WO2010054183A2 - Colorants, compositions et procédés d’utilisation associés - Google Patents

Colorants, compositions et procédés d’utilisation associés Download PDF

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WO2010054183A2
WO2010054183A2 PCT/US2009/063531 US2009063531W WO2010054183A2 WO 2010054183 A2 WO2010054183 A2 WO 2010054183A2 US 2009063531 W US2009063531 W US 2009063531W WO 2010054183 A2 WO2010054183 A2 WO 2010054183A2
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dye
halo
substrate
acceptor
alkyl
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WO2010054183A3 (fr
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Xiangzhi Song
James W. Foley
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Harvard University
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Harvard University
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    • 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
    • C09B19/00—Oxazine 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
    • 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
    • C09B21/00—Thiazine 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/14—Styryl dyes
    • C09B23/145—Styryl dyes the ethylene chain carrying an heterocyclic residue, e.g. heterocycle-CH=CH-C6H5
    • 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
    • C09B55/00—Azomethine dyes
    • C09B55/009—Azomethine dyes, the C-atom of the group -C=N- being part of a ring (Image)
    • 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
    • C09B57/007—Squaraine dyes
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the invention relates to donor-acceptor dyes, compositions, and related methods of use.
  • Donor-acceptor dyes are well known and of considerable commercial importance. These dyes can be characterized by the presence of one or more electron donating auxochrome groups, which typically contain a heteroatom having a non- bonded pair of electrons such as O, N or S. These electron donating groups are directly connected via a sigma and a partial pi-bond to a simple or complex electron accepting moiety, most often a ring system (Griffiths, J. Colour and Constitution of Organic Molecules, London, Academic Press, 1976, Chapters 6 & 7). In the absence of a donor group the acceptor moiety is typically colorless, or at most light yellow, and vice versa.
  • the donor-acceptor (DA) family is made up of a large number of different classes of dyes having a wide variety of properties (Griffiths, J. Colour and Constitution of Organic Molecules, London, Academic Press, 1976, Chapters 7 & 9). Many DA dyes are non-fluorescent and are used for coloring materials; examples include the anthraquinones, triarylmethanes and aminoazo dyes. Some DA dyes are photosensitizers such as the phenothiazine dye Methylene Blue and are used in photomedicine and in photopolymerization systems.
  • DA dyes include those that fluoresce when illuminated with light of the appropriate wavelength; examples include but are not limited to rhodamines, oxazines and squaraines. Fluorescent dyes can be useful in situations where a small amount of dye must give an intense signal, such as in biological systems where it is desirable to perturb the system as little as possible.
  • Donor-acceptor dyes can be composed of one donor and one acceptor, such as the laser dye DCM; two donors and one acceptor, such as the fluorescent dye Rhodamine B; or three donors and one acceptor, such as the cationic colorant Crystal Violet.
  • An exemplary "two donor-one acceptor" dye is Capri Blue:
  • the dimethylamino groups constitute the two electron donors, each of which is directly attached via a sigma bond and a partial pi bond to a xanthene ring system, which constitutes the complex electron acceptor moiety.
  • a photon electron density flows from the two amino moieties to the xanthene ring to give an excited state species.
  • relaxation of the excited state either by the generation of heat (internal conversion) or light (fluorescence) corresponds to the reverse process.
  • This type of degradation can be undesirable for one or more of the following reasons: (1) it can cause a significant blue shift in the dye's color; (2) the resulting NH groups can be acidic and reactive in alkaline environments; and (3) the resulting NH groups can react with reactive bioconjugation groups and therefore preclude their use if degradation occurs during storage but before bioconjugation.
  • An additional property of dyes with dialkylamino donors that can be undesirable is their propensity to induce decreasing fluorescence quantum yields as the temperature of the environment rises.
  • the inventors have discovered novel dyes, compositions comprising the dyes, and methods of using the dyes.
  • the invention features a donor- acceptor dye of formula (I),
  • the acceptor is a moiety that can accept electron density from the donor moiety under the influence of light of the appropriate wavelength.
  • the compound of formula (I) is not
  • the compound of formula (I) is not
  • the compound of formula (I) is not
  • the compound of formula (I) is not
  • the compound is a closed shell compound.
  • acceptor moiety comprises at least ten contiguous pi-bonded carbon, nitrogen, oxygen and sulfur atoms (e.g., at least ten, at least twelve, at least thirteen, or at least fourteen).
  • the compound is a fluorescent compound.
  • the compound is a zwitterion.
  • the compound is a salt.
  • R 1 is C 2 - 4 alkylenyl, C 2 - 4 heteroalkylenyl, C 2 - 4 alkenylenyl, arylenyl, or heteroarylenyl; each of which is optionally substituted with 1-4 R 5 .
  • R 1 is C 2 - 4 alkylenyl, such as C 2 - 4 alkylenyl substituted with 1-4 R 5 .
  • R 1 is C 2 ⁇ heteroalkylenyl, such as C 2 _ 4 heteroalkylenyl substituted with 1-4 R 5 .
  • R 1 is alkenylenyl, such as alkenylenyl substituted with 1-4 R 5 .
  • R 1 is arylenyl, such as arylenyl substituted with 1-4 R 5 .
  • R 1 is arylenyl substituted with 1 R 5 (e.g.
  • R 1 is unsubstituted.
  • R 2 is C 2 _ 4 alkylenyl, C 2 _ 4 heteroalkylenyl, C 2 _ 4 alkenylenyl, arylenyl, or heteroarylenyl; each of which is optionally substituted with 1-4 R 5 .
  • R 2 is alkylenyl, such as alkylenyl substituted with 1-4 R 5 .
  • R 2 is C 2 ⁇ heteroalkylenyl, such as C 2 _ 4 heteroalkylenyl substituted with 1-4 R 5 .
