WO2016116111A1 - Colorants fluorescents de type acridine et de type xanthénium substitués - Google Patents

Colorants fluorescents de type acridine et de type xanthénium substitués Download PDF

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WO2016116111A1
WO2016116111A1 PCT/DK2016/050016 DK2016050016W WO2016116111A1 WO 2016116111 A1 WO2016116111 A1 WO 2016116111A1 DK 2016050016 W DK2016050016 W DK 2016050016W WO 2016116111 A1 WO2016116111 A1 WO 2016116111A1
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group
alkyl
formula
compound
amino
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Thomas Just SØRENSEN
Bo V. LAURSEN
Marco SANTELLA
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Københavns Universitet
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems 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 to carbon atoms of the ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems 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 to carbon atoms of the ring system
    • C07D219/06—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems 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 to carbon atoms of the ring system
    • C07D219/08—Nitrogen atoms
    • 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
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/06—Hydroxy derivatives of triarylmethanes in which at least one OH group is bound to an aryl nucleus and their ethers or esters
    • 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
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • 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
    • 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
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D15/00—Woodstains
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials

Definitions

  • the present invention relates to a new class of substituted acridinium- and xanthenium-like fluorescent dyes having a 9-x-antracenium rings system.
  • the new substituted fluorescent dyes may be attached to a linker, conjugated to carrier molecule such as e.g. a protein, a nucleic acid, a lipid, or a saccharide, or deposited or immobilised on solid support materials.
  • carrier molecule such as e.g. a protein, a nucleic acid, a lipid, or a saccharide, or deposited or immobilised on solid support materials.
  • the substituted fluorescent dyes are useful for various purposes, including use for fluorescence imaging and in sensors for monitoring or determining analytes.
  • Fluorescent dyes are widely used as tracers for localization of biological structures by fluorescence microscopy, for quantification of analytes by fluorescence immunoassay, for flow cytometric analysis of cells, for measurement of physiological state of cells and other applications.
  • advantages of fluorescent agent over other types of absorption dyes include the detectability of emission at a wavelength distinct from the excitation, the orders of magnitude of greater detectability of fluorescence emission over light absorption, the generally low level of fluorescence background in most biological samples, and the measurable intrinsic spectral properties of fluorescence polarization, lifetime and excited state energy transfer.
  • Different fluorescence compounds such as unsubstituted or substituted rhodamine, xanthenes and acridine compounds, may be used in biological application and/or for measuring an analyte.
  • the compound is claimed to have a high stability towards ortho substitution.
  • the third amino group ⁇ para aminophenyl) attached to the basic acridine structure is an electron donating group. This is known to lower the fluorescence quantum yield, which according to the present invention is undesired.
  • WO 2009/103798 relates to phenyl acridinium compounds suitable for colouring keratinous fibers.
  • the compounds comprise an amino group (para aminophenyl), optionally substituted with e.g. Ci-C 6 alkyl. Up to four alkoxy groups may be present in the phenyl rings but a basic acridine structure having additional two amino and/or sulfur groups is not disclosed.
  • US20060020141 relates to derivatives of rhodamine compounds and similar basic compounds having least -COO " or -S0 3 ⁇ substituents in the phenyl group attached to the acridinium or xanthenium structure.
  • the colorant compositions particular suitable for use in phase change inks.
  • Fluorescence of many art- recognized commercial dyes such as Alexa Fluor 488, Alexa Fluor 514, and Alexa Fluor 555, provides a high absorption but have a limited quantum yield in solution.
  • the moderate quantum yield decreases the brightness, and thus the detection sensitivity or requires use of disproportionately larger quantities of the fluorescent dyes.
  • the present invention provides a novel class of fluorescent compounds of Formula (I).
  • Compound of Formula (I) of the present invention have generally a high molar absorptivity (£ ma x greater than 40,000 cm 1 M "1 ) and high quantum yields ( ⁇ ).
  • the compounds of the present invention provides at least a high molar absorptivity/absorption coefficient, and/or an improved quantum yield, which render the compounds of the present invention and their conjugates highly useful for e.g. biological assays.
  • thee present invention relates to a fluorescent dye compound of Formula (I)
  • X is NR 1A , O, S, S0 2 , SO, Se0 2 , SeO, Te0 2 , SiR 1B R lc , CO, or CR 1B R 1C ,
  • Y 1 is NR 2A R 2B , or SR 2C , OR 2C , or heterocyclyl,
  • Y 2 is NR 2D R 2E , SR 2F , OR 2F , or hete
  • Z is H, aryl, phenyl, Ci-C 6 alkyl
  • R 1A , R 1B , and R 1C are independently selected from the group comprising hydrogen, C1-C12 alkyl, C1-C12 alkanoic acid, phenyl, benzoic acid, C 2 -Ci 2 alkenyl, C 2 - C 12 alkynyl, isothiocyante, Ci-C 6 isothiocyante, Ci-C 6 aminoalkyl, or Ci-C 6 alkylsulphonate,
  • R 2A , R 2B , R 2C , R 2D , R 2E , and R 2F are independently selected from the group comprising hydrogen, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, phenyl, C 4 -C 8 cycloalkyl, Ci-C 6 aminoalkyl, Ci-C 6 alkylenesulphonate, methylenesulfonate, ethylenesulfonate, C 1 -C6 alkylsulphonyl, triflouromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, sulfonyl
  • R 3 is selected from the group comprising H, halogen, SR 4 , OR 4 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, aryl, N0 2 , NHCOCR 4 , CN, COOH, COOR 4 , S0 3 H, isothiocyante, CHO, or COR 4 , optionally, Ci-C 6 alkyleneN0 2 , Ci-C 6 alkyleneNHCOCR 4 , Ci-C 6 alkyleneCN, Ci-C 6 alkyleneCOOH, Ci-C 6 alkyleneCOOR 4 , Ci-C 6 alkyleneS0 3 H, Ci-C 6 alkyleneisothiocyante, Ci-C 6 alkyleneCHO, or Ci-C 6 alkyleneCOR 4 .
  • R 4 is selected from the group comprising Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, phenyl, C 4 -C 8 cycloalkyi, Ci-C 6 aminoalkyi, Ci-C 6 alkylenesulphonate, methylenesulfonate, ethylenesulfonate, C1-C6 alkylsulphonyl, triflouromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, sulfonyl, sulfonyloxy, thioacyl, thiol, thiocarbonyl, Ci-C
  • R 5A , R 5B , R 5C , and R 5D are independently selected from the group comprising Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, phenyl, C 4 -C 8 cycloalkyi, Ci-C 6 aminoalkyi, Ci-C 6 alkylenesulphonate, methylenesulfonate, ethylenesulfonate, Ci-C 6 alkylsulphonyl, triflouromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, sulfonyl, sulfonyloxy, thio
  • R 6A , R 6B , R 6C , and R 6D are independently selected from the group comprising H, halogen, SR 4 , OR 4 , Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, aryl, N0 2 , CN, COOH, COOR 4 , S0 3 H, CHO, or COR 4 Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, phenyl, C 4 -C 8 cycloalkyi, Ci-C 6 aminoalkyi, Ci-C 6 alkylenesulphonate, methylenesulfonate, ethylenesulfonate, C1-C6 alkylsulphonyl, triflouromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulf
  • R 6E , and R 6F are independently selected from R 3 ,
  • R 1B and R 1C may together with the silicium or carbon atom to which they are attached form a 5 or 6 membered cyclic or heterocyclic group, R and R may together with the nitrogen atom to which they are attached form a 5 or 6 membered heterocyclic group,
  • R 2B and R 6B may together with the nitrogen atom to which they are attached form a 5 or 6 membered heterocyclic group
  • R 2C and R 6C may together with the nitrogen atom to which they are attached form a 5 or 6 membered heterocyclic group
  • R 2D and R 6D may together with the nitrogen atom to which they are attached form a 5 or 6 membered heterocyclic group
  • the 5 or 6 membered heterocyclic group may independently be substituted once or more with Ci-C 6 alkyl, or Ci-C 6 alkylsulfonate, eaCh Z , R 1A , R 1 B , R R 2A ? R 2B ? R2 C ? R2D; R2E? R2 F ? R3; R 4 ? R5A; R5B; R5C; R5D;
  • R 6A , R 6B , R 6C , R 6D , R 6E , or R 6F may optionally be substituted with one or more substituents independently selected from the group comprising halogen, cyano, nitro, Ci-C 6 alkoxy, Ci-C 6 alkoxy carboxy, sulphonate, Ci-C 6 -alkylsulphonate, or cholesterol, and
  • Compound of Formula (I) of the present invention have generally a high molar absorptivity (£ ma x greater than 40,000 cm 1 M "1 ) and high quantum yields ( ⁇ ).
