WO2003087052A2 - Colorants et composes fluorescents - Google Patents

Colorants et composes fluorescents Download PDF

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Publication number
WO2003087052A2
WO2003087052A2 PCT/US2003/010995 US0310995W WO03087052A2 WO 2003087052 A2 WO2003087052 A2 WO 2003087052A2 US 0310995 W US0310995 W US 0310995W WO 03087052 A2 WO03087052 A2 WO 03087052A2
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groups
group
reactive
compound
aromatic
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PCT/US2003/010995
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WO2003087052A3 (fr
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Ewald A. Terpetschnig
Leonid Patsenker
Anatolij Tatarets
Irina Fedyunyaeva
Igor Borovoy
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Terpetschnig Ewald A
Leonid Patsenker
Anatolij Tatarets
Irina Fedyunyaeva
Igor Borovoy
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Application filed by Terpetschnig Ewald A, Leonid Patsenker, Anatolij Tatarets, Irina Fedyunyaeva, Igor Borovoy filed Critical Terpetschnig Ewald A
Priority to AU2003224912A priority Critical patent/AU2003224912A1/en
Publication of WO2003087052A2 publication Critical patent/WO2003087052A2/fr
Priority to US10/724,580 priority patent/US7250517B2/en
Publication of WO2003087052A3 publication Critical patent/WO2003087052A3/fr
Priority to US10/986,446 priority patent/US7411068B2/en

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Definitions

  • the invention relates to compounds based on squaric, croconic, or rhodizonic acid, among others.
  • the invention relates to compounds useful as dyes and/or luminescent labels or tracers.
  • a luminescent compound, or luminophore is a compound that emits light.
  • a luminescence method is a method that involves detecting light emitted by a luminophore, and using properties of that light to understand properties of the luminophore and its environment.
  • Luminescence methods may be based on chemiluminescence and/or photoluminescence, among others, and may be used in spectroscopy, microscopy, immunoassays, and hybridization assays, among others.
  • Photoluminescence is a particular type of luminescence that involves the abso ⁇ tion and subsequent re-emission of light.
  • a luminophore In photoluminescence, a luminophore is excited from a low-energy ground state into a higher-energy excited state by the abso ⁇ tion of a photon of light. The energy associated with this transition is subsequently lost through one or more of several mechanisms, including production of a photon through fluorescence or phosphorescence.
  • Photoluminescence may be characterized by a number of parameters, including extinction coefficient, excitation and emission spectrum, Stokes' shift, luminescence lifetime, and quantum yield.
  • An extinction coefficient is a wavelength-dependent measure of the absorbing power of a luminophore.
  • An excitation spectrum is the dependence of emission intensity upon the excitation wavelength, measured at a single constant emission wavelength.
  • An emission spectrum is the wavelength distribution of the emission, measured after excitation with a single constant excitation wavelength.
  • a Stokes' shift is the difference in wavelengths between the maximum of the emission spectrum and the maximum of the abso ⁇ tion spectrum.
  • a luminescence lifetime is the average time that a luminophore spends in the excited state prior to returning to the ground state.
  • a quantum yield is the ratio of the number of photons emitted to the number of photons absorbed by a luminophore.
  • Luminescence methods may be based upon or influenced by a variety of spectral properties, including but not limited to extinction coefficient, excitation and emission spectra, Stokes' shift, and quantum yield, and such methods may involve characterizing fluorescence intensity, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), fluorescence lifetime (FLT), total internal reflection fluorescence (TIRF), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP), as well as the analogous methods based upon phosphorescence.
  • FP fluorescence polarization
  • FRET fluorescence resonance energy transfer
  • FLT fluorescence lifetime
  • TIRF total internal reflection fluorescence
  • FCS fluorescence correlation spectroscopy
  • FRAP fluorescence recovery after photobleaching
  • Luminescence methods offer several significant potential strengths. First, luminescence methods may be very sensitive, because modern detectors, such as photomultiplier tubes (PMTs) and charge-coupled devices (CCDs), are capable of detecting very low levels of emitted light. Second, luminescence methods may be very selective, because the luminescence signal may come almost exclusively from the luminophore.
  • PMTs photomultiplier tubes
  • CCDs charge-coupled devices
  • the luminophore may have an extinction coefficient and/or quantum yield that is too low to permit detection of an adequate signal in a particular method.
  • the luminophore also may have a Stokes' shift that is too small to permit detection of emission light without significant detection of excitation light.
  • the luminophore also may have an excitation spectrum that does not permit it to be excited by a particular wavelength-limited light source, such as a laser or an arc lamp.
  • the luminophore also may be relatively nonphotostable, so that it is readily bleached and rendered nonluminescent.
  • the luminophore also may have an excitation and/or emission spectrum that overlaps with the well-known autoluminescence of biological and other samples; such autoluminescence is particularly significant at wavelengths below about 600 nm.
  • the luminophore may also be expensive, especially if it is difficult to manufacture. Summary of the Invention
  • the invention provides compounds useful as labels and tracers, reactive synthetic intermediates useful for the preparation of such compounds, as well as photoluminescent compounds and dyes in general, and general methods of synthesizing and using such compounds and dyes, among others.
  • Figure 1 is a plot of the abso ⁇ tion spectra of Compound 13 as measured in chloroform and methanol.