  • R 2 is heteroalkylenyl substituted with 1 R 5 (e.g., wherein R 5 is oxo, halo, -NO 2 , -CN, -
  • R is alkenylenyl, such as alkenylenyl substituted with 1-4 R 5 .
  • R 2 is arylenyl, such as arylenyl substituted with 1-4 R 5 .
  • R 2 is arylenyl substituted with 1 R 5 (e.g.
  • R 2 is unsubstituted.
  • the donor is selected from 7-azabicyclo[2.2.1]heptyl, 7- azabicyclo[2.2.1]heptenyl, 7-azabicyclo[2.2.1]heptadienyl, 8-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]octenyl, 8-azabicyclo[3.2.1]octadienyl, 9-azabicyclo[3.3.1]nonyl, 9-azabicyclo[3.3.1]nonenyl, 9-azabicyclo[3.3.1]nonadienyl, 7-aza-[b]- benzobicyclo[2.2.1]heptyl, and 7-aza-[b,e]-dibenzobicyclo[2.2.1]heptyl.
  • n 1, e.g., the dye may have one donor moiety.
  • the acceptor is
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • a is 1.
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • R d is alkyl
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • c is 1.
  • R 11 is -SO 2 R g (e.g., wherein R g is hydroxyl or halo.
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • the acceptor is:
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • R , 16 is not positioned ortho to the donor moiety.
  • D is O.
  • h is 1.
  • R 18 is -OR d (e.g., wherein R d is alkyl.)
  • the acceptor is wherein: i is 0, 1 or 2; j is 0, or 1; k is O, 1, 2, 3 or 4;
  • R h is hydrogen or alkyl; wherein at least one of R 20 , R 21 , R 22 or R 23 comprises a negatively charged moiety, or the dye further comprises a negatively charged counterion Z " ; and
  • Z " is an anion such as halide, acetate, tosylate, azide, tetrafluoroborate, tetraphenylborate, hexafluorophosphate, phosphate, sulfate, perchlorate, trifluoromethanesulfonate or hexafluoroantimonate.
  • E is O. In some embodiments, R 20 is not positioned ortho to the donor moiety. In some embodiments, j is 1. In some embodiments, R 21 is halo. In some embodiments, k is 1. In some embodiments, R is halo. In some embodiments, R h is hydrogen or alkyl.
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • G is O, S, CR 26 R 27 , SiR 28 R 29 , or NR 30 ;
  • Z " is an anion such as halide, acetate, tosylate, azide, tetrafluoroborate, tetraphenylborate, hexafluorophosphate, phosphate, sulfate, perchlorate, trifluoromethanesulfonate or hexafluoroantimonate.
  • R 24 and R 25 are not positioned ortho to the donor moieties.
  • G is O or NR 30 .
  • R 1 is hydrogen or alkyl.
  • n is 2, e.g., the dye may have two donor moieties.
  • the acceptor is wherein: p is 0, 1 or 2; q is 0, 1 or 2;
  • Z " is an anion such as halide, acetate, tosylate, azide, tetrafluoroborate, tetraphenylborate, hexafluorophosphate, phosphate, sulfate, perchlorate, trifluoromethanesulfonate or hexafluoroantimonate.
  • R 32 and R 33 are not positioned ortho to the donor moieties.
  • M is O or S.
  • the acceptor is:
  • Q is O, S, CR 42 R 43 , SiR 44 R 45 , or NR 46 ;
  • R 40 and R 41 are not positioned ortho to the donor moieties.
  • Q is O or NR 37 .
  • the acceptor is
  • the acceptor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • n 3 e.g., the dye may have three donor moieties.
  • the acceptor is:
  • Z " is an anion such as halide, acetate, tosylate, azide, tetrafluoroborate, tetraphenylborate, hexafluorophosphate, phosphate, sulfate, perchlorate, trifluoromethanesulfonate or hexafluoroantimonate.
  • the acceptor is:
  • the dye is selected from:
  • the dye has a photo stability of at least 1.5 times that of a reference dye when subjected to irradiation, as measured by a decrease in optical density (e.g., wherein the reference dye is a dye having an amino, alkylamino or dialkylamino donor moiety).
  • the dye further comprises a reactive moiety. In some embodiments, the dye further comprises a linker and a reactive moiety. In some embodiments, the dye has the following formula (II): [Formula (I) ]—L z -X
  • the linker and reactive moiety are attached to the acceptor. In some embodiments, the linker and reactive moiety are attached to the donor. In some embodiments, the reactive moiety is selected from the group consisting of Michael acceptor (e.g., an ⁇ , ⁇ -unsaturated carbonyl moiety), carboxylic acid or an activated derivative thereof (e.g., a succinimidyl-containing group), maleimido-containing group, isothiocyanate, sulfonic acid or activated derivative thereof (e.g., a sulfonyl chloride), aldehyde, ketone, carbonyl azide, iodoacetamide, alkyne and azide.
  • Michael acceptor e.g., an ⁇ , ⁇ -unsaturated carbonyl moiety
  • carboxylic acid or an activated derivative thereof e.g., a succinimidyl-containing group
  • maleimido-containing group e.g., a succinimidyl
  • the reactive moiety is directly attached to the dye. In some embodiments, the reactive moiety is attached to the dye via a linker. In some embodiments, the linker is selected from the group consisting of:
  • a' is an integer between 1 and 20; the linker as drawn may be positioned between the donor and acceptor in either direction; and the alkyl chains may have varying degrees of unsaturation.
  • the linker is -(CH 2 ) a — , -(CH 2 -CH 2 -O) a — , Or-NH-CH 2 - CH 2 -O-CH 2 -CH 2 -O-CH 2 -COO-.
  • the dye is conjugated to a substrate. In some embodiments, the dye is conjugated to a substrate via a covalent bond. In some embodiments, the dye is conjugated to a substrate via a linker and a reactive moiety.
