazoles
  • C07D263/56—Benzoxazoles; Hydrogenated benzoxazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/04—Indoles; Hydrogenated indoles
  • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
  • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
  • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
  • C07D311/78—Ring systems having three or more relevant rings
  • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
  • C07D311/82—Xanthenes
  • C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
  • C07D311/88—Nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/06—Peri-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
  • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
  • C07D493/10—Spiro-condensed systems
• • 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
  • G01N33/533—Production of labelled immunochemicals with fluorescent label
• • 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
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

• the present invention relates to a novel class of energy transfer dyes, their preparation and their use as labels in biological systems.
• UK Patent No. 2301 833 B discloses, inter alia, that complexes including: i) a first fluorochrome having first absorption and emission spectra; ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of the second fluorochrome being longer than the wavelength of the emission maximum of the first fluorochrome, and a portion of the absorption spectrum of the second fluorochrome overlapping a portion of the emission spectrum of the first fluorochrome; iii) at least one linker group chosen from the group consisting of alkyl chains containing from 1 to 20 carbon atoms, which may optionally include from 1 to 8 oxygen atoms as polyether linkages, or from 1 to 8 nitrogen atoms as polyamine linkages, or from 1 to 4 CO-NH groups as polyamide linkages and up to 2 bicyclo[2,2,2]octyl groups, for covalently attaching the first and second fluorochromes for transfer of resonance energy transfer between the first and second fluo
• a cassette includes a covalently linked structure or complex with at least two fluorescent dye moieties, a linker group, and preferably a reactive group for attaching the complex to a biological material or other target material.
• the reactive group is chosen to be suitable for forming a covalent linkage with a functional group on a particular target material.
• the dyes are selected so that the emission spectrum of one dye overlaps the absorption spectrum of a second dye, so that energy transfer can occur between the dyes.
• the present invention provides an energy transfer dye of the formula (I):
• R 1 is a first dye suitable as an acceptor or donor in an energy transfer arrangement
• R 5 is a second dye that is suitable as a donor or acceptor in an energy transfer arrangement with the first dye
• A comprises a chain that contains 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 linearly linked atoms selected from carbon, nitrogen and oxygen.
• the chain may optionally be substituted, if desired, with groups as known to those skilled in the art which do not prevent energy transfer, for example, by C, 23 or4 linear or branched alkyl or phenyl, optionally substituted with 1,2,3, or 4 substituents independently selected from OH, halo, methyl or ethyl) groups).
• A is a chain of linearly linked atoms;
• R 3 is a hydrogen or a reactive or functional group suitable for attaching the energy transfer dye to a target material, e.g., a biological material as noted above; and 3 D comprises an atom or group for attaching R 5 to the linker chain A, in which the covalent linkage between A and R 5 does not include a sulphur atom. Also, preferably no sulphur atom is present in D, for example, as a side group not in the direct covalent linkage between R 5 and A.
• R 1 and R 5 are a fluorescein/rhodamine pair there are preferably
• A is a C 67 89 10 11 12 , 3 14 15 16 or l7 hydrocarbon chain.
• R 3 may be any group suitable for attaching the energy transfer dye to a target material, preferably a target biological material and, as such, will be well known to those skilled in the art.
• R 3 is selected from the group consisting of carboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite, and groups covalently reactive with carboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite.
• Suitable dyes for R 1 may be dyes which contain reactive or functional groups capable of linking with S.
• the attachment group D may be any group, other than sulphur, suitable for connecting R 5 with A.
• D is PO 3 or NH-CO.
• the dye moieties, e.g., R 1 and R 5 , of the present energy transfer dyes are fluorophores which are selected, as further indicated herein, to be able to participate in an energy transfer arrangement.
• the energy transfer dyes of this invention have a total molecular weight of less than 10,000 or 5,000 daltons, more preferably less than 3,000 or 2000 daltons, still more preferably less than 1 ,500 or 1 ,200 daltons.
• energy 4 transfer arrangement In connection with the energy transfer dyes of the present invention, by “energy 4 transfer arrangement” is meant that two fluorescent dyes are selected having absorption and emission spectra suitable for energy transfer between the dyes, and located with sufficient physical proximity and linkage such that photoexitation of a first dye (the donor) results in the transfer of energy from the first dye to the second dye (the acceptor). Additional energy transfers involving one or more additional dye moieties can also be created.
• an “energy transfer dye” refers to a fluroescent dye complex having at least two dye moieties which can participate in energy transfer between those two dye moieties.
• acceptor in an energy transfer arrangement is meant a dye moiety which absorbs energy at a wavelength emitted by a donor dye moiety, i.e., the absorption spectrum of the acceptor overlaps the emission spectrum of the donor.