  • the chosen solvent system is preferably MeCN, MeOH, DCM, or 10% DMSO.
  • X is selected as S, S0 2 , SO, Se0 2 , SeO, Te0 2 , SiR 1B R lc , CO or CR 1B R 1C .
  • a redshift of the emitted light is generally obtained. This is preferred as the background signal from absorbance and emission from biological tissue are low.
  • fluorescence compounds dyes providing fluorescence in the red or near infrared spectrum are beneficial.
  • the selected R 3 groups may not be strong electron donating groups, such as NH 2 , NR 2 , or OH, as the amount quantum yieldO, decreases.
  • R 5A , R 5B , R 5C , and R 5D are methyl.
  • Y 1 is NR 2A R 2B
  • Y 2 is NR 2D R 2E
  • R 2A , R 2B , R 2D , and R 2E are all ethyl.
  • the counter ion may preferably be selected from the group comprising CI “ , Br “ , BF 4 “ , B(C 6 H 6 ) 4 “ , PF 6 “ , HS0 4 “ , S0 4 2” , TRISPHAT, CF 3 S0 3 “ , and CH3SO3 “ , preferably Br “ , BF 4 “ , B(C 6 H 6 ) 4 “ , PF 6 “ , TRISPHAT, CF3SO3 “ , and CH3SO3 “ , or even more preferably BF 4 " , PF 6 “ , ⁇ , AsF 6 “ , SbF 6 “ , CI0 4 “ , N0 3 _ , or TRISPHAT.
  • other counter ions may according to the present invention also be suitable.
  • compounds of Formula (I) are generally found to be photostable as well as chemical stable during use to a degree exceeding that of the systems without the substituted hydroxyl groups. Further, they generally show good long-term stability
  • At least one, two or three groups of R 5A , R 5B , R 5C , and R 5D are a lower molecule or lower alkyl, such as methyl or ethyl.
  • Compounds of Formula (I) is substituted with one or more sulphonate groups. This enhances the water solubility and resistance against quenching upon conjugation and increases photostability.
  • compounds of Formula (I) are chemically reactive, and are substituted by at least one reactive group.
  • the reactive group functions as the site of attachment for another moiety, such as a linker, L, a carrier molecule or a solid support, wherein the reactive group chemically reacts with an appropriate reactive or functional group on the linker, carrier molecule or solid support.
  • the present invention relates to fluorescent dye Compounds of Formula (II)
  • R 6C , R 6D , R 6E , and R 6F have the same meanings as given above, Z is H, aryl, phenyl, Ci-C 6 alkyl, or at least one of the subgroups R 1A , R 1B , R 1C , R 2A , R 2B , R 2C , R 2D , R 2E , R 2F , R 3 , R 4 , R 5A , R 5B , R 5C , R 5D , R 6A , R 6B , R 6C , R 6D , R 6E , or R 6F is a linker L or modified to be a linker L, the linker L is attach to a reactive group, Rx, to form -L-Rx or the linker L is attach to a conjugated substance, Sc, to form -L-Sc,
  • each L is optionally the same or different and is a covalent linkage
  • each Rx is optionally the same or different and is a reactive group
  • each Sc is optionally the same or different and is a conjugated substance
  • each L is independently a single covalent bond, or L is a covalent linkage having 1-24 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon- nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorusnitrogen bonds.
  • Rx is independently selected from the group comprising carboxylic acid, alkyl halide, acrylamide, activated ester of a carboxylic acid, hydroxy, aldehyde, sulfonate, amine, antigen, anhydride, aniline, aryl halide, azide, aziridine, boronate, carboxylic acid, carbodiimide, diazoalkane, epoxide, glycol, haloacetamide, halotriazine, hydrazine, hydroxylamine, imido ester, isocyanate, isothiocyanate, ketone, maleimide, phosphoramidite, sulfonyl halide, or thiol group, and
  • Sc is independently selected from the group comprising amino acid, peptide, protein, carbohydrate, monosaccharide, disaccharide, polysaccharide, nucleotide, nucleic acid polymer, antibody, avidin, streptavidin, lectin, growth factor, actin, toxin, phycobiliprotein, antibody, steroid, vitamin, ion- complexing moiety, nucleotide, nucleic acid polymer, hapten, drug, lipid, lipid assembly, synthetic polymers, polymeric microparticle, nonbiological organic polymer, polymeric microparticle, metabolite, cell, cellular systems, cellular fragment, subcellular particle, animal cell, plant cell, bacterium, yeast, bacteria, bacterial particle, virus, virus component, virus particle, other cellular components, or protist,
  • compounds of Formula (I) and compounds of Formula (II) further comprise a reactive group, which is selected from an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a photoactivatable group, a reactive platinum complex, a silyl halide, a sulfonyl halide, and a thiol.
  • a reactive group which is selected from an
  • the reactive group is selected from the group consisting of carboxylic acids, succinimidyl ester of carboxylic acids, hydrazides, amines, isothiocyanates, HOBT active esters, pentafluorophenols, azides, alkynes and maleimides.
  • the reactive group may be attached to any appropriate site on compounds of Formula (I).
  • compounds of Formula (I) and compounds of Formula (II) having a reactive group can be attached to a wide variety of linkers, carrier molecules or solid supports that contain or are modified to contain functional groups with suitable reactivity, resulting in chemical attachment of the compounds of Formula (I) and compounds of Formula (II).
  • the choice of the reactive group used to attach the compounds of Formula (I) and compounds of Formula (II) to the linker, conjugated carrier molecule or solid support typically depends on the reactive or functional group on the substance to be conjugated and the type or length of the linkage desired.
  • the types of functional groups typically present on the organic or inorganic substances include, but are not limited to, amines, amides, thiols, alcohols, phenols, aldehydes, ketones, phosphates, imidazoles, hydrazines, hydroxyla mines, disubstituted amines, halides, epoxides, silyl halides, carboxylate esters, sulfonate esters, purines, pyrimidines, carboxylic acids, olefinic bonds, or a combination of these groups.
  • a single type of reactive site may be available on the substance (typical for polysaccharides or silica), or a variety of sites may occur (e.
  • the present invention relates to compounds of Formula (I) and compounds of Formula (II), wherein Sc is a member of a specific binding pair that is associated non-covalently with the complementary member of the specific binding pair, the specific binding pair may be selected from the following pairs:
  • the present invention relates to compounds of Formula (I) and compounds of Formula (II) attached to a solid support, wherein the solid support is preferably selected from the group comprising a microfluidic chip, a silicon chip, a microscope slide, a microplate well, cuvette, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead, sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, starch, or a sol-gel based matrix.