  • the dye has the following formula (III):
  • the substrate is a biomolecule (e.g., an amino acid, a polypeptide, a nucleic acid, an antibody, or an antigen), a pharmaceutical agent, a metabolite, a diagnostic agent, a controlled substance, a toxin, biotin, a polymer, or a textile (e.g., an article of clothing).
  • the substrate has been previously labeled with another dye.
  • the invention features a composition comprising a dye of formula (I) and an additional component.
  • the component is a solvent.
  • the component is a substrate.
  • the substrate is a biomolecule (e.g., an amino acid, a polypeptide, a nucleic acid, an antibody, or an antigen), a pharmaceutical agent, a metabolite, a diagnostic agent, a controlled substance, a toxin, biotin, a polymer, or a textile (e.g., an article of clothing).
  • the substrate has been previously labeled with another dye.
  • the component is a reagent (e.g., an acid, a base, a reducing agent, or a coupling agent).
  • the component is a textile (e.g., an article of clothing).
  • the composition comprises a plurality of components. In some embodiments, the composition features a dye of formula (II).
  • the invention features a kit comprising a dye of formula (I).
  • the kit further comprises a container (e.g., a vial). In some embodiments, the kit further comprises instructions for use of the dye. In some embodiments, the kit further comprises a substrate. In some embodiments, the kit further comprises a reference standard. In some embodiments, the kit comprises a dye of formula (II).
  • the invention features a method of labeling a substrate, the method comprising mixing a dye of formula (I) with the substrate under conditions sufficient to label the substrate, thereby labeling the substrate.
  • the dye is of formula (II).
  • the substrate is a biomolecule (e.g., an amino acid, a polypeptide, a nucleic acid, an antibody, or an antigen), a pharmaceutical agent, a metabolite, a diagnostic agent, a controlled substance, a toxin, biotin, a polymer, or a textile (e.g., an article of clothing).
  • the substrate has been previously labeled with another dye.
  • the dye is of formula (III), the biomolecule of formula (III) is an antigen, and the substrate is an antibody. In some embodiments, the dye is of formula (III), the biomolecule of formula (III) is an antibody, and the substrate is an antigen. In some embodiments, the method further comprises evaluating the labeled substrate. In some embodiments, the amount of labeled substrate is evaluated qualitatively. In some embodiments, the amount of labeled substrate is evaluated quantitatively. In some embodiments, the amount of labeled substrate is evaluated in comparison to a control or reference standard.
  • the substrate is labeled in an imagewise manner.
  • the invention features a method of staining an object with a dye of formula (I), the method comprising contacting the dye with the object, thereby staining the object.
  • the object is a cell or group of cells.
  • the object is a polymer.
  • the object is a textile (e.g., an article of clothing).
  • the dyes described herein can have one or more beneficial properties, including properties that are improved over one or more dyes known in the art.
  • Exemplary properties described herein may include one or more of the following: (1) colors and extinction coefficients comparable to or better than those dyes known in the art; (2) increased resistance to photooxidative dealkylation; (3) fluorescence quantum yields that are as good or better than the best known members in their class, regardless of the substitution pattern or rigidity of the electron donor moieties; (4) fluorescence quantum yields that are unaffected or little affected by temperature; (5) are as readily synthesized as are dyes known in the art; and (6) improved solubilities over those known in the art.
  • FIG. 1 is the absorption spectrum of dye 221SR as a function of time of illumination by a 1.7W, 514 nm CW argon laser in anaerobic trifluoroethanol.
  • the structure of the dye is given in Example 20.
  • FIG. 2 is the absorption spectrum of dye 331SR as a function of time of illumination by a 1.7W, 514 nm CW argon laser in anaerobic trifluoroethanol.
  • the structure of the dye is given in Example 20.
  • FIG. 3 is the absorption spectrum of control dye TMSR as a function of time of illumination by a 1.7W, 514 nm CW argon laser in anaerobic trifluoroethanol.
  • the structure of the dye is given in Example 20.
  • FIG. 4 shows the absorption spectra of sulfonaphthylrhodamine dyes 221SNR and control TMSNR as a function of time of illumination by a 1.7W, 514 nm CW argon laser in anaerobic trifluoroethanol.
  • the structures of the dyes are given in Example 24.
  • FIG. 5 is a plot of the optical density at wavelength of maximum absorption vs. time of illumination by a IW bank of green LEDs for dyes 221RE, 221SR,
  • alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C 1 - C 12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it.
  • arylalkyl refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Examples of “arylalkyl” include benzyl, 2-phenylethyl, 3- phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.
  • alkylhydroxy refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by a hydroxyl group.
  • haloalkyl refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl).
  • alkylamino refers to an -NH(alkyl) or -N(alkyl) 2 radical.
  • alkylenyl refers to a divalent alkyl, e.g., - CH 2 -, -CH 2 CH 2 -, and -CH 2 CH 2 CH 2 -.
  • heteroalkyl refers to an alkyl moiety in which one or more of the - CH 2 - groups has been replaced with a heteroatom such as O, S or NH.
  • heteroalkylenyl refers to a divalent heteroalkyl, e.g., -CH 2 -O-CH 2 -, -CH 2 -CH 2 -NH- CH 2 -, and -CH 2 -CH 2 -S-CH 2 -CH 2 -.
  • alkenyl refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and having one or more double bonds.
  • alkenyl groups include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups.
  • One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent.
  • alkynyl refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds.
  • alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3- hexynyl.
  • One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent.
  • aryl refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted (e.g., by one or more substituents).
  • aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.
  • arylenyl refers to a divalent aryl, e.g., -C 6 H 4 -.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom can be substituted (e.g., by one or more substituents).
  • heteroarylenyl refers to a divalent heteroaryl, e.g., -C 5 H 3 N-.
