Fluorescent dyes with large stokes shift

Abstract

The present disclosure generally relates to novel and readily available fluorescent compounds with large Stokes shift (LSS) and thermostable fluorescence for extending the multiplex capabilities of fluorescence-based nucleic acid detection techniques. In addition, conjugates, probes, and FRET pairs comprising these fluorescent compounds, as well as methods for amplifying and detecting target nucleic acids using these fluorescent compounds and methods for labeling are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 356,433, filed on June 28, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to novel and readily available fluorescent compounds with large Stokes shift (LSS) and thermally stable fluorescence for use in expanding the multiplexing capabilities of fluorescence-based nucleic acid detection techniques. Background Art

[0004] The polymerase chain reaction (PCR) has become a ubiquitous tool in biomedical research, disease monitoring, and diagnosis. Amplification of nucleic acid sequences by PCR is described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188. PCR is now well known in the art and has been extensively described in the scientific literature. See PCR Applications, ((1999) Innis et al., ed., Academic Press, San Diego), PCR Strategies, ((1995) Innis et al., ed., Academic Press, San Diego); PCR Protocols, ((1990) Innis et al., ed., Academic Press, San Diego) and PCR Technology, ((1989) Erlich, ed., Stockton Press, New York). "Real-time" PCR assays are able to simultaneously amplify and detect and / or quantify the starting amount of a target sequence. The basic principle of using the 5′ to 3′ nuclease activity of a DNA polymerase is to amplify and detect and / or quantify the starting amount of a target sequence. Real-time PCR assays are described in Holland et al., (1991) Proc. Natl. Acad. Sci. 88: 7276-7280 and U.S. Pat. No. 5,210,015. Real-time PCR without nuclease activity (nuclease-free assay) has been described in U.S. Patent Publication No. 20100143901A1. The use of fluorescent probes in real-time PCR is described in U.S. Pat. No. 5,538,848.

[0005] Typical real-time PCR protocols with fluorescent probes include the use of labeled probes specific for each target sequence. Preferably, the probes are labeled with one or more fluorescent moieties that absorb and emit light at specific wavelengths. Upon hybridization with the target sequence or its amplicon, the probes exhibit detectable changes in fluorescence emission due to probe hybridization or hydrolysis.

[0006] However, the main challenge of real-time assays remains the ability to analyze many targets in a single tube. In almost every field of medicine and diagnostics, the number of target loci is increasing rapidly. For example, in forensic DNA identification, pathogenic microorganism detection, multi-locus genetic disease screening, and multi-gene expression studies, multiple loci must be analyzed.

[0007] Commercial fluorescence-based automated polymerase chain reaction (PCR) devices can detect multiple targets in a single reaction vessel (multiplexed assays) by distinguishing light from fluorophores of different colors. The dyes are selected in a way that minimizes their spectral overlap. Each fluorophore in the set can be excited by light at or near the absorption maximum, and the emitted light (fluorescence) is detected at or near the fluorescence maximum. Individual fluorophores can be distinguished by limiting the wavelength (band) range of excitation and emission by optical filters. The specific combination of excitation bands and simultaneously detected emission bands defines an optical channel, and each channel allows the identification of one PCR target.

[0008] The maximum number of achievable optical channels depends on many interrelated factors, such as the available spectral range, excitation light intensity, fluorophore brightness, fluorophore spectral width, filter bandwidth, and detection factor sensitivity. State-of-the-art fluorescence detection-based PCR devices use four to six optical filters per excitation and emission pathway. Thus, using standard fluorophores, four to six individual PCR targets can be distinguished. Summary of the invention

[0009] The present disclosure relates to dyes with large Stokes shifts, such as Stokes shifts of about 50 nm or greater, about 60 nm or greater, about 70 nm or greater, about 80 nm or greater, about 90 nm or greater, etc.). Herein, it has been determined that incorporation of specific linker moieties into the dyes of the present disclosure allows for easy tuning of their spectral properties, such as their excitation and emission wavelengths. In addition, by selecting appropriate linker moieties, the dyes of the present disclosure can be converted to their respective activated derivatives, such as their respective NHS-esters; or modified to incorporate functional groups capable of participating in click chemistry chemical reactions for biomolecule labeling. In addition, the introduction of protecting groups allows the dyes to be derivatized into phosphoramidites, such as those compatible with nucleic acid solid phase synthesis and phosphoramidite chemistry.

[0010] In addition, it has been determined that the dyes of the present disclosure exhibit excellent brightness in combination with thermally stable fluorescence. It has also been surprisingly found that the dyes of the present disclosure can be easily obtained from inexpensive starting materials in a single, high-yield reaction step. These benefits and other benefits are further described herein.

