Fluorescent dyes with large Stokes shifts

Abstract

The present disclosure generally relates to novel and readily accessible fluorescent compounds having large Stokes shifts (LSS) and thermostable fluorescence for expanding the multiplexing capabilities of fluorescence-based nucleic acid detection technologies. Further provided are conjugates, probes, and FRET pairs comprising these fluorescent compounds, as well as methods and labeling methods for amplification and detection of target nucleic acids utilizing these fluorescent compounds.

Description

Technical Field

[0001] Cross - reference to Related Applications This disclosure claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 356,433, filed Jun. 28, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to novel and readily accessible fluorescent compounds having large Stokes shifts (LSS) and thermally stable fluorescence for expanding the multiplexing capabilities of fluorescence - based nucleic acid detection technologies.

Background Art

[0003] Polymerase chain reaction (PCR) has become a universal tool in biomedical research, disease monitoring, and diagnosis. Amplification of nucleic acid sequences by PCR is described in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188. PCR is currently well known in the art and is widely described in the scientific literature. See PCR Applications, ((1999) Innis et al., eds., Academic Press, San Diego), PCR Strategies, ((1995) Innis et al., eds., Academic Press, San Diego); PCR Protocols, ((1990) Innis et al., eds., Academic Press, San Diego), and PCR Technology, ((1989) Erlich, ed., Stockton Press, New York). "Real-time" PCR assays can simultaneously perform amplification and detection and / or quantification of the starting amount of a target sequence. The basic TaqMan® real-time PCR assay that uses the 5' to 3' nuclease activity of DNA polymerase is described in Holland et al., (1991) Proc. Natl. Acad. Sci. 88:7276-7280 and U.S. Patent No. 5,210,015. Real-time PCR without nuclease activity (nuclease-free assay) is described in U.S. Patent Application Publication No. 20100143901. The use of fluorescent probes in real-time PCR is described in U.S. Patent No. 5,538,848.

[0004] A typical real-time PCR protocol using a fluorescent probe involves the use of a labeled probe specific for each target sequence. The probe is preferably labeled with one or more fluorescent moieties that absorb and emit light at a specific wavelength. When hybridized to the target sequence or its amplicon, the probe exhibits a detectable change in fluorescence emission as a result of probe hybridization or hydrolysis.

[0005] However, the main challenge of real-time assays remains the ability to analyze multiple targets within a single tube. In virtually every area of medicine and diagnostics, the number of target loci is rapidly increasing. For example, to name a few, in forensic DNA profiling, detection of pathogenic microorganisms, screening for multi-locus genetic diseases, and multi-gene expression studies, multiple loci must be analyzed.

[0006] Commercially available fluorescence-based devices for automated polymerase chain reaction (PCR) can detect multiple targets within a single reaction vessel (multiplexing) by distinguishing light from different-colored fluorophores. The dyes are selected to minimize their spectral overlap. All fluorophores within an ensemble can be excited with light at or near their absorption maxima, and the emitted light (fluorescence) is detected at or near the fluorescence maxima. By restricting the range of wavelengths (bands) of excitation and emission with optical filters, individual fluorophores can be distinguished. A specific combination of the excitation band and the emission band detected simultaneously defines an optical channel, each enabling the identification of one PCR target.

[0007] The maximum number of achievable optical channels depends on a number of interrelated factors such as the available spectral range, excitation light intensity, fluorophore brightness, fluorophore spectral width, filter bandwidth, and detector sensitivity. State-of-the-art PCR devices with fluorescence-based detection technology use 4 - 6 optical filters per excitation and emission path. Thus, with standard fluorophores, 4 - 6 individual PCR targets can be distinguished. SUMMARY OF THE INVENTION

[0008] The present disclosure relates to dyes having a large Stokes shift, such as a Stokes shift of about 50 nm or more, about 60 nm or more, about 70 nm or more, about 80 nm or more, about 90 nm or more. It has been determined herein that by incorporating certain linker moieties into the dyes of the present disclosure, facile adjustment of their spectroscopic properties, such as their excitation and emission wavelengths, becomes possible. Further, by selecting an appropriate linker moiety, the dyes of the present disclosure can be converted into their respective activated derivatives, such as their respective NHS-esters; or modified to incorporate functional groups that can participate in click chemistry chemical reactions for biomolecule labeling. Further, the introduction of a protecting group enables the derivatization of the dyes to phosphoramidites, such as those compatible with solid-phase synthesis of nucleic acids and phosphoramidite chemistry.

[0009] Furthermore, the dyes of the present disclosure have been found to exhibit excellent brightness in combination with thermally stable fluorescence. Surprisingly, it has also been discovered that the dyes of the present disclosure are readily accessible from inexpensive starting materials in a single high-yield reaction step. These and other advantages are further described herein.

[0010] A first aspect of the present disclosure is a compound having formula (I),

Chemical formula

[0011] In some embodiments, R 1 is 9-fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, monomethoxytrityl (MMT), DMS and p-toluenesulfonamide. In some embodiments, R 1 is H. In some embodiments, the first carbon atom of R 2 is a primary carbon atom. In some embodiments, the first carbon atom of R 2 is a secondary carbon atom. In some embodiments, R 2The first carbon atom of [is a tertiary carbon atom. In some embodiments, the group capable of participating in the "click chemistry reaction" may be selected from the group consisting of bicyclo[6.1.0]nonyne) group ("BCN"), dibenzocyclooctyne ("DBCO"), alkene, trans-cyclooctene ("TCO"), maleimide, aldehyde, ketone, azide, tetrazine, thiol, 1,3-nitrone, hydrazine, and hydroxylamine. In some embodiments, the group capable of participating in the "click chemistry reaction" is DBCO, TCO or azide. In some embodiments, the thiol-reactive group is selected from the group consisting of haloacetyl, maleimide, iodoacetamide, aziridine, acryloyl, arylating agent, vinyl sulfone, methanethiosulfonate, pyridyldisulfide and TNB-thiol. In some embodiments, the thiol-reactive group is maleimide. In some embodiments, the amine-reactive group is selected from the group consisting of NHS ester, isothiocyanate, acyl azide, sulfonyl chloride, sulfodichlorophenol, pentafluorophenol, tetrafluorophenol, 4-sulfo-2,3,5,6-tetrafluorophenyl, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, fluorophenol ester, sulfochlorophenol, carbodiimide, phthalimide, benzotriazole, imide ester and anhydride. In some embodiments, the carbonyl-reactive group is selected from the group consisting of hydrazine, hydrazine derivatives and amines. In some embodiments, R 2is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group, which is substituted with -CO2-maleimide or -C2-CO2-maleimide; a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-NHS ester or -C2-CO2-NHS ester; and a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-hydrazine or -C2-CO2-hydrazine, and is selected from the group consisting of. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group, which is -Me, -Et, -CO2 - , -CO2-(thiol-reactive group), -CO2-(amine-reactive group), -CO2-(carboxy-reactive group), -C2-CO2 - , -C2-CO2-(thiol-reactive group), -C2-CO2-(amine-reactive group), -C2-CO2-(carboxy-reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group substituted with BCN, DBCO, azide, or TCO. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, or halogen. In some embodiments, R 2 is selected from the following:

Chemical formula

[0012] In some embodiments, R 2 is -phosphoramidite or -O-phosphoramidite. In some embodiments, R 1are 9-fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, monomethoxytrityl (MMT), DMS, and p-toluenesulfonamide; R 2 is -phosphoramidite or -O-phosphoramidite.

[0013] A second aspect of the present disclosure is a compound selected from the group consisting of

Chemical formula

Chemical formula

[0014] A third aspect of the present disclosure is a compound selected from the group consisting of

Chemical formula

[0015] A fourth aspect of the present disclosure is a compound having formula (IA),

Chemical formula

[0016] In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with -CO2-maleimide or -C2-CO2-maleimide; a C1-C8 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with -CO2-NHS ester or -C2-CO2-NHS ester; and a C1-C8 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with -CO2 hydrazine or -C2-CO2 hydrazine, selected from the group consisting of. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a C1-C8 branched or unbranched alkyl group substituted with one or more of a group capable of participating in a "click chemistry" reaction. In some embodiments, the group capable of participating in a "click chemistry" reaction is selected from the group consisting of azide, DBCO, TCO, maleimide, and tetrazine. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, halogen, -D, or a C1-C8 branched or unbranched alkyl group substituted with one or more of a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2- is a C1-C8 branched or unbranched alkyl group substituted with one or more of -OH, halogen or -D. In some embodiments, R 2 is a phosphoramidite or -O-phosphoramidite. In some embodiments, R 2 is selected from:

Chemical formula

[0017] In some embodiments, the compounds of the fourth aspect of the present disclosure have a Stokes shift of at least about 70 nm. In some embodiments, the compounds of the fourth aspect of the present disclosure have a Stokes shift of at least about 80 nm. In some embodiments, the compounds of the fourth aspect of the present disclosure have a Stokes shift of at least about 90 nm. In some embodiments, the compounds of the fourth aspect of the present disclosure are thermally stable over a temperature range of about 25°C to about 100°C. In some embodiments of the third and fourth aspects, [X] - is selected from the group consisting of chloride, bromide, iodide, sulfate, benzenesulfonate, p-toluenesulfonate, p-bromobenzenesulfonate, methanesulfonate, trifluoromethanesulfonate, phosphate, perchlorate, tetrafluoroborate, hexafluorophosphate, tetraphenylboride, nitrate; and anions of aromatic or aliphatic carboxylic acids.

[0018] The fifth aspect of the present disclosure is a conjugate comprising (i) a specific binding entity and (ii) a dye moiety derived from a compound having any one of formulas (I), (IA), and (IB) (such as any of the compounds described herein). In some embodiments, the specific binding entity is a protein. In some embodiments, the protein is an antibody, an antibody fragment, or an enzyme. In some embodiments, the specific binding entity is an oligonucleotide. In some embodiments, the oligonucleotide comprises from about 5 to about 60 mers. In some embodiments, the dye moiety is coupled to the 5' end of the oligonucleotide. In some embodiments, the dye moiety is coupled to the 3' end of the oligonucleotide. In some embodiments, the dye moiety is derived from any one of the compounds having formula (IA).

[0019] The sixth aspect of the present disclosure is a conjugate comprising (i) a hapten and (ii) a dye moiety derived from a compound having any one of formulas (I), (IA), and (IB). In some embodiments, the hapten is pyrazole; a nitrophenyl compound; benzofurazan; triterpene; urea; thiourea; rotenone or a rotenone derivative; oxazole; thiazole; coumarin or a coumarin derivative; or a cyclolignan.

[0020] The seventh aspect of the present disclosure is a conjugate having formula (II),

Chemical formula

[0021] In some embodiments, the protein is an antibody, such as a primary antibody or a secondary antibody. In some embodiments, the oligonucleotide comprises from about 5-mer to about 60-mer. In some embodiments, the oligonucleotide comprises from about 5-mer to about 40-mer. In some embodiments, the oligonucleotide comprises from about 5-mer to about 20-mer. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 25 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 20 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y has the structure of formula (IIIC),

Chemical formula

[0022] In some embodiments, R 3 is a C1-C6 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -CO2-, -C2-CO2-, -D or halogen. In some embodiments, R 3 is a C1-C4 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -CO2-, -C2-CO2-, -D or halogen.

[0023] An eighth aspect of the present disclosure is a conjugate having any one of formula (IIC) or (IID),

Chemical formula

Chemical formula

[0024] In some embodiments, the first carbon atom of R 3 is a primary carbon atom. In some embodiments, the first carbon atom of R 3 is a secondary carbon atom. In some embodiments, R 3The first carbon atom of is a tertiary carbon atom. In some embodiments, Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 20 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, R 1 is H; and R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group, which is substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2, and -OH. In some embodiments, R 1 is H; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; and Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 30 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, R 1 is H; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; and Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 25 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, R 1 is H; R 3is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 20 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, R 1 is H; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 15 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, R 1 is H; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 10 carbon atoms and optionally having one or more heteroatoms selected from O, N or S.

[0025] A ninth aspect of the present disclosure is a kit comprising (i) a first conjugate (described herein) comprising a first oligonucleotide coupled to a dye moiety derived from a compound having any one of formulas (I), (IA) and (IB), and (ii) a second conjugate comprising an oligonucleotide coupled to a quencher). In some embodiments, the first conjugate has any one of formulas (IIC) or (IID). In some embodiments, the first conjugate is directly coupled to the dye moiety. In some embodiments, the first conjugate is indirectly coupled to the dye moiety via a linker (e.g., a substituted or unsubstituted linker having 5 to about 40 carbon atoms), for example.

[0026] The tenth aspect of the present disclosure is a probe having formula (IV): [Dye 1]-[Y] a -[5'-oligonucleotide-3']-[Y] a -[Dye 2] (IV), wherein one of [Dye 1] or [Dye 2] is derived from any one of the compounds of formula (I), (IA), and (IB); the other of [Dye 1] or [Dye 2] is a quencher; the oligonucleotide is an oligonucleotide having about 5 to about 60 mers; each Y is independently a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 2 to about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and a is 0, 1, or 2.

[0027] In some embodiments, one of [Dye 1] or [Dye 2] is derived from a compound having formula (IA): [Chemical formula] wherein R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, or halogen.

[0028] In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, or halogen. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, or halogen. In some embodiments, the first carbon atom of R 2 is a primary carbon atom. In some embodiments, R 2The first carbon atom of [compound name] is a secondary carbon atom. In some embodiments, R 2 The first carbon atom of [compound name] is a tertiary carbon atom. In some embodiments, R 2 is selected from the following:

Chemical formula

[0029] The eleventh aspect of the present disclosure is a conjugate having formula (V), [(oligomer 1)(dye)]-linker-[(oligomer 2)(Q1)] (V), wherein oligomers 1 and 2 are each different and are oligonucleotides having from about 5 mers to about 30 mers; the dye is derived from a compound having any one of formulas (I), (IA), and (IB); Q1 is a quencher; and the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having from about 5 to about 30 carbon atoms.

[0030] In some embodiments, the dye is derived from a compound having formula (IA),

Chemical formula

[0031] In some embodiments, the dye has a Stokes shift of at least about 70 nm. In some embodiments, the dye has a Stokes shift of at least about 80 nm. In some embodiments, the dye has a Stokes shift of at least about 90 nm. In some embodiments, at least one of oligomers 1 and 2 comprises LNA, L-LNA, or PNA. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 25 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 20 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 15 carbon atoms.

[0032] In a twelfth aspect of the present disclosure, a kit comprising: (i) a conjugate having any one of formulas (IIC) and (IID); and (ii) a compound having formula (VIII), [Oligomer 3]-[Q2] (VIII), wherein oligomer 3 is an oligonucleotide having 5 to 30 mers; and Q2 is a quencher.