  • Figure 2 is a combination plot showing the abso ⁇ tion and emission spectra of Compound 14 measured in methanol.
  • Figure 3 is the abso ⁇ tion spectrum of Compound 14 measured in chloroform.
  • Figure 4 is a plot of the abso ⁇ tion spectrum of Compound 15 as measured in chloroform.
  • Figure 5 is a plot of the abso ⁇ tion spectrum of Compound 17 as measured in chloroform.
  • Figure 6 is a plot of the abso ⁇ tion spectrum of Compound 18 as measured in chloroform.
  • Figure 7 is a plot of the abso ⁇ tion spectrum of Compound 22 as measured in chloroform.
  • Figure 8 is a plot of the abso ⁇ tion spectrum of Compound 26 as measured in chloroform.
  • Figure 9 is a plot of the abso ⁇ tion and emission spectra of Compound 28 as measured in water.
  • Figure 10 is a bar graph showing the relative photostability of
  • aliphatic groups include linear or branched hydrocarbon substituents that are optionally fully saturated, or include one or more elements of unsaturation. Unsaturated aliphatic groups are typically comparatively unreactive, and may be referred to as alkanes, alkyl groups, or paraffins. Aliphatic groups inco ⁇ orating carbon-carbon double bonds are typically quite reactive, and may be referred to as alkenes, alkadienes, or olef ⁇ ns. Aliphatic groups inco ⁇ orating carbon-carbon triple bonds are typically highly reactive, and may be referred to as alkynes or acetylenes. In complex structures, the aliphatic groups may inco ⁇ orate branched chains or cross- linked elements.
  • alicyclic groups include hydrocarbon substituents that inco ⁇ orate closed rings. Alicyclic substituents may include rings in boat conformations, chair conformations, or resemble bird cages. Alicyclic groups typically have properties resembling those of aliphatic groups and should not be confused with aromatic ring structures, such as compounds including a six- membered benzene ring.
  • Alicyclic groups may include cycloparaffins or cycloalkanes (saturated hydrocarbons), cycloolefins or cycloalkenes that typically include one or more carbon-carbon double bonds), or cycloacetylenes or cycloalkynes (also referred to as cyclynes) that typically include one or more carbon-carbon triple bonds.
  • cycloparaffins may also be referred to as naphthenes, and includes cyclopropane, cyclohexane, and cyclopentane, among others.
  • the class of cycloolefins includes cyclopentadiene and cyclooctatetraene, among others.
  • Alicyclic groups are derived from petroleum or coal tar, and many can be synthesized by various methods. Alicyclic groups may optionally include heteroalicyclic groups, that include one or more heteroatoms, typically nitrogen, oxygen, or sulfur.
  • aromatic groups include substituents that inco ⁇ orate one or more unsaturated cyclic hydrocarbons. A typical aromatic group substituent is phenyl (or covalently bound benzene), which is characterized by a 6-membered delocalized aromatic ring. Most aromatic groups are reactive and chemically versatile. As used herein, aromatic groups includes heteroaromatic ring substituents, that include one or more heteroatoms such as nitrogen, oxygen, or sulfur, among others.
  • Heteroaromatic ring systems typically include one or more 5- or 6-membered rings, and include examples such as pyridines, pyrroles, furans, thiophenes, and purines. Many aromatic groups may be derived from petroleum and coal tar.
  • Any substituent of the compounds of the invention may be further substituted one or more times by any of a variety of substituents, including without limitation, F, CI, Br,
  • alkyl substituents include hydrocarbon chains having 1-22 carbons, more typically having 1-6 carbons, sometimes called "lower alkyl.”
  • a substituent is further substituted by a functional group that'is ionically charged, such as for example a carboxylic acid, sulfonic acid, or a quaternary ammonium group, the ionic substituent may serve to increase the overall hydrophilicity of the compound.
  • the compounds of the invention are optionally further substituted by a reactive functional group, or a conjugated substance, as described below.
  • functional groups such as “carboxylic acid,” “sulfonic acid,” and “phosphoric acid” include the free acid moiety as well as the corresponding metal salts of the acid moiety, and any of a variety of esters or amides of the acid moiety, including without limitation alkyl esters, aryl esters, and esters that are cleavable by intracellular esterase enzymes, such as alpha- acyloxyalkyl ester (for example acetoxymethyl esters, among others).
  • the compounds of the invention may be depicted in structural descriptions as possessing an overall charge, it is to be understood that the compounds depicted include an appropriate counterion or counterions to balance the formal charge present on the compound. Further, the exchange of counterions is well known in the art and readily accomplished by a variety of methods, including ion-exchange chromatography and selective precipitation, among others.
  • the compounds of the invention include those described by the formula:
  • Z is a four, five, or six-member aromatic ring
  • A, B, C, D, E, and F are substituents of Z.
  • substituent F is absent
  • substituents E and F are absent, as shown below.
  • substituents A, B, C, D, E, and F when present, may be present on ring Z in any order, although some arrangements of ring substituents may be preferred for selected embodiments. Where substituents B and C are at adjacent positions on ring Z, substituents A, D, E, and F are typically neutral. Where substituents B and C are separated by one of substituents A, D, E, or F, at least one of substituents A, D, E, and F is typically negatively charged.