[0011] The first aspect of the present disclosure is a compound having formula (I):

[0012]

[0013] in

[0014] R 1 is H or a protecting group;

[0015] R 2 For -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 -(carboxyl reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a group capable of participating in a "click chemistry" reaction. 1 -C 8 a branched or unbranched alkyl, a branched or unbranched heteroalkyl, or a cycloalkyl group; and

[0016] [X] - is the counter anion, provided that when R 2 When it has a negative charge, [X] - Does not exist.

[0017] In some embodiments, R 1 is 9-fluorenylmethylcarbamate, tert-butylcarbamate, benzylcarbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, monomethoxytrityl (MMT), DMS and p-toluenesulfonamide. In some embodiments, R 1 is H. In some embodiments, R 2 The first carbon atom of is a primary carbon atom. 2 The first carbon atom of is a secondary carbon atom. 2The first carbon atom of is a tertiary carbon atom. In some embodiments, the group capable of participating in the "click chemistry reaction" is selected from the group consisting of the following items: bicyclo [6.1.0] nonyne) group ("BCN"), dibenzocyclooctyne ("DBCO"), olefins, 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 the following items: haloacetyl, maleimide, iodoacetamide, aziridine, acryloyl, arylating agent, vinyl sulfone, methanethiosulfonate, pyridyl disulfide 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, ethylene oxide, carbonate, aryl halide, fluorophenol ester, sulfochlorophenol, carbodiimide, phthalimide, benzotriazole, imidoester 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 2 Selected from the group consisting of: 2 -Maleimide or -C 2 -CO 2 -Maleimide substituted 1 -C 8 branched or unbranched alkyl, branched or unbranched heteroalkyl or cycloalkyl group; 2 -NHS ester or -C 2 -CO 2 -NHS ester substituted C 1 -C 8 branched or unbranched alkyl, branched or unbranched heteroalkyl or cycloalkyl group; and -CO 2 -Hydrazine or -C 2 -CO 2 -Hydrazine substituted C 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -CO 2 -(thiol reactive group), -CO 2 -(amine reactive group), -CO 2 -(carboxyl reactive group), -C 2 -CO2 - , -C 2 -CO 2 -(thiol reactive group), -C 2 -CO 2 -(amine reactive group), -C 2 -CO 2 -(carboxyl reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a group capable of participating in a "click chemistry" reaction. 1 -C 6 In some embodiments, R 2 C is substituted by BCN, DBCO, azide or TCO 1 -C 6 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 6 In some embodiments, R 2 Selected from:

[0018]

[0019]

[0020] In some embodiments, R 2 is -phosphoramidite or -O-phosphoramidite. In some embodiments, R 1 is 9-fluorenylmethylcarbamate, tert-butylcarbamate, benzylcarbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, monomethoxytrityl (MMT), DMS and p-toluenesulfonamide; and R 2 It is -phosphoramidite or -O-phosphoramidite.

[0021] A second aspect of the present disclosure is a compound selected from the group consisting of:

[0022]

[0023] The dye is

[0024]

[0025] And wherein [X]- is a counter anion.

[0026] The third aspect of the present disclosure is a compound selected from the group consisting of:

[0027]

[0028] Where [X]- is the counter anion.

[0029] A fourth aspect of the present disclosure is a compound having formula (IA):

[0030]

[0031] R 2 For -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 -(carboxyl reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a group capable of participating in a "click chemistry" reaction. 1 -C 8 branched or unbranched alkyl, branched or unbranched heteroalkyl or cycloalkyl group; and [X]- is a counter anion, provided that when R 2 When it has a negative charge, [X]- does not exist.

[0032] In some embodiments, R 2 Selected from the group consisting of: 2 -Maleimide or -C 2 -CO 2 -Maleimide substituted 1 -C 8 branched or unbranched alkyl, branched or unbranched heteroalkyl or cycloalkyl group; 2 -NHS ester or -C 2 -CO 2 -NHS ester substituted C 1 -C 8 branched or unbranched alkyl, branched or unbranched heteroalkyl or cycloalkyl group; and -CO 2 -Hydrazine or -C2 -CO 2 -Hydrazine substituted C 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 8 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 For -Me, -Et, -CO 2 - , -OH, halogen, -D or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, halogen or -D 1 -C 8 In some embodiments, R 2 is -phosphoramidite or -O-phosphoramidite. In some embodiments, R 2 Selected from:

[0033]

[0034]

[0035] In some embodiments, the compound of the fourth aspect of the present disclosure has a Stokes shift of at least about 70nm. In some embodiments, the compound of the fourth aspect of the present disclosure has a Stokes shift of at least about 80nm. In some embodiments, the compound of the fourth aspect of the present disclosure has a Stokes shift of at least about 90nm. In some embodiments, the compound of the fourth aspect of the present disclosure is thermally stable in 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, tetraphenylborate ion, nitrate, and an anion of an aromatic or aliphatic carboxylic acid.