[0033] A thirteenth aspect of the present disclosure is a FRET pair comprising a first member having formula (VIIA) and a second member having formula (VIIB), [Dye 1]-[Y]a -[5’-oligonucleotide 1-3’] (VIIA), [5’-oligonucleotide 2-3’]-[Y] a -[Dye 2] (VIIB), wherein, one of Dye 1 or Dye 2 is derived from a compound having any one of formula (I), (IA), and (IB); the other of Dye 1 or Dye 2 is a quencher; each Y is independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; a is 0, 1, or 2; and oligonucleotide 1 and oligonucleotide 2 are different.

[0034] In some embodiments, each Y is independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, one of Dye 1 or Dye 2 is derived from a compound having formula (IA), [Chemical formula] wherein, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D or halogen. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, or halogen. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, or halogen. In some embodiments, R 2The first carbon atom of [the relevant entity] is a primary carbon atom. In some embodiments, R 2 The first carbon atom of [the relevant entity] is a secondary carbon atom. In some embodiments, R 2 The first carbon atom of [the relevant entity] is a tertiary carbon atom. In some embodiments, R 2 is selected from the following: [Chemical formula]

[0035] A fourteenth aspect of the present disclosure is a method for amplifying and detecting a target nucleic acid in a sample, comprising the following steps: (a) contacting a sample containing the target nucleic acid in a single reaction vessel with (i) a pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to the opposite strand of a sub-sequence of the target nucleic acid; and (ii) an oligonucleotide probe comprising an annealing portion and a tag portion, the tag portion comprising a nucleotide sequence non-complementary to the target nucleic acid sequence, the annealing portion comprising a nucleotide sequence at least partially complementary to the target nucleic acid sequence, the oligonucleotide probe hybridizing to the region of the sub-sequence of the target nucleic acid to which the pair of oligonucleotide primers binds, and the probe further comprising an interaction dual-label comprising a dye derived from a compound having formula (I) located on the tag portion and a first quencher portion located on the annealing portion, the dye being separated from the first quencher portion by a nuclease-sensitive cleavage site; and prior to step (b) (enumerated below), the tag portion is reversibly and temperature-dependently bound to a quenching oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe, hybridizing to the tag portion, and the quenching oligonucleotide comprising at least a second quencher portion capable of quenching the dye on the tag portion when the quenching oligonucleotide is bound to the tag portion; (b) Following step (a), amplifying the target nucleic acid by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5'-to-3' nuclease activity, such that during the extension step of each PCR cycle, the nuclease activity of the polymerase enables cleavage and separation of the tag portion from the first quencher portion on the annealing portion of the probe; (c) Measuring one or more signals from the dye at a first temperature at which the quenching oligonucleotide is bound to the tag portion; (d) Measuring one or more signals from the dye at a second temperature higher than the first temperature, at which the quenching oligonucleotide is not bound to the tag portion; and (e) Obtaining a calculated signal value by subtracting the median or average value of the one or more signals detected at the first temperature from the median or average value of the one or more signals detected at the second temperature; Thereby, a calculated signal value higher than the threshold signal value enables determination of the presence of the target nucleic acid.

[0036] In some embodiments, the PCR amplification of step (b) can reach the end point beyond the logarithmic phase of amplification. In some embodiments, the tag portion includes modifications such that it cannot be extended by the nucleic acid polymerase. In some embodiments, the tag portion of the oligonucleotide probe, or the quenching oligonucleotide, or both the tag portion and the quenching oligonucleotide contain one or more nucleotide modifications. In some embodiments, the one or more nucleotide modifications are selected from the group consisting of locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA), 2'-O alkyl substitution, L-enantiomer nucleotides, and combinations thereof.

[0037] A fifteenth aspect of the present disclosure is a method for directly labeling a dye with an oligonucleotide having a terminal amine, comprising: (i) obtaining a dye comprising a dye core and having a cyano group disposed at the meso position of the dye core; (ii) contacting the obtained dye with an oligonucleotide having a terminal amine in the presence of a base and a solvent, wherein the linker is located between the oligonucleotide and the terminal amine, and the linker is a C1-C8 alkyl group, heteroalkyl group, cycloalkyl group or heterocycloalkyl group substituted with one or more of -Me, -Et, -CO2-, -C2-CO2-, -D or halogen.

[0038] In some embodiments, the base is selected from the group consisting of N,N-diisopropylethylamine (DIPEA), cesium carbonate, potassium carbonate, sodium carbonate, tributylamine (TBA), N,N-dicyclohexylmethylamine, 2,6-di-tert.-butylpyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]nona-5-ene (DBN), 1,1,3,3-tetramethylguanidine (TMG), and 2,2,6,6-tetramethylpiperidine. In some embodiments, the solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), sulfolane, N-butylpyrrolidone, γ-butyrolactone, δ-butyrolactone, N-methylpyrrolidone, N,N-dimethylformamide, sulfolane, and silene. In some embodiments, the oligonucleotide comprises from about 5-mers to about 60-mers. In some embodiments, the oligonucleotide comprises from about 5-mers to about 40-mers. In some embodiments, the oligonucleotide comprises from about 5-mers to about 30-mers. In some embodiments, the oligonucleotide comprises from about 5-mers to about 20-mers. In some embodiments, the oligonucleotide comprises from about 5-mers to about 15-mers. In some embodiments, the oligonucleotide comprises LNA, L-LNA or PNA. In some embodiments, the dye having a cyano group located at the meso position of the dye core is an R800 perchlorate dye.

Brief Description of the Drawings

[0039] For a general understanding of the features of the present disclosure, reference is made to the drawings. In the drawings, like reference numerals are used throughout to identify like elements.

[0040]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0041] Conversely, it should also be understood that in any method recited in the claims herein that includes a plurality of steps or acts, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited, unless expressly indicated otherwise.

[0042] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" is defined inclusively such that "comprising A or B" means including A, B, or both A and B.

[0043] As used in this specification and the claims, it should be understood that "or" has the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is to be construed as inclusive, that is, including at least one of the elements or items in the list, but also including a plurality and optionally additional items not listed in the list. Only terms such as "only one of" or "exactly one of" or, when used in the claims, "consisting of", which are clearly indicated to the contrary, refer to exactly one element of a number or list of elements. Generally, the term "or" as used herein is to be construed as indicating an exclusive alternative (i.e., "one or the other but not both") only when preceded by an exclusive term such as "either", "only one of", "only one of only" or "exactly one of". "Consisting essentially of", when used in the claims, shall have the ordinary meaning as used in the field of patent law.

[0044] Terms such as "comprising", "including", "having", etc. are used interchangeably and have the same meaning. Similarly, "comprises", "includes", "has", etc. are used interchangeably and have the same meaning. Specifically, each term is defined in accordance with the general definition of U.S. patent law of "comprising", and thus is construed as an open term meaning "at least the following", and is also construed not to exclude additional features, limitations, aspects, etc. Thus, for example, a "device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c. Further, steps and processes may be outlined herein in a particular order, but those skilled in the art will recognize that the ordered steps and processes may vary.

[0045] As used in the specification and claims of this specification, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of every element specifically listed within the list of elements, nor does it exclude any combinations of elements within the list of elements. This definition also allows for the possibility that elements other than those specifically identified within the list of elements referred to by the phrase "at least one" may optionally exist, whether or not they are related to those specifically identified elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") can, in one embodiment, refer to at least one, optionally two or more, of A and no B (and optionally includes elements other than B), in another embodiment can refer to at least one, optionally two or more, of B and no A (and optionally includes elements other than A), and in yet another embodiment can refer to at least one, optionally two or more, of A and at least one, optionally two or more, of B (and optionally includes other elements), and so on.

[0046] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0047] As used in this specification, the symbol

Chemical Structure

[0048] As used herein, the term "alkyl" includes saturated aliphatic groups including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl group-substituted cycloalkyl groups, and cycloalkyl group-substituted alkyl groups. The term alkyl further includes alkyl groups that may contain one or more heteroatoms such as oxygen, nitrogen, sulfur, or phosphorus atoms that replace one or more carbons of the hydrocarbon backbone. In certain embodiments, straight-chain or branched-chain alkyl has 8 or fewer carbon atoms in its backbone (e.g., 1-C8 for straight-chain and C1-C8 for branched-chain). Further, the term alkyl includes both "unsubstituted alkyl" and "substituted alkyl", the latter referring to an alkyl moiety having a substituent that replaces a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties.

[0049] As used herein, the term "amine-reactive group" refers to a reagent or group that can form a covalent bond with an amine group or another molecule.

[0050] As used herein, the term "antibody" refers to an immunoglobulin or an immunoglobulin-like molecule, and by way of example, but not limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate (such as in mammals such as humans, goats, rabbits and mice), and antibody fragments that specifically bind to a molecule of interest (or a group of molecules that are very similar to the molecule of interest) to substantially eliminate binding to other molecules. An antibody further refers to a polypeptide ligand that includes at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. An antibody may be composed of heavy and light chains, each of which has a variable region called a variable heavy (VH) region and a variable light (VL) region. Collectively, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. The term antibody includes intact immunoglobulins, as well as variants and portions thereof well known in the art.

[0051] As used herein, "C" where "a" and "b" are integers a -C b"a" to "b" refers to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl, cycloalkynyl or aryl group, or the total number of carbon atoms and heteroatoms in a heteroalkyl, heterocyclyl, heteroaryl or heteroaricyclic group. That is, the ring of an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl or heteroaricyclic group can contain "a" to "b" carbon atoms. Thus, for example, a "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2, CH3CH2CH(CH3)- and (CH3)3C-.

[0052] As used herein, the term "carbonyl-reactive group" refers to a reagent or group that can form a covalent bond with a carbonyl group or another molecule.

[0053] As used herein, the term "click chemistry" refers to a chemical philosophy uniquely defined by the groups of Sharpless and Meldal, and describes chemistry tuned to rapidly and reliably generate substances by joining small units together. "Click chemistry" has been applied to a collection of reliable and autonomous organic reactions (Kolb, H.C., Finn, M.G.; Sharpless, K.B. Angew. Chem. Int. Ed. 2001, 40, 2004 - 2021). For example, copper-catalyzed azide-alkyne [3+2] cycloaddition has been identified as a reliable molecular bond in water (Rostovtsev, V.V.; et al. Angew. Chem. Int. Ed. 2002, 41, 2596 - 2599), and has been used to reinforce the investigation of some types of biomolecular interactions (Wang, Q.; et al. J. Am. Chem. Soc. 2003, 125, 3192 - 3193; Speers, A.E.; et al. J. Am. Chem. Soc. 2003, 125, 4686 - 4687; Link, A.J.; Tirrell, D.A. J. Am. Chem. Soc. 2003, 125, 11164 - 11165; Deiters, A.; et al. J. Am. Chem. Soc. 2003, 125, 11782 - 11783). Furthermore, applications to organic synthesis (Lee, L.V.; et al. J. Am. Chem. Soc. 2003, 125, 9588 - 9589), drug discovery (Kolb, H.C.; Sharpless, K.B. Drug Disc. Today 2003, 8, 1128 - 1137; Lewis, W.G.; et al. Angew. Chem. Int. Ed. 2002, 41, 1053 - 1057), and surface functionalization (Meng, J.-C.; et al. Angew. Chem. Int. Ed. 2004, 43, 1255 - 1260; Fazio, F.; et al. J. Am. Chem. Soc. 2002, 124, 14397 - 14402; Collman, J.P.; et al. Langmuir 2004, ASAP, in press; Lummerstorfer, T.; Hoffmann, H.J. Phys. Chem. B 2004, in press) have also emerged.Generally, "click chemistry" promotes reactions with modular applications that are broad in scope, have high chemical yields, produce harmless by-products, are chemoselective, require simple reaction conditions, use starting materials and reagents that are readily available, are solvent-free or use mild solvents (such as water), are easy to separate products, have a large thermodynamic driving force favorable for reaction with a single reaction product, and have high atom economy. While certain general criteria may be inherently subjective, it is not necessary to meet all criteria.

[0054] As used herein, the term "conjugate" refers to two or more molecules or moieties (including macromolecules or supramolecular molecules) that are covalently attached to a larger construct. In some embodiments, the conjugate includes one or more biomolecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins, etc.) covalently attached to one or more other molecular moieties.

[0055] As used herein, the terms "couple", "coupled", or "coupling" refer to the joining, bonding (such as covalent bonding), or linking of one molecule or atom to another molecule or atom.

[0056] As used herein, "derivative" is used according to its plain ordinary meaning in chemistry and biology and refers to a compound that is structurally similar to another compound (i.e., the so-called "reference" compound), but whose composition is different, for example, when an atom is replaced by an atom of a different element, or in the presence of a particular functional group, or when a particular functional group is replaced by another functional group, or in the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is similar or equivalent in function and appearance to the reference compound, but whose structure or origin is neither similar nor equivalent.

[0057] As used herein, the term "heteroatom" means including boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si). As described herein, in some embodiments, a "heterocyclic ring" may contain one or more heteroatoms. In other embodiments, an aliphatic group may contain one or more heteroatoms or may be substituted with one or more heteroatoms.

[0058] As used herein, the term "oligonucleotide" refers to a linear oligomer of natural or modified nucleoside monomers linked by phosphodiester bonds or analogs thereof. Examples of oligonucleotides include deoxyribonucleosides, ribonucleosides, their anomeric forms, peptide nucleic acids (PNA), etc., which can specifically bind to a target nucleic acid. Usually, the monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from several monomer units (e.g., 3 - 4) to dozens of monomer units (e.g., 40 - 60). When an oligonucleotide is represented by a sequence of letters such as "ATGCCTG", unless otherwise specified, the nucleotides are in the 5'-3' order from left to right, where "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents deoxythymidine, and "U" represents uridine which is a ribonucleoside. Usually, oligonucleotides contain four natural deoxynucleotides, but they can also contain ribonucleosides or unnatural nucleotide analogs as described above. When an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity (e.g., single-stranded DNA, RNA / DNA duplex, etc.), the selection of an appropriate composition of the oligonucleotide or polynucleotide substrate is within the knowledge of a person skilled in the art.

[0059] As used herein, the term "phosphoramidite" refers to a trivalent phosphorus group typically used in oligonucleotide synthesis. A detailed description of the chemistry used to form oligonucleotides by the phosphoramidite method is provided in U.S. Patent Nos. 4,458,066 and 4,415,732 to Caruthers et al., Caruthers et al., Genetic Engineering, 4:1-17 (1982); Users Manual Model 392 and 394 Polynucleotide Synthesizers, pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (1991) (each of which is incorporated herein by reference in its entirety).

[0060] As used herein, the term "primary antibody" refers to an antibody that specifically binds to a target protein antigen in a tissue sample. The primary antibody is typically the first antibody used in immunohistochemical techniques.

[0061] As used herein, the term "protecting group" refers to a moiety that, when attached to a reactive group in a molecule, masks, reduces, or prevents the reactivity thereof. A "protected" molecule has one or more reactive groups (e.g., hydroxyl, amino, thiol, etc.) protected by a protecting group. Examples of protecting groups can be found in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York, 1999, Harrison and Harrison et al. Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996), and "Protection of Nucleosides for Oligonucleotide Synthesis", Current Protocols in Nucleic Acid Chemistry, ed. by Boyle, A.L., John Wiley & Sons, Inc., 2000, New York, N.Y. (all of which are hereby incorporated by reference in their entirety).