  • A, B, C, D, E, and F are independently selected from a variety of elements and groups, including but not necessarily limited to O, S, Se, Te, C(R a )(R b ), N-R c , N(R d )(R e ), W 1 , and W 2 , as described below.
  • Each A substituent on the Z ring is typically selected from the group consisting of O, N-R c , S, Se, Te, and C(R a )(R b ), where each of R a , R b and R c is selected from the group consisting of carbonic acid, cyano, carboxamide, carbonic ester, nitro, reactive groups, aliphatic aromatic, reactive aromatic, aromatic and aliphatic amine groups;
  • the B and C substituents typically are 5-membered heterocyclic ring systems that may be additionally fused to a 6-membered aromatic ring.
  • Each B and C may be bound directly to the Z ring system, or may be bound via a polymethine linkage that includes 1-7 methylene moieties.
  • Each B and C ring system optionally includes one or more heteroatoms selected from O, S, Se, Te, N-R h , and C(R i )(R j ), where R h is H, an aliphatic group, an alicyclic group, an aromatic group, or a reactive aliphatic group.
  • R 1 and R J may include aliphatic and reactive aliphatic groups.
  • Each heterocyclic ring system B and C may be substituted by one or more of H, aliphatic groups, alicyclic groups, aromatic groups, linked carriers, reactive groups capable of covalent attachment to a carrier, spacers bound to one or more reactive groups capable of covalent attachment to a carrier, parts of a condensed aromatic or heterocyclic ring, and parts of a substituted condensed aromatic or heterocyclic ring, and ionic substituents capable of increasing the hydrophilicity of the entire compound.
  • B and C may be covalently bound via a linkage that includes multiple nonhydrogen atoms.
  • suitable heterocyclic ring systems for the B and C substituents include:
  • each of R R 2 , R 3 and R is independently selected from the group consisting of H, aliphatic groups, alicyclic groups, aromatic groups, linked carriers, reactive groups capable of covalent attachment to a carrier, spacers bound to one or more reactive groups capable of covalent attachment to a carrier, parts of a condensed aromatic or heterocyclic ring, and parts of a substituted condensed aromatic or heterocyclic ring.
  • either of A and B may be substituted by one or more ionic substituents capable of increasing the hydrophilicity of the entire compound.
  • each X 1 , X 2 , X 3 , and X 4 shown above may be independently selected from the group consisting of N, O, S, and C-R k , where R k is selected from the group consisting of H, aliphatic groups, alicyclic groups, aromatic groups, linked carriers, reactive groups capable of covalent attachment to a carrier, spacers bound to one or more reactive groups capable of covalent attachment to a carrier, ionic substituents capable of increasing the hydrophilicity of the entire compound, parts of a condensed aromatic or heterocyclic ring, and parts of a substituted condensed aromatic or heterocyclic ring.
  • the B and C substituents may also be selected from a group consisting of
  • each of B and C may be
  • n is independently 0, 1, or 2.
  • n 0, the heterocyclic ring is bound to ring Z via a monomethine linkage.
  • n 1, the heterocyclic ring is bound to ring Z via a trimethine linkage.
  • n 2
  • the heterocyclic ring is bound to ring Z via a pentamethine linkage.
  • the selection of a monomethine, trimethine, or pentamethine linkages permits the spectral properties of the resulting compound to be altered according to the characteristics desired.
  • shifting from a monomethine to a trimethine, to a pentamethine linkage in a W 1 or W 4 substituent typically results in a shifting of the abso ⁇ tion and emission wavelengths of the resulting compounds to progressively longer wavelengths.
  • Each ring substituent Y is independently O, S, Se, Te, N-R h , or C(R )(R ), where each R is independently hydrogen, or an aliphatic, alicyclic, or aromatic group that is optionally substituted by a reactive functional group R x .
  • Each R' and R J is independently an aliphatic group that is optionally substituted by R x .
  • Y may be selected from the group consisting of O, S, N-R h , and C(R i )(R j ).
  • the substituents R h , R and R j may be independently selected from alkyls having 1-6 carbons, more typically methyl or ethyl.
  • Each substituent R , R and R is independently hydrogen, or an aliphatic, alicyclic, or aromatic group, a reactive functional group R x , or a conjugated substance S c .
  • R 1 in combination with an adjacent R 2 , or R 2 in combination with an adjacent R 3 forms a fused aromatic or heterocyclic ring that is itself optionally further substituted.
  • the ring members X 1 , X 2 , X 3 , and X 4 are independently selected from the group consisting of N, 0, S, and C-R k , where R k is hydrogen, or an aliphatic, alicyclic, or aromatic group that is optionally substituted by a reactive functional group R x . or a conjugated substance So or an ionic substituent capable of increasing the hydrophilicity of the resulting compound.
  • each of substituents D, E, and F is selected from the group consisting of O, S, Se, Te, N-R c , N(R d )(R e ), and C(R f )(R g ), where each of R c , R d , and R e is an aliphatic group, a heteroatom-substituted aliphatic group, a polyether, or an aromatic group, each of which may be further substituted by a reactive functional group R x .