[0036] A 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 of formula (I), formula (IA), and formula (IB) (such as any of those 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 between about 5 and about 60 polymers. 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 of the compounds having formula (IA).

[0037] A 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 formula (I), formula (IA), and formula (IB). In some embodiments, the hapten is a pyrazole; a nitrophenyl compound; a benzofuran; a triterpene; urea; a thiourea; rotenone or a rotenone derivative; an oxazole; a thiazole; a coumarin or a coumarin derivative; or a cyclolignan.

[0038] The seventh aspect of the present disclosure is a conjugate having formula (II):

[0039]

[0040] in

[0041] R 1 is H or a protecting group;

[0042] R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 8 an alkyl, heteroalkyl or cycloalkyl group;

[0043] The "specific binding entity" is an oligonucleotide or a protein;

[0044] Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is 0, 1 or 2.

[0045] In some embodiments, protein is an antibody, for example, an antibody or a secondary antibody. In some embodiments, the oligonucleotide comprises between about 5 polymers and about 60 polymers. In some embodiments, the oligonucleotide comprises between about 5 polymers and about 40 polymers. In some embodiments, the oligonucleotide comprises between about 5 polymers and about 20 polymers. In some embodiments, Y is a branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and about 20 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, Y has a structure of formula (IIIC):

[0046]

[0047] Where R 3 and R 4 Each of is independently a bond or a group selected from carbonyl, amide, imide, ester, ether, -NH, -N-, thiol or thiol;

[0048] R 5 For key, C 1 -C 12 The alkyl or heteroalkyl groups include, and wherein R 5 can include a carbonyl, imide or thione;

[0049] R a and R b are independently H or methyl;

[0050] g and h are independently 0 or an integer in the range of 1 to 4; i is 0, 1 or 2.

[0051] In some embodiments, R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 6 In some embodiments, R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2-, -D or halogen substituted with one or more of C 1 -C 4 Alkyl, heteroalkyl or cycloalkyl groups.

[0052] The eighth aspect of the present disclosure is a conjugate having a structure of any one of Formula (IIC) and Formula (IID):

[0053]

[0054] in

[0055] R 1 is H or a protecting group;

[0056] R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 8 an alkyl, heteroalkyl or cycloalkyl group;

[0057] The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer;

[0058] Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and

[0059] a is 0, 1 or 2.

[0060] In some embodiments, R 3 The first carbon atom of is a primary carbon atom. 3 The first carbon atom of is a secondary carbon atom. 3 The first carbon atom of is a tertiary carbon atom. In some embodiments, Y is a branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C1 -C 8 In some embodiments, R 1 is H; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 30 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 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 25 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 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 15 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, R 1is H; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 10 carbon atoms and optionally having one or more heteroatoms selected from O, N or S.

[0061] The ninth aspect of the present disclosure is a kit comprising (i) a first conjugate comprising a first oligonucleotide coupled to a dye moiety derived from a compound having any one of formula (I), formula (IA) and formula (IB); and (ii) a second conjugate comprising an oligonucleotide coupled to a quencher. In some embodiments, the first conjugate has any one of formula (IIC) or formula (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, such as through a joint (e.g., a substituted or unsubstituted joint having between 5 and about 40 carbon atoms).

[0062] The tenth aspect of the present disclosure is a probe having formula (IV):

[0063] [Dye 1]-[Y] a -[5′-oligonucleotide-3′]-[Y] a -[Dye 2] (IV),

[0064] in

[0065] One of [Dye 1] or [Dye 2] is derived from a compound of any one of Formula (I), Formula (IA) and Formula (IB); and the other of [Dye 1] or [Dye 2] is a quencher;

[0066] The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer;

[0067] each Y is independently a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and

[0068] a is 0, 1 or 2.

[0069] In some embodiments, one of [Dye 1] or [Dye 2] is derived from a compound having formula (IA):

[0070]

[0071] Where R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 8 Branched or unbranched alkyl groups.

[0072] In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 6 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 4 In some embodiments, R 2 The first carbon atom of is a primary carbon atom. 2 The first carbon atom of is a secondary carbon atom. 2 The first carbon atom of is a tertiary carbon atom. 2 Selected from:

[0073]

[0074] An eleventh aspect of the present disclosure is a conjugate having formula (V):

[0075] [(oligo 1)(dye)]-linker-[(oligo 2)(Q1)](V),

[0076] in

[0077] Oligo 1 and oligo 2 are each different and are oligonucleotides having between about 5 and about 30 mers;

[0078] The dye is derived from a compound having any one of formula (I), formula (IA) and formula (IB);

[0079] Q1 is a quencher; and

[0080] The linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 30 carbon atoms.