[0062] As used herein, the term "reactive group" or "reactive functional group" refers to a functional group that can chemically associate, interact, hybridize, hydrogen bond, or couple with a functional group of a different moiety. In some embodiments, a "reaction" between two reactive groups or two reactive functional groups can mean that a covalent bond is formed between the two reactive groups or two reactive functional groups, or that the two reactive groups or two reactive functional groups associate with each other, interact with each other, hybridize with each other, hydrogen bond with each other, etc. Thus, in some embodiments, a "reaction" includes binding events such as the binding of a hapten to an anti-hapten antibody, or a guest molecule that associates with a supramolecular host molecule.

[0063] As used herein, the term "secondary antibody" in this specification refers to an antibody that specifically binds to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent (e.g., a label, an enzyme, etc.) if a subsequent reagent is present. A secondary antibody is generally the second antibody used in immunohistochemical techniques.

[0064] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules characterized by binding to each other while substantially excluding binding to other molecules (e.g., a specific binding pair can have a binding constant that is at least 10 3 M -1 times greater, 10 4 M -1 times greater or 10 5 M -1 times greater). Specific examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidin such as streptavidin, and protein A). A specific binding moiety can also include a molecule (or a portion thereof) that is specifically bound by such a specific binding protein.

[0065] As used herein, the term "Stokes shift" refers to the difference (in units of wavelength or frequency) between the positions of the band maxima of the absorption and emission spectra of the same electronic transition (fluorescence and Raman are two examples). When a system (whether a molecule or an atom) absorbs a photon, it gains energy and enters an excited state. One way for the system to relax is to emit a photon and thus lose that energy.

[0066] Most small molecule fluorophores are thought to exhibit Stokes shifts on the order of 10 - 25 nm. Fluorophores with significantly large Stokes shifts are collectively referred to as "large Stokes shift" (LSS) dyes, "high Stokes shift" dyes, or "megastokes" dyes. Two photophysical mechanisms have been discussed in the literature to explain the occurrence of large Stokes shifts. The molecular geometry mechanism is based on the conformational relaxation of the excited state fluorophore and the resulting rearrangement of the surrounding solvent dipoles. The Stokes shift grows as the difference between the (equilibrated) molecular geometries and dipole moments in the ground and excited states increases. Large Stokes shift fluorescence for the electronic mechanism is due to intramolecular charge transfer (ICT) in the excited state. A common problem with small Stokes shift fluorophores is internal quenching of fluorescence. Such self - quenching is caused by spectral overlap of excitation and emission and is common especially at high fluorophore concentrations. LSS dyes have better - separated spectral bands and minimize photon re - absorption. The probability that a fluorophore is excited outside its main excitation peak is not zero. As a result, fluorescence from one dye necessarily contributes to the total light detected in multiple emission channels. This spectral "crosstalk" or "bleed - through" can be compensated for computationally to some extent by using a given correction factor. Additionally, scattering of the excitation light increases the background fluorescence in adjacent channels. LSS dyes make it possible to reduce or even avoid crosstalk and scattering from other fluorophores. LSS dyes are particularly useful in experimental environments where many fluorophores produce a strong background signal. Such large spectral separation as in LSS dyes allows for more effective filtering of the excitation light, thereby enhancing the sensitivity of target detection. LSS dyes provide access to fluorescence data from optical channels that were previously inaccessible. Facilitated by the wide spectral separation and used in combination with standard fluorophores, LSS dyes make it possible to enhance the multiplexing ability of fluorescence PCR devices.In this way, the LSS label enables the implementation of additional channels to established 4-6 color devices. In principle, the 21 channels can be composed from combinations of filters of a 6-color device. However, in practice, the number of channels is limited by the commercial availability of LSS dyes with a sufficiently large Stokes shift. Based on the 150 nm Stokes shift of currently commercially available LSS dyes, nine additional channels can be implemented. The channels of standard dyes are highlighted in light gray, while dark gray indicates channels for which no suitable LSS dye is currently available. Alternatively, resonance energy transfer (RET) probes can also be used to generate a large “virtual” Stokes shift and access these channels.

[0067] Whenever a group or moiety is described as "substituted" or "optionally substituted", the group can be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as "substituted or unsubstituted" if it is already substituted, the substituent can be selected from one or more of the indicated substituents. When no substituents are indicated, an indicated "optionally substituted" or "substituted" group can be substituted with one or more groups individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroaricyclic, aralkyl, heteroaralkyl, (heteroaricyclic)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, amino, ether, amino (e.g., a mono-substituted amino group or a di-substituted amino group), and protected derivatives thereof. Any of the above groups may contain one or more heteroatoms including O, N or S. For example, if a moiety is substituted with an alkyl group, the alkyl group may contain a heteroatom selected from O, N or S (e.g., -(CH2-CH2-O-CH2-CH2)-).

[0068] As used herein, the term "thiol-reactive group" refers to a thiol group or a reagent or group that can form a covalent bond with another molecule.

[0069] Summary The present disclosure relates to dyes such as dyes having a large Stokes shift. The present disclosure also relates to conjugates and probes comprising one or more of the disclosed dyes. The present disclosure also provides a kit comprising one or more of the disclosed dyes; or one or more conjugates comprising one or more of the disclosed dyes. The dyes of the present disclosure can be used with any fluorescence-based PCR platform equipped with a compatible optical filter. Conjugates comprising or derived from one of the dyes disclosed herein are also compatible with PCR using TAGS (Temperature assisted generation of signal) technology, provided that the dye exhibits stable fluorescence at temperatures up to 100° C. (see U.S. Pat. Nos. 11,028,433, 11,034,997, and 11,345,958; and U.S. Patent Application Publication No. 2021 / 0269857, the disclosures of which are incorporated herein by reference in their entireties).

[0070] A common problem with fluorophores having a "small" Stokes shift is internal quenching of fluorescence. Such self-quenching is caused by spectral overlap of excitation and emission and is common particularly at high fluorophore concentrations. "Large" Stokes shift dyes, such as those of the present disclosure, generally have more well-separated spectral bands and minimize photon reabsorption.

[0071] The probability that a fluorophore is excited outside its main excitation peak is thought not to be zero. As a result, fluorescence from one dye necessarily contributes to the total light detected in multiple emission channels. This spectral "crosstalk" or "bleed-through" can be computationally compensated for to some extent by using a given correction factor. Additionally, scattering of the excitation light increases the background fluorescence in adjacent channels. "Large" Stokes shift dyes, such as those of the present disclosure, enable reduction of crosstalk and scattering from other fluorophores. "Large" Stokes shift dyes, such as those of the present disclosure, are particularly useful in experimental environments where many fluorophores produce a strong background signal. The large spectral separation of "large" Stokes shift dyes, such as those of the present disclosure, is thought to enable more effective filtering of the excitation light, thereby enhancing the sensitivity of target detection.

[0072] "Large" Stokes shift dyes, such as those of the present disclosure, also provide access to fluorescence data from optical channels that were previously inaccessible. Facilitated by their broad spectral separation, when used in combination with standard fluorophores, "large" Stokes shift dyes, such as those of the present disclosure, enable an increase in the multiplexing ability of fluorescence PCR devices by adding additional channels to established 4- to 6-color instruments. In principle, 21 channels are available from the combination of filters of a 6-color instrument. However, in practice, the number of channels is limited by the availability of dyes with appropriate spectral characteristics and a sufficiently large Stokes shift.

[0073] Dye or dye precursor The present disclosure provides a compound having formula (I),

Chemical formula

[0074] In some embodiments, [X] is chloride, bromide, iodide, sulfate, benzenesulfonate, p-toluenesulfonate, p-bromobenzenesulfonate, methanesulfonate, trifluoromethanesulfonate, phosphate, perchlorate, tetrafluoroborate, hexafluorophosphate, tetraphenylboride, nitrate, and the anion of an aromatic or aliphatic carboxylic acid. In some embodiments, R 1 is H. In other embodiments, R 1 is 9-fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, monomethoxytrityl (MMT), DMS, and p-toluenesulfonamide. In some embodiments, the first carbon atom of R 2 is a primary carbon atom. In other embodiments, the first carbon atom of R 2 is a secondary carbon atom. In still other embodiments, the first carbon atom of R 2 is a tertiary carbon atom. In some embodiments, R 2 is unsubstituted. In other embodiments, R 2 contains one substitution. In other embodiments, R 2 contains two or more substitutions. In some embodiments, R 2includes a heteroalkyl group having a heteroatom selected from O, N, or S. In some embodiments, R 2 includes a heteroalkyl group having an O heteroatom. In some embodiments, a group capable of participating in a "click chemistry reaction" may be selected from a bicyclo[6.1.0]nonyne group ("BCN"), dibenzocyclooctyne ("DBCO"), alkene, trans-cyclooctene ("TCO"), maleimide, aldehyde, ketone, azide, tetrazine, thiol, 1,3-nitrone, hydrazine, or hydroxylamine. In some embodiments, R 1 is H; R 2is one of BCN, DBCO, TCO, azide, tetrazine, or maleimide. In some embodiments, the compound of formula (I) ends with one of a thiol-reactive group, an amine-reactive group, or a carboxy-reactive group. In some embodiments, the thiol-reactive group is selected from haloacetyl, maleimide, iodoacetamide, aziridine, acryloyl, arylating agent, vinyl sulfone, methanethiosulfonate, pyridyl disulfide, TNB-thiol, and disulfide reducing agent. In some embodiments, the thiol-reactive group can include maleimide. In some embodiments, the thiol-reactive group can include haloacetyl. In some embodiments, the thiol-reactive group can include aziridine. In some embodiments, the thiol-reactive group can include acryloyl. In some embodiments, the thiol-reactive group can include arylating agent. In some embodiments, the thiol-reactive group can include vinyl sulfone. In some embodiments, the thiol-reactive group can include pyridyl disulfide. In some embodiments, the thiol-reactive group can include TNB-thiol. In some embodiments, the thiol-reactive group can include disulfide reducing agent. In some embodiments, the amine-reactive group is selected from NHS esters (e.g., NHS, sulfo-NHS, N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide), isothiocyanates, acyl azides, sulfonyl chlorides, sulfodichlorophenol, pentafluorophenol, tetrafluorophenol, 4-sulfo-2,3,5,6-tetrafluorophenyl, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, fluorophenol esters, sulfochlorophenol, uranyl, carbodiimides, phthalimides, benzotriazoles, imide esters, anhydrides, etc. In some embodiments, the carbonyl-reactive group is hydrazine, a hydrazine derivative, or an amine. In some embodiments, R 2is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-maleimide. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-maleimide. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-maleimide. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -C2-CO2-maleimide. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -C2-CO2-maleimide. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -C2-CO2-maleimide. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-NHS ester. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-NHS ester. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-NHS ester. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -C2-CO2-NHS ester. In some embodiments, R 2is a C1-C6 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with a -C2-CO2-NHS ester. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with a -C2-CO2-NHS ester. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with a -CO2-hydrazine. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with a -CO2-hydrazine. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with a -CO2-hydrazine. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with a -C2-CO2-hydrazine. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with a -C2-CO2-hydrazine. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with a -C2-CO2-hydrazine. In some embodiments, R 2 is -Me, -Et, -CO2 - -OH, -phosphoramidite, -O-phosphoramidite, -D, or a C1-C8 branched or unbranched alkyl group substituted with one or more groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 -, -OH, -D, halogen, or a C1-C8 branched or unbranched alkyl group substituted with one or more of groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, or halogen. In some embodiments, R2 is a C1-C6 branched or unbranched alkyl group substituted with BCN, DBCO, or TCO. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group, which is -Me, -Et, -CO2 - , -CO2-(thiol-reactive group), -CO2-(amine-reactive group), -CO2-(carboxy-reactive group), -C2-CO2 - , -C2-CO2-(thiol-reactive group), -C2-CO2-(amine-reactive group), -C2-CO2-(carboxy-reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or one or more of groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, or a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, halogen, or a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C6 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, or halogen. In some embodiments, R 2is a C1-C6 branched or unbranched alkyl group substituted with BCN, DBCO, or TCO. In some embodiments, R 2 is -Me, -Et, -CO2 - , -CO2-(thiol-reactive group), -CO2-(amine-reactive group), -CO2-(carboxy-reactive group), -C2-CO2 - , -C2-CO2-(thiol-reactive group), -C2-CO2-(amine-reactive group), -C2-CO2-(carboxy-reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a C1-C4 branched or unbranched alkyl group, branched or unbranched heteroalkyl group, or cycloalkyl group substituted with one or more of the groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, or a C1-C4 branched or unbranched alkyl group substituted with one or more of the groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -D, halogen, or a C1-C4 branched or unbranched alkyl group substituted with one or more of the groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -D, or a C1-C4 branched or unbranched alkyl group substituted with one or more of halogen. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group substituted with BCN, DBCO, or TCO. In some embodiments, R 2 is selected from the following:

Chemical formula

[0075] Examples of the compound of formula (I) include, but are not limited to: [Chemical] In the formula, "Dye" is [Chemical] as follows.

[0076] In some embodiments, the compound of formula (I) [Chemical] includes.

[0077] In some embodiments, the compound of formula (I) has the structure of formula (IA), [Chemical] wherein R 2 and [X] are as defined above.

[0078] In some embodiments, the first carbon atom of R 2 is a primary carbon atom. In other embodiments, the first carbon atom of R 2 is a secondary carbon atom. In still other embodiments, the first carbon atom of R 2 is a tertiary carbon atom. In some embodiments, R 2 is substituted with a group capable of participating in a "click chemistry reaction" such as BCN, DBCO, TCO, maleimide, aldehyde, ketone, azide, tetrazine, thiol, 1,3-nitrone, hydrazine, or hydroxylamine. In some embodiments, R 2 is substituted with an azide moiety. In some embodiments, R 2 is substituted with a DBCO moiety. In some embodiments, R 2 is substituted with a TCO moiety. In some embodiments, R 2is substituted with a maleimide moiety. In some embodiments, the compound of formula (IA) terminates with one of a thiol-reactive group, an amine-reactive group, or a carboxy-reactive group. In some embodiments, the thiol-reactive group is selected from haloacetyl, maleimide, iodoacetamide, aziridine, acryloyl, arylating agent, vinyl sulfone, methanethiosulfonate, pyridyldisulfide, TNB-thiol, and disulfide reducing agent. In some embodiments, the amine-reactive group is selected from NHS esters (e.g., NHS, sulfo-NHS, N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide), isothiocyanate, acyl azide, sulfonyl chloride, sulfodichlorophenol, pentafluorophenol, tetrafluorophenol, 4-sulfo-2,3,5,6-tetrafluorophenyl, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, fluorophenol ester, sulfochlorophenol, uranyl, carbodiimide, phthalimide, benzotriazole, imido ester, anhydride, etc. In some embodiments, the carbonyl-reactive group is hydrazine, a hydrazine derivative, or an amine. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-maleimide. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -C2-CO2-maleimide. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -CO2-NHS ester. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with -C2-CO2-NHS ester. In some embodiments, R 2is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with a group capable of participating in a "click chemistry" reaction. In some embodiments, the group capable of participating in a "click chemistry" reaction is BCN, DBCO or TCO. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, or a C1-C8 branched or unbranched alkyl group substituted with one or more of the groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -D, halogen, or a C1-C8 branched or unbranched alkyl group substituted with one or more of the groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, halogen, or a C1-C8 branched or unbranched alkyl group substituted with one or more of -D. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, or a C1-C8 branched or unbranched alkyl group substituted with one or more of the groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -D, halogen, or a C1-C8 branched or unbranched alkyl group substituted with one or more of the groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, halogen, or a C1-C8 branched or unbranched alkyl group substituted with one or more of -D. In some embodiments, R 2 is -Me, -Et, -CO2 -, -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a C1-C4 branched or unbranched alkyl group substituted with one or more groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, -D, halogen, or a C1-C4 branched or unbranched alkyl group substituted with one or more groups capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is -Me, -Et, -CO2 - , -OH, halogen, or a C1-C4 branched or unbranched alkyl group substituted with one or more of -D.