  • the substituents R and R s are independently selected from the group consisting of acyl, carboxylic acid, cyano, sulfonic acid, phosphoric acid, carboxylic acid esters, carboxylic acid amides, sulfonic acid esters, arylsulphonyl, carbamoyl, heteroaromatic groups, phosphate, esters of phosphate, and aliphatic amine groups.
  • R and R 8 taken in combination, may form a ring structure, typically a 5- or 6-membered ring.
  • 5- and 6- membered rings include, but are not limited to, pyrazolidine dione rings, barbituric acid, thiobarbituric acid, isoxazolone ring, pyrazolone ring, pyridone ring, rhodanine ring, pyrrolotriazole ring, pyrazolotriazole ring, etc.
  • the most preferable are pyrazolidine dione ring systems or rhodanine ring systems.
  • the compounds of the invention are optionally substituted, either directly or via a substituent, by one or more chemically reactive functional groups that may be useful for covalently attaching the compound to a desired substance.
  • Each reactive group, or R x may be bound to the compound directly by a single covalent bond, or may be attached via a covalent spacer or linkage, L, and may be depicted as -L-R x .
  • the covalent linkage L typically includes stable chemical bonds, such as, without limitation, carbon-carbon bonds, carbon-nitrogen bonds, nitrogen- nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds.
  • L may include any number of nonhydrogen atoms, but typically includes less than about 20 nonhydrogen atoms, that may include carbon, nitrogen, and sulfur.
  • a reactive group R x is typically selected so as to react with, and thereby form a covalent bond with, a complementary functional group on a substance to be labeled (resulting in a conjugated substance, or S c , as discussed below).
  • the types of functional groups present on the molecule or substance to be labeled may include, but are not limited to, amines, thiols, alcohols, phenols, aldehydes, ketones, phosphates, imidazoles, hydrazines, hydroxylamines, disubstituted amines, halides, epoxides, sulfonate esters, purines, pyrimidines, carboxylic acids, or a combination of these groups.
  • Amines, thiols, and alcohols are typically available and appropriately cross-reactive for the labeling of a desired molecule or substance.
  • the reactive functional group of the invention may be selected from the following functionalities: activated carboxylic esters, acyl azides, acyl halides, acyl halides, acyl nitriles, acyl nitriles, aldehydes, ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, azindines, boronates, carboxylic acids, carbodiimides, diazoalkanes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, maleimides, phosphoramidites, silyl halides, sulfonate esters, and sulfonyl halides.
  • the following reactive functional groups are useful for the preparation of labeled molecules or substances, and are therefore suitable reactive functional groups for the pu ⁇ oses of the compounds of the invention.
  • N-hydroxysuccinimide esters, isothiocyanates, and sulfonyl chlorides which may form stable covalent bonds with amines, including amines in proteins and amine-modified nucleic acids
  • Carboxylic acid functional groups including various derivatives of carboxylic acids, including N-hydroxybenzotriazole esters, thioesters, p- nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters, and acyl imidazoles.
  • Alkyl halides including chloromethyls
  • Haloacetamides including iodoacetamides and chloroacetamides
  • the compound of the invention including a synthetic precursor compound, may be covalently or noncovalently associated with one or more substances. Covalent association may occur through various mechanisms, including a reactive functional group as described above, and may involve a covalent linkage, L, separating the compound or precursor from the associated substance (which may therefore be referred to as -L-Sc).
  • the association may occur through various mechanisms, including inco ⁇ oration of the compound or precursor into or onto a solid or semisolid matrix, such as a bead or a surface, or by nonspecific interactions, such as hydrogen bonding, ionic bonding, or hydrophobic interactions (such as Nan der Waals forces).
  • the associated carrier may be selected from the group consisting of polypeptides, polynucleotides, polysaccharides, beads, microplate well surfaces, metal surfaces, semiconductor and non-conducting surfaces, nano-particles, and other solid or semi-solid surfaces.
  • the associated or conjugated substance may be associated with or conjugated to more than one compound of the invention, which may be the same or different.
  • methods for the preparation of dye-conjugates of biological substances is well-known in the art. See, for example, Haugland et al., MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Eighth Edition (1996) hereby inco ⁇ orated by reference.
  • the association or conjugation of a chromophore or luminophore to a substance imparts the spectral properties of the chromophore or luminophore to the substance.
  • Useful substances for preparing conjugates according to the present invention include, but are not limited to, amino acids, peptides, proteins, nucleosides, nucleotides, nucleic acids, carbohydrates, lipids, ion-chelators, nonbiological polymers, cells, and cellular components.
  • the substance to be conjugated may be protected on one or more functional groups.
  • the peptide may be a dipeptide or larger, and typically includes 5 to 36 amino acids.
  • the conjugated substance may be a protein, it may be an enzyme, an antibody, or a phycobiliprotein.
  • the conjugated substance may be a nucleic acid polymers, such as for example DNA oligonucleotides, RNA oligonucleotides (or hybrids thereof), or single- stranded, double-stranded, triple-stranded, or quadruple-stranded DNA, or single-stranded or double-stranded RNA.
  • One class of carriers includes carbohydrates that are polysaccharides, such as dextrans.
  • the resulting conjugate may be useful as an ion indicator, particularly where the luminescence properties of the conjugate are altered by binding a target ion.