  • halo or halogen refers to any radical of fluorine, chlorine, bromine or iodine.
  • substituted refers to a group “substituted” on an alkyl, alkylenyl, alkenyl, alkenylenyl, alkynyl, aryl, arylenyl, heteroaryl or heteroarylenyl group at any atom of that group. Any atom can be substituted.
  • partial pi-bond refers to a bond in which there is direct p-orbital overlap between the two atoms but the resulting pi bond does not necessarily contain two electrons as is the case in an isolated carbon-carbon double bond.
  • donor refers to a moiety that readily releases electrons to an acceptor.
  • acceptor refers to a moiety that accepts the electron density from the donor moiety.
  • complex acceptor refers to a moiety that accepts electron density from the donor moiety and can provide extended conjugation.
  • donor- acceptor dye abbreviated DA dye refers to a dye comprising a donor and an acceptor as defined above, wherein the donor is directly connected to the acceptor via a sigma and partial pi-pond as defined above.
  • close-shell molecule refers to a molecule in which all of the ground state electrons are paired.
  • optical density refers to a measure of the amount of light transmitted through a solution of an organic molecule using a spectrophotometer.
  • photostability refers to the % change in OD of a solution of the dye under the influence of irradiation.
  • the dyes described herein are closed shell compounds. Included herein are dyes that are zwitterions or salts. In some embodiments, the dye has a photostability of at least 1.5 times that of a reference dye (e.g., a reference dye having an alkylamino or dialkylamino donor moiety) when subjected to irradiation, as measured by a decrease in optical density.
  • a reference dye e.g., a reference dye having an alkylamino or dialkylamino donor moiety
  • the dyes described herein can have one or more donor moieties (e.g., 1, 2, or
  • the dyes may be represented by the generic structures below, in which D represents a donor and A is an acceptor. In the cases in which multiple donors are employed, the donors may be same or different.
  • the donor moieties of the dyes herein generally include one or more apex-N- substituted azabicycloalkanes and alkenes, for example, as shown in formula (I) below.
  • R 1 and R 2 of the donor moiety represent two branches of an azabicyclic ring system.
  • R 1 and R 2 are each independently carbon chains (e.g., C2-4 alkylenyl, C2-4 heteroalkylenyl, C2-4 alkenylenyl) or a cyclic moiety (e.g., arylenyl or heteroarylenyl).
  • Each of R 1 and R 2 can be independently substituted or unsubstituted, for example, as described herein.
  • one or both branches are alkylenyl.
  • one or both branches are alkenylenyl.
  • one or both branches are aryl, e.g., phenyl.
  • R 1 and R 2 may be the same or different.
  • R 1 and R 2 can in some instances be substituted with one or more substituents.
  • substituents include alkyl groups, oxo groups, alcohols and carboxylic acids.
  • the bridgehead carbons may also be substituted, as provided above.
  • Each of R 3 and R 4 can be independently further substituted.
  • Exemplary donor moieties include the following:
  • an acceptor moiety is a moiety that can accept electron density from the donor moiety under the influence of light of the appropriate wavelength.
  • exemplary acceptor moieties include at least ten contiguous pi-bonded carbon, nitrogen, oxygen and sulfur atoms (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, or 18).
  • a dye can include one or more donor moieties such as those described herein.
  • the acceptor moiety can include an aromatic moiety.
  • the acceptor moiety is a phenoxazine, phenothiazine, xanthene, coumarin, naphthalene, aryl azo compound, triarylmethane or squaraine.
  • exemplary acceptor moieties include the following:
  • Exemplary dyes include those described in the examples.
  • a dye described herein may be zwitterionic.
  • a dye described herein may be a salt.
  • a dye described herein may have a positive charge on the acceptor moiety, and thus also include a negatively charged counterion such as halide, acetate, tosylate, azide, tetrafluoroborate, tetraphenylborate, phosphorus hexafluoride, phosphate, sulfate, perchlorate, trifluoromethanesulfonate or hexafluoroantimonate.
  • a dye described herein includes a substituent, for example, on a donor or acceptor moiety (e.g., the acceptor moiety), which allows the dye to form a bond with a substrate.
  • a dye described herein can include a reactive moiety, which allows the dye to form a bond such as a covalent, hydrogen, ionic or electrostatic bond with a substrate.
  • the reactive moiety can be directly attached to the dye, for example, through a covalent bond, or can be attached to the dye through a linker (e.g., a linker including a carbon chain).
  • Formula (II) Exemplary dyes that include a reactive moiety are provided in Formula (II) below, in which L is a linker, z is an integer between 0 and 20, and X is a reactive moiety. [Formula (I)]— L z -X
  • the reactive moiety can be any group which can react with a substrate.
  • exemplary reactive moieties include Michael acceptors, carboxylic acids or an activated derivative thereof, maleimido-containing groups, isothiocyanates, sulfonic acids or activated derivatives thereof, aldehydes, ketones, carbonyl azides, iodoacetamides, alkynes and azides.
  • the linker moiety can be any group that attaches the reactive moiety to the dye.
  • exemplary linker moieties include: -(C1-C4 alkylenyl) a — , -[(Ci-C 4 alkylenyl)- O]a-, -NH-(Ci-C 4 alkylenyl) a -NH- -NH-(Ci-C 4 alkylenyl) a -CO- -NH-(Ci-C 4 alkylenyl) a .-COO- -NH-(C 1 -C 4 alkylenyl) a -SO 2 NH- -CO-(C 1 -C 4 alkylenyl) a -CO- -CO-(Ci-C 4 alkylenyl) a -COO- -CO-(C 1 -C 4 alkylenyl) a -SO 2 NH- -COO-(
  • a dye described herein is conjugated to a substrate (e.g., a biomolecule such as an antibody, antigen or nucleic acid), or an organic compound (e.g., a pharmaceutical compound, a metabolite, a controlled substance, or a toxin).