[0081] In some embodiments, the dye is derived from a compound having Formula (IA):

[0082]

[0083] Where R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 6 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 4 In some embodiments, R 2 The first carbon atom of is a primary carbon atom. 2 The first carbon atom of is a secondary carbon atom. 2 The first carbon atom of is a tertiary carbon atom. 2 Selected from:

[0084]

[0085] In certain embodiments, dye has a Stokes shift of at least about 70nm. In certain embodiments, dye has a Stokes shift of at least about 80nm. In certain embodiments, dye has a Stokes shift of at least about 90nm. In certain embodiments, at least one of oligomer 1 and oligomer 2 comprises LNA, L-LNA or PNA. In certain embodiments, joint is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group with carbon atoms between about 5 and about 25. In certain embodiments, joint is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group with carbon atoms between about 5 and about 20. In certain embodiments, joint is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group with carbon atoms between about 5 and about 15.

[0086] A twelfth aspect of the present disclosure is a kit comprising: (i) a conjugate having any one of formula (IIC) and formula (IID); and (ii) a compound having formula (VIII):

[0087] [Oligo 3]-[Q2] (VIII),

[0088] in

[0089] Oligo 3 is an oligonucleotide having between 5 and 30 mers; and Q2 is a quencher.

[0090] 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):

[0091] [Dye 1]-[Y] a -[5′-oligonucleotide 1-3′] (VIIA),

[0092] [5′-Oligonucleotide 2-3′]-[Y] a -[Dye 2] (VIIB),

[0093] in

[0094] One of Dye 1 or Dye 2 is derived from a compound having any one of Formula (I), Formula (IA) and Formula (IB);

[0095] The other of Dye 1 or Dye 2 is a quencher;

[0096] each Y is independently a branched or unbranched, straight chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S;

[0097] a is 0, 1, or 2; and

[0098] Oligonucleotide 1 and oligonucleotide 2 are different.

[0099] In some embodiments, each Y is independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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):

[0100]

[0101] Where R 2 For -Me, -Et, -CO 2 -C substituted with one or more of -OH, -D or halogen 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 6 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 4 In some embodiments, R 2 The first carbon atom of is a primary carbon atom. 2 The first carbon atom of is a secondary carbon atom. 2 The first carbon atom of is a tertiary carbon atom. 2 Selected from:

[0102]

[0103] A fourteenth aspect of the present disclosure is a method for amplifying and detecting a target nucleic acid in a sample, the method comprising the following steps:

[0104] (a) contacting the sample containing the target nucleic acid with (i) a pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to opposite strands of a subsequence of the target nucleic acid in a single reaction vessel; and (ii) an oligonucleotide probe comprising an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence that is not complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence that is at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid defined by the pair of oligonucleotide primers, wherein the probe further comprises a nucleotide sequence derived from a polypeptide having formula (I) located on the tag portion. ) and a first quencher moiety located on the annealing moiety and wherein the dye is separated from the first quencher moiety by a nuclease-sensitive cleavage site; and wherein prior to step (b) (as described below), the tag moiety is reversibly bound in a temperature-dependent manner to a quencher oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag moiety of the oligonucleotide probe and bound to the tag moiety by hybridization, wherein the quencher oligonucleotide comprises at least a second quencher moiety capable of quenching the dye on the tag moiety when the quencher oligonucleotide is bound to the tag moiety;

[0105] (b) after 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 can cleave and separate the tag portion from the first quencher portion on the annealing portion of the probe;

[0106] (c) measuring one or more signals from the dye at a first temperature at which the quencher oligonucleotide is bound to the tag moiety;

[0107] (d) measuring one or more signals from the dye at a second temperature higher than the first temperature, at which second temperature the quencher oligonucleotide does not bind to the tag moiety; and

[0108] (e) obtaining a calculated signal value by subtracting a median or average value of the one or more signals detected at the first temperature from a median or average value of the one or more signals detected at the second temperature;

[0109] A calculated signal value therein above a threshold signal value allows determining the presence of the target nucleic acid.

[0110] In some embodiments, the PCR of step (b) is allowed to amplify to an end point beyond the amplification logarithmic phase. In some embodiments, the tag portion comprises a modification that makes it impossible to be extended by a nucleic acid polymerase. In some embodiments, the tag portion of the oligonucleotide probe or the quencher oligonucleotide or both the tag portion and the quencher oligonucleotide contain one or more nucleotide modifications. In some embodiments, 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-enantiomeric nucleotides and combinations thereof.

[0111] The fifteenth aspect of the present invention is a method for directly labeling a dye with an oligonucleotide having a terminal amine, wherein the method comprises: (i) obtaining a dye comprising a dye core and having a cyano group located at the middle position of the dye core; (ii) contacting the obtained dye with the oligonucleotide having the terminal amine in the presence of a base and a solvent, wherein a linker is located between the oligonucleotide and the terminal amine, wherein the linker is a -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 8 Alkyl, heteroalkyl, cycloalkyl or heterocycloalkyl groups.