[0079] Examples of the compounds of formula (IA) include, but are not limited to, those shown in Tables 1a, 1b and 1c.

[0080]

Table 1a

[0081]

Table 1b

[0082]

Table 1c

[0083] In some embodiments, the compound of formula (I) has the structure of formula (IB),

Chemical formula

[0084] In some embodiments, R 2 's first carbon atom is a primary carbon atom. In some embodiments, R 2 's first carbon atom is a secondary carbon atom. In some embodiments, R 2 's first carbon atom is a tertiary carbon atom. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with a -CO2-NHS ester. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with a -C2-CO2-NHS ester. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with a -CO2-maleimide. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with a -C2-CO2-maleimide. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group substituted with a group capable of participating in a "click chemistry" reaction. In some embodiments, the group capable of participating in a "click chemistry" reaction is BCN, DBCO or TCO. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, or a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 -is a C1-C8 branched or unbranched alkyl group substituted with one or more of -OH, -D, halogen, or a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - -OH, halogen, or -D. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - -OH, -phosphoramidite, -O-phosphoramidite, -D, or a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - -OH, -D, halogen, or a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - -OH, halogen, or -D. In some embodiments, R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2-, -OH, -D, halogen, or a group capable of participating in a "click chemistry" reaction. In some embodiments, R 2 is a C1-C4 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - -OH, halogen, or -D. In some embodiments, R 2 is -phosphoramidite or -O-phosphoramidite.

[0085] Non-limiting examples of the compounds of formula (IB) are shown in Tables 2a, 2b and 2c:

[0086] [Table 2a]

[0087] [Table 2b]

[0088] [Table 2c]

[0089] In some embodiments, R 1 is trifluoroacetamide, and R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a group capable of participating in a "click chemistry" reaction. In other embodiments, R 1 is trifluoroacetamide, and R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, -D, halogen, or a group capable of participating in a "click chemistry" reaction. In yet other embodiments, R 1 is trifluoroacetamide, and R 2 is a C1-C8 branched or unbranched alkyl group substituted with one or more of -Me, -Et, -CO2 - , -OH, halogen, or -D. In some embodiments, R 1 is trifluoroacetamide, and R 2 is -phosphoramidite or -O-phosphoramidite. In some embodiments, R 1 is trifluoroacetamide, and R 2 is selected from the group consisting of: [Chem.]

[0090] In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 50 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 55 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 60 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 65 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 70 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 75 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 80 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 85 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 90 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 95 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 100 nm. In some embodiments, any one of the compounds of formula (I), (IA) and (IB) has a Stokes shift of at least about 105 nm. In some embodiments, the compound of formula (I) has a Stokes shift of at least about 110 nm.

[0091] [Table 3]

[0092] Table 3 above summarizes the spectroscopic properties including the Stokes shifts of some compounds having formula (IA). The column "Amino linker reagent" corresponds to the primary amines that react with the R800 dye to give compound R and 1a - 1s of the present disclosure. The absorption maximum and emission maximum, and the resulting Stokes shift are shown in nanometers. The UPLC peak area of the fluorescence peak was divided by the peak area of the absorption peak to obtain an estimated brightness value of the dye. Since compound 1b was unstable and compound 1s did not show properties towards the LSS dye (11 nm Stokes shift), the brightness data was not determined (n.d.).

[0093] In some embodiments, the dyes having any one of formulas (I), (IA), and (IB) are thermally stable. For example, the applicant has surprisingly discovered that the dyes of formulas (I), (IA), and (IB) exhibit thermally stable fluorescence over a temperature range of about 25°C to about 100°C. This is shown in FIG. 24 which shows fluorescence as a function of temperature for compounds R, 1i, 1k, 1j, and 1n. No significant decrease in fluorescence was observed up to 100°C. For compounds R and 1n, a slightly significant drift towards higher fluorescence can be explained by the increased solubility at high temperatures due to their higher hydrophobicity compared to compounds 1i, 1k, 1j which are carboxylic acids. In some embodiments, the dyes having any one of formula (IA) are thermally stable over a temperature range of about 25°C to about 100°C. In some embodiments, compounds 1a - 1s are thermally stable over a temperature range of about 25°C to about 100°C as described herein.

[0094] Conjugate The present disclosure also provides conjugates comprising one or more of the compounds of formula (I), (IA) and (IB) and a specific binding entity, or derived therefrom. In some embodiments, one or more of the compounds having formula (I), (IA) and (IB) are directly coupled to the specific binding entity. In some embodiments, one or more of the compounds having formula (I), (IA) and (IB) are indirectly coupled to the specific binding entity. In some embodiments, the indirect coupling is via one or more linkers.

[0095] In some embodiments, the conjugate comprises a compound derived from any one of formula (I), (IA) and (IB) directly or indirectly coupled to the specific binding entity. In some embodiments, the "specific binding entity" is an oligonucleotide, an antibody, an antibody fragment, biotin or streptavidin. In some embodiments, the antibody is a primary antibody. In some embodiments, the antibody is a secondary antibody.

[0096] In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 60 mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 55 mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 50 mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 45 mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 40 mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 35 mers. In some embodiments, the oligonucleotide comprises from about 5 to about 30 mers. In some embodiments, the oligonucleotide comprises from about 5 to about 25 mers. In some embodiments, the oligonucleotide comprises from about 5 to about 20 mers. In some embodiments, the oligonucleotide comprises from about 5 to about 15 mers.

[0097] In some embodiments, the "specific binding entity" is an oligonucleotide, and the dye having the formula (I) is directly or indirectly coupled to the 5'-end of the oligonucleotide. In some embodiments, the "specific binding entity" is an oligonucleotide, and the dye having the formula (I) is directly or indirectly coupled to the 3'-end of the oligonucleotide.

[0098] In some embodiments, a conjugate comprising a compound derived from any one of formulas (I), (IA), and (IB) and a specific binding entity has the structure of formula (II),

Chemical formula

[0099] In embodiments where the specific binding entity is an oligonucleotide, the dye moiety of the conjugate can be coupled to either the 5' end or the 3' end of the oligonucleotide. In some embodiments, the oligonucleotide comprises from about 5 mers to about 40 mers, whether or not it is attached to a dye moiety at the 5' end or the 3' end. In some embodiments, R 3 is a C1-C6 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -CO2-, -C2-CO2-, -D, or halogen. In some embodiments, R 3 is a C1-C4 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -CO2-, -C2-CO2-, -D, or halogen.

[0100] In some embodiments, Y may include a carbonyl, amine, ester, ether, amide, imine, thione, or thiol group. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 25 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 20 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 15 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 10 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y has the structure of formula (IIIA), [Chemical Formula] wherein d and e are each independently an integer in the range of 2 to 20; Q is a bond, O, S, or N(R c )(R d ); R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ); R c and R d are independently CH3 or H; and A and B are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.

[0101] In some embodiments, d and e are integers in the range of 2 to 6. In some embodiments, d and e are integers in the range of 2 to 10. In other embodiments, d and e are integers in the range of 2 to 5. In some embodiments, both d and e are 1. In some embodiments, A and B are independently branched or unbranched, linear or cyclic, substituted or unsubstituted saturated or unsaturated groups having 1 to 6 carbon atoms and optionally having one or more O, N or S heteroatoms. Independently, they are branched or unbranched, linear or cyclic, substituted or unsubstituted saturated or unsaturated groups having 1 to 4 carbon atoms and optionally having one or more O, N or S heteroatoms. In some embodiments, Y has the structure of formula (IIIB),

Chemical formula

[0102] In some embodiments, Y has the structure of formula (IIIC),

Chemical formula

[0103] In some embodiments, R a and R b are each H. In some embodiments, Y is derived from: 5'-amino-modifier C6-TFA (GLEN RESEARCH catalog number 10-1916) amino-modifier C6 dT (GLEN RESEARCH catalog number 10-1039) amino-L-threoninol amidite

Chemical formula

[0104] In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-. In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; R 3 is a C1-C6 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-. In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; R 3 is a C1-C4 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-. In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; R 3is a C1-C6 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; R 3 is a C1-C4 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S.

[0105] In some embodiments, the conjugate of formula (II) has the structure of either formula (IIA) or (IIB), [Dye]-[Y] a -[5'-oligonucleotide-3'] (IIA) or [5'-oligonucleotide-3']-[Y] a -[Dye] (IIB), wherein the dye is derived from any one of formula (I), (IA) or (IB); Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having from 2 to about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is 0, 1, or 2; and the oligonucleotide is an oligonucleotide having from about 5 to about 60 mers.

[0106] In some embodiments, the dye is derived from any one of Compounds 1a - 1s (see Tables 1a, 1b, and 1c herein). In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 20 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 10 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, Y has the structure of any one of Formulas (IIIA), (IIIB), and (IIIC) described herein. In some embodiments, the oligonucleotide comprises from about 5 -mers to about 60 -mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 55 -mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 50 -mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 45 -mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 40 -mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 35 -mers. In some embodiments, the oligonucleotide comprises from about 5 to about 30 -mers. In some embodiments, the oligonucleotide comprises from about 5 to about 25 -mers. In some embodiments, the oligonucleotide comprises from about 5 to about 20 -mers. In some embodiments, the oligonucleotide comprises from about 5 to about 15 -mers.

[0107] In some embodiments, the conjugate of formula (II) has the structure of either formula (IIC) or (IID),

Chemical formula

Chemical formula

[0108] In some embodiments, the first carbon atom of R 3 is a primary carbon atom. In some embodiments, the first carbon atom of R 3 is a secondary carbon atom. In some embodiments, the first carbon atom of R 3 is a tertiary carbon atom. In some embodiments, R 3 is a C1-C6 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -CO2-, -C2-CO2-, -D, or halogen. In some embodiments, R 3is a C1-C4 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -CO2-, -C2-CO2-, -D or halogen. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 25 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 20 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 15 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 10 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, Y may include a carbonyl, amine, ester, ether, amide, imine, thione or thiol group. In some embodiments, Y has the structure of formula (IIIA), [Chemical formula] wherein d and e are each independently an integer in the range of 2 to 20; Q is a bond, O, S or N(R c )(R d ); R a and R b are independently H, a C1-C4 alkyl group, F, Cl or N(R c )(R d ); R c and R dis independently CH3 or H; A and B are independently branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated groups having between 1 and 12 carbon atoms and optionally having one or more O, N or S heteroatoms. In some embodiments, d and e are integers in the range of 2 to 6.

[0109] In some embodiments, d and e are integers in the range of 2 to 10. In other embodiments, d and e are integers in the range of 2 to 5. In some embodiments, both d and e are 1. In some embodiments, A and B are independently branched or unbranched, linear or cyclic, substituted or unsubstituted saturated or unsaturated groups having between 1 and 6 carbon atoms and optionally having one or more O, N or S heteroatoms. Independently, they are branched or unbranched, linear or cyclic, substituted or unsubstituted saturated or unsaturated groups having between 1 and 4 carbon atoms and optionally having one or more O, N or S heteroatoms. In some embodiments, Y has the structure of formula (IIIB),

Chemical formula

Chemical formula

[0110] In some embodiments, R 1 is H; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-. In some embodiments, R 1 is H; R 3 is a C1-C6 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-. In some embodiments, R 1 is H; R 3 is a C1-C4 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-. In other embodiments, R 1 is H; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In other embodiments, R 1 is H; R3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 20 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In other embodiments, R 1 is H; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 15 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In yet other embodiments, R 1 is H; R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 10 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S. In further embodiments, R 1 is H; R 3is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -D, -C2-CO2-, -CO2-, and -OH-; Y has the structure of any one of formula (IIIA), formula (IIIB), and formula (IIIC). In some embodiments, the oligonucleotide comprises from about 5-mers to about 60-mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 55-mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 50-mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 45-mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 40-mers. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises from about 5 to about 35-mers. In some embodiments, the oligonucleotide comprises from about 5 to about 30-mers. In some embodiments, the oligonucleotide comprises from about 5 to about 25-mers. In some embodiments, the oligonucleotide comprises from about 5 to about 20-mers. In some embodiments, the oligonucleotide comprises from about 5 to about 15-mers.

[0111] The present disclosure also relates to conjugates comprising a compound of formula (I) and a hapten or an enzyme (e.g., alkaline phosphatase; horseradish peroxidase). In some embodiments, the compound having formula (I) is directly coupled to the hapten or enzyme. In some embodiments, the compound having formula (I) is indirectly coupled to the hapten or enzyme. In some embodiments, the indirect coupling is via one or more linkers. In some embodiments, the hapten is a pyrazole (e.g., nitropyrazole); a nitrophenyl compound; a benzofurazan; a triterpene; a urea (e.g., phenylurea); a thiourea (e.g., phenylthiourea); rotenone or a rotenone derivative; an oxazole (e.g., oxazole sulfonamide); a thiazole (e.g., thiazole sulfonamide); a coumarin or a coumarin derivative; or a cyclo lignan. In some embodiments, the hapten is dinitrophenyl, biotin, digoxigenin, and fluorescein, and any derivatives or analogs thereof. Other haptens are described in U.S. Patent Nos. 8,846,320, 8,618,265, 7,695,929, 8,481,270, and 9,017,954, the disclosures of which are incorporated herein by reference in their entireties.