  • the substrate can be conjugated directly to the dye or can be conjugated through a reactive moiety, for example, a reactive moiety described above.
  • the reactive moiety can be attached to the dye directly (e.g., through a covalent bond) or through a linker such as a linker described herein.
  • Exemplary dye conjugates are provided in formula (III) below, where b' is 1, 2 or 3. f Formula (11)1— substrate (III)
  • the dye is synthesized by reaction of the acceptor bearing an appropriate leaving group with the donor amine, e.g., apex-N-substituted azabicyclic amine, according to the following scheme:
  • LG is a leaving group and n is 1, 2 or 3.
  • the acceptor is an aromatic moiety and the leaving group is a halogen such as fluorine, chlorine or bromine, and the reaction proceeds via nucleophilic aromatic substitution.
  • the acceptor is not commercially available with the appropriate halogen substituents, it may be synthesized by methods known to those skilled in the art, such as chlorination with phosphorus pentachloride.
  • the appropriate azabicyclic amine is reacted with an aromatic molecule that is not the acceptor itself but rather a precursor to the desired acceptor, according to the following scheme:
  • reaction 1 the precursor is an aromatic moiety and the leaving group is a halogen such as fluorine, chlorine or bromine, and the reaction proceeds via nucleophilic aromatic substitution.
  • reaction 2 comprises acid- catalyzed condensation, as described in Example 2 below.
  • donor moieties e.g., apex-N-substituted azabicyclic amines
  • Those that are not commercially available may be synthesized using methods that will be known to those of ordinary skill in the art.
  • methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art.
  • Synthetic chemistry transformations useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M.
  • Described herein are methods of using the dyes and compositions described herein.
  • Exemplary methods include methods of labeling a substrate with a dye described herein.
  • the substrate may be a biomolecule such as a polypeptide or nucleic acid, or an organic molecule such as a pharmaceutical agent, metabolite, controlled substance or toxin.
  • the dyes described herein can be used in a variety of methods, for example, where the labeling of a substrate is desirable.
  • Exemplary methods and techniques appropriate for the dyes described herein include: fluorescence microscopy, F ⁇ rster resonance energy transfer (FRET), fluorescent immunoassays (FIA), flow cytometry, fluorescence-activated cell sorting (FACS), single-molecule fluorescence spectroscopy, DNA sequencing, fluorescence in-situ hybridization (FISH), quantitative real-time polymerase chain reaction (qPCR), DNA microarrays, dye lasers, stains, photodynamic therapy (PDT), inkjet applications and clothing dyes.
  • FRET F ⁇ rster resonance energy transfer
  • FFA fluorescent immunoassays
  • FACS fluorescence-activated cell sorting
  • DNA sequencing fluorescence in-situ hybridization
  • FISH fluorescence in-situ hybridization
  • qPCR quantitative real-time polymerase chain reaction
  • DNA microarrays dye lasers, stains, photo
  • the dyes described herein e.g., the dyes of formulas (I), (II) and (III), could be used in fluorescence microscopy.
  • the specimen such as a cell or group of cells containing a substrate labeled with a dye described herein, is illuminated with light of a specific wavelength which is absorbed by the dye, causing it to emit a longer wavelength of light (of a different color than the absorbed light).
  • the illumination light may be separated from the much weaker emitted fluorescence through the use of an emission filter.
  • the photostability of the dyes described herein can provide an advantage to researchers using fluorescence microscopy.
  • a dye described herein can allow the use of a lower concentration of the dye and also provide for longer time periods of illumination.
  • FRET F ⁇ rster resonance energy transfer
  • FRET results from a distance-dependent interaction between the electronic excited states of two molecules in which excitation is transferred from a donor fluorophore to an acceptor fluorophore without emission of a photon.
  • the process of energy transfer results in a reduction (quenching) of fluorescence intensity and excited state lifetime of the donor fluorophore and, where the acceptor is a fluorophore, can produce an increase in the emission intensity of the acceptor fluorophore.
  • FRET is a useful tool to quantify molecular dynamics in biophysics and biochemistry, such as protein-protein interactions, protein-DNA interactions, and protein conformational changes.
  • one of them is labeled with a donor fluorophore and the other with an acceptor fluorophore, and these fluorophore-labeled molecules are mixed. When they are dissociated, the donor fluorophore emission is detected upon the donor fluorophore excitation.
  • the acceptor fluorophore emission is predominantly observed because of the intermolecular FRET from the donor fluorophore to the acceptor fluorophore.
  • the dyes described herein e.g., the dyes of formulas (I), (II) and (III), could be used in a Fluorescent Immunoassay, abbreviated FIA.
  • FIA Fluorescent Immunoassay
  • the dyes described herein e.g., the dyes of formulas (I), (II) and (III), could be used in flow cytometry, which is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.
  • a beam of light of a single wavelength often supplied by a laser, is directed onto a hydro-dynamically focused stream of fluid.
  • a number of detectors are aimed at the point where the stream passes through the light beam and one or more fluorescent detectors.
  • Each suspended particle passing through the beam scatters the light in some way, and fluorescent dyes found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source.
  • This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to derive various types of information about the physical and chemical structure of each individual particle.
  • Fluorescence-activated cell sorting The dyes described herein, e.g., the dyes of formulas (I), (II) and (III), could be used in fluorescence-activated cell sorting (FACS), which is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
  • FACS fluorescence-activated cell sorting
  • Single-molecule fluorescence The dyes described herein, e.g., the dyes of formulas (I), (II) and (III), could be used in single-molecule fluorescence experiments.
  • Single-molecule fluorescence has emerged as a useful tool for probing various processes which cannot be fully understood on the bulk level, using the fluorescence of a molecule to record information pertaining to its environment, structure, and position. The technique affords the ability to obtain information otherwise not available due to ensemble averaging of a bulk material.