  • the associated or conjugated substance may be a member of a specific binding pair, and therefore useful as a probe for the complementary member of that specific binding pair, each specific binding pair member having an area on the surface or in a cavity which specifically binds to and is complementary with a particular spatial and polar organization of the other.
  • the conjugate of a specific binding pair member may be useful for detecting and optionally quantifying the presence of the complementary specific binding pair member in a sample, by methods that are well known in the art.
  • Representative specific binding pairs may include ligands and receptors, and may include but are not limited to the following pairs: antigen — antibody, biotin-avidin, biotin-streptavidin, IgG-protein A, IgG-protein G, carbohydrate- lectin, enzyme-enzyme substrate; DNA-antisense DNA, and RNA-antisense RNA.
  • the associated or conjugated substance may include proteins, carbohydrates, nucleic acids, and nonbiological polymers such as plastics, among others.
  • Selected compounds of the invention may exhibit advantageous photoluminescence properties.
  • B and C may be chosen from W 1 and/or W 4 , and A, B, C, D, E, and F typically are present in any order.
  • one of B and C may be selected from among W 2 , W 3 , W 5 and W 6 , thereby producing a compound that may exhibit shorter wavelength spectral properties.
  • B and C are adjacent, then each of B and C may be a W 1 , W 2 , or W 3 moiety, and each of A, D, E, and F is a neutral substituent.
  • B and C are separated by one of A, D, E, or F, then one of B and C may be a W , W , or W moiety, and one of B and C may be a W 4 , W 5 , or W 6 moiety, and one of A, D, E, and F is a negatively charged substituent. If B and C are separated by two of A, D, E, and F, which is possible only in the six-member ring, then each of B and C is a W 4 , W 5 , or W 6 moiety, and each of A, D, E, and F is neutral.
  • N 1 through N 4 represent the structures A, D, E, and F as defined above, in any order.
  • substituents A, B, C, D, E, and F may include at least one of S, Se, Te, and C(R a) (R b ).
  • the compound may include at least one heteroatom in X 1 through X 4 of one or more of W 1 or W 2 .
  • the compound may also include a reactive functional group and/or a carrier and/or a conjugated substance.
  • the compound of the invention has utility as a synthetic precursor or synthetic intermediate in the preparation of additional useful compounds, the
  • a representative precursor in which Z is a four-member ring is represented by the formula
  • R 13 may be an alkyl having 1-6 carbons).
  • V 2 and V 3 may be O, S, NR 14 , and
  • R 14 and R 15 may independently be CN
  • Selected compounds of the invention also may include pairs, triplets, and higher numbers of compounds conjugated together to form a single compound. Such “tandem compounds” may exhibit desirable spectral properties, such as enhanced Stokes' shifts, or longer-wavelength abso ⁇ tion or emission. Such tandems also may be useful for applications requiring energy transfer, as well as other methods and applications.
  • Selected combinations of subunit compounds are given below, where each A, B, C, D, E, F, and Z independently have the meanings provided above, and U represents a covalent linkage or bridge, such as may be formed by the reaction of a compound having a reactive functional group with an appropriate functional group on another compound.
  • the particular ring size for Z and the particular substituents may vary between members of the tandem compounds. In particular, such compounds may be symmetrical (where the subunits are equivalent) or unsymmetrical (where the subunits are inequivalent).
  • One set of tandem compounds in particular includes bis-squaric acid derivatives (a coupled pair of compounds where each Z is a four-membered ring).
  • bis-squaraine derivatives where the two individual squaraine ring structures are covalently linked by a mono-or poly-methylene bridge, as represented by the formula
  • the B-U-C coupling includes a vinyl bridge and E and F are absent.
  • One or more hydrogens of the methylene bridge may be substituted by F, CI, Br, I, cyano, sulfonic acid, and carboxylic acid, among others.
  • the bis-squaric acid derivatives may contain a larger delocalized pi-electron system, shifting their abso ⁇ tion and emission maxima towards longer wavelengths when compared to the analogous mono-squaraine compounds.
  • Bridged cyanine dyes have been described previously (US 5,571,388; US 3,864,644; US 5,800,995, WO01/02374 and US 3,821,233). However, none of the dyes previously described has included a bridging moiety that produces a twist with respect to the planes of the cyanine heterocyclic systems. In 1986 Y. Kobayashi et al. (Bull. Chem. Soc. Jpn. 59, 311-312) described a twisted cyanine dye structure for a squaraine dye as evidenced by a crystal structure analysis. Analogously bridged cyanine dyes exhibiting a twisted conformation may therefore exhibit unique spectral and photophysical properties e.g. quantum yields, spectral band widths etc. Examples of bridging cyanine dyes may include compounds having the formula:
  • B and C are independently 5-membered heterocyclic ring systems that are optionally fused to an additional 6-membered aromatic ring, as discussed above, BRIDGE includes a conjugated covalent linkage, and SPACER includes a covalent linkage.
  • Such cyanine compounds may include the following examples:
  • n 1 to 6.
  • the compounds of the invention may possess utility as labelling agents or tracers.
  • the compounds may possess strong absorbance, and therefore utility as chromophores, or they may exhibit luminescence, and therefore possess utility as luminescence labels.