[0112] TaqMan® probe The present disclosure also provides a TaqMan® probe in which the first dye of the TaqMan® probe is derived from any one of the compounds of formula (I), (IA) and (IB), and the second dye is a quencher. For example, as known in the art, a TaqMan® assay may be performed using a TaqMan® probe. As used herein, the terms "TaqMan® probe" and "hydrolysis probe" may be understood interchangeably. In some embodiments, the first dye and the quencher derived from the compound having formula (I) are located near the ends of the probe, and in some such embodiments, the compound having formula (I) is located near the 5'-end and the quencher is located near the 3'-end. The term "3'-end" may be understood in the broadest sense understood in the art. Further, the terms "3'-end" and "3'-terminus" may be understood interchangeably as known in the art. Also, the terms "3'-end" and "3'-terminus" as used herein may refer to the 5'-end of the nucleotide chain, but it should be understood that it does not exclude the addition of another molecular moiety (e.g., fluorophore, quencher, linker, etc.) to the 3'-end of the probe at the 3'-end.

[0113] A TaqMan® probe can hybridize to its target sequence. Further, a composition comprising a TaqMan® probe can further comprise a pair of primers, such as one forward primer and one reverse primer. These primers are generally unlabeled. Further, generally, the forward primer binds upstream of the band and the reverse primer binds downstream of the band, such that the TaqMan® probe binds to a sequence that is part of the strand to be amplified. A PCR reaction well known in the art is performed. Thus, the target DNA is melted and then conditions are selected that allow annealing of the primers and probe to the target DNA. Subsequently, conditions are selected that allow DNA polymerase to amplify the DNA strand between the primers. In the context of a TaqMan® assay, the DNA polymerase generally has 5' to 3' exonuclease activity. Also, the DNA polymerase can be Taq polymerase or a functional variant thereof. When the DNA polymerase reaches the TaqMan® probe, the 5' end is cleaved. Thereby, the compound having an orb derived from formula (I), or the quencher bound to the 5' terminal nucleotide(s) is also cleaved. In some embodiments, the compound having or derived from formula (I) is cleaved. As a result, the compound having or derived from formula (I) and the quencher can diffuse in different directions. The spatial distance between both can be significantly increased and the fluorescence generated by the compound having or derived from formula (I) is significantly increased as it is no longer quenched by the dark quencher. Also, the TaqMan® assay may be analyzed in real time. The TaqMan® assay can also be performed during a real-time (life-time) PCR method. It can also be performed quantitatively in a qPCR reaction.

[0114] The TaqMan® assay using the probes of the present disclosure can be used for allele discrimination, genotyping, bacterial identification assays, DNA quantification, and determination of the amount of virus in clinical specimens, gene expression assays, and verification of microarray results. It can also be used for allele discrimination, genotyping, and bacterial identification assays. Genotyping can be, for example, single nucleotide polymorphism (SNP) genotyping, and thus includes determination of the genotype at a defined locus of interest in a sample, where the locus is a single base. Alternatively, genotyping can be copy number variant (CNV) genotyping. A copy number variant (CNV) is a segment of DNA in which a difference in copy number (the copy number of a DNA sequence or a part thereof) is found by comparison of two or more genomes. As described above, the sequences (and the loci of various SNPs and CNVs) can be obtained from databases such as The Database of Genomic Variants (DGV), NCBI dbSNP database, UCSC Genome Bioinformatics Site, DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER), HapMap Project, Sanger Institute Copy Number Variation Project and the Human Structural Variation Project.

[0115] The present disclosure provides a probe having the structure of formula (IV), for example, a TaqMan® probe, [Dye 1]-[Y] a -[5'-oligonucleotide-3']-[Y] a -[Dye 2] (IV), wherein, one of [Dye 1] or [Dye 2] is derived from any one of formula (I), (IA), or (IB); the other of Dye 1 or Dye 2 is a quencher; the oligonucleotide is an oligonucleotide having from about 5 to about 60 mers; Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having from 2 to about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is 0, 1, or 2.

[0116] In some embodiments, the quencher is a molecule that reduces the fluorescence intensity of Dye 1 or Dye 2. In other embodiments, the quencher is selected from Deep Dark Quencher DDQ-I, DABCYL, Eclipse® Dark quencher, Iowa Black® FQ, Iowa Black® RQ, Black Hole Quencher® series (BHQ-0, BHQ-1, BHQ-2, BHQ-3), QSY-7, DDQ-II, Iowa Black® RQ, QSY-21, Black Berry quencher (BBQ-650, available from LGC Biosearch); IDT dual quencher (ZEN quencher; TAO quencher); Onyx quencher (available from Milipore Sigma), and TAMRA quencher. In some embodiments, one of Dye 1 or Dye 2 has a Stokes shift of at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, etc. In some embodiments, one of [Dye 1] or [Dye 2] is derived from a compound having Formula (IA). In other embodiments, the dye is derived from a compound having Formula (IA), wherein R 2 is

Chemical formula

[0117] TAGS probe The present disclosure further provides a conjugate having the structure of Formula (V), [(Oligomer 1)(Dye)]-linker-[(Oligomer 2)(Q1)] (V), wherein Oligomers 1 and 2 are each different and are oligomers having from about 5-mers to about 30-mers; The dye is derived from any one of (I), (IA) or (IB); Q1 is a quencher; and The linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having from about 5 to about 40 carbon atoms.

[0118] In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 30 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 25 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 20 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 15 carbon atoms. In some embodiments, at least one of Oligomer 1, Oligomer 2, or the linker comprises a nuclease-sensitive cleavage site. In some embodiments, Oligomers 1 and 2 can include DNA, L-DNA, RNA, L-RNA, LNA, L-LNA, PNA (peptide nucleic acid, described in U.S. Patent No. 5,539,082 to Nielsen et al.), BNA (bridged nucleic acid, e.g., 2’,4’-BNA(NC) [2’-O,4’-C-aminomethylene-bridged nucleic acid] described in Rahman et al., J. Am. Chem. Soc. 2008;130(14):4886-96, L-BNA, etc. (where “L-XXX” refers to the L-enantiomer of the sugar unit of the nucleic acid), or any other known modifications and alterations of nucleotide bases, sugars, or the phosphodiester backbone. In some embodiments, one of Oligomer 1 or Oligomer 2 comprises or consists entirely of L-DNA. In other embodiments, Oligomer 1 comprises or consists entirely of L-DNA. In still other embodiments, Oligomer 1 consists entirely of L-DNA. In some embodiments, the linker is an unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 30 carbon atoms. In other embodiments, the linker is an unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 25 carbon atoms. In still other embodiments, the linker is an unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having from about 5 to about 25 carbon atoms.In some embodiments, the dye has a Stokes shift of at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, etc. In some embodiments, the dye is derived from a compound having formula (IA). In other embodiments, the dye is derived from a compound having formula (IA), wherein R 2 is [Chemical formula] as follows.

[0119] The present disclosure further provides an intermediate having the structure of formula (VI), (group capable of participating in click chemistry reaction)-(C2-C8)-O-[(oligonucleotide)(dye)](VI), wherein the oligonucleotide is an oligonucleotide having from about 5 to about 60 mers; and the dye is derived from any one of (I), (IA) or (IB).

[0120] In some embodiments, the dye has a Stokes shift of at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, etc. In some embodiments, the dye is derived from a compound having formula (IA). In other embodiments, the dye is derived from a compound having formula (IA), wherein R 2 is [Chemical formula] as follows.

[0121] Kit The present disclosure also provides kits comprising at least two compounds having any one of formula (IA). The present disclosure also provides kits comprising at least three compounds having any one of formula (IA). The present disclosure also provides kits comprising at least four compounds having any one of formula (IA). The present disclosure also provides kits comprising at least five compounds having any one of formula (IA). The present disclosure also provides kits comprising at least six compounds having any one of formula (IA). The present disclosure also provides kits comprising seven or more compounds having any one of formula (IA).

[0122] In some embodiments, at least one of the compounds having formula (IA) included in any kit has a Stokes shift greater than about 50 nm. In some embodiments, at least one of the compounds having formula (IA) included in any kit has a shift greater than about 60 nm. In some embodiments, at least one of the compounds having formula (IA) included in any kit has a Stokes shift greater than about 70 nm. In some embodiments, at least one of the compounds having formula (IA) included in any kit has a Stokes shift greater than about 80 nm. In some embodiments, at least one of the compounds having formula (IA) included in any kit has a Stokes shift greater than about 90 nm. In some embodiments, at least one of the compounds having formula (IA) included in any kit has a Stokes shift greater than about 100 nm. In some embodiments, at least one of the compounds having formula (IA) included in any kit has a Stokes shift greater than about 110 nm.

[0123] FRET pair The present disclosure also provides a kit comprising a FRET pair. FRET is a form of molecular energy transfer (MET), which is a process in which energy passes non-radiatively between a donor molecule and an acceptor molecule. FRET results from the properties of certain chemical compounds; when exposed to light of a specific wavelength, they emit light (i.e., fluoresce) at a different wavelength. Such compounds are called fluorophores or fluorescent labels. In FRET, energy passes non-radiatively over a long distance (e.g., 10 to 100 angstroms) between a donor molecule, which can be a fluorophore, and an acceptor molecule, which can be a quencher or another fluorophore. The donor absorbs a photon and transfers this energy non-radiatively to the acceptor (Forster, 1949, Z. Naturforsch. A4: 321 - 327; Clegg, 1992, Methods Enzymol. 211: 353 - 388).

[0124] When two fluorophores with overlapping excitation and emission spectra are in proximity, excitation of one fluorophore causes light to be emitted and fluorescence to occur at a wavelength that is absorbed and stimulates the second fluorophore. In other words, the excited state energy of the first (donor) fluorophore is transferred to an adjacent second (acceptor) fluorophore by resonance-induced dipole-dipole interaction. As a result, the lifetime of the donor molecule decreases and its fluorescence is quenched, while the fluorescence intensity of the acceptor molecule is enhanced and depolarized. When the excited state energy of the donor moves to a non-fluorophore acceptor, the fluorescence of the donor is then quenched without subsequent emission of fluorescence by the acceptor. In this case, the acceptor functions as a quencher.

[0125] Pairs of molecules that can participate in FRET are called FRET pairs. For energy transfer to occur, the donor and acceptor molecules typically must be in proximity (e.g., up to a maximum of 70 - 100 angstroms) (Clegg, 1992, Methods Enzymol. 211:353 - 388; Selvin, 1995, Methods Enzymol. 246:300 - 334). The efficiency of energy transfer rapidly decreases as the distance between the donor and acceptor molecules increases. Effectively, this means that FRET can occur most efficiently up to a distance of about 70 angstroms.

[0126] In some embodiments of the present disclosure, the FRET pair includes a first member comprising a dye of formula (I) or a dye derived therefrom directly or indirectly coupled to a first oligonucleotide; and a second member comprising a second oligonucleotide directly or indirectly bound to a quencher. In some embodiments, the first member of the FRET pair includes a conjugate having any one of formula (IIA), (IIB), (IIC), or (IID).

[0127] In some embodiments, the FRET pair includes a first member having formula (VIIA) and a second member having formula (VIIB), [Dye 1]-[Y] a -[5'-Oligonucleotide 1 - 3'] (VIIA), [5'-Oligonucleotide 2 - 3']-[Y] a -[Dye 2] (VIIB), wherein, one of Dye 1 or Dye 2 is derived from any one of formula (I), (IA), or (IB), the other of Dye 1 or Dye 2 is a quencher; each Y is independently a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; a is 0, 1, or 2; and Oligonucleotide 1 and oligonucleotide 2 are different.

[0128] In some embodiments, one of Dye 1 or Dye 2 has a Stokes shift of at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, etc. In some embodiments, one of Dye 1 or Dye 2 is derived from formula (IA), wherein R 2 is

Chemical formula

[0129] Any quencher may be used in the compositions described herein without limitation, as long as it reduces the fluorescence intensity of the dye of formula (I) used or a dye derived therefrom. Quenchers commonly used in FRET include, but are not limited to, Deep Dark Quencher DDQ-I, DABCYL, Eclipse® Dark Quencher, Iowa Black® FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black® RQ, QSY-21, and Black Hole Quencher® BHQ-3. Quenchers for use in the compositions provided herein may be obtained commercially, for example, from Eurogentec (Belgium), Epoch Biosciences (Bothell, Wash.), Biosearch Technologies (Novato, Calif.), Integrated DNA Technologies (Coralville, Iowa), and Life Technologies (Carlsbad, Calif.).

[0130] In some embodiments, each Y is independently a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, each Y is independently a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 25 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, each Y is independently a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 20 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, each Y is independently a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to about 15 carbon atoms and optionally having one or more heteroatoms selected from O, N or S.

[0131] In some embodiments, oligonucleotides 1 and 2 comprise nucleotide modifications selected from locked nucleic acids (LNA), peptide nucleic acids (PNA), bridged nucleic acids (BNA), 2'-O-alkyl substitutions, L-enantiomer nucleotides, or combinations thereof. In some embodiments, the nucleotide modification comprises LNA.

[0132] In some embodiments, the present disclosure provides a method for determining a genotype at a locus of interest in a sample containing genetic material, the method comprising contacting the genetic material with a first probe having formula (VIIA) and a second probe having formula (VIIB); and detecting the binding of one of the first and second probes to the genetic material, thereby determining the genotype at the locus. In some embodiments, the first and second probes each have a 5' end opposite the 3' end and a predetermined number of nucleotides (e.g., 4, 6, 8, 10, 12, 16, 20 nucleotides) consisting of at least one DNA nucleotide and a predetermined number of locked nucleic acid nucleotides (e.g., at least 5 of 2, 3, 4, 5, 6, 7, 8 locked nucleotides). In some embodiments, the nucleotides of the first probe include a first discrimination position, the nucleotides of the second probe include a second discrimination position at the same nucleotide position as the first discrimination position in the first probe, the first discrimination position includes a nucleobase different from the second discrimination position, and the nucleobases at the other nucleotides of the first and second probes are the same.

[0133] TAGS probe The present disclosure also provides a kit comprising (i) a conjugate having formula (V) and (ii) a conjugate having formula (VIII), [(oligomer 1)(dye)]-linker-[(oligomer 2)(Q1)] (V), [oligomer 3]-[Q2] (VIII), wherein, the dye is derived from any one of (I), (IA) or (IB); oligomers 1, 2, and 3 are each different and are oligomers having from about 5-mers to about 30-mers; Q1 and Q2 are the same or different quenchers; and the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having from about 5 to about 40 carbon atoms.

[0134] In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having from about 5 to about 30 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having from about 5 to about 25 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having from about 5 to about 20 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having from about 5 to about 15 carbon atoms. In some embodiments, one of Oligomer 1 or Oligomer 2 comprises or consists entirely of L-DNA. In other embodiments, Oligomer 1 comprises or consists entirely of L-DNA. In still other embodiments, Oligomer 1 consists entirely of L-DNA. In other embodiments, Oligomer 2 comprises or consists entirely of L-DNA. In still other embodiments, Oligomer 2 consists entirely of L-DNA. In some embodiments, Q1 and Q2 are the same. In other embodiments, Q1 and Q2 are different. In some embodiments, the dye has a Stokes shift of at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, etc. In some embodiments, the dye in the kit is derived from formula (IA) wherein R 2 is [Chemical formula] is as follows.