[0112] 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] non-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), cyclopentane, N-butylpyrrolidone, γ-valerolactone, δ-valerolactone, N-methylpyrrolidone, N, N-dimethylformamide, cyclopentane and Cyrene. In some embodiments, the oligonucleotide comprises between about 5 polymers and about 60 polymers. In some embodiments, the oligonucleotide comprises between about 5 polymers and about 40 polymers. In some embodiments, the oligonucleotide comprises between about 5 polymers and about 30 polymers. In some embodiments, the oligonucleotide comprises between about 5 polymers and about 20 polymers. In some embodiments, the oligonucleotide comprises between about 5 polymers and about 15 polymers. In some embodiments, the oligonucleotide comprises LNA, L-LNA or PNA. In some embodiments, the dye having a cyano group located at the middle position of the dye core is R800 perchlorate dye. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] For a general understanding of the features of the present disclosure, reference is made to the drawings, in which like reference numerals are used throughout to identify like elements.

[0114] Figure 1 A method of preparing a branched DNA probe to incorporate the fluorescent dye of the present disclosure is exemplified.

[0115] Figure 2 Optical channels and dye assignments for the Cobas x800 PCR instrument are shown. The center wavelengths of the excitation (vertical) and emission filters (horizontal) are in nanometers. The fields on the diagonal correspond to the optical channels that can be accessed using standard fluorophores (COU, FAM, HEX, JA270, Cy5.5). Compound R, Compound 1a to Compound 1k ("RLS 1") are LSS dyes that are spectrally suitable for the 435nm / 580nm channel. Compound 11 to Compound 1r ("RLS2") are large Stokes shift dyes that are spectrally suitable for the 495nm / 580nm channel.

[0116] Figure 3A , Figure 3B and Figure 3C Analytical data for R800 dye (upper panel) and compound R (lower panel) are summarized. Shown are the chromatograms, mass spectra in positive ion mode, and absorbance spectra.

[0117] Figure 4A The chromatogram at the absorption maximum for compound R is shown.

[0118] Figure 4B The absorption and fluorescence emission spectra for compound R are shown.

[0119] Figure 4C The mass spectrum in positive ion mode is shown for compound R.

[0120] Figure 5A The chromatogram at the absorption maximum is shown for compound 1a.

[0121] Figure 5B Absorption and fluorescence emission spectra are shown for compound 1a.

[0122] Figure 5C The mass spectrum in positive ion mode is shown for compound 1a.

[0123] Figure 6 The mass spectrum in positive ion mode is shown for compound 1b.

[0124] Fig. 7A The chromatogram at the absorption maximum for compound 1c is shown.

[0125] Figure 7B Absorption and fluorescence emission spectra are shown for compound 1c.

[0126] Figure 7C The mass spectrum in positive ion mode is shown for compound 1c.

[0127] Fig. 8A The chromatogram at the absorption maximum for compound 1d is shown.

[0128] Figure 8B Absorption and fluorescence emission spectra are shown for compound 1d.

[0129] Figure 8C The mass spectrum in positive ion mode is shown for compound 1d.

[0130] Fig. 9A The chromatogram at the absorption maximum for compound 1e is shown.

[0131] Fig. 9B Absorption and fluorescence emission spectra are shown for compound 1e.

[0132] Fig. 9C The mass spectrum in positive ion mode is shown for compound 1e.

[0133] Fig. 10AThe chromatogram at the absorption maximum for compound 1f is shown.

[0134] Fig. 10B Absorption and fluorescence emission spectra are shown for compound 1f.

[0135] Fig. 10C The mass spectrum in positive ion mode is shown for compound 1f.

[0136] Fig.11A The chromatogram at the absorption maximum for compound 1g is shown.

[0137] Fig. 11B Absorption and fluorescence emission spectra are shown for compound 1g.

[0138] Fig. 11C The mass spectrum in positive ion mode is shown for compound 1g.

[0139] Fig. 12A The chromatogram at the absorption maximum for compound 1h is shown.

[0140] Fig. 12B Absorption and fluorescence emission spectra are shown for compound 1h.

[0141] Fig. 12C The mass spectrum in positive ion mode is shown for compound 1h.

[0142] Fig.13A The chromatogram at the absorption maximum for compound li is shown.

[0143] Fig. 13B Absorption and fluorescence emission spectra are shown for compound 1i.

[0144] Fig. 13C The mass spectrum in positive ion mode is shown for compound 1i.

[0145] Fig.14A The chromatogram at the absorption maximum for compound 1j is shown.

[0146] Fig. 14B Absorption and fluorescence emission spectra are shown for compound 1j.

[0147] Fig. 14C The mass spectrum in positive ion mode is shown for compound 1j.

[0148] Fig.15A The chromatogram at the absorption maximum for compound 1 k is shown.

[0149] Fig. 15B Absorption and fluorescence emission spectra are shown for compound 1k.