  • the solid support is preferably selected from the group
  • the present invention relates to compounds of Formula (I) is used in a fluorescent probe for a bioimaging and biosensing application.
  • the present invention relates to a method for determining an analyte of a sample, the method comprising :
  • the analyte is selected from the group comprising H + (pH), Na + , K + , Ca 2+ , 0 2 , C0 2 , H 2 0 2 , metal ions, metabolites, cells or biological derivatives thereof and other suitable analytes.
  • Figure 1 UV-Vis absorptions spectra of 2a, 3a, 4 and 5 recorded in MeCN.
  • Figure 2 Emission picture of 2a (shown as A) and 4 (shown as C) as cell staining fluorescent probe.
  • Figure 3 Emission picture of permeabilized and non-permabilized cells stained with 2a, and 4, and relatives normalized emission spectra of 2a, and 4, as cell staining fluorescent probe.
  • Figure 4 UV-Vis absorbance spectra recorded in of compound 8 recorded at acidic pH (shown as A2-acridinium) and basic pH (shown as A 2 -acridine).
  • Figure 5 Photostability of compound 2A, 3A, 4 and 5.
  • X-axis represents the number of times the same area has been images in a scanning confocal microscope.
  • the synthetic strategy originates from functionalization of the easily accessible (DMP)(TMP) 2 C + .
  • Compound 1, A 2 DMP + may be synthetized by a two-fold nucleophilic aromatic substitution reaction of diethyl amine to the methoxy groups in the para position of the starting material (Scheme 1).
  • the chemical structure of 1 is not symmetric; thus upon ortho amino ring- closure, it is possible to obtain two different products from different positions when functionalized, (Scheme 1, position a, b). Upon substitution at position a, an acridinium orange like system is generated.
  • Compound 1 may be reacted with primary amines. The reaction is quenched with aqueous solution of KPF 6 and subsequent precipitation with the desired salt. It was found that only the acridinium derivative arising from substitution in position a is formed. In the case of S 2 -DMP + , the selectivity is not 100% if following the same procedure as described above, cf. Scheme 2
  • the A 2 -acridiniums may be subjected to further ring closure reaction in orderin the A 2 -ADOTAs + , compounds 3a-c, Scheme 4.
  • Compounds of Formula (I) provide interesting optical properties with applications as fluorescent probes. Presence of an acid is e.g . useful for further conjugation with bio-macromolecules. The nature of the compounds of Formula (I) permits the introduction of functional groups during nucleophilic aromatic substitution.
  • Carboxylic acid may be introduced by reacting A 2 -DMP + with amines having a carboxylic acid.
  • 4-amino benzoic and y-amino butyric acid may be chosen as reagents, Scheme 7.
  • the dyes 6 and 7 may be further functionalized to generate an active NHS- ester derivative that can be easily conjugated with macromolecules.
  • the formation of the NHS-ester is well-known for the skilled person and already developed for trianguleniums system Shumilov, D. et al. Methods and Applications in Fluorescence 2014, 2, 024009).
  • Compounds of Formula (I) are useful for e.g. cell staining.
  • Compounds of Formula (I) with a suitable functional group may be attached to the carboxylic moiety.
  • the synthesis of the 7-C molecule may be achieved by reacting the acid 7 with a slight excess of cholesterol in presence of triphenylphosphine and diethyl azodicarboxylate following the known Mitsunobu reaction.
  • Alkyl refers to monovalent saturated aliphatic hydrocarbyl groups.
  • Ci-C 6 -alkyl and “Ci-Ci 2 -alkyl”, unless otherwise indicated, denotes an alkyl group with 1 to 6 or 1 to 12 carbon atoms, respectively.
  • suitable alkyl groups include straight or branched Ci-C6-alkyl, which, unless otherwise indicated, denotes an alkyl group with 1 to 6 carbon atoms.
  • Such suitable Ci-C 6 -alkyl groups include, for example, methyl, ethyl, propyl, e.g. n-propyl and isopropyl, butyl, e.g. n-butyl, /so-butyl, sec-butyl and tert-butyl, pentyl, e.g. n-pentyl, and hexyl (e.g. n-hexyl).
  • suitable alkyl groups include linear Ci-Ci 2 -alkyl, which, unless otherwise indicated, refers to straight alkyl chains of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms.
  • Alkenyl refers to aliphatic hydrocarbyl groups having at least one double bond.
  • C 2 -C 6 -alkenyl unless otherwise indicated, may be interpreted similarly to the term “alkyl”.
  • Suitable alkenyl groups include, for example, ethenyl, propenyl, 1-butenyl, and 2-butenyl.
  • Alkynyl refers to aliphatic hydrocarbyl groups having at least one double bond.
  • C 2 -C 6 -alkynyl unless otherwise indicated, may be interpreted similarly to the term “alkyl”.
  • Alkenyl groups contain at least 1 triple bond.
  • halogen denotes fluorine (F), chlorine (CI), bromine (Br) and iodine (I), preferably F, CI or Br.
  • the compounds of Formula (I) and compounds of Formula (I) may be substituted with one, two, three, four, five, six or even more halogens, preferable CI or Br, more preferable CI.
  • Ci-C 6 -alkoxy refers to the group -0-Ci-C 6 -alkyl wherein Ci-C 6 -alkyl is defined herein.
  • Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, f-butoxy, sec-butoxy, and n-pentoxy.
  • Ci-Ci 2 alkanoic acid refers to the group of C 1 -C 12 alkyl COOH.
  • Acyl refers to the groups H-C(O)-, alkyl-C(O)-, alkenyl-C(O)-, alkynyl-C(O)-, cycloalkyl-C(O)-, cycloalkenyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)-, and heterocyclic- C(O)-, wherein alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as otherwise defined herein.
  • Acyl includes the "acetyl" group CH 3 C(0)-.
  • Acylamino refers to the groups -NRC(0 )alkyl, -NRC(0)cycloalkyl, - NRC(0)cycloalkenyl, - NRC(0)alkenyl, -NRC(0)alkynyl, -NRC(0)aryl, NRC(0)heteroaryl, and -NRC(O) heterocyclic, wherein R is hydrogen or alkyl and wherein alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as otherwise defined herein,
  • Alkyloxy refers to the groups alkyl-C(0)0-, alkenyl-C(0)0-, alkynyl-C(0)0-, aryl-C(0)0-, cycloalkyl-C(0)0-, cycloalkenyl-C(0)0-, heteroaryl-C(0)0-, and heterocyclic-C(0)0-, wherein alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as otherwise defined herein.
  • Aminocarbonyl refers to the group -C(0)NR 2A R 2B , where R 2A R 2B is as defined herein, optionally R 2A R 2B may be joined together with the nitrogen bound thereto to form a heterocyclic group.
  • Aminothiocarbonyl refers to the group -C(S) NR 2A R 2B , where R 2A R 2B is as defined herein, optionally R 2A R 2B may be joined together with the nitrogen bound thereto to form a heterocyclic group.
  • Aminocarbonylamino refers to the group -NR 4 C(0)N 2A R 2B , optionally R 2A R 2B may be joined together with the nitrogen bound thereto to form a heterocyclic group.
  • Aminothiocarbonylamino refers to the group -NR 4 C(S) NR 2A R 2B 3 , optionally p2Ap2B ma y j 0 j nec j together with the nitrogen bound thereto to form a heterocyclic group.