• donor in an energy transfer arrangement is meant a dye moiety which absorbs energy from light, and emits light at frequencies at least partially within the absorption spectrum of an acceptor dye moiety.
• linear or branched alkyl is meant a straight-chain or branched saturated aliphatic hydrocarbon. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.
• halo is meant fluoro, chloro, bromo or iodo.
• target material refers to a compound or structure to which a energy transfer dye is to be covalently attached or to which such a dye is attached.
• biological material is meant a compound produced by or present in an organism, including but not limited to polypeptides, nucleic acid molecules, carbohydrates, and lipids. Such compounds may be derivatised to include a group suitable for covalent attachment of an energy transfer dye.
• the term does not mean that the dyes of the present invention must be used with intact organisms, as often the dyes will be used with extracts, such as nucleic acid extracts, or samples, including preserved samples such as tissue sections, or in nucleic acid sequencing reactions.
• the energy transfer dye is of the formula (II):
• R 1 is a first dye suitable as an acceptor or donor in an energy transfer arrangement
• R 2 is hydrogen, C, 23 or4 linear or branched alkyl or phenyl (optionally substituted as above);
• R 3 is hydrogen or a reactive or functional group other than thiol
• R 4 is hydrogen, C* 23 or4 linear or branched alkyl, or substituted or unsubstituted phenyl;
• R 5 is a second dye that is suitable as a donor or acceptor in an energy transfer arrangement with the first dye;
• m is 1,2 or 3;
• n is 1,2,3,4,5,6,7,8, or 9.
• the donor dye is a fluorescein or cyanine dye.
• R 1 contains a reactive or functional group suitable for covalent attachment of the dye to a thiol- containing component of A.
• preferred reactive groups include iodoacetamido- and maleimido- groups.
• the acceptor is a rhodamine or cyanine dye.
• R 5 contains a reactive or functional group suitable for attachment of the dye to a corresponding functional or reactive group component of A.
• dyes which contain a carboxyl or activated carboxyl group are preferred.
• Suitable fluorescein donor dyes include but are not limited to 5- and 6- carboxyfluorescein and 6-carboxy-4' ,5 ' -dichloro-2' ,7'-dimethoxyfluorescein.
• Suitable cyanine donor dyes include but are not limited to CyA (3-( - carboxypentyl)-3'-ethyl-5, 5 '-dimethyl oxacarbocyanine), Cy3 (3-( -carboxypentyl)-l'- ethyl-3 ,3 ,3 ' ,3 ' -tetramethyl-5 ,5 ' -disulphonato-carbocyanine) .
• Suitable rhodamine acceptor dyes include, but are not limited to 6- carboxyrhodamine (Rhodamine 110), 5-carboxyrhodamine-6G (R6G-5 or REG-5), 6- 6 carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA or TMR), 6-carboxy-X-rhodamine (ROX).
• 6- carboxyrhodamine Rhodamine 110
• 5-carboxyrhodamine-6G R6G-5 or REG-5
• 6- 6 carboxyrhodamine-6G R6G-6 or REG-6
• TAMRA N,N,N',N'-tetramethyl-6-carboxyrhodamine
• ROX 6-carboxy-X-rhodamine
• Suitable cyanine acceptor dyes include but are not limited to, Cy3.5 (3-( - carboxypentyl)- 1 ' -ethyl-3 ,3 ,3 ' ,3 ' -tetramethyl-4,5 ,4' ,5 ' -( 1 ,3 -disulphonato)dibenzo- carbocyanine), Cy5 (l-( -carboxypentyl)- 1' -ethyl-3, 3,3', 3' -tetramethyl-5, 5' -disulphonato- dicarbocyanine, Cy5.5 (l-( -carboxypentyl)-l'-ethyl-3,3,3',3'-tetramethyl-4,5,4',5'-(l,3- disulphonato)-dibenzo-dicarbocyanine, Cy7 (l-( -carboxypentyl)- 1' -ethyl-3, 3,
• Cyanine dyes suitable for use in the energy transfer dyes of the present invention are disclosed in US Patent No. 4,268,486 (Waggoner et al; incorporated herein by reference in its totality including any drawings). The above and additional dyes are described, for example, in Southwick et al., 1990, Cytometry 11 :418-430; Mujumdar et al, 1993, Bioconjugate Chemistry 4:105-111; and Waggoner and Ernst, Fluorescent Reagents for Flow Cytometry, Part 1 : Principles of Clinical Flow Cytometry (1993).
• the complexes may contain a third dye, e.g.