  • a dye described herein may provide simultaneous high photostability and fluorescence quantum yields that are useful in single-molecule experiments.
  • DNA sequencing is a biological method for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Determining DNA sequences is useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the large-scale sequencing of the human genome, as well as the sequencing of many animal, plant, and microbial genomes.
  • DNA sequencing is often accomplished using 'dye-terminator sequencing', which allows the sequencing to be performed in a single reaction.
  • the reaction requires a DNA template, a DNA primer, a DNA polymerase, the four deoxynucleotides, and four fluorescently-labeled dideoxynucleotides.
  • Each of the four dideoxynucleotides is labeled with a different fluorescent dye, each fluorescing at a different wavelength. These serve as chain-terminating nucleotides, lacking the 3'-OH group required for the formation of a new phosphodiester bond.
  • FISH fluorescence in situ hybridization
  • FISH is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. It uses fluorescently-labeled oligonucleotide probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can then be used to determine where the fluorescent probe has bound to the chromosomes.
  • FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
  • Quantitative real-time polymerase chain reaction The dyes described herein, e.g., the dyes of formulas (I), (II) and (III), could be used in quantitative real time polymerase chain reaction (qPCR).
  • qPCR quantitative real time polymerase chain reaction
  • the procedure follows the general principle of polymerase chain reaction.
  • the amplified DNA is generally quantified as it accumulates in the reaction in real time after each amplification cycle.
  • Two common methods of quantification are the use of fluorescent dyes that intercalate with double- stranded DNA, and the use of fluorescently-labeled DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.
  • a DNA microarray is a multiplex technology used in molecular biology and in medicine. It includes an arrayed series of thousands of microscopic spots of DNA oligonucleotides, each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element, which is used as a probe hybridize a cDNA or cRNA sample (called a target) under high- stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target. DNA microarrays can be used to measure changes in expression levels or to detect single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • the dyes described herein e.g., the dyes of formulas (I), (II) and (III), could be used as laser dyes.
  • a dye laser is a laser that uses an organic dye as the lasing medium, usually as a liquid solution. Compared to gases and most solid state lasing media, a dye can usually be used for a much wider range of wavelengths.
  • the dye solution is typically circulated from a large reservoir and through a thin dye jet that serves as the gain medium, so that the dye molecules are used only for a short time within the pump and laser beam and have a long time to recover before they are used again.
  • Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. In biochemistry it involves adding a class -specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells, for instance), or organelles within individual cells. Staining is not limited to biological materials, it can also be used to study the morphology of other materials for example the lamellar structures of semicrystalline polymers or the domain structures of block copolymers.
  • the dyes described herein could be used in photodynamic therapy (PDT).
  • PDT photodynamic therapy
  • a targeted biomolecule pathogen or diseased tissue is selectively stained with a dye that is relatively nontoxic in the dark.
  • the surrounding healthy tissue contains relatively little dye.
  • the PDT dye either becomes, or generates, a toxin which can inactivate the targeted substrate.
  • the toxin that is generated by the PDT agent is a reactive oxygen species such as singlet oxygen, superoxide or hydrogen peroxide. Killing of the biomolecule, pathogen or diseased tissue only occurs where light and dye are simultaneously present.
  • the cleansing organs such as the liver, spleen and kidneys, which remove dye from the blood and consequently can contain considerable quantities of dye, are not damaged because they reside in dark parts of the animal and the dye therefore does not become activated.
  • InkJet applications The dyes described herein, e.g., the dyes of formulas (I), (II) and (III), could be used in inkjet applications. InkJet printers are important in numerous applications in the generation of documents as well as photographs.
  • the dye is dissolved in an appropriate solvent (usually aqueous with one or more cosolvents) and applied via inkjet to a substrate in an imagewise manner. Each individual pixel of ink is applied via one or more ink nozzles.
  • the dyes described herein e.g., the dyes of formulas (I), (II) and (III), could be used as clothing dyes. Imparting a specific color to a garment is typically accomplished in one of two ways. In the first procedure, a material web is produced from color-dyed threads. Each thread is pulled through a dyeing bath in a circulating fashion within a line dyeing machine. A second procedure involves dyeing of the competed garments in drum dyeing machines. Digital printing methods such as inkjet printing (see above) are becoming increasingly important for printing of textiles as well.
  • a dye described herein may be part of a composition.
  • Exemplary compositions include a dye of formula (I), (II) or (III) and an additional component such as a solvent, substrate or reagent.
  • the compositions described herein may include a substrate that is to be labeled with a dye described herein.
  • Exemplary substrates include those described herein, for example, a component of interest in an assay such as a biological assay.
  • the substrate may be a biomolecule such as a polypeptide or nucleic acid.
  • the substrate may be an organic molecule described herein such as a pharmaceutical agent or an analyte.
  • compositions described herein may include a solvent.
  • the solvent may be water, buffer, or an organic solvent such as DMSO.
  • the composition may include multiple solvents, for example in the case that the dye and the substrate are not soluble in the same solvent.
  • the substrate is a polypeptide dissolved in a buffer and the dye is dissolved in an organic solvent such as DMSO, and the two are admixed in order to label the substrate.
  • compositions described herein may include a reagent.
  • a composition can include a reagent which can facilitate the reaction with one or more components in the composition, such as a reaction between a dye and a substrate.
  • the reagent is an acid, base or reducing agent.
  • the reagent is a coupling agent such as a carbodiimide, which would facilitate labeling of a substrate with a dye described herein.
  • a composition described herein is subjected to energy, for example, energy sufficient to cause the dye to fluoresce.
  • the compositions described herein can be used in the methods described herein. Exemplary methods include methods of labeling a substrate with a dye described herein.
  • a dye described herein can be provided in a kit.