  • Aminocarbonyloxy refers to the group -0-C(O)NFr A R , optionally R A R B may be joined together with the nitrogen bound thereto to form a heterocyclic group.
  • Aminosulfonyl refers to the group -S0 2 NR 2A R 2B , where R 2A R 2B is as defined herein, optionally R 2A R 2B may be joined together with the nitrogen bound thereto to form a heterocyclic group.
  • Aminosulfonyloxy refers to the group -0-SO 2 NR 2A R 2B , where R 2A R 2B is as defined herein, optionally R 2A R 2B may be joined together with the nitrogen bound thereto to form a heterocyclic group.
  • Aminosulfonylamino refers to the group -NR 4 -S0 2 NR 2A R 2B , where R 2A R 2B is as defined herein, optionally R 2A R 2B may be joined together with the nitrogen bound thereto to form a heterocyclic group.
  • Aryl refers to a monovalent aromatic carbocyclic group of from 5 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic (e.g., 2- benzoxazolinone, 2H-1 ,4-benzoxazin- 3(4H)-one-7-yl, and the like) provided that the point of attachment is at an aromatic carbon atom.
  • Preferred aryl groups include phenyl and naphthyl.
  • Carboxyl or “carboxy” refers to -COOH.
  • Carboxyl ester or “carboxy ester” refers to the groups -C(0)0-alkyl, -C(0)0- alkenyl, -C(0)0-alkynyl, -C(0)0-aryl, -C(0)0-cycloalkyl, -C(0)0-cycloalkenyl, - C(0)0-heteroaryl, and -C(0)0-heterocyclic, wherein alkyl, alkenyl, alkynyl, cycloalkyi, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • (Carboxyl ester)amino refers to the group -NR-C(0 )0-alkyl, - NR-C(0)0- alkenyl, -NR-C(0)0-alkynyl, -NR-C(0)0-aryl, -NR-C(0)0-cycloalkyl, -NR-C(0)0- cycloalkenyl, -NR-C(0)0-heteroaryl, and -NR-C(0)0-heterocyclic, wherein alkyl, alkenyl, alkynyl, cycloalkyi, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • (Carboxyl ester)oxy refers to the group -0-C(0)0-alkyl, s-0-C(0)0-alkenyl, -0-C(0)0-alkynyl, -0-C(0)0-aryl, -0-C(0)0-cycloalkyl, -0-C(0)0-cycloalkenyl, -O- C(0)0-heteroaryl, and 0-C(0)0-heterocyclic, wherein alkyl, alkenyl, alkynyl, cycloalkyi, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
  • suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.
  • Heteroaryl refers to an aromatic group of from 5 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring.
  • Such heteroaryl groups can have a single ring (e.g., pyridinyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl) wherein the condensed rings may or may not be aromatic and/or contain a heteroatom provided that the point of attachment is through an atom of the aromatic heteroaryl group.
  • Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
  • Heterocycle or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl” refers to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged systems, from 1 to 10 carbon atoms and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur or oxygen within the ring wherein, in fused ring systems, one or more the rings can be cycloalkyl, aryl or heteroaryl provided that the point of attachment is through the non-aromatic ring.
  • the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, sulfinyl, sulfonyl moieties.
  • heterocycle and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquinoline, 4,5,6,
  • "5- or 6-membered heterocyclyl containing at least one nitrogen or sulphur atom” include, but are not limited to, benzoefuran, indole, pyrrolidine, pyrrole, thiolane, thiophene, imidazolidine, pyrazolidine, imidazole, pyrazole, oxazolidine, isoxazolidine, oxazole, isoxazole, thiazolidine, isothiazolidine, thiazole, isothiazole, dithiolane, triazoles, furazan, oxadiazole, thiadiazole, dithiazole, tetrazole, piperidine, pyridine, thiane, thiopyran, piperazine, diazines, morpholine, oxazine, thiomorpholine, thiazine, dithiane, dithiine, triazine, or tetrazine.
  • Sulfonyl refers to the divalent group -S(0) 2 -.
  • Alkylsulfonyl refers to the group -S(0) 2 -alkyl wherein alkyl is as defined herein.
  • the alkyl group is a small group having less than 6 carbon atoms, more preferable the alkyl group is methyl or ethyl.
  • “Sulfonyloxy” refers to the group -OS0 2 -alkyl, -OS0 2 -alkenyl -OS0 2 - cycloalkyl, -OS0 2 -cycloalkenyl, -OS0 2 -aryl, -OS0 2 -heteroaryl, and -OS0 2 - heterocyclic, wherein alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • Thioacyl refers to the groups H-C(S)-, alkyl-C(S)-, alkenyl-C(S)-, alkynyl- C(S)-, cycloalkyl-C(S)-, cycloalkenyl-C(S)-, aryl-C(S)-, heteroaryl-C(S)-, and heterocyclic-C(S)-, wherein alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • Thiol refers to the group -SH.
  • Alkylthio refers to the group -S-alkyl wherein alkyl is as defined herein.
  • impermissible substitution patterns e.g., methyl substituted with 5 fluoro groups.
  • impermissible substitution patterns are well known to the skilled person.
  • carrier molecule or “conjugated substance” as used herein refers to a biological or a non-biological component that is or becomes attached to a compound of Formula (I) of the present invention.
  • compounds of Formula (I) when conjugated to a carrier molecule, contain at least one group, -L-Rx, where Rx is the reactive group that is attached to the fluorophore by a covalent linkage L or directly to the fluorophore (compounds of Formula (I)).
  • the covalent linkage attaching compounds of Formula (I) to Rx contains multiple intervening atoms that serve as a spacer.
  • the dyes with a reactive group (Rx) fluorescently label a wide variety of organic or inorganic substances that contain or are modified to contain functional groups with suitable reactivity, resulting in chemical attachment of the conjugated substance (Sc), represented by -L-Sc.
  • the reactive group and functional group are typically an electrophile and a nucleophile that can generate a covalent linkage.
  • the reactive group is a photoactivatable group.
  • the conjugation reaction between the reactive dye and the substance to be conjugated results in one or more atoms of the reactive group Rx to be incorporated into a new linkage L attaching compounds of Formula (I) to the conjugated substance Sc.
  • alkyl halides alcohols/phenols ethers
  • alkyl sulfonates alcohols/phenols ethers
  • aryl halides mines arvl amines aziridines thiols thioethers boronates glycols boronate esters carboxylic acids amines/anilines carboxamides carboxylic acids alcohols esters
  • halotriazines amines/anilines aminotriazines halotriazines alcohols/phenols triazinyl ethers imido esters amines/anilines amidines
  • isocya nates alcohols/phenols urethanes
  • esters carboxylic acids esters
  • Activated esters generally have the formula -COQ, where Q is a good leaving group (e.g. succinimidyloxy (-OC 4 H 4 0 2 ) sulfosuccinimidyloxy (-OC 4 H 3 0 2 -SO 3 H), 1-oxybenzotriazolyl (-OC 6 H 4 N 3 ); or an aryloxy group or aryloxy substituted one or more times by electron withdrawing substituents such as nitro, sulfo, fluoro, chloro, cyano, or trifluoromethyl, or combinations thereof, used to form activated aryl esters; or a carboxylic acid activated by a carbodiimide to form an anhydride or mixed anhydride -OCORa or - OCNR a NHR b , where R a and R b , which may be the same or different, are d-C 6 alkyl, Ci-C 6 perfluoroalkyl, or
  • the covalent linkage L binds the reactive group Rx or conjugated substance Sc to the fiuorophore (Compounds of Formula (I)), either directly (L is a single bond) or with a combination of stable chemical bonds, optionally including single, double, triple or aromatic carbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen- nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorus-nitrogen bonds.