[0150] Fig. 15C The mass spectrum in positive ion mode is shown for compound 1k.

[0151] Fig.16A The chromatogram at the absorption maximum for compound 11 is shown.

[0152] Fig. 16B Absorption and fluorescence emission spectra are shown for compound 11.

[0153] Fig. 16C The mass spectrum in positive ion mode is shown for compound 11.

[0154] Fig.17A The chromatogram at the absorption maximum for compound 1m is shown.

[0155] Fig. 17B Absorption and fluorescence emission spectra are shown for compound 1m.

[0156] Fig. 17C The mass spectrum in positive ion mode is shown for compound 1m.

[0157] Fig.18A The chromatogram at the absorption maximum for compound In is shown.

[0158] Fig.18B Absorption and fluorescence emission spectra are shown for compound 1n.

[0159] Fig. 18C The mass spectrum in positive ion mode is shown for compound 1n.

[0160] Fig.19A The chromatogram at the absorption maximum for compound 1o is shown.

[0161] Fig.19B Absorption and fluorescence emission spectra are shown for compound 1o.

[0162] Fig.19C The mass spectrum in positive ion mode is shown for compound 1o.

[0163] Fig. 20A The chromatogram at the absorption maximum for compound 1p is shown.

[0164] Fig. 20B Absorption and fluorescence emission spectra are shown for compound 1p.

[0165] Fig. 20C The mass spectrum in positive ion mode is shown for compound 1p.

[0166] Fig.21AThe chromatogram at the absorption maximum for compound 1q is shown.

[0167] Fig.21B Absorption and fluorescence emission spectra are shown for compound 1q.

[0168] Fig. 21C The mass spectrum in positive ion mode is shown for compound 1q.

[0169] Fig.22A The chromatogram at the absorption maximum for compound 1r is shown.

[0170] Fig. 22B Absorption and fluorescence emission spectra are shown for compound 1r.

[0171] Fig. 22C The mass spectrum in positive ion mode is shown for compound 1r.

[0172] Fig.23A The chromatogram at the absorption maximum for compound 1s is shown.

[0173] Fig. 23B Absorption and fluorescence emission spectra are shown for compound 1s.

[0174] Fig.23C The mass spectrum in positive ion mode is shown for compound 1s.

[0175] Fig.24 The thermal stability of the fluorescence for compound R (open line), compound 1i (solid line), compound 1k (dashed line), compound 1j (dotted line) and 1n (dashed line) is shown. Each sample is excited at its respective absorption maximum and the fluorescence signal at the emission maximum is plotted as a function of temperature. For simplicity, the data have not been normalized to concentration.

[0176] Fig.25 Direct labeling of 36-mer DNA modified with R800 dye to BHQ-2 and amino is shown. DNA labeling is achieved without the need for a carboxylic acid functional group because the dye linker moiety is derived from a DNA amino modifier. All chromatograms are obtained using the same mobile phase gradient. The first chromatogram shows the retention time of the pre-purified DNA starting material. The second chromatogram shows the retention time of the R800 dye. The third chromatogram shows the separation of the labeling reaction and the labeled DNA dye conjugate. The fourth figure shows the purified DNA dye conjugate. The superposition of DNA absorption spectra before and after the labeling reaction is shown in the upper right figure. The lower right figure shows the deconvoluted mass spectrum of the DNA-dye conjugate in the negative ion mode.

[0177] Fig.26AChromatograms of three different 5'-azido-modified DNA sequences of different lengths (15mer, 16mer and 37mer) labeled with R800 dye are shown. The DNA-dye conjugates are obtained by direct labeling, i.e., no active ester or click chemistry functional groups are required. In this way, the amino linker of the DNA becomes an integral part of the dye structure. All chromatograms were obtained using the same mobile phase gradient.

[0178] Fig.26B Shown for Fig.26A Absorption spectrum (left) and deconvoluted mass spectrum (right) of the DNA sequence in negative ion mode.

[0179] Fig. 27 5′-azido 15-mer DNA labeled with compound 1j is shown. The DNA-1j conjugate is obtained by forming an amide bond between the amino functional group at the 5′-penultimate position of DNA and the active ester of compound 1j generated in situ. The chromatogram shows the retention time of the DNA-1j conjugate. Since compound 1j is a racemic mixture of enantiomers, the labeled DNA is a mixture of diastereomers and is separated as a bimodal. The absorption spectra for the left and right peaks are shown in the two middle figures. The figure below shows the deconvoluted mass spectrum in the negative ion mode of the DNA-1j conjugate.

[0180] Fig.28 The LSS dye (solid line) prepared in Example 6 is shown. PCR growth curve of DNA probe (replicate). Fluorescence was detected in the LSS channel ( Figure 2 , channel RLS 1, 435nm / 580nm). The same The PCR growth curve was obtained for the sequence (dashed line). Fluorescence was detected in the Cy5.5 channel ( Figure 2 , 580 / 700nm) and superimposed in the graph for comparison. DETAILED DESCRIPTION

[0181] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are presented unless indicated to the contrary.