• R 2 is hydrogen or methyl and preferably hydrogen. It will be appreciated by those skilled in the art that when m is other than 1 , there will be several R 2 groups present. In such a situation the R 2 groups may be the same or different. Preferably R 2 is hydrogen.
• R 4 is preferably hydrogen or methyl, preferably hydrogen.
• n is other than 1 then the R 4 groups may be the same or different.
• R 3 is as hereinbefore defined and is preferably a carboxyl or activated carboxyl group such as succinimidyl ester or sulpho-succinimidyl ester.
• m + n is 7,8,9 or 10 when R'/R 5 is a fluorescein/rhodamine pair and 3,4 or
• R 1 and R 5 are cyanine dyes.
• m is 1,2 or 3, preferably 1.
• n is 1,2,3,4,5,6,7,8, or 9 and preferably 5 for a fluorescein/rhodamine pair.
• Preferred compounds of the present invention are: 7 6-carboxyfluorescein-S-CH 2 -CH-NH-CO- (CH 2 ) 5 -NH-CO-ROX
• the present invention relates to a biological material containing an energy transfer dye of the formula (I) or (II).
• Suitable biological materials include, but are not limited to, antibodies, antigens, peptides, proteins, carbohydrates, lipids, nucleotides, oxy or deoxy polynucleic acids and cells which may be derivatised, if necessary so that they contain one or more groups suitable for attachment of an energy transfer dye, e.g., amino, hydroxy, thiophosphoryl, sulphydryl or carboxy groups.
• the present invention provides a method for the preparation of an energy transfer dye of the present invention using at least three coupling reactions:
• the term "coupling” refers to the formation of a covalent bond(s) linking two components, for example, linking a dye moiety with the A portion of the energy transfer dye.
• the dye R 1 will normally contain a substituent suitable for reaction with a thiol group or will be modified to contain such a group.
• iodoacetamide is a suitable substituent for fluorescein dyes
• maleimido is a suitable substituent for cyanine dyes.
• the fluorescent labeling energy transfer dyes may be used to form reagents by covalently binding the dyes to carrier materials such as polymer particles, cells, glass beads, antibodies, proteins, peptides, enzymes, carbohydrates, lipids and nucleotides or nucleic acids (DNA and RNA) and analogues which contain or have been derivatised to include at least one first reactive group capable of forming a covalent bond with the functional group on the labeling complex (or functional group capable of forming a covalent bond with a reactive group on the complex, as described above) and at least one second reactive group (or functional group, as the case may be), having specificity for, and being capable of forming a covalent bond with, a target biological compound, such as antibodies, cells, drugs, antigens, bacteria, viruses and other micro-organisms.
• carrier materials such as polymer particles, cells, glass beads, antibodies, proteins, peptides, enzymes, carbohydrates, lipids and nucleotides or nucleic acids (DNA and RNA)
• the carrier has functional groups
• said functional groups may be antibody or DNA suited for attachment to antigen or a complementary DNA sequence, respectively.
• the carrier material may be a polymer particle or an antigen suitable for attachment to DNA or an antibody for example.
• Techniques for covalently binding fluorochromes to carrier materials such as those mentioned are well known in the art and readily available in the literature.
• the carrier material can further include nucleotides derivatised to contain one of amino, sulphydryl, carboxyl, carbonyl or hydroxyl groups, and oxy or deoxy polynucleic acids derivatised to contain one of amino, thiophosphoryl, sulphydryl, carboxyl, carbonyl or hydroxyl groups.
• the functional groups on the carrier material which are complementary to i.e. capable of forming covalent bonds with, the reactive groups of the labeling complexes of the invention include amino, sulphydryl, carboxyl, carbonyl and hydroxyl groups.
• the present invention also relates to labeling processes in which, in a first step, an energy transfer dye of the present invention covalently reacts with and thereby labels a first component and then uses the labeled first component to bind with a second component to form a labeled second component.
• the first component may be one member of a specific binding pair, (a specific binding partner).
• the fluorescently labeled specific binding partner is then used as a probe for binding to a second member of the specific binding pair (the second component) for which it has specific affinity.
• the specific binding pairs may include a wide variety of molecules which are arbitrarily termed ligands and receptors.
• An example of such ligand-receptor pairs includes an antibody and the corresponding antigen for which the antibody is specific.
• the second step of the procedure may be used to determine the amount of labeled antibodies which are attached to that type of cell by determining the intensity of the fluorescence of the cells.
• monoclonal antibodies and other components covalently labeled in a first step with the fluorescent compounds of the present invention could be used as antigen probes. Numerous other examples are known to those skilled in the art.