  • L typically includes ether, thioether, carboxamide, sulfonamide, urea, urethane or hydrazine moieties.
  • L moieties have 1-20 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S; and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.
  • L is a combination of single carbon-carbon bonds and carboxamide or thioether bonds.
  • the longest linear segment of the linkage L preferably contains 410 nonhydrogen atoms, including one or two heteroatoms.
  • Further examples of L include substituted or unsubstituted polymethylene, arylene, alkylarylene, arylenealkyl, or arylthio.
  • L contains 1-6 carbon atoms; in another, L is a thioether linkage.
  • L is or incorporates the formula -(CH2)a(CONH(CH 2 )b)z-, where a has any value from 0-5, b has any value from 1-5 and z is 0 or 1.
  • L is or incorporates a substituted platinum atom as described in U.S. Patent No. 5,714,327 to Houthoff et al. (1998).
  • the -L-Rx and -L-Sc moieties may be directly bounded to the fiuorophore at any of R 1A , R 1B , R 1C , R 2A , R 2B , R 2C , R 2D , R 2E , R 2F , R 3 , R 4 , R 5A , R 5B , R 5C , R 5D , R 6A , R 6B , R 6C , R 6D , R 6E , or R 6F , preferably at one of R 1A , R 1B , R 1C , R 2A , R 2B , R 2C , R 2D , R 2E , R 2F , or R 3 , more preferably at R 1A , R 1B , R 1C , R 2A , R 2B , R 2C , R 2D , R 2E , or R 2F , or -L-Rx and -L
  • R 1A , R 1B , R 1C , R 2A , R 2B , R 2C , R 2D , R 2E , R 2F , R 3 , R 4 , R 5A , R 5B , R 5C , R 5D , R 6A , R 6B , R 6C , R 6D , R 6E , or R 6F is an -L-Rx or -L-Sc moiety.
  • exactly one of R 1A , R 1B , or R 1C is an -L-Rx or -L-Sc moiety.
  • the choice of the reactive group used to attach the fiuorophore to the substance to be conjugated typically depends on the functional group on the substance to be conjugated and the type or length of covalent linkage desired.
  • the types of functional groups typically present on the organic or inorganic substances include, but are not limited to, amines, thiols, alcohols, phenols, aldehydes, ketones, phosphates, imidazoles, hydrazines, hydroxyla mines, disubstituted amines, halides, epoxides, sulfonate esters, purines, pyrimidines, carboxylic acids, or a combination of these groups.
  • a single type of reactive site may be available on the substance (typical for polysaccharides), or a variety of sites may occur (e.g. amines, thiols, alcohols, phenols), as is typical for proteins.
  • a conjugated substance may be conjugated to more than one fluorophore, which may be the same or different, or to a substance that is additionally modified by a hapten, such as biotin.
  • Rx will react with an amine, a thiol, an alcohol, an aldehyde or a ketone.
  • Rx is an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine (including hydrazides), an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a sulfonyl halide, or a thiol group.
  • Rx is a carboxylic acid a succinimidyl ester, an amine, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group or an azidoperfluorobenzamido group.
  • the dye becomes chemically reactive after illumination with light of an appropriate wavelength.
  • Rx is a succinimidyl ester of a carboxylic acid
  • the reactive dye is particularly useful for preparing dye-conjugates of proteins or oligonucleotides.
  • Rx is a maleimide
  • the reactive dye is particularly useful for conjugation to thiol-containing substances.
  • RN is a hydrazide
  • the reactive dye is particularly useful for conjugation to periodate-oxidized carbohydrates and glycoproteins, and in addition is an aldehyde-fixable polar tracer for cell microinjection.
  • the reactive dyes of the invention are useful for the preparation of any conjugated substance that possesses a suitable functional group for covalent attachment of the fluorophore.
  • dye-conjugates include, among others, conjugates of synthetic polymers, polymeric microparticles, antigens, steroids, vitamins, drugs, haptens, metabolites, toxins, environmental pollutants, amino acids, peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates, lipids, ion- complexing moieties, and nonbiological polymers.
  • conjugates of cells, cellular systems, cellular fragments, or subcellular particles include, among others, virus particles, bacterial particles, virus components, biological cells (such as animal cells, plant cells, bacteria, yeast, or protists), or cellular components.
  • Sulfonated reactive dyes typically label reactive sites at the cell surface in cell membranes, organelles, or cytoplasm.
  • the conjugated substance is an amino acid, peptide, protein, tyramine, polysaccharide, ion-complexing moiety, nucleotide, nucleic acid polymer, hapten, drug, hormone, lipid, lipid assembly, polymer, polymeric microparticle, biological cell or virus.
  • conjugates of biological polymers such as peptides, proteins, oligonucleotides and/or nucleic acid polymers are also labeled with a second fluorescent or non-fluorescent dye, including an additional dye of the present invention, to form an energy-transfer pair.
  • the conjugated substance (Sc) is an amino acid (including those that are protected or are substituted by phosphates, carbohydrates, or C1-C22 carboxylic acids), or is a polymer of amino acids such as a peptide or protein.
  • Preferred conjugates of peptides contain at least five amino acids, more preferably 5 to 36 amino acids.
  • Preferred peptides include, but are not limited to, neuropeptides, cytokines, toxins, protease substrates, and protein kinase substrates.
  • Preferred protein conjugates include enzymes, antibodies, lectins, glycoproteins, histones, albumins, lipoproteins, avidin, streptavidin, protein A, protein G, phycobiliproteins and other fluorescent proteins, hormones, toxins and growth factors.
  • the conjugated protein is an antibody, an antibody fragment, avidin, streptavidin, a toxin, a lectin, a hormone, or a growth factor.
  • the conjugated substance is a toxin, it is a neuropeptide or a phallotoxin, such as phalloidin.
  • the conjugated substance is a phycobiliprotein
  • it is typically a phycoerythrin, a phycocyanin, or an allophycocyanin, preferably B- or R- phycoerythrin, or an allophycocyanin.
  • the phycobiliprotein is optionally chemically cross-linked, particularly a cross-linked allophycocyanin.
  • the dye of the invention acts as a donor dye, and the phycobiliprotein acts as the ultimate acceptor, permitting excitation with a 488 nm light source with very long wavelength fluorescence.
  • X is S0 2 , SO, Se0 2 , SeO, Te0 2 , SiR 1B R lc , CO or CR ⁇ 1 , thereby introducing a redshift, the dye is an acceptor dye and the phycobiliprotein is the initial donor dye.
  • the dye- phycobiliprotein conjugate exhibits an effective Stokes shift of 100 nm, with maximal excitation of the dye at 485-515 nm, and maximal fluorescence emission of the phycobiliprotein at 620 nm or greater.
  • These energy transfer pairs optionally comprise a chemically reactive group or a conjugated substance, typically attached via the phycobiliprotein, to facilitate use as detectable labels or tracers.
  • the phycobiliprotein is optionally labeled with additional fluorophores, which may be the same or different, that function as additional energy transfer dyes, donor dyes, or ultimate emitter dyes.
  • the conjugated substance (Sc) is a nucleic acid base, nucleoside, nucleotide or a nucleic acid polymer, including those that were modified to possess an additional linker or spacer for attachment of the dyes of the invention, such as an alkynyl linkage (US Pat. 5,047,519), an aminoallyl linkage (US Pat. 4,711,955) or other linkage.