[0135] In some embodiments, the present disclosure provides a kit for detecting two or more target nucleic acid sequences in a sample, which includes the following: (a) Two or more pairs of oligonucleotide primers having sequences complementary to each strand of the two or more target nucleic acid sequences; (b) At least one oligonucleotide probe comprising two distinct portions: (i) An annealing moiety that contains a sequence at least partially complementary to one of two or more target nucleic acid sequences and anneals within one of the two or more target nucleic acid sequences, the annealing moiety containing a first quencher moiety; and (ii) A tag moiety that is attached to the 5'-end or 3'-end of the annealing moiety or is attached via a linker between the 5'-end and 3'-end of the annealing moiety, the tag moiety containing a nucleotide sequence non-complementary to one of the two or more target nucleic acid sequences, the tag moiety containing a compound derived from formula (IA), the detectable signal of which can be quenched by the first quencher moiety on the annealing moiety, the compound derived from formula (IA) being separated from the first quencher moiety by a nuclease-sensitive cleavage site; (c) At least one quenching oligonucleotide that contains a nucleotide sequence at least partially complementary to the tag moiety of the oligonucleotide probe and hybridizes to the tag moiety to form a duplex, the quenching oligonucleotide containing a second quencher moiety that quenches the detectable signal generated by the compound derived from formula (IA) on the tag moiety when the quenching oligonucleotide hybridizes to the tag moiety.

[0136] In some embodiments, the tag portion is attached to the 5' end of the annealing portion. In some embodiments, the tag portion is attached via a linker between the 5' end and the 3' end of the annealing portion. In some embodiments, the tag portion of the oligonucleotide probe or the quenching oligonucleotide or both the tag portion of the oligonucleotide probe and the quenching oligonucleotide contain one or more nucleotide modifications. In some embodiments, the one or more nucleotide modifications include nucleotide modifications selected from locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA), 2'-O alkyl substitution, L-enantiomer nucleotide, or combinations thereof. In some embodiments, the nucleotide modification includes LNA. In some embodiments, the nucleotide modification includes PNA. In some embodiments, the nucleotide modification includes BNA. In some embodiments, the nucleotide modification includes L-enantiomer nucleotide. In some embodiments, the nucleotide modification includes L-enantiomer LNA (L-LNA). In some embodiments, the nucleotide modification includes 2'-O alkyl substitution. In some embodiments, the nucleotide modification includes 2'-O methyl substitution (2'-OMe).

[0137] A method for amplifying and detecting a target nucleic acid in a sample, comprising the following steps: (a) A sample containing the target nucleic acid is placed in a single reaction vessel, (i) a pair of oligonucleotide primers, each of which can hybridize to the opposite strand of a sub-sequence of the target nucleic acid; (ii) An oligonucleotide probe comprising an annealing portion and a tag portion, wherein the tag portion contains a nucleotide sequence non-complementary to the target nucleic acid sequence, the annealing portion contains a nucleotide sequence at least partially complementary to the target nucleic acid sequence, hybridizes to a region of a sub-sequence of the target nucleic acid to which a pair of oligonucleotide primers binds, and the probe further comprises an interaction dual label including a compound having (or derived from) formula (IA) located on the tag portion and a first quencher portion located on the annealing portion, and the compound having (or derived from) formula (IA) is separated from the first quencher portion by a nuclease-sensitive cleavage site; contacting step, and Before step (b), the tag portion reversibly binds in a temperature-dependent manner to a quenching oligonucleotide containing a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe, binds to the tag portion by hybridization, and the quenching oligonucleotide contains at least a second quencher portion capable of quenching a compound having (or derived from) formula (IA) on the tag portion when the quenching oligonucleotide binds to the tag portion; (b) Subsequently to step (a), amplifying the target nucleic acid by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5'-to-3' nuclease activity, whereby during the extension step of each PCR cycle, the nuclease activity of the polymerase enables cleavage and separation of the tag portion from the first quencher portion on the annealing portion of the probe; (c) Measuring an inhibition signal from a compound having (or derived from) formula (IA) at a first temperature at which the quenching oligonucleotide is bound to the tag portion; (d) Raising the temperature to a second temperature at which the quenching oligonucleotide does not bind to the tag portion; (e) Measuring a temperature correction signal from a compound having (or derived from) formula (IA) at the second temperature; (f) Obtaining a calculated signal value by subtracting the suppression signal detected at the first temperature from the temperature correction signal detected at the second temperature; (g) Repeating steps (b) to (f) in a plurality of PCR cycles; (h) Measuring the calculated signal values from the plurality of PCR cycles and detecting the presence of the target nucleic acid.

[0138] A method for amplifying and detecting a target nucleic acid in a sample, comprising the following steps: (a) A sample containing the target nucleic acid is placed in a single reaction vessel, (i) A pair of oligonucleotide primers, each of which can hybridize to the opposite strand of a sub-sequence of the target nucleic acid; (ii) An oligonucleotide probe comprising an annealing portion and a tag portion, wherein the tag portion contains a nucleotide sequence non-complementary to the target nucleic acid sequence, the annealing portion contains a nucleotide sequence at least partially complementary to the target nucleic acid sequence, the oligonucleotide probe hybridizes to the region of the sub-sequence of the target nucleic acid bound by the pair of oligonucleotide primers, and the probe further comprises an interaction dual label comprising a compound having (or derived from) formula (IA) located on the tag portion and a first quencher portion located on the annealing portion, and the compound having (or derived from) formula (IA) is separated from the first quencher portion by a nuclease-sensitive cleavage site; contacting step, and Before step (b), the tag portion reversibly binds in a temperature-dependent manner to a quenching oligonucleotide containing a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe, hybridizes to the tag portion, and the quenching oligonucleotide contains at least a second quencher portion capable of quenching the compound having (or derived from) formula (IA) on the tag portion when the quenching oligonucleotide binds to the tag portion; (b)Following step (a), amplifying the target nucleic acid by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5'-to-3' nuclease activity, such that during the extension step of each PCR cycle, the nuclease activity of the polymerase enables cleavage and separation of the tag portion from the first quencher portion on the annealing portion of the probe; (c)Measuring one or more signals from a compound having formula (IA) (or derived therefrom) at a first temperature at which the quenching oligonucleotide is bound to the tag portion; (d)Measuring one or more signals from a compound having formula (IA) (or derived therefrom) at a second temperature higher than the first temperature at which the quenching oligonucleotide is not bound to the tag portion; (e)Obtaining a calculated signal value by subtracting the median or average value of the one or more signals detected at the first temperature from the median or average value of the one or more signals detected at the second temperature; Thereby, a calculated signal value higher than the threshold signal value enables determination of the presence of the target nucleic acid.

[0139] In some embodiments, the PCR amplification of step (b) can reach an end point beyond the logarithmic phase of amplification. In some embodiments, the tag portion comprises a modification such that it cannot be extended by the nucleic acid polymerase. In some embodiments, the tag portion of the oligonucleotide probe or the quenching oligonucleotide or both the tag portion and the quenching oligonucleotide contain one or more nucleotide modifications. In some embodiments, the one or more nucleotide modifications are selected from the group consisting of locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA), 2'-O alkyl substitution, L-enantiomer nucleotides, and combinations thereof.

[0140] Other methods of using TAGS probes are described in U.S. Pat. Nos. 11,028,433, 11,034,997, and 11,345,958; and U.S. Patent Application Publication No. 2021 / 0269857, the disclosures of which are hereby incorporated by reference in their entireties.

[0141] Synthesis The present disclosure provides methods for synthesizing a compound of any one of formulas (I), (IA), and (IB) and its derivatives and analogs. The present disclosure also provides methods for synthesizing intermediates.

[0142] Summary R 1 Compounds of formulas (I) and (IB) in which R is a protecting group enable the formation of stable amino protection under the conditions of solid-phase synthesis using phosphoramidite chemistry in the preparation of nucleic acids. For example, the amino group can be protected as trifluoroacetamide (TFA, trifluoroacetyl protecting group) to obtain compounds 2a - 2s in which R 1 is the TFA protecting group (see Tables 2a - 2c). In some embodiments, the TFA group can be cleaved between gaseous, aqueous ammonia, or a primary amine (such as methylamine, propylamine, tert-butylamine, etc.), which are common deprotection conditions in the solid-phase synthesis of nucleic acid analogs. Alternatively, the amine can be protected as benzyl carbamate (benzyl chloroformate, Cbz or Z protecting group), or as 9-fluorenylmethyl carbamate (Fmoc protecting group).

[0143] Dye Synthesis and Accessibility A single high-yield reaction from the inexpensive, commercially available rhodamine 800 perchlorate dye can be used as a starting material (fluorophore having a julolidine core structure, CAS number [137993-41-0]) with a primary amine.

Chemical Formula

[0144] Compounds 1b, 1c, 1h - 1j, 1k, 1o, 1r, and 1s (see Tables 1a - 1c) can be used for the labeling of biomolecules either by in situ activation of the carboxylic acid or by means of the corresponding NHS - esters. The NHS - esters are prepared by using trifluoroacetic anhydride (TFAA) and N - hydroxysuccinimide (NHS) in the presence of a base.

Chem.

[0145] The alcohol functional groups of Compounds 2h, 2j, 2m, and 2q (see Tables 2a - 2c) can be directly converted to the corresponding phosphoramidites for the 5’ - modification of nucleic acids and nucleic acid analogs.

Chem.

[0146] The NHS - esters of Compounds 2b, 2c, 2h - 1j, 2k, 2o, 2r, and 2s (see Tables 2a - 2c) can be converted to the corresponding phosphoramidites for the internal modification of nucleic acids and nucleic acid analogs according to the following synthetic sequence:

Chem.

[0147] Compounds 1g and 2g (see Tables 1a and 2a) find further use in copper - catalyzed click chemistry [Cu(I) - catalyzed azide - alkyne 1,3 - dipolar cycloaddition, CuAAC]. Compound 1g can be used for the solution labeling of biomolecules, while Compound 2g allows for the introduction of an alkyne group during solid - phase synthesis for on - column labeling.

[0148] Results R 2 With reference to the compounds having formula (1A) where R is butylamine, the absorption and fluorescence maxima as well as the brightness of Compounds 1a - 1s were analyzed. The results are shown in Table 3 of this specification. Unexpectedly, R 1It has been discovered that the configuration of the carbon directly bonded to the amino group has a great influence on the fluorescence characteristics of the dye core. The absorption maximum of the dye changed up to about 106 nm, the fluorescence changed up to about 35 nm, corresponding to a Stokes shift of about 11 nm to about 92 nm.

[0149] A common problem with normal fluorophores having a "small" Stokes shift is internal quenching of fluorescence. Such self-quenching is caused by spectral overlap of excitation and emission and is common especially at high fluorophore concentrations. LSS dyes such as those described herein generally have better separated spectral bands and minimize photon reabsorption.

[0150] The probability that a fluorophore is excited outside its main excitation peak is not zero. As a result, the fluorescence from one dye necessarily contributes to the total light detected in multiple emission channels. This spectral "crosstalk" or "bleed-through" can be compensated computationally to some extent by using a predetermined correction factor. Additionally, scattering of the excitation light increases the background fluorescence in adjacent channels. The dyes of the present disclosure (e.g., those having formula (IA)) enable even reduction or elimination of crosstalk and scattering from other fluorophores. The dyes of the present disclosure (e.g., those having formula (IA)) are particularly useful in experimental environments where many fluorophores produce a strong background signal. A large spectral separation such as that of the dyes of the present disclosure (e.g., those having formula (IA)) allows for more effective filtering of the excitation light, thereby enhancing the sensitivity of target detection (see, e.g., FIG. 2).

[0151] In addition, the dyes of the present disclosure (e.g., having formula (IA)) are also thought to provide access to fluorescence data from optically inaccessible channels. Facilitated by their broad spectral separation and used in combination with standard fluorophores, the dyes of the present disclosure enable an increase in the multiplexing ability of fluorescence PCR devices by adding additional channels to established 4-6 color devices (see Figure 2). In principle, 21 channels are available from the combination of filters of a 6-color device. However, in practice, the number of channels is limited by suitable spectral characteristics and commercial availability with a sufficiently large Stokes shift.

[0152] Activation of the dye and coupling of the activated dye to a DNA molecule The present disclosure also provides a method of activating a compound of formula (I) and subsequently coupling the activated compound to an oligomer. The following schematic illustrates the solution labeling of DNA molecules with DMT-MM.

Chemical formula

[0153] wherein, "Dye" is

Chemical formula

[0154] Synthesis of TAGS probes The present disclosure also provides a method of synthesizing TAGS probes, which comprise compounds having formula (I) and which are thermally stable up to about 100 °C. The use of such TAGS probes is described in this specification as well as in U.S. Pat. Nos. 11,028,433, 11,034,997, and 11,345,958; and U.S. Patent Application Publication No. 2021 / 0269857, the disclosures of which are incorporated herein by reference in their entirety.

[0155] In some embodiments, the probe (such as those having formula (V)) is synthesized by first preparing 5'-N3-modified DNA.

Chemical Formula

[0156] Next, the 5'-N3-modified DNA is coupled to an oligonucleotide containing a quencher and a first reactive group, for example, a reactive group capable of participating in a "click chemistry" reaction (e.g., DBCO). Then, when reacted with the oligonucleotide containing the quencher and the first reactive group, the 5'-N3-modified DNA is "clicked" in place, and the probe shown in formula (V) is obtained.

[0157] The present disclosure also provides a method for directly coupling an oligonucleotide having a terminal amine group to a cyano moiety present at the meso position of a dye core in order to provide any one of the compounds having, for example, formula (VIIA) or (VIIB) (see, for example, Example 6 herein). In some embodiments, a linker is present between the terminal amine group and the oligonucleotide. In some embodiments, the linker is a C1-C8 branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group having one or more substituents (e.g., -Me, -Et, -CO2-, -C2-CO2-, -D, or halogen).

Chemical Formula

[0158] R 3 is a C1-C8 alkyl group, heteroalkyl group, or cycloalkyl group substituted with one or more of -Me, -Et, -CO2-, -C2-CO2-, -D or halogen; and the oligonucleotide is an oligonucleotide having about 5 to about 60 mers.

[0159] In some embodiments, the base is N,N-diisopropylethylamine (DIPEA), cesium carbonate, potassium carbonate, sodium carbonate, tributylamine (TBA), N,N-dicyclohexylmethylamine, 2,6-di-tert-butylpyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]nona-5-ene (DBN), 1,1,3,3-tetramethylguanidine (TMG), or 2,2,6,6-tetramethylpiperidine. In some embodiments, the solvent is dimethyl sulfoxide (DMSO), sulfolane, N-butylpyrrolidone, γ-valerolactone, δ-valerolactone, N-methylpyrrolidone, N,N-dimethylformamide, sulfolane, and silene. In some embodiments, the reaction is carried out at a temperature in the range of about 20°C to about 70°C. In some embodiments, the reaction is carried out for a time in the range of about 60 minutes to about 72 hours. In some embodiments, the dye is rhodamine. In some embodiments, the dye is rhodamine 800.