  • the luminescent compounds of the invention typically are utilized by illumination of the compound at an appropriate wavelength, followed by observation of a detectable luminescence response. Excitation of the compound may be accomplished using any light source capable of exciting the selected compound of the invention. Typically, the light source is capable producing light at or near the wavelength of maximal abso ⁇ tion of the selected compound.
  • Appropriate illumination sources include, but are not limited to, incandescent bulbs, fluorescent bulbs, arc lamps, ultraviolet or visible wavelength emission lamps, mercury arc lamps, xenon lamps, argon-ion lasers, diode lasers, and Nd-YAG lasers.
  • the illumination source typically is inco ⁇ orated in an instrument or apparatus, such as a microplate reader, fluorometer, flow cytometer, fluorescence microscope, or other instrument.
  • detectable luminescence response is meant a change in, or occurrence of, a luminescence signal that is detectable either by direct visual observation or instrumentally.
  • the detectable response is a change in luminescence, such as a change in the intensity, excitation or emission wavelength distribution of luminescence, luminescence lifetime, luminescence polarization, or a combination thereof.
  • the detectable luminescence response is observed instrumentally, it typically is observed using CCD cameras, video cameras, photographic film, laser-scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, or fluorescence microplate readers.
  • the detectable luminescence response may be simply detected, or it may be quantified. Where it is quantified, the intensity, wavelength, or other spectral property of the luminescence response typically is compared to a calibration standard that may be the result of a calibration curve, a calculation of an expected response, or a luminescent reference material such as a luminescent-labeled microparticle. Where the luminescent compound is used in conjunction with a sample, the sample may be a solution containing one or more biomolecules that are either biological in origin or have been synthetically prepared. The sample optionally is a biological sample that is prepared from a blood sample, urine sample, a swipe, a smear, or other physiological sampling method.
  • the sample may be derived from an environmental sample, such as an air sample, a water sample, or a soil sample.
  • the sample typically is aqueous, but may contain biologically compatible organic solvents, buffering agents, inorganic salts, or other components known in the art for assay solutions.
  • the compounds of the invention may be provided in the form of kits for performing selected assays or experiments. These kits optionally include chemically reactive forms of one or more compounds of the invention that permit the user to label substances of interest and develop individual assays.
  • the kit optionally inco ⁇ orates additional reagents, including but not limited to buffering agents, luminescence calibration standards, enzymes, enzyme substrates, nucleic acid stains, labeled antibodies, or other additional luminescence detection reagents.
  • the compound of the invention is optionally present in pure form, as a concentrated stock solution, or in a prediluted solution ready for use in the appropriate assay.
  • the kit may be designed for use in an automated and/or high-throughput assay, and so is designed to be fully compatible with microplate readers, microfluidic methods, and/or other automated high-throughput methods.
  • the compounds of the invention may be synthesized according to, but not limited to, the procedures shown herein.
  • Selected benzoxazole-based squaraine compounds may be prepared using a two-step synthetic procedure as shown below. In particular, this method may be useful for preparing unsymmetrical benzoxazole-based squaraine dyes.
  • Benzoxazol-based squaraine dyes such as 13 and 14 have not been reported in the literature. The reason for that might very well be explained by the lower reactivity of the 2-methyl-benzoxazolium iodide as compared to methylene bases such as the indolenine or the benzothiozole based heterocycles:
  • monosubstituted squarate intermediates typically exhibit a much higher reactivity when compared to squaric acid or esters of squaric acid.
  • the reactivity of mono-substituted squaraine derivatives such as Compounds 4 or 7, as shown below, permits the synthesis of mono- benzoxazole squaraine Compounds 13 and 14, for which previously described synthetic methods are unsuccessful.
  • dyes that absorb in the range between 580 nm and 630 nm that combine high extinction coefficients and good quantum yields.
  • Texas RedTM a well known commercial dye, has an abso ⁇ tion maximum around 590 nm and emission around 610 nm and exhibits high quantum yields and good extinction coefficients but lacks photostabilty.
  • BodipyTM dyes and AlexaTM dyes are alternatives for this wavelength region but their chromophores also exhibit a limited ability to absorb light efficiently (e ⁇ 70,000 - 100,000).
  • the mono-benzoxazole derivative 13 has an extinction coefficient 6 ⁇
  • Bis- benzoxazole squaraine dyes may exhibit even shorter abso ⁇ tion maxima.
  • the mono-benzoxazole-substituted squaraine dyes of this invention may have abso ⁇ tion maxima between 500-700 nm, exhibit very high extinction coefficients (as high as 200,000), and/or bright fluorescence in organic solvents and in aqueous media in the presence of protein.
  • the spectral data as well as a plot of the relative photostability of Compound 14 compared to a known bis-indolenine squaraine dye Compound I (structure provided below) is shown in Figure 10.
  • Compounds 20 and 21 show abso ⁇ tion maxima that make them perfectly suitable for excitation with a 532-nm, frequency-doubled Nd-Yag laser. Their extinction coefficients, in the order of e ⁇ 100,000, are comparable to those of commercially available dyes in this wavelength range.
  • Water-soluble groups may be introduced by quarternization of the five-ring N- atoms by a sulfonic acid group connected by a covalent spacer as exemplified in Compound 27:
  • Compound 27 shows an extraordinarily large Stokes' shift of about 65 nm in water (see Figure 9). Large Stokes' shifts are desirable because they help to increase the sensitivity of the fluorescence measurement.