  • the kit includes (a) a dye described herein, e.g., a composition that includes a dye described herein, and, optionally (b) a reference standard, and (c) informational material.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the use of a dye described herein for the methods described herein.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of the dye, molecular weight of the dye, concentration, date of expiration, batch or production site information, and so forth.
  • the informational material relates to methods for labeling a substrate with the dye.
  • the informational material can include instructions to label a substrate with a dye described herein.
  • the informational material can include instructions to label the reference standard provided in the kit.
  • the informational material of the kits is not limited in its form.
  • the informational material, e.g., instructions is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet.
  • the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording.
  • the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about a dye described herein and/or its use in the methods described herein.
  • the informational material can also be provided in any combination of formats.
  • a dye described herein can be provided in any form, e.g., solution, dried or lyophilized form. It is preferred that a dye described herein be substantially pure.
  • the liquid solution may be an aqueous solution or an organic solution.
  • reconstitution generally is by the addition of a suitable solvent.
  • the solvent e.g., water, buffer or DMSO, can optionally be provided in the kit.
  • the kit can include one or more containers for the composition containing a dye described herein.
  • the kit contains separate containers, dividers or compartments for the composition and informational material.
  • the composition can be contained in a bottle or vial, and the informational material can be contained in a plastic sleeve or packet.
  • the separate elements of the kit are contained within a single, undivided container.
  • the composition is contained in a bottle or vial that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more samples of a dye described herein.
  • the kit includes a plurality of ampules, vials or bottles, each containing a sample of a dye described herein.
  • the containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
  • step (b) A 125 mg sample of the compound prepared in step (a) was placed in 4 mL of aqueous 48% HI and heated on a steam bath 7 hrs. The colorless solution was poured onto an excess of solid potassium carbonate and the resulting mixture extracted three times with methylene chloride. The methylene chloride extracts were washed with IN aqueous HCl and twice with brine too ensure the counter ion of the dye was chloride. After drying this solution over sodium sulfate, the solution was poured onto 50 g of silica gel and placed in a crystallizing dish which was then exposed to ambient conditions in a laboratory hood. The silica gel rapidly turned a dark blue color.
  • step (b) A 120 mg sample of the compound prepared in step (a) was placed in 5 mL of aqueous 48% HI and heated on a steam bath 6 hrs. The colorless solution was poured onto an excess of solid potassium carbonate and the resulting mixture extracted three times with methylene chloride. The methylene chloride extracts were washed with IN aqueous HCl and twice with brine too ensure the counter ion of the dye was chloride. After drying this solution over sodium sulfate, the solution was poured onto 4.5 g of silica gel and placed in a crystallizing dish which was then exposed to ambient conditions in a laboratory hood. The silica gel rapidly turned a dark blue color.
  • This compound was prepared using substantially the same method as used in Examples 1 and 2 with the exception that the amine used in step (a) was the hydrochloride salt of 9-azabicyclo[3.3.1]nonane.
  • Phenothiazine (1.541 g) was taken up in 75 mL ether. To this solution was added dropwise 0.8 mL Br 2 in 11 mL glacial acetic acid over 5 minutes. A precipitate rapidly formed which was isolated by filtration. The solid was washed with ether until the washings were colorless and air dried. Yield was 3.040 g (90.1%). A 1.881 g sample of this phenothiazinium bromide was placed in a 100 mL flask and diluted with 20 mL methanol. 1.677 g 7-azabicyclo[2.2.1]heptane in 10 mL methanol was added dropwise. The reaction was stirred at room temperature overnight.
  • a pure sample of the desired compound was obtained by preparative TLC and had a wavelength absorption maximum at 652 nm in ethanol.
  • This compound was prepared using substantially the same method used in Example 5 except that 8-azabicyclo[3.2.1]cyclooctane was used as the secondary amine. Purity was established using HPLC, TLC and NMR spectroscopy. The structure was further verified by high resolution mass spectroscopy.
  • This compound was prepared using substantially the same method used in Example 5 except that 9-azabicyclo[3.3.1]nonane was the secondary amine used. Purity was established using HPLC, TLC and NMR spectroscopy. The structure was further verified by high resolution mass spectroscopy.
  • the reaction mixture was diluted with 75 mL H 2 O and extracted with 3 x 70 mL ethyl acetate. The organic layers were combined, dried over MgSO 4 , and concentrated to give an orange-brown oil. The oil was chromatographed on a silica gel column eluted with 40% CH 2 Cl 2 in hexanes. The desired product came off the column as a yellow band. The product was concentrated to give 1.30 g of a yellow oil (40%).
  • step (c) A 0.378 g sample of the product of step (b), 7- azabicyclo[2.2.1]heptane-para-benzaldehyde, was taken up in a solution of 0.143 g KOH pellets (85% KOH) in 5 mL CH 3 OH. To this solution was added 0.378 g dicyano-pyran, the product of step (a), and the reaction mixture was stirred overnight at 24°C. It was then heated to reflux for 1/2 hour. TLC eluted with 1%
  • CH 3 OH/CH 2 C1 2 showed fluorescent orange -red dye.
  • the mixture was diluted with 80 mL H 2 O and extracted with 3 x 75 mL CH 2 Cl 2 . Organic layers were then dried over MgSO 4 , concentrated, and chromatographed on a silica gel column and eluted with 1% CH 3 OH/CH 2 C1 2 . Product-containing fractions showed evidence of aldehyde contamination by NMR analysis.
  • the material was again subjected to column chromatography using neutral alumina as solid phase and eluting with 40% hexanes/CH 2 Cl 2 to yield 94 mg of dye.
  • the dye was dissolved in 5 mL CH 2 Cl 2 , and 15 mL hexanes were added to the mixture.