Examples

[0160] The following examples are presented to illustrate embodiments of the present disclosure that are presently preferred to practice. It is understood that the examples are illustrative only and that the present disclosure is not considered to be limited except as set forth in the appended claims.

[0161] Abbreviations AU = absorbance unit; COU = coumarin; CPG = controlled pore glass; dATP = 2'-deoxyadenosine 5'-triphosphate; dCTP = 2'-deoxycytidine 5'-triphosphate; dGTP = 2'-deoxyguanosine 5'-triphosphate; DBCO = dibenzocyclooctyne modification; DCM = dichloromethane; DIPEA = N,N-diisopropylethylamine; DMSO = dimethyl sulfoxide; DMT-MM = 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salt; dUTP = 2'-deoxyuridine 5'-triphosphate; EDTA = ethylenediaminetetraacetic acid; eq. = molar equivalent; EtOH = ethanol, EU = emission unit; FAM = fluorescein; HAA = hexylammonium acetate buffer, HCl = hydrochloride; HEX = hexachloro-fluorescein; LSS = large Stokes shift; MeCN = acetonitrile; n.d. = not determined; qPCR = real-time polymerase chain reaction; RT = room temperature; R800 = rhodamine 800; SPE = solid phase extraction; TE = Tris / EDTA mixture; TEA = triethylammonium; TEAA = triethylammonium acetate; TEAB = triethylammonium bicarbonate; UPLC-MS = ultra-high performance liquid chromatography coupled to a mass spectrometer.

[0162] General materials and methods R800 perchlorate dye [137993-41-0] and 1-bicyclo[1.1.1]pentylamine hydrochloride were obtained from MilliporeSigma (Burlington, MA, U.S.A.). trans-4-Aminocyclohexane-carboxylic acid was obtained from TCI America (Portland, Oregon, U.S.A.). d9-Butylamine was obtained from C / D / N Isotopes Inc. (Pointe-Claire, QC, Canada). 3-Aminobicyclo[1.1.1]pentane-1-carboxylic acid HCl was obtained from AA Blocks Inc. (San Diego, CA, U.S.A.). Bicyclo[2.2.2]octan-1-amine HCl was obtained from 1Click Chemistry (Kendall Park, NJ, U.S.A.). 4-Aminobicyclo[2.2.2]octan-1-ol HCl and 4-aminobicyclo[2.2.2]octane-1-carboxylic acid were obtained from Absolute Chiral (San Diego, CA, U.S.A.). Reagents and materials for chemical DNA synthesis were obtained from Glen Research (Sterling, VA, U.S.A.). TEAB buffer was obtained as a ready-made solution (1.0 M, pH 8.5) and used without further dilution. TEAA and HAA buffers were prepared by diluting commercially available stock solutions (Glen Research, Sterling, VA, U.S.A.) with water to a final concentration of 100 mM. Other reagents were obtained from MilliporeSigma (Burlington, MA, U.S.A.) unless otherwise specified. Dry solvents on activated molecular sieves for chemical reactions were obtained from Acros Organics (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Solvents for chromatography (HPLC grade) were obtained from MilliporeSigma (Burlington, MA, U.S.A.) or VWR (Radnor, PA, U.S.A.). Ultrapure water was obtained from a Milli-Q® purification system (MilliporeSigma) having a resistivity of at least 18.2 MΩ·cm at 25°C.

[0163] Chemical reactions were carried out using an Eppendorf ThermoMixer (registered trademark) C (Enfield, CT, U.S.A.). Microwave-assisted reactions were performed using a Discover (registered trademark) SP microwave system manufactured by CEM (Matthews, NC, U.S.A.) equipped with a focused single-mode reaction chamber (2.45 GHz) inside a heavy-walled glass vial (2.0 mL or 10.0 mL). The reaction temperature was monitored with a built-in IR temperature sensor and maintained constant by automatic power control. The microwave-assisted reactions were stirred under active cooling with compressed air. Flash column chromatography was performed using an automated flash chromatography system (CombiFlash (registered trademark) Rf + Lumen) manufactured by Teledyne-Isco (Lincoln, NE, U.S.A.). Substitution of the dye counterions was achieved by standard ion-exchange procedures such as ion exchange, SPE, liquid-liquid extraction, or precipitation from organic solvents.

[0164] DNA oligomers with 3'-modifications were synthesized on CPG pre-loaded with spacer C3, phosphate, or BHQ-2 (Black Hole Quencher®). DNA sequences with primary amino modifications were synthesized by solid-phase DNA synthesis using amino-modifying phosphoramidites. For 5'-end modification, 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (5'-amino-C6) was used. For internal modification, 5'-dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (amino-C6-dT) was used. If necessary due to the sequence context, amino-C6-dT can also be used at the 5'-end. DNA sequences with 5'-azide modifications were prepared by synthesizing 5'-bromo-modified DNA with bromo-hexyl phosphoramidite and subsequently performing the conversion from bromo to azide on the solid support. A solution of sodium azide and sodium iodide in DMSO (100 mM each) was added to the CPG, and the mixture was heated at 65 °C for 1.0 h to effect Br / N3 exchange (quantitative reaction). After DNA synthesis and on-column modification, the DNA was cleaved, deprotected, desalted, and precipitated by standard methods. The latter two steps were performed to ensure the removal of trace amounts of amines. The aqueous solution of the DNA sequence was dried using a rotary vacuum concentrator (SpeedVac® Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) or a freeze dryer (Labconco freeze dryer with an ice capacity of 4.5 L and a collector temperature of -105 °C; Labconco Corp., Kansas City, MO, U.S.A.).

[0165] UPLC analysis was performed on a Waters I-class ACQUITY UPLC (Waters Corporation, Milford, Massachusetts, USA) equipped with a diode array, fluorescence, and mass spectrometry (ZSpray™) detector. A Waters Oligonucleotide BEH C18 column (130 Å, 1.7 μm, 2.1×50 mm) was used with a suitable gradient of TEAA buffer (100 mM, pH 7.0) in MeCN at 1.0 ml / min. Chromatograms were recorded at 260 nm for DNA and at the absorption maxima of the respective dyes or dye-labeled probes. Semipreparative HPLC purification was carried out on a Waters 600 HPLC at 10.0 ml / min using a 996 photodiode array detector and a Waters XBridge™ BEH C18 OBD Prep column (130 Å, 5 μm, 19.0×250 mm). Prior to injection, the samples were filtered through a Teflon® syringe filter (0.22 μm). Absorption spectra were obtained using a NanoDrop One C spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) with background correction at 750 nm. Fluorescence spectra and thermal stability of the fluorescence data were recorded on a Cary Eclipse fluorescence spectrophotometer equipped with a temperature controller (Agilent Technologies, Santa Clara, CA, USA).

[0166] Example 1: General procedure for preparing compounds R, 1a - 1s The R800 perchlorate dye (100 mM in 1.0 equivalent DMSO, 10 μL) in DMSO was converted with a primary amine (100 mM in 5.0 equivalent DMSO) in the presence of a base in a reaction mixer at 50 °C. For n-butylamine, d6-butylamine, ethanolamine, 2-methoxyethylamine, 2-(2-aminoethoxy)ethanol, propargylamine, tert.-butylamine and 2-amino-2-methyl-1-propanol, the base was DIPEA (1.0 equivalent). For amines with carboxylic acid functionality (glycine, β-alanine, L-alanine, L-leucine, L-valine, L-(+ / -)-3-aminobutyric acid, trans-4-aminocyclohexanecarboxylic acid, 4-aminobicyclo[2.2.2]octane-1-carboxylic acid), cesium carbonate (2.0 equivalents) was used as the base. For amines obtained as HCl salts (1-bicyclo[1.1.1]pentylamine HCl, 3-aminobicyclo[1.1.1]pentane-1-carboxylic acid HCl, bicyclo[2.2.2]octane-1-amine HCl), the amount of cesium carbonate was increased to neutralize the acid (5.0 equivalents). The identity of the dye products was confirmed by mass spectrometry in the positive ion mode.

[0167] The spectroscopic properties of the dye products R, 1a - 1s were determined by UPLC-MS analysis. A sample (0.5 μL) of the reaction solution was diluted with MeCN (29.5 μL) and separated on a C18 stationary phase using a gradient of TEAA buffer mobile phase and MeCN (40 - 70% MeCN in 2.0 minutes). For compound 1p, the gradient was extended by 30 seconds. The absorption and emission spectra were recorded by injecting an amount that gave approximately 0.5 - 1.0 AU at the absorbance maximum of each dye.

[0168] To determine the fluorescence per absorption unit as a measure of dye brightness, sample amounts were injected into the UPLC to obtain approximately 0.1 AU at the absorbance maximum of each dye. Fluorescence emission was measured by excitation at the absorption maximum (3D fluorescence mode, 1.0 PMT gain, 1.0 second time constant) and in a 100 nm fluorescence emission window centered at the emission maximum. The peak area of the fluorescence peak was divided by the peak area of the absorption peak.

[0169] Results: The analytical data of the reaction of n-butylamine with R800, which gives compound R, are shown in FIGS. 3A, 3B, and 3C. The detailed analytical data of compounds 1a - 1s are shown in FIGS. 4 - 23 (A: chromatogram; B: excitation & emission spectra; C: mass spectrum). The spectroscopic data of compounds R and 1a - 1s are summarized in Table 1. Compound 1b was unstable and not further analyzed (FIG. 6). Surprisingly, compound 1s showed typical spectral characteristics of a "normal" fluorescent dye rather than an LSS dye (a Stokes shift of about 11 nm, FIG. 23B). This result is in contrast to compound 1g, which showed a Stokes shift of 81 nm (FIG. 11B). In summary, these results demonstrated that the linker moiety has a significant impact on the spectroscopic properties of the dye core.

[0170] Example 2: Synthesis of Compound 1j

Chem.

[0171] Results: The success of the synthesis of compound 1j was demonstrated by the analytical data in FIG. 14 (A: chromatogram; B: excitation and emission spectra; C: mass spectrum). In Example 7, compound 1j was used for DNA labeling (FIG. 27).

[0172] Example 3: Synthesis of Compound 1k

Chem.

[0173] Results: The success of the synthesis of compound 1k was demonstrated by the analytical data in Figure 15 (A: chromatogram; B: excitation and emission spectra; C: mass spectrum).

[0174] Example 4: Synthesis of compound 1q

Chemical formula

[0175] Results: The successful synthesis of Compound 1g was demonstrated by the analytical data in Figure 21 (A: chromatogram; B: excitation and emission spectra; C: mass spectrum).

[0176] Example 5: Thermal stability of fluorescence A small sample of the LSS dye in DMSO was diluted to 10% DMSO concentration with TEAA buffer (0.1 M, pH 7.0, 0.5 mL). The fluorescence signal was recorded as a function of temperature by exciting each LSS dye at its excitation maximum and recording the fluorescence at the emission maximum from 25 °C to 100 °C at a heating rate of 1 °C / min.

[0177] Results: Fluorescence as a function of temperature for compounds R, 1i, 1k, 1j, and 1n is shown in Figure 24. The results show no significant decrease in fluorescence up to 100 °C, demonstrating the thermal stability of the fluorescence for the LSS dyes of the present disclosure. For compounds R and 1n, a slightly prominent drift to higher fluorescence could be explained by the increased solubility at high temperature due to their higher hydrophobicity compared to compounds 1i, 1k, 1j which are carboxylic acids.

[0178] Example 6: Direct Labeling of DNA with R800 Dye

Chemical formula

[0179] Results: The effectiveness of the direct labeling method has been demonstrated by the successful derivatization of various amino-modified DNA probe sequences with R800. Analytical data of the labeling reaction of TaqMan® probe sequences with internal BHQ-2 are shown in FIG. 25. This labeled DNA was used in Example 10. Analytical data of three 5'-azide-modified DNA sequences are shown in FIG. 26A (chromatogram) and FIG. 26B (absorption and mass spectra). These results showed that site-specific labeling of DNA can be achieved without the need for active esters or click chemistry functionalities. In this approach, the molecular composition of the amino-linker of DNA was incorporated such that it became an essential part of the dye structure.

[0180] Example 7: Labeling of DNA with LSS Dye Carboxylic Acid After automated solid-phase synthesis and standard work-up procedures, the analytical amount of the crude DNA was analyzed by UPLC-MS to determine the percentage of the amino-modified target sequence. The total DNA amount was determined spectrophotometrically using the calculated extinction coefficient of the DNA sequence at 260 nm.

[0181] DNA precipitation: The crude DNA obtained from solid-phase synthesis was desalted against water by standard methods (NAP-25 or SPE) and dried in a rotary vacuum concentrator. In a reaction tube, the DNA was redissolved in water (0.1 mL) using a warm water bath. Sodium iodide was added to a final concentration of 10.0 M. Absolute ethanol (200 proof, 30.0 mL) was added and the tube was vortexed. The suspension was centrifuged in a tabletop centrifuge at maximum speed (5.0 min). The supernatant was carefully decanted and discarded. Excess sodium iodide was removed by washing the pellet with dry ethanol. The pellet was dried under high vacuum and redissolved in TEAB buffer (pH 8.5) for immediate use in the labeling reaction.

[0182] Activation of Fluorescent Dye Carboxylic Acid: In a reaction vial, the carboxylic acid dye (1.0 equivalent) was dissolved in dry DMSO (8 mM). DIPEA (2.0 equivalents) was added and the solution was mixed briefly. In a separate glass vial, the tetrafluoroborate of DMT-MM (2.0 equivalents) was weighed, the dye carboxylic acid solution was added, and then it was mixed vigorously until all the solids dissolved.

[0183] The reaction mixture was shaken at room temperature for 15 minutes.

[0184] Labeling Reaction: The activated dye solution (3.0 equivalents) was rapidly mixed with the amino-modified DNA (1.0 equivalent primary amine), and the labeling reaction was carried out at room temperature in a reaction mixer for 30 minutes. The progress of the reaction was monitored by UPLC analysis, and a sample of the reaction mixture (1.0 μL) was diluted with water (19.0 μL) before UPLC injection (7.0 μL).

[0185] Purification: The labeled DNA was purified by reverse-phase liquid chromatography using a suitable gradient of TEAA buffer (0.1 M, pH 7.0) and MeCN. The combined product fractions were concentrated in a centrifugal vacuum concentrator and desalted by size exclusion chromatography. The purified DNA probe (0.1 mM) was lyophilized and redissolved in TE buffer (10.0 mM Tris·HCl, 1.0 mM EDTA) for qPCR.

[0186] Results: The successful labeling of amino-modified DNA with LSS dye 1j was demonstrated by the analytical data in Figure 27 (A: chromatogram; B: excitation and emission spectra; C: mass spectrum). Since compound 1j was a racemic mixture of enantiomers, the labeled DNA was a mixture of diastereomers and was separated as a doublet peak.