  • heterocyclic five-membered rings for example oxazole rings
  • the heterocyclic five-membered rings may be readily derivatized using a variety of functional groups that will have permit the alteration, or 'tuning,' of abso ⁇ tion and emission wavelengths, solubility, and the functionality of the dye, among other properties.
  • the oxazole ring might be replaced by an oxadiazole ring to achieve shorter wavelength abso ⁇ tion, or by any other suitable heterocycle as described herein, in order to fine tune or change the spectral and photophysical properties and photostability of the dye.
  • Non- fluorescent dyes are:
  • these compounds may become non- emissive.
  • One of the possible uses of such compounds are as "dark" acceptor dyes in energy transfer based assays, where a fluorescent donor labeled antibody binds to an acceptor-labeled antigen. Binding between two energy- transfer partners is generally accompanied by a change in the donor emission. The high extinction coefficients makes these dyes very effective acceptor dyes in energy transfer experiments.
  • Nitro groups may also be introduced into the molecule via substitution of the central squaraine ring:
  • the high extinction coefficients of the subject dyes make them well- suited as effective quencher dyes for use in energy-transfer based assays or as labels for use in abso ⁇ tion based applications.
  • the twisted bridged cyanine dyes described above may be synthesized as exemplified below. These compounds typically include 4 main structural elements:
  • a heterocyclic base (I) in which Y is substituted to contain a linker that includes a functional group (OH, -NH 2 ) can be synthesized according to US patent application No. 2002/0077487 Al.
  • a heterocyclic component (II) that is substituted with a linker that includes one functional group may be synthesized according to Hamilton et al. (US Patent No. 6,140,494).
  • a non-conjugated bridging element e.g. a cyanuric acid derivative
  • a conjugated bridging element between the two heterocycles (squaraine bridge, or a cyanine bridge), the conjugated bridge could also be included as a part of elements I or II (e.g. structure lib or lie below).
  • Cyanuric acid contains 3 reactive chlorine functionalities. It is well described in the literature (K. Venkataraman. The chemistry of synthetic dyes, Vol. 4, Academic Press, New York and London, 1971) and in patents (US Patent No. 1,625,530) that each of the three chlorine substituents shows a very distinct reactivity that may be activated at a selected temperature.
  • the first Cl- atom typically reacts at 0 - 5 ° C, the second at 20 - 40 ° C, and the third one at 70 - 100 ° C.
  • the coupling reactions may be conducted in aqueous, organic or mixed organic-aqueous media, in solution or in suspension in the presence of base to neutralize the HCl.
  • cyanuric chloride couples to primary or secondary amino groups, but it can also be utilized for coupling with hydroxy or mercapto groups although those chemical bonds are less stable.
  • the 3 rd chlorine may be reacted at 70 - 100 ° C with an amino- group containing linker that also may include a reactive group for covalent attachment to biomolecules or another ionic group improve on water-solubility among other substituents.
  • an amino- group containing linker that also may include a reactive group for covalent attachment to biomolecules or another ionic group improve on water-solubility among other substituents. Examples of reactive functional groups are given above.
  • the heterocyclic components are fused using the conjugated bridging element.
  • different reaction conditions might be used.
  • the two moieties are refluxed in a mixture of BuOH and toluene.
  • malonaldehyde dianil hydrochloride is used as the bridging element the reaction may be done in acetic anhydride in presence of triethylamine at 110 ° C.
  • Another way of bridging squaraine dyes is by coupling two squaraine moieties via a methine bridge.
  • methine bridge There are several ways to intoduce a methine bridge into squaraine dyes.
  • One way is to synthesize the methine-bridged disquaric acid derivative by first reducing dibutyl squarate in a Grignard reaction according to J. Am. Chem. Soc. 92 (19), 5749-5750, (1970) to 3- butoxy-4-methyl-3-cyclobutene- 1 ,2-dione: 1 ) CH 3 MgBr
  • the methine-bridged bisquaric acid derivatives can be further used in a reaction with a heterocyclic base in BuOH/toluene among other solvent systems to synthesize the squaraine dye (see below):
  • indolenine based bridged disquaraine dyes of this invention could be synthesized.
  • the bis-squaraine compounds are expected to exhibit abso ⁇ tion and emission maxima in the range betwee 750 and 900 nm.
  • This wavelength range is currently still open for new discoveries as most of the cyanine dyes that absorb in this wavelength range exhibit shortcomings such as very low photostability in addition to low emission quantum yields. Examples
  • the obtained gum is column purified (Silica gel 60, 0-5% methanol — chloroform) to give triethylammonium 3- cyanoimino-4-oxo-2-(l,3,3-trimethyl-2,3-dihychO-lH-2-indolylidenmethyl)-l- cyclobuten-1 -olate (5) as an oiled yellow solid. Yield: 0.96 g (78%).
  • the raw product is column purified (Silica gel 60, 0-2% methanol — chloroform) to give triethylammonium 3-dicyanomethylene-4-oxo-2-(l,3,3-trimethyl-2,3-dihydro- lH-2-indolylidenmethyl)-l-cyclobuten-l -olate (7) as an orange crystals. Yield is 98%.