  • the conjugated nucleotide is a nucleoside triphosphate or a deoxynucleoside triphosphate or a dideoxynucleoside triphosphate.
  • nucleic acid polymer conjugates are labeled, single- or multi- stranded, natural or synthetic DNA or HNA, DNA or RNA oligonucleotides, or DNA/RNA hybrids, or incorporate an unusual linker such as morpholine derivatized phosphates (AntiVirals, Inc., Corvallis OR), or peptide nucleic acids such as N-(2- aminoethyl)glycine units.
  • an unusual linker such as morpholine derivatized phosphates (AntiVirals, Inc., Corvallis OR), or peptide nucleic acids such as N-(2- aminoethyl)glycine units.
  • the nucleic acid is a synthetic oligonucleotide, it typically contains fewer than 50 nucleotides, more typically fewer than 25 nucleotides.
  • oligonucleotide-primed DNA polymerization such as by using the polymerase chain reaction or through primer extension, or by terminal-transferase catalyzed addition of a labeled nucleotide to a 3'-end of a nucleic acid polymer.
  • the dye is attached via one or more purine or pyrimidine bases through an amide, ester, ether or thioether bond; or is attached to the phosphate or carbohydrate by a bond that is an ester, thioester, amide, ether or thioether.
  • dye conjugate of the invention is simultaneously labeled with a hapten such as biotin or digoxigenin, or to an enzyme such as alkaline phosphatase, or to a protein such as an antibody.
  • Nucleotide conjugates of the invention are readily incorporated by a DNA polymerase and can be used for in situ hybridization and nucleic acid sequencing (e.g., US Pats. 5,332,666; 5,171,534; and 4,997,928; and WO Appl. 94/05688).
  • the conjugated substance (Sc) is a carbohydrate that is a mono-, di-, or polysaccharide.
  • Sc is a carbohydrate it is a polysaccharide, such as a dextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch, agarose and cellulose.
  • the carbohydrate is a polysaccharide that is a lipopolysaccharide.
  • Preferred polysaccharide conjugates are dextran or FICOLL conjugates.
  • the conjugated substance is a lipid (typically having 6-60 carbons), including glycolipids, phospholipids, sphingolipids, and steroids.
  • the conjugated substance is a lipid assembly, such as a liposome.
  • the lipophilic moiety may be used to retain the conjugated substances in cells, as described in US Pat. 5,208,148.
  • Conjugates having an ion-complexing moiety serve as indicators for calcium, sodium, magnesium, potassium, or other biologically important metal Ions.
  • Preferred ion-complexing moieties are crown ethers, including diaryldiaza crown ethers (US Pat. 5.405,975); BAPTA chelators (US Pat. 5,453,517, US Pat. 5,516,911, and US Pat. 5,049,673); APTHA chelators (AM. J. PHYSIOL. 256, C540 (1989)); or pyridine- and phenanthroline-based metal ion chelators (U.S. Patent No. 5,648,270).
  • the ion-complexing moiety is a diaryldiaza crown ether or BAPTA chelator.
  • the ion indicators are optionally conjugated to plastic or biological polymers such as dextrans or microspheres to improve their utility as sensors.
  • the dye itself acts as an indicator of H + at pH values within about 1.5 pH units of the individual dye's pKa.
  • An example of such indicator dye is Compound 8, cf. also Figure 4.
  • conjugates of non-biological materials include dye-conjugates of organic or inorganic polymers, polymeric films, polymeric wafers, polymeric membranes, polymeric particles, polymeric microparticles including magnetic and non-magnetic microspheres, conducting and non-conducting metals and non-metals, and glass and plastic surfaces and particles.
  • Conjugates are optionally prepared by copolymerization of a sulfonated dye that contains an appropriate functionality while preparing the polymer, or by chemical modification of a polymer that contains functional groups with suitable chemical reactivity.
  • the conjugated substance comprises a glass or silica, which may be formed into an optical fiber or other structure.
  • Conjugates typically result from mixing appropriate sulfonated reactive dyes and the substance to be conjugated in a suitable solvent in which both are soluble.
  • the dyes of the invention are readily soluble in aqueous solutions, facilitating conjugation reactions with most biological materials. For dyes that are photoactivated, conjugation also requires illumination.
  • Labeled members of a specific binding pair are typically used as fluorescent probes for the complementary member of that specific binding pair, each specific binding pair member having an area on the surface or in a cavity that specifically binds to and is complementary with a particular spatial and polar organization of the other.
  • Preferred specific binding pair members are proteins that bind non-covalently to low molecular weight ligands, such as biotin, drug-haptens and fluorescent dyes (such as an anti-fluorescein antibody).
  • Such probes optionally contain a covalently bound moiety that is removed by an enzyme or light, or R" is H and the compound fluoresces following oxidation. Representative specific binding pairs are shown in Table 2.
  • biotin avidin or streptavidin or anti-biotin
  • Hormone hormone receptor Ion chelator
  • IgG is an immunoglobulin t aDNA and aRNA are the antisense (complementary) strands used for hybridization.
  • compounds of Formula (I) are substituted by a blocking moiety that substantially alters the fluorescence of the fluorophore, where the subsequent removal of the blocking moiety restores the fluorescence of the parent dye.
  • cleavage of the blocking moiety from the dye is accomplished by enzymatic activity, making the blocked dye an enzyme substrate (for example as described by Mangel et al., U.S. Patent No. 4,557,862 (1985)).
  • the blocking moiety is a photolabile caging group, such as a substituted or unsubstituted derivative of o-nitroarylmethine (including a-carboxy o- itroarylmethine (U.S. Patent No. 5,635,608 to Haugland et al. (1997)) and bis-(5-t- butoxycarbonylmethoxy)-2-nitrobenzyl), of 2methoxy-5-nitrophenyl, or of desyl.
  • o-nitroarylmethine including a-carboxy o- itroarylmethine (U.S. Patent No. 5,635,608 to Haugland et al. (1997)) and bis-(5-t- butoxycarbonylmethoxy)-2-nitrobenzyl
  • Enzymes that may be detected or quantitated using appropriately blocked dyes include microsomal dealkylases (for example, cytochrome P450 enzymes), glycosidases (for example - galactosidase, -glucosidase, a-fucosidase, glucosaminidase), phosphatases, sulfatases, esterases, lipases, guanidinobenzoatases and others. Conjugates of rhodol dyes that are amino acid or peptide amides are typically useful as peptidase substrates.
  • microsomal dealkylases for example, cytochrome P450 enzymes
  • glycosidases for example - galactosidase, -glucosidase, a-fucosidase, glucosaminidase
  • phosphatases for example - galactosidase, -glucosidase, a-f
  • the resulting dye-conjugate is useful as a substrate for peroxidase enzymes (as described in U.S. Patent No. 5,196,306 to Bobrow et al. (1993)).
  • the reduced derivatives of compounds of Formula (I) serve as substrates for enzymes that take up electrons, or in the detection of chemical oxidizing agents, reactive oxygen species or nitric oxides. Sulfonation of compounds of Formula (I) provides improved water solubility for these substrates.
  • two or more sulfonic acid moieties may be attached to Compounds of Formula (I) or Compounds of Formula (II).
  • the dye compounds of the invention are generally utilized by combining Compounds of Formula (I) or Compounds of Formula (II) as described above with the sample of interest under conditions selected to yield a detectable optical response.
  • the term "dye compound" is used herein to refer to reactive and non-reactive compounds of Formula (I) and their conjugates.
  • the dye compound typically forms a covalent or non-covalent association or complex with an element of the sample, or is simply present within the bounds of the sample or portion of the sample.