• additional ligand-receptor pairs include, for example, biotin-(strept)avidin, hormone receptor- hormone, DNA-complementary DNA, DNA-RNA, DNA-binding protein, enzyme- substrate, and the like. It is to be understood that any two molecules which possess a specific binding affinity may be employed, so that the energy transfer dyes of the present invention may be used for labeling one member of a specific binding pair which in turn may be used in the detection of the complementary member.
• the energy transfer dyes of the present invention provide a valuable set of fluorescent labels which are particularly useful for multiparameter analysis and importantly, are sufficiently low in molecular weight to permit materials labeled with the fluorescent complexes to penetrate cell structures. As such, the dyes are well suited for use with DNA probes. Multiparameter analysis can be performed on multiple samples to 10 detect the presence of target biological compounds. Each sample is labeled by well known labeling methods with a different dye or energy transfer dye.
• one sample suspected of containing a target biological compound is incubated with a single fluorochrome, such as fluorescein, Cascade Blue, a BODIPY dye, or one of the monomethine rigidized dyes, or CY3O(SO 3 ) 2 , or CY3(SO 3 ) 2 , all emitting in the 500-575nm wavelength range (green to orange).
• a second sample suspected of containing the target biological compound (the same compound or a different compound as that in sample 1), is incubated with an energy transfer dye of the invention, for example fluorescein-CY3NH 2 , which will absorb light at 488nm and emits fluorescence at 574nm (orange).
• Additional samples suspected of containing another target compound are incubated with other dyes of the invention, such as fluorescein-CY3-CY5 and fluorescein- CY3-CY7, both of which absorb light at 488nm, but emit fluorescence at 672nm and 782nm respectively (red to near infra-red).
• fluorescent labels such as fluorescein-CY3-CY5 and fluorescein- CY3-CY7, both of which absorb light at 488nm, but emit fluorescence at 672nm and 782nm respectively (red to near infra-red).
• each differently labeled sample will fluoresce a different color at the emission wavelength of its particular label, allowing the individual labels to be distinguished from each other.
• the fluorescent energy transfer labeling dyes of the present invention can be used for a variety of immunofluorescent techniques, including direct and indirect immunoassays, and other known fluorescent detection methods.
• the conditions of each labeling reaction e.g. pH, temperature and time are known in the art, but generally room temperature is preferred. If reacting with an amine, pH 9.4 is preferred. The pH is adjusted depending on the optimum reaction conditions for the particular reactive groups according to known techniques.
• the energy transfer dyes of the present invention and the reagents that can be made from them offer a wide variety of fluorescent labels with large Stokes' shifts. Those in the art will recognize that the dyes of the invention can be used in a variety of fluorescence applications over a wide range of the visible spectrum.
• the following mixture was prepared in a microcentrifuge tube: 300 1 of 0.25M L- cysteine (free base), 200 1 1M potassium phosphate, pH 8, 800 1 of a solution of 50mg/ml 5-iodoacetamido-fluorescein (Molecular Probes) in DMF, and 700 1 of water.
• the reaction mixture was incubated at room temperature for 1 hour protected from light.
• rhodamine acceptor dyes may be substituted for ROX in the above reaction to generate four different energy transfer dye cassettes for use in sequencing applications.
• the product energy transfer dye from v) was purified by reverse phase HPLC using a DeltaPak column (15 micron, 300A, C18 reverse phase, 7.8 x 300mm) and a gradient of triethylammonium acetate, pH 7.0 and 50% acetonitrile in triethylammonium acetate, pH 7.0. Fractions having absorbance at both 496nm and 576nm were collected, pooled and dried overnight in a SpeedVac apparatus. The product was redissolved in water and evaluated spectroscopically using a Perkin Elmer LS-50B Luminescence Spectrometer. With excitation at 488nm, a strong peak was observed at 603nm, characteristic of the ROX emission and indicating excellent energy transfer. The absorbance spectrum showed bands characteristic of both fluorescein and ROX.
• the cysteine carboxyl of the above energy transfer dye may be converted to an NHS ester derivative by the following method.
• the above acid derivative is dissolved in DMF at a concentration of lOmg/ml.
• To the stirred solution in a round bottomed flask is added 1.5 mole equivalents of 13 dicyclohexylcarbodiimide (DCC) and 1.5 mole equivalents of N-hydroxysuccinimide.
• DCC dicyclohexylcarbodiimide
• N-hydroxysuccinimide N-hydroxysuccinimide
• the activated dye is then precipitated from solution by adding four volumes of ethyl acetate.
• the resulting pellet is rinsed 2 times with ethyl acetate and finally dried under vacuum.
• the NHS ester may now be coupled to a suitable target material (e.g. an amino-link oligonucleotide) using standard conditions.

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