  • the raw product is column purified (Silica gel 60, 0-20% methanol — dichloromethane) to give 2-[3-hydroxy-4- oxo-2-( 1 ,3 ,3 -trimethyl-2,3 -dihydro- lH-2-indolylidenmethyl)-2- cyclobutenyliden]-l, 3-indanedione (8). Yield: 120 mg (39 %). R f 0.31 (Sorbfil, chloroform— methanol, 10: 1).
  • the raw product is column purified (Silica gel 60, 0-5% methanol — chloroform) to give triethylammonium 3- dicyanomethylene-2-(3-methyl-2,3-dihydro- 1 ,3-benzothiazol -2-ylidenmethyl)- 4-oxo-l-cyclobuten-l -olate (10). Yield: 190 mg (69 %).
  • a mixture of 40 mg (0.12 mmol) 3-butoxy-4-(l,3,3-trimethyl-2,3- dihydro-lH-2-indolylidenmethyl)-3-cyclobutene-l,2-dione (4) and 50 mg (0.13 mmol) 2,3-dimethyl-5-(4-phenylphenyl)-l,3-oxazol-3-ium iodide (1) is refluxed for 8.5 hours in a mixture of 5 ml 1-butanol and 3 ml toluene using a Dean-Stark trap. The solvent is removed under reduced pressure by a rotary evaporator.

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Abstract

L'invention concerne des colorants, des produits intermédiaires réactionnels utilisés dans la synthèse de composés luminescents et non luminescents, et des procédés de synthèse et d'utilisation de ces composés. Ces composés luminescents et non luminescents peuvent être constitués notamment d'acide squarique, d'acide croconique, et d'acide rhodizonique, et de leurs analogues.
PCT/US2003/010995 1998-04-08 2003-04-10 Colorants et composes fluorescents WO2003087052A2 (fr)

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US10/986,446 US7411068B2 (en) 1998-04-08 2004-11-10 Luminescent compounds

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WO2005118839A1 (fr) 2004-05-28 2005-12-15 Ge Healthcare Uk Limited Procede et reactif permettant de mesurer l'activite enzymatique de la nitroreductase
WO2008094637A2 (fr) 2007-01-30 2008-08-07 Seta Biomedicals. Llc Composés luminescents
CN103173213A (zh) * 2013-03-11 2013-06-26 中南大学 一种次氯酸根离子荧光探针及其合成方法和应用
US9841428B2 (en) 2014-09-26 2017-12-12 Seta Biomedicals, Llc Viscosity-sensitive dyes and method
CN108070275A (zh) * 2016-11-10 2018-05-25 中国科学院化学研究所 方酸染料类化合物、制备方法及用途
US11091646B2 (en) 2017-08-14 2021-08-17 Seta Biomedicals, Llc Luminescent squaraine rotaxane compounds
CN114516821A (zh) * 2018-10-09 2022-05-20 宁波卢米蓝新材料有限公司 一种含有多元环的化合物、应用及有机电致发光器件

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US5571388A (en) * 1984-03-29 1996-11-05 Li-Cor, Inc. Sequencing near infrared and infrared fluorescense labeled DNA for detecting using laser diodes and suitable labels thereof
US6140494A (en) * 1996-04-19 2000-10-31 Amersham Pharmacia Biotech Uk Limited Squarate dyes and their use in fluorescent sequencing method

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005118839A1 (fr) 2004-05-28 2005-12-15 Ge Healthcare Uk Limited Procede et reactif permettant de mesurer l'activite enzymatique de la nitroreductase
US7579140B2 (en) 2004-05-28 2009-08-25 Ge Healthcare Uk Limited Method and reagent for measuring nitroreductase enzyme activity
JP4838239B2 (ja) * 2004-05-28 2011-12-14 ジーイー・ヘルスケア・ユーケイ・リミテッド ニトロレダクターゼ酵素活性の測定法及び試薬
US8378120B2 (en) 2004-05-28 2013-02-19 Ge Healthcare Uk Limited Method and reagent for measuring nitroreductase enzyme activity
WO2008094637A2 (fr) 2007-01-30 2008-08-07 Seta Biomedicals. Llc Composés luminescents
WO2008094637A3 (fr) * 2007-01-30 2008-11-20 Ewald A Terpetschnig Composés luminescents
CN103173213A (zh) * 2013-03-11 2013-06-26 中南大学 一种次氯酸根离子荧光探针及其合成方法和应用
US9841428B2 (en) 2014-09-26 2017-12-12 Seta Biomedicals, Llc Viscosity-sensitive dyes and method
CN108070275A (zh) * 2016-11-10 2018-05-25 中国科学院化学研究所 方酸染料类化合物、制备方法及用途
CN108070275B (zh) * 2016-11-10 2020-07-07 中国科学院化学研究所 方酸染料类化合物、制备方法及用途
US11091646B2 (en) 2017-08-14 2021-08-17 Seta Biomedicals, Llc Luminescent squaraine rotaxane compounds
CN114516821A (zh) * 2018-10-09 2022-05-20 宁波卢米蓝新材料有限公司 一种含有多元环的化合物、应用及有机电致发光器件
CN114516821B (zh) * 2018-10-09 2024-02-13 宁波卢米蓝新材料有限公司 一种含有多元环的化合物、应用及有机电致发光器件

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