  • step (c) A sample of the dichlorosulfofluoran prepared in step (b) was treated with excess 8-azabicyclo[3.2.1]octane in DMSO and heated on steam bath for 30 min. The resulting magenta solution was poured into water and extracted with methylene chloride. The organic solution of dye was dried over sodium sulfate and applied to a silica gel coated glass plate. The plate was developed with a solvent mixture of methanol-methylene chloride. The isolated magenta dye was determined to be pure by TLC.
  • the targeted sulforhodamine-4'carboxylic acid dye has an absorption maximum at 568 nm in methanol, an emission maximum at 587 nm and a fluorescence lifetime of 3.5 nsec at room temperature in the same solvent.
  • the sample was further purified by preparative TLC to give 0.2 g of pure product (40% yield).
  • the dye In methanol the dye has an absorption maximum at 547 nm, an emission maximum at 572 nm and a fluorescence lifetime of 4.02 nsec.
  • step (c) A mixture of 23 mg (0.05 mmol) of the dichloro compound prepared in step (b) was heated on a steam bath with 200 mg (2.0 mmol) of 7- azabicyclo[2.2.1]heptane in 5 mL of DMSO for 1 hr. Cold water (40 mL) was added to the cooled red mixture. The aqueous mixture was extracted with 50 mL of methylene chloride and the organic layer dried over sodium sulfate. The targeted dye was purified by flash chromatography using silica gel as solid phase and 5% methanol /methylene chloride as solvent to give 18 mg of pure dye (63%). The dye has an absorption maximum at 550 nm, an emission maximum at 575 nm and a fluorescence lifetime of 4.59 nsec in methanol at room temperature.
  • the dye was prepared using the same method used in Example 12 with the exception that the starting dye was the compound prepared in Example 14.
  • the targeted dye has an absorption maximum at 558 nm, an emission maximum at 582 nm and a fluorescence lifetime of 4.22 nsec in methanol at room temperature.
  • step (b) The phenylenediamine prepared in step (a) was treated with an excess of 8- hydroxyquinoline and ammonium per sulfate in water at room temperature. After approximately 5 min the blue solution was extracted with methylene chloride. The organic layer was concentrated and the targeted dye was purified by preparative TLC. The desired dye has an absorption maximum at 654 nm in methylene chloride. Addition of CuBr 2 to a methylene chloride solution of the title dye gave a copper(II) chelate compound having an absorption maximum at 714 nm. Similarly, the nickel(II) chelate of the title dye was observed to have an absorption maximum at 735 nm.
  • step (b) An excess of the compound prepared in step (a) was dissolved in a mixture of toluene and n-butanol and treated with a small amount of squaric acid. The solution was heated at reflux temperature for 5 min. The resulting blue solution was evaporated to dryness and the residue purified by preparative TLC.
  • the targeted dye has an absorption maximum at 671 nm, an emission maximum at 682 nm and a fluorescence lifetime of 0.36 nsec in acetone at room temperature.
  • the desired dye is an orange solid having an absorption maximum at 490 nm and a less intense band at 530 nm in ethanol, an emission maximum at 572 nm with a shoulder at 604 nm and a fluorescence lifetime of 0.08 nsec. Unlike highly fluorescent 2:1 adducts, this 1:1 adduct does not appear to be fluorescent to the eye when in solution.
  • This material had the formula:
  • step (b) A sample of the compound prepared in step (a) was treated with excess 8- azabicyclo[3.2.1]octane in DMSO and heated on a steam bath for 1 hr during which time the solution turned from orange to magenta. The mixture was treated with water and the dye extracted into methylene chloride. The dye was purified by TLC using silica gel as solid phase and methanol/methylene chloride as eluent. In methanol the targeted dye has an absorption maximum at 558 nm, an emission maximum at 576 nm and a fluorescence lifetime of 3.72 nsec.
  • Table 1 contains the absorption maximum, fluorescence maximum and the fluorescence quantum yields, ⁇ , at both temperatures.
  • Two of the new dyes of this invention, 221SR and 321SR, have improved fluorescence quantum yields as compared to the control TMSR. Moreover, all of the new dyes are less adversely affected by a temperature increase from 20 to 60 0 C.
  • Table 2 contains the absorption maximum, fluorescence maximum and the fluorescence quantum yields, ⁇ , at both temperatures.
  • Example 22 The compounds 221Ox, 321Ox and JuIOx having formulae depicted in
  • Example 21 were dissolved in methanol and two drops of each were placed in parallel columns on a silica gel TLC plate. One column was protected from exposure to light by a foil cover while the other was exposed to ambient light provided by fluorescent room lighting at bench top level.
  • Control dye JuIOx was deposited at two levels, one relatively dilute and one more concentrated. After 63 hours of exposure to room light the spots of JuIOx, which were originally cyan in color, had turned into a greenish color whereas the 321Ox and 221Ox appeared to retain their original color.
  • the two dyes of this invention are considerably more stable to light- induced degradation under the conditions of the experiment than is the known laser dye JuIOx.
  • the dye having the donor group of the present invention is significantly more stable to laser-induced photodegradation.

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Abstract

L’invention concerne des colorants donneur-accepteur, des compositions et des procédés d’utilisation associés.
PCT/US2009/063531 2008-11-07 2009-11-06 Colorants, compositions et procédés d’utilisation associés Ceased WO2010054183A2 (fr)

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WO2016116510A1 (fr) * 2015-01-20 2016-07-28 Cynora Gmbh Molécules organiques à substitution napthyle destinées à être utilisées dans des composants optoélectroniques
WO2025007415A1 (fr) * 2023-07-03 2025-01-09 北京大学 Colorant fluorescent substitué par un cycle aza[3.2.1] ponté, et son procédé de préparation et son utilisation
US12605468B2 (en) 2020-01-24 2026-04-21 Oregon Health & Science University Oxazine-based fluorophore compounds for nerve-specific imaging

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