  • the sample is then illuminated at a wavelength selected to elicit the optical response.
  • staining of the sample is used to determine a specified characteristic of the sample by further comparing the optical response with a standard or expected response.
  • the dye compounds of the invention are typically used in an aqueous, mostly aqueous or aqueous-miscible solution prepared according to methods generally known in the art.
  • concentration of dye compound is dependent upon the experimental conditions and the desired results, but typically ranges from about one nanomolar to one millimolar or more.
  • concentration is determined by systematic variation until satisfactory results with minimal background fluorescence are accomplished.
  • the dye compounds are most advantageously used to stain samples with biological components.
  • the sample may comprise heterogeneous mixtures of components (including intact cells, cell extracts, bacteria, viruses, organelles, and mixtures thereof) or a single component or homogeneous group of components (e.g. natural or synthetic amino acid, nucleic acid or carbohydrate polymers, or lipid membrane complexes).
  • These dyes are generally non-toxic to living cells and other biological components, within the concentrations of use, although those sulfonated dyes that are additionally substituted one or more times by Br or I are efficient photosensitizers.
  • the dye compound is combined with the sample in any way that facilitates contact between the dye compound and the sample components of interest.
  • the dye compound or a solution containing the dye compound is simply added to the sample, sulfonated compounds of Formula (I) tend to be impairment to membranes of biological cells, but once inside viable cells are typically well retained.
  • Treatments that permeabilize the plasma membrane such as shock treatments or high extracellular ATP can be used to introduce dye compounds into cells.
  • the dye compounds are inserted into cells by pressure microinjection, scrape loading, patch clamp methods, phagocytosis, or by osmotic lysis of pinocytic vesicles.
  • Compounds of Formula (I) that incorporate an amine or a hydrazine residue can be microinjected into cells, where they can be fixed in place by aldehyde fixatives such as formaldehyde or giutaraldehyde. This makes these dyes useful for intracellular applications such as neuronal tracing.
  • Dye compounds that possess a lipophilic substituent, such as phospholipids, will noncovalently incorporate into lipid assemblies, e.g. for use as probes for membrane structure; or for incorporation in liposomes, lipoproteins, films, plastics, lipophilic microspheres or similar materials; or for tracing.
  • Lipophilic compounds of Formula (I) are useful as fluorescent probes of membrane structure, wherein the sulfonic acid moiety permits trapping of the probe at or near the membrane's surface.
  • two or more sulfonic acid moieties may be attached to Compounds of Formula (I) or Compounds of Formula (II).
  • Chemically reactive dye compounds will covalently attach to a corresponding functional group on a wide variety of materials.
  • Using dye compounds (including photoreactive versions) to label reactive sites on or within cells permits the determination of their presence or quantity, accessibility, or their spatial and temporal distribution in the sample.
  • the relative impermeance of the dyes of the invention to membranes of biological cells give them utility as fluorescent probes for assessing the topography of protein distribution in living cells, or as an indicator of single cell viability.
  • the negative charge of the dye compounds at neutral pH also facilitates the electrophoretic separation of dye-conjugates of carbohydrates, drugs and other low molecular weight compounds for analysis by capillary zone electrophoresis (CZE), HPLC or other separation techniques. Precipitation of the conjugate is minimized, even after labeling with multiple fluorophores, since compounds of Formula (I) are fully ionized at neutral pH.
  • CZE capillary zone electrophoresis
  • the sample is optionally combined with one or more additional detection reagents.
  • An additional detection reagent typically produces a detectable response due to the presence of a specific cell component, intracellular substance, or cellular condition, according to methods generally known in the art. Where the additional detection reagent has, or yields a product with, spectral properties that differ from those of the subject dye compounds, multi-color applications are possible.
  • the additional detection reagent is a dye or dye-conjugate of the present invention having spectral properties that are detectably distinct from those of the other staining dye.
  • the compounds of the invention that are dye conjugates are used according to methods extensively known in the art: e.g. use of antibody conjugates in microscopy and immunofluorescent assays; and nucleotide or oligonucleotide conjugates for nucleic acid hybridization assays and nucleic acid sequencing (e.g., US Patent Nos. 5,332,666 to Prober, et al. (1994); 5,171,534 to Smith, et al. (1992); 4,997,928 to Hobbs (1991); and WO Appl. 94/05688 to Menchen, et al..
  • Dye-conjugates of multiple independent dyes of the invention possess utility for multi-color applications.
  • Compounds of Formula (I) may exhibit no crossreactivity with antifluorescein.
  • anti-fluorescein antibodies, conjugated to fluorescent compounds of Formula (I) to amplify fluorescein labels results in both amplification of signal and photostabilization, due to the high photostability of the dyes of the present invention.
  • Labeled antibodies are also useful for multi-color applications, as the use of a red fluorescent anti-fluorescein antibody in conjunction with fluorescein labeling results in a bright, photostable red fluorescent signal.
  • Assays using fluorescent compounds of Formula (I) or compounds of Formula (II) involve contacting a sample with a compound of Formula (I) or compounds of Formula (II) and measuring the fluorescence.
  • the presence of an analyte that interacts with the compound of Formula (I) may alter the fluorescence of the compound of Formula (I) in many different ways. Essentially any change in fluorescence caused by the analyte may be used to determine the presence of the analyte and, optionally the concentration of the analyte, in the sample.
  • the change may take one or more of several forms, including a change in the intensity of the fluorescence and/or quantum yield and/or in fluorescent lifetime. These changes may be either in the positive or negative direction and may be of a range of magnitudes, which preferably will be detectable as described below.
  • a change in quantum yield and/or fluorescent lifetime caused by an analyte may be used as the basis for detecting the presence of an analyte in a sample and may optionally be used to determine the concentration of the analyte.
  • the presence of an analyte in a sample is detected by contacting the sample with a compound of Formula (I) or compounds of Formula (II) that is sensitive to the presence of the analyte.
  • the fluorescence of the solution is then determined using a suitable device, preferably a spectrofluorometer.
  • the fluorescence of the solution may be compared against a set of standard solutions containing known quantities of the analyte or reference dyes.
  • Comparison to standards may be used to calculate the concentration of the analyte, i.e. the analyte.
  • the analyte may be essentially any substance described above.
  • the concentration or amount of the analyte may change over time and the fluorescent signal may serve to monitor those changes.
  • the particular form of the analyte that interacts with compound of Formula (I) or compounds of Formula (II) may be produced or consumed by a reaction occurring in the solution, in which case the fluorescence signal may be used to monitor reaction kinetics.
  • Assays using fluorescent compounds of Formula (I) or compounds of Formula (II) involve contacting a sample with a compound of Formula (I) or compounds of Formula (II) and measuring the fluorescence.
  • the presence of an analyte that interacts with the compound of Formula (I) may alter the fluorescence of the compound of Formula (I) in many different ways. Essentially any change in fluorescence caused by the analyte may be used to determine the presence of the analyte and, optionally the concentration of the analyte, in the sample.
  • the change may take one or more of several forms, including a change in the intensity of the fluorescence and/or quantum yield and/or in fluorescent lifetime. These changes may be either in the positive or negative direction and may be of a range of magnitudes, which preferably will be detectable as described below.
  • a change in quantum yield and/or fluorescent lifetime caused by an analyte may be used as the basis for detecting the presence of an analyte in a sample and may optionally be used to determine the concentration of the analyte.
  • the presence of an analyte in a sample is detected by contacting the sample with a compound of Formula (I) or compounds of Formula (II) that is sensitive to the presence of the analyte.