[0182] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" is defined as inclusive, such as "comprising A or B" means comprising A, B, or A and B.

[0183] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one element in a number of elements or a list of elements, but also including more than one element, and optionally including additional unlisted items. Only terms indicating the contrary, such as "only one of" or "exactly one of", or "consisting of..." used in the claims, will refer to the inclusion of exactly one element in a number of elements or a list of elements. In general, the term "or" used herein should be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both") only when it is preceded by an exclusive term such as "or", "one of", "only one", or "exactly one". "Substantially consisting of..." used in the claims should have the ordinary meaning used in the field of patent law.

[0184] As used herein, the terms "including", "comprising", "having", and the like are used interchangeably and have the same meaning. Similarly, "including", "comprising", "having", and the like are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "including" under ordinary U.S. patent law, and therefore each term may be understood as an open term, meaning "at least the following", and may also be interpreted as not excluding 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 involving steps a, b, and c" means that the method includes at least steps a, b, and c. In addition, although the steps and processes may be outlined herein in a particular order, those skilled in the art will recognize that the order steps and processes may vary.

[0185] As used herein in the specification and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood as at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. In addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, the definition also allows other elements to be optionally present, whether or not they are related to the specifically identified elements. Thus, as a 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 refer to at least one, optionally including more than one A, without B (and optionally including elements other than B) in one embodiment; in another embodiment, at least one, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0186] References throughout this specification to "one embodiment," "an embodiment," etc., mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in different places in 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.

[0187] As used herein, the symbol It refers to the position where one part is bonded to another part.

[0188] 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), cycloalkyl groups substituted with alkyl groups, and cycloalkyl groups substituted with 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, a straight or branched chain alkyl has 8 or fewer carbon atoms in its backbone (e.g., for a straight chain, C 1 -C 8 , for the branched chain, C 1 -C 8). In addition, the term alkyl includes both "unsubstituted alkyl" and "substituted alkyl," the latter referring to alkyl moieties having substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphino, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxyl, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfonylamino, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0189] As used herein, the term "amine-reactive group" refers to an agent or group that can form a covalent bond with an amine group or another molecule.

[0190] As used herein, the term "antibody" refers to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to: IgA, IgD, IgE, IgG and IgM and combinations thereof; similar molecules produced during the immune response of any vertebrate (e.g., in mammals such as humans, goats, rabbits and mice, etc.); and antibody fragments that specifically bind to a target molecule (or a group of highly similar target molecules) and substantially exclude binding to other molecules. "Antibody" further refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region, which specifically recognizes and binds to an epitope of an antigen. An antibody can be composed of a heavy chain and a light chain, each of which has a variable region, referred to as a variable heavy chain (VH) region and a variable light chain (VL) region. The VH region and the VL region are jointly responsible for binding to the antigen recognized by the antibody. The term "antibody" also includes complete immunoglobulins and variants and portions thereof well known in the art.

[0191] As used herein, C a To C b (where "a" and "b" are integers) 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 heteroalicyclic group. That is, the ring of an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heteroalicyclic group may contain from "a" to "b" (inclusive) carbon atoms. Thus, for example, "C 1 To C 4"Alkyl" refers to all alkyl groups having 1 to 4 carbons, i.e., CH 3 -、CH 3 CH 2 -、CH 3 CH 2 CH 2 -、(CH 3 ) 2 CH--、CH 3 CH 2 CH 2 CH 2 , CH 3 CH 2 CH(CH 3 )- and (CH 3 ) 3 C-.

[0192] As used herein, the term "carbonyl-reactive group" refers to an agent or group that can form a covalent bond with a carbonyl group or another molecule.

[0193] As used herein, the term "click chemistry" refers to a chemical principle independently defined by the research groups of Sharpless and Meldal, describing a chemical process tailored to rapidly and reliably generate substances by linking small units together."Click chemistry" has been applied to a set of reliable and autonomous organic reactions (Kolb, HC; Finn, MG; Sharpless, KB Angew. Chem. Int. Ed. 2001, 40, 2004-2021). For example, the copper-catalyzed azide-alkyne [3+2] cycloaddition reaction was identified as a reliable molecular ligation in water (Rostovtsev, VV et al., Angew. Chem. Int. Ed. 2002, 41, 2596-2599), which has been used in studies to enhance various types of biomolecular interactions (Wang, Q. et al., J. Am. Chem. Soc. 2003, 125, 3192-3193; Speers, AE et al., J. Am. Chem. Soc. 2003, 125, 4686-4687; Link, AJ; Tirrell, DAJ Am. Chem. Soc. 2003, 125, 11164-11165; Deiters, A. et al., J. Am. Chem. Soc. 2003, 125, 11782-11783). In addition, it has also appeared in organic synthesis (Lee, LV et al., J. Am. Chem. Soc. 2003, 125, 9588-9589), drug discovery (Kolb, HC; Sharpless, KB Rug Disc. Today 2003, 8, 1128-1137; Lewis, WG 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, JP et al., Langmuir 2004, ASAP, in press; Lummerstorfer, T.; Hoffmann, HJ Phys. Chem. B 2004, in press).Generally speaking, "click chemistry" promotes reactions that are widely applicable in a modular manner, obtains high chemical yields, generates harmless byproducts, has chemical specificity, requires simple reaction conditions, uses readily available starting materials and reagents, uses no solvents or uses mild solvents (e.g., water), easily isolates the products, has a high thermodynamic driving force to favor the reaction to obtain a single reaction product, and obtains high atom economy. Although certain general criteria may be subjective in nature, and not all criteria need to be met.

[0194] As used herein, the term "conjugate" refers to two or more molecules or moieties (including macromolecules or supramolecules) covalently linked 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) covalently linked to one or more other molecular moieties.

[0195] As used herein, the term "coupled" or "coupled" refers to the binding, bonding (eg, covalent bonding), or connection of one molecule or atom to another molecule or atom.

[0196] As used herein, the term "derivative" is used according to its ordinary meaning in the fields of chemistry and biology, and refers to a compound that is structurally similar to another compound (the so-called "reference" compound) but has a different composition (e.g., an atom is replaced by an atom of a different element, or a specific functional group is present, or one functional group is replaced by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound is different). Therefore, an analog is a compound that is similar or equivalent in function and appearance to a reference compound, but has a different structure or origin.

[0197] As used herein, the term "heteroatom" refers to boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si). In some embodiments, as described herein, a "heterocycle" may contain one or more heteroatoms. In other embodiments, an aliphatic group may contain one or more heteroatoms or be substituted with one or more heteroatoms.

[0198] As used herein, the term "oligonucleotide" refers to a linear oligomer of natural or modified nucleoside monomers connected by a phosphodiester bond or its analogs. Oligonucleotides include deoxyribonucleosides, ribonucleosides, their end group isomer forms, peptide nucleic acids (PNA), etc. that can specifically bind to target nucleic acids. Typically, monomers are connected by phosphodiester bonds or their analogs to form oligonucleotides, and their size ranges from several monomer units, such as 3-4, to tens of monomer units, such as 40-60. Whenever an oligonucleotide is represented by a letter sequence, such as "ATGCCTG", it should be understood that the nucleotides are in 5'-3' order from left to right, and unless otherwise specified, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents deoxythymidine, and "U" represents ribonucleoside uridine. Typically, oligonucleotides contain four natural deoxynucleotides; however, they can also contain ribonucleosides or non-natural nucleotide analogs, as described above. When the enzyme has a specific oligonucleotide or polynucleotide substrate requirement for activity, such as single-stranded DNA, RNA / DNA duplex, etc., then the selection of an appropriate composition for the oligonucleotide or polynucleotide substrate is well within the skill of the art.

[0199] As used herein, the term "phosphoramidite" refers to a trivalent phosphorus group commonly used in oligonucleotide synthesis. A detailed description of the chemistry used to form oligonucleotides by the phosphoramidite method is provided in Caruthers et al., U.S. Patent Nos. 4,458,066 and 4,415,732; Caruthers et al., Genetic Engineering, 4: 1-17 (1982); Users Manual Model 392 and 394 Polynucleotide Synthesizers, pp. 6-1 to pp. 6-22, Applied Biosystems, Part No. 901237 (1991), each of which is incorporated by reference as a whole.

[0200] As used herein, the term "primary antibody" refers to an antibody that specifically binds to a target protein antigen in a tissue sample. A primary antibody is typically the first antibody used in an immunohistochemistry procedure.

[0201] As used herein, the term "protecting group" refers to a portion that masks, reduces or prevents the reactivity when attached to a reactive group in a molecule. 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 TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd Edition John Wiley & Sons, New York, 1999, Harrison and Harrison et al. Compendium of Synthetic Organic Methods, Vol. 1-8 (John Wiley and Sons, 1971-1996), and "Protection of Nucleosides for Oligonucleotide Synthesis," Current Protocols in Nucleic Acid Chemistry, Boyle, AL Editor, John Wiley & Sons, Inc., 2000, New York, NY, all of which are incorporated herein by reference in their entirety.

[0202] As used herein, the term "reactive group" or "reactive functional group" refers to a functional group that is capable of chemically associating, interacting, hybridizing, hydrogen bonding, or coupling with a functional group of a different moiety. In some embodiments, a "reaction" between two reactive groups or two reactive functional groups may mean the formation of a covalent bond between the two reactive groups or two reactive functional groups