[0187] Example 8: Preparation of Dye-Labeled DNA Probe Using Click Chemistry The dye-labeled DNA probes can also be prepared by strain-promoted azide-alkyne cycloaddition between DNA-binding DBCO or BCN and azide-modified dyes. A DNA probe containing a 5'-DBCO modification, internal BHQ-2, and a 3'-C3 spacer blocker was prepared by solid-phase DNA synthesis and purification by standard methods. DNA (1.0 equivalent, 100 μM, 50 mM TEAA buffer, pH 7.0) and dye azide (1.1 equivalents, 100 μM, 50 mM TEAA buffer, pH 7.0) were mixed and held at 40 °C in a shaker for 2 hours. Excess dye was removed by ethanol precipitation. UPLC analysis showed quantitative labeling of the DNA. The above protocol has been used to prepare several DNA conjugates with common fluorescent dyes. The LSS dye with an azide linker is expected to yield DNA-LSS dye conjugates in the same manner.

[0188] Example 9: Preparation of Branched DNA Probes Using Click Chemistry Branched DNA probes for thermal multiplexing were prepared by strain-promoted azide-alkyne cycloaddition between an oligonucleotide with 5'-BHQ-2 and internal DBCO modification and a 5'-azide-modified oligonucleotide with a dye at the second-to-last position (e.g., the labeled DNA from Example 6). Both DNA sequences were mixed in a 1:1.1 stoichiometric ratio in TEAA buffer (50 mM, pH 7.0) and held at 40 °C in a reaction mixer for 2 hours. UPLC analysis showed a quantitative click reaction with some remaining DNA excess that was removed by HPLC purification.

[0189] Example 10: PCR Amplification Using Dye-Labeled DNA Probes All qPCR components were prepared using nuclease-free water. A reaction mixture with a total volume of 50 μL was prepared by mixing three components called master mixture (20 μL), buffer mixture (20 μL), and dNTP mixture (10 μL). The master mixture contained tricine buffer (pH 8.2), manganese acetate, potassium acetate, glycerol, DMSO, surfactant, target DNA (5000 copies / reaction), polymerase aptamer, forward primer DNA and reverse primer DNA, as well as polymerase enzyme, and TaqMan® probe. The dNTP mixture contained dATP, dCTP, dGTP (each 2.0 mM), and dUTP (4.0 mM). Each qPCR with 5 pmol of target was prepared in duplicate in the wells of a 96-well plate. The TaqMan® probe was a DNA sequence having the large Stokes shift (LSS) dye and BHQ-2 quencher prepared in Example 6 (analysis data of FIG. 25). For comparison, another qPCR contained a TaqMan® probe having the same sequence, which was labeled with Cy5.5. The plate was sealed and subjected to an amplification cycle using a LightCycler® 480 system (Fritz Hoffmann-La Roche, Basel, Switzerland). The growth curve was analyzed from fluorescence data collected with an appropriate combination of excitation and emission channels.

[0190] Results: FIG. 28 shows the PCR amplification curves measured in their respective optical channels as described in FIG. 2 for TaqMan® probes having LSS and Cy5.5 dyes. Specifically, fluorescence for the LSS-labeled probe was detected in channel RLS 1 (435 nm excitation, 580 nm emission), and fluorescence of the Cy5.5-labeled probe was detected in the Cy5.5 channel (580 nm excitation, 700 nm emission). Both amplification curves were overlaid in FIG. 28 for comparison. The LSS dye signal showed a fluorescence signal greater than that of the conventional Cy5.5 dye. This experiment demonstrates the general applicability and compatibility of the LSS dye as a bright reporter in TaqMan® PCR, and it is expected that other LSS dye variants in the present disclosure will similarly generate qPCR signals.

[0191] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Aspects of the above embodiments can be modified, as needed, to employ concepts of various patents, applications, and publications to provide further alternative embodiments.

[0192] Although the present disclosure has been described with reference to several exemplary embodiments, it should be understood that many other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of the present disclosure. More specifically, reasonable variations and modifications are possible in the parts and / or arrangements of the components of the combination configurations of the foregoing disclosure, drawings, and the subject matter of the appended claims without departing from the spirit of the present disclosure. In addition to variations and modifications in the parts and / or arrangements of the components, alternative uses will also be apparent to those skilled in the art.

Claims

1. A compound having formula (I), 【Chemistry 1】 During the ceremony, R １ is either H or a protecting group; R ２ is a C １ -C ８ branched alkyl group, -CO₂-, -OH, -D, or a C₁-C₆ unbranched alkyl group, C₁-C₈ branched or unbranched heteroalkyl group, or cycloalkyl group substituted with one or more of halogen, which is -Me, -Et, -CO ２ -, -CO ２ -(thiol-reactive group), -CO ２ -(amine-reactive group), -CO ２ -(carboxy-reactive group), -C ２ -CO ２ -, -C ２ -CO ２ -(thiol-reactive group), -C ２ -CO ２ -(amine-reactive group), -C ２ -CO ２ -(carboxy-reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, or halogen, or substituted with one or more of groups capable of participating in a "click chemistry" reaction, where the groups capable of participating in the "click chemistry" reaction are selected from the group consisting of bicyclo[6.1.0]nonyne) group ("BCN"), dibenzocyclooctyne ("DBCO"), alkene, trans-cyclooctene ("TCO"), maleimide, aldehyde, ketone, azide, tetrazine, thiol, 1,3-nitrone, hydrazine, and hydroxylamine; and [X] － is a counter anion, however R ２ If it has a negative charge, [X] － It does not exist, and here, The thiol-reactive group is selected from the group consisting of haloacetyl, maleimide, iodoacetamide, aziridine, acryloyl, arylating agent, vinyl sulfone, methanethiosulfate, pyridyl disulfide, and TNB-thiol. The amine-reactive group is selected from the group consisting of NHS esters, isothiocyanates, acyl azides, sulfonyl chlorides, sulfodichlorophenols, pentafluorophenols, tetrafluorophenols, 4-sulfo-2,3,5,6-tetrafluorophenyls, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, fluorophenol esters, sulfochlorophenols, carbodiimides, phthalimides, benzotriazoles, imide esters, and anhydrides. A compound in which the carbonyl reactive group is selected from the group consisting of hydrazine, hydrazine derivatives, and amines.

2. R ２ However, -CO ２ - Maleimide or - C ２ -CO ２ - C substituted with maleimide １ -C ８ Branched alkyl groups, C1-C6 unbranched alkyl groups substituted with one or more -CO2-, -OH, -D, or halogens, C1-C8 branched or unbranched heteroalkyl groups, or cycloalkyl groups; -CO ２ -NHS ester or -C ２ C substituted with -CO2-NHS ester １ -C ８ Branched or unbranched alkyl groups, branched or unbranched heteroalkyl groups, or cycloalkyl groups; and -CO ２ -Hydrazine or -C ２ -CO ２ - C substituted with hydrazine １ -C ８ The compound according to claim 1, selected from the group consisting of a branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group.

3. The compound according to claim 1, R ２ is -Me, -Et, -CO ２ － C substituted with one or more -OH, -D, or halogens １ -C ６ It is a branched alkyl group, or a C1-C6 unbranched alkyl group substituted with one or more -CO2-, -OH, -D, or halogens; or R ２ However, the group consists of the following: 【Chemistry 2】 A compound selected from among them.

4. A compound selected from the group consisting of the following, 【Transformation 3】 In the formula, "Dye" means 【Chemistry 4】 And, [X] － Is it a counter-anion? or A compound selected from the group consisting of the following, 【Transformation 5】 [X] － Is it a counter-anion? or A compound having formula (IA), 【Transformation 6】 In the formula, R ２ This is a C1-C8 branched alkyl group, a C1-C6 unbranched alkyl group substituted with one or more -CO2-, -OH, -D, or halogens, a C1-C8 branched or unbranched heteroalkyl group, or a cycloalkyl group, which is -Me, -Et, -CO ２ － , -CO ２ - (thiol reactive group), -CO ２ - (amine-reactive group), -CO ２ - (carboxyl-reactive group) - C ２ -CO ２ － , -C ２ -CO ２ - (thiol reactive group), C ２ -CO ２ - (amine-reactive group), -C ２ -CO ２ It is substituted with one or more groups that can participate in a "click chemistry" reaction, where the groups that can participate in a "click chemistry" reaction are selected from the group consisting of bicyclo[6.1.0]nonine) group ("BCN"), dibenzocyclooctin ("DBCO"), alkenes, trans-cyclocten ("TCO"), maleimides, aldehydes, ketones, azides, tetrazines, thiols, 1,3-nitrones, hydrazines, and hydroxylamines; [X] － is a counter anion, however R ２ If it has a negative charge, [X] － The compound according to claim 1 does not exist.

5. A conjugate comprising (i) a specific binding entity and (ii) a dye moiety containing any one of the compounds described in any one of claims 1 to 4.

6. A conjugate having formula (II), 【Transformation 7】 During the ceremony, R １ is either H or a protecting group; R ３ -Me, -Et, -CO ２ -, -C ２ -CO ２ C substituted with -, -D, or one or more halogens １ -C ８ It is an alkyl group, heteroalkyl group, or cycloalkyl group; The aforementioned "specific binding entity" is an oligonucleotide or a protein; Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 2 to about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is a conjugate, which is 0, 1, or 2.

7. The conjugate according to claim 6, wherein Y has the structure of formula (IIIIC), 【Transformation 8】 In the formula, R ３ and R ４ Each of these is independently a bond or group selected from carbonyl, amide, imide, ester, ether, -NH, -N-, thion, or thiol; R ５ C １ -C １２ It is an alkyl group or heteroalkyl group, R ５ It may contain a carbonyl, imine, or thion; R ａ and R ｂ It is independently either H or methyl; g and h are independent integers ranging from 0 to 4; i is a conjugate, which is 0, 1, or 2.

8. A conjugate having either formula (IIC) or (IID), 【Chemistry 9】 【Chemistry 10】 During the ceremony, R １ is either H or a protecting group; R ３ -Me, -Et, -CO ２ -, -C ２ -CO ２ C substituted with -, -D, or one or more halogens １ -C ８ It is a branched or unbranched alkyl group, a branched or unbranched heteroalkyl group, or a cycloalkyl group; The oligonucleotide is an oligonucleotide having approximately 5 to 60 mergers; Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 2 to about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is a conjugate, which is 0, 1, or 2.

9. A kit comprising: (i) a first conjugate comprising a first oligonucleotide coupled to a dye portion comprising any one of the compounds described in any one of claims 1 to 4; and (ii) a second conjugate comprising an oligonucleotide coupled to a quencher capable of reducing the fluorescence intensity of the dye portion.

10. A probe having formula (IV), [Pigment 1] - [Y] ａ -[5'-oligonucleotide-3']-[Y] ａ - [Dye 2] (IV), During the ceremony, Either [Dye 1] or [Dye 2] comprises any one of the compounds described in any one of claims 1 to 4; the other of dye 1 or dye 2 is a quencher capable of reducing the fluorescence intensity of the dye portion; The oligonucleotide is an oligonucleotide having approximately 5 to 60 mergers; Each Y is independently a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group having 2 to about 40 carbon atoms and optionally one or more heteroatoms selected from O, N, or S; and a is a probe, which is 0, 1, or 2.

11. A conjugate having formula (V), [(Oligomer 1)(Pigment)]-Linker-[(Oligomer 2)(Q1)] (V), During the ceremony, Oligomers 1 and 2 are different and are oligonucleotides having approximately 5 to 30 m² each; The dye comprises one of the compounds described in any one of claims 1 to 4; Q1 is a quencher that can reduce the fluorescence intensity of the dye portion; and A linker is a conjugate, which is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having approximately 5 to 30 carbon atoms.

12. The conjugate according to claim 11, wherein at least one of oligomers 1 and 2 comprises LNA, L-LNA, or PNA.

13. A kit comprising (i) the conjugate described in claim 11; and (ii) a compound having formula (VIII), [Oligomer 3]-[Q2] (VIII), During the ceremony, Oligomer 3 is an oligonucleotide having 5 to 30 members; and Q2 is a quencher that can reduce the fluorescence intensity of the dye portion, in this kit.

14. A FRET pair comprising a first member having formula (VIIA) and a second member having formula (VIIB), [Pigment 1] - [Y] ａ -[5'-oligonucleotide 1-3'] (VIIA), [5'-oligonucleotide 2-3']-[Y] ａ - [Dye 2] (VIIB), During the ceremony, Dye 1 or dye 2 comprises one of the compounds described in any one of claims 1 to 4; The other of dye 1 or dye 2 is a quencher that can reduce the fluorescence intensity of the dye portion; Each Y is independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to about 40 carbon atoms and optionally one or more heteroatoms selected from O, N, or S; a is 0, 1, or 2; and Oligonucleotide 1 and oligonucleotide 2 are different FRET pairs.

15. A method for amplifying and detecting a target nucleic acid in a sample, (a) The sample containing the target nucleic acid is subjected to a single reaction in a single reaction vessel. (i) A pair of oligonucleotide primers, each of which is capable of hybridizing to the opposite strand of the subsequence of the target nucleic acid; (ii) an oligonucleotide probe comprising an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence non-complementary to the target nucleic acid sequence, and the annealing portion comprises a nucleotide sequence at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid to which the pair of oligonucleotide primers binds, and the probe further comprises an interaction double label comprising a dye containing one of the compounds according to any one of claims 1 to 4 located on the tag portion and a first quencher portion located on the annealing portion, wherein the dye is separated from the first quencher portion by a nuclease-sensitive cleavage site; a step of contacting the oligonucleotide probe; Prior to step (b), a contact step is performed, wherein the tag portion is temperature-dependently and reversibly bound to a quenching oligonucleotide having a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe, and is bound to the tag portion by hybridization, and the quenching oligonucleotide includes at least a second quencher portion that can quench the dye on the tag portion when the quenching oligonucleotide is bound to the tag portion; (b) An amplification step following step (a), wherein the target nucleic acid is amplified by a polymerase chain reaction (PCR) using a nucleic acid polymerase having 5' to 3' nuclease activity, thereby enabling the nuclease activity of the polymerase to cleave and separate the tag portion from the first quencher portion on the annealing portion of the probe during the extension step of each PCR cycle; (c) A step of measuring one or more signals from the dye at a first temperature in which the quenching oligonucleotide is bound to the tag portion; (d) A step of measuring one or more signals from the dye at a second temperature higher than the first temperature, in which the quenching oligonucleotide is not bound to the tag portion; (e) A step of obtaining a calculated signal value by subtracting the median or average value of the one or more signals detected at the first temperature from the median or average value of the one or more signals detected at the second temperature; A method wherein a calculated signal value higher than a threshold signal value enables the determination of the presence of the target nucleic acid.

16. The method according to claim 15, wherein one or more nucleotide modifications are selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), cross-linked nucleic acids (BNA), 2'-O alkyl substitutions, L-enantiomerized nucleotides, and combinations thereof.

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