  • the fluorescence of the solution is then determined using a suitable device, preferably a spectrofluorometer.
  • the fluorescence of the solution may be compared against a set of standard solutions containing known quantities of the analyte or reference dyes. Comparison to standards may be used to calculate the concentration of the analyte, i.e. the analyte.
  • the analyte may be essentially any substance described above.
  • the concentration or amount of the analyte may change over time and the fluorescent signal may serve to monitor those changes.
  • the particular form of the analyte that interacts with compound of Formula (I) or compounds of Formula (II) may be produced or consumed by a reaction occurring in the solution, in which case the fluorescence signal may be used to monitor reaction kinetics.
  • the sample may be illuminated with a wavelength of light selected to give a detectable optical response and observed with a means for detecting the optical response.
  • Appropriate illuminating equipment includes, but is not limited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers and laser diodes. These illumination sources are optionally integrated into laser scanners, fluorescence microplate readers, standard or minifluorometers, or chromatographic detectors.
  • a detectable optical response means a change in or occurrence of, an optical signal that is detectable either by observation or instrumentally.
  • the detectable response is a change in fluorescence, such as a change in the intensity, excitation or emission wavelength distribution of fluorescence, fluorescence lifetime, fluorescence polarization, or a combination thereof.
  • the degree and/or location of staining, compared with a standard or expected response, indicates whether and to what degree the sample possesses a given characteristic.
  • the optical response may optionally be detected by visual inspection, or by use of any of the following devices: CCD cameras, video cameras, photographic film, laser-scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or by means for amplifying the signal such as photomultiplier tubes.
  • CCD cameras CCD cameras
  • video cameras photographic film
  • laser-scanning devices fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or by means for amplifying the signal such as photomultiplier tubes.
  • solid support refers to a matrix or medium that is substantially insoluble in liquid phases and capable of binding a molecule or particle of interest.
  • Solid supports of the current invention include semi-solid supports and are not limited to a specific type of support.
  • Useful solid supports include solid and semisolid matrixes, such as sol-gels, aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtitre plates or microplates), membranes, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports.
  • solid and semisolid matrixes such as sol-gels, aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtitre plates or microplates), membranes, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports.
  • useful solid supports include silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides such as Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead, starch and the like.
  • polysaccharides such as Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,
  • compound 1 300 mg, 0.42 mmol was dissolved in 15 mL acetonitrile and y-aminobutyric acid (0.174g, 1.69 mmol) and l,8-Diazabicycloundec-7-ene (0.51g, 3.3 mmol) were added. The mixture was heated to reflux for lh. The reaction progress was followed by MALDI-TOF specctrometry analysis until presence of the product was observed. The reaction mixture was cooled to room temperature and poured onto 0.2 M KPF 6 (100 mL). The product was extracted with DCM (3x50 mL) .
  • the synthesis of the 7-C molecule was achieved by reacting the acid 7 with a slight excess of cholesterol in presence of triphenylphosphine and diethyl azodicarboxylate following the known Mitsunobu reaction.
  • the emission maximum is at 530 nm.
  • Compound 4 showed staining properties only in permeable cells and seemed to be in the cellular compartment.
  • the emission maximum is at 565nm in accordance to solution studies.
  • the absorbance properties of compound 8 was investigated, cf. Figure 4.
  • Compound 8 may exist in two different forms, as cationic species in acidic medium or as neutral acridine species in basic medium.
  • the absorbance spectra of the two species were recorded at different pH values.
  • ⁇ max of the acridinium species was found at 492 nm, which are similar to compounds of 2a-c.
  • ⁇ max of the lowest energy transition result in a blueshift compared to the A2 acridine specie, which appears at 438 nm. Both spectra resemble the acridinium type spectrum with respect to bandshape.
  • the two inter-convertible states can be used as pH sensors.
  • the two states are interconvertible upon protonation/deprotonation. Presence of a shoulder on the absorbance band for the neutral form (red) was observed and it may be attributed to lack of fully deprotonation of the acridinium dye. Thus, compound 8 may be used as pH sensor.
  • the prepared dyes have been measured in various solvents.
  • the dyes present considerate fluorescent emissive properties with high quantum yield and lifetimes of the range of 2 - 4 ns, cf. Table 3.
  • Table 3 Optical properties of the new c ass of dyes.
  • the novel Compounds of Formula (I) has a higher brightness, £ ma x ⁇ .
  • detection of analytes even in small amounts may be improved.
  • the efficient emitted light from compound 2a and 4, £ ma x-0 is improved when compared to corresponding compounds having two O-bridges, i.e. 2a vs. 3a and 4 vs. 5, cf. Table 4.
  • O-bridging of acridine a-like compounds seems to lower the observed response
  • compound 4 has a much higher effective observed response than Rhodamine B and Alexa Fluor 555 at A em of 560-580 nm .

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Abstract

La présente invention concerne une nouvelle classe de colorants fluorescents de type acridinium et de type xanthénium substitués comportant un système de cycles 9-x-antracénium. Les nouveaux colorants fluorescents substitués peuvent être reliés à un lieur, conjugués à une molécule de support telle que, par exemple, une protéine, un acide nucléique, un liquide ou un saccharide, où déposés ou immobilisés sur des matériaux de support solides. Les colorants fluorescents substitués sont utiles pour différentes applications, comprenant l'utilisation pour l'imagerie par fluorescence et dans des capteurs pour surveiller ou déterminer des analytes.
PCT/DK2016/050016 2015-01-21 2016-01-20 Colorants fluorescents de type acridine et de type xanthénium substitués WO2016116111A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018043579A1 (fr) * 2016-08-31 2018-03-08 国立大学法人名古屋大学 Composé de phospha-rhodamine ou sel correspondant et colorant fluorescent l'utilisant
WO2018181529A1 (fr) * 2017-03-28 2018-10-04 国立大学法人名古屋大学 Composé de phospha-rhodol, sel de celui-ci, et colorant fluorescent l'utilisant
WO2019057451A1 (fr) 2017-08-29 2019-03-28 Universität Basel Colorants organiques à petites molécules

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WO2007049057A2 (fr) * 2005-10-28 2007-05-03 Iti Scotland Limited Nouveaux colorants fluorescents et utilisation de ceux-ci

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WO2007049057A2 (fr) * 2005-10-28 2007-05-03 Iti Scotland Limited Nouveaux colorants fluorescents et utilisation de ceux-ci

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LAURSEN, B.W. ET AL.: "2,6,10-Tris(dialkylamino)trioxatriangulenium salts: a new promising fluorophore. Ion-pair formation and aggregation in non-polar solvents", PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, vol. 4, no. 8, 2005, pages 568 - 575, XP002754806 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018043579A1 (fr) * 2016-08-31 2018-03-08 国立大学法人名古屋大学 Composé de phospha-rhodamine ou sel correspondant et colorant fluorescent l'utilisant
JPWO2018043579A1 (ja) * 2016-08-31 2019-06-24 国立大学法人名古屋大学 ホスファローダミン化合物若しくはその塩、並びにそれを用いた蛍光色素
JP7013024B2 (ja) 2016-08-31 2022-01-31 国立大学法人東海国立大学機構 ホスファローダミン化合物若しくはその塩、並びにそれを用いた蛍光色素
WO2018181529A1 (fr) * 2017-03-28 2018-10-04 国立大学法人名古屋大学 Composé de phospha-rhodol, sel de celui-ci, et colorant fluorescent l'utilisant
WO2019057451A1 (fr) 2017-08-29 2019-03-28 Universität Basel Colorants organiques à petites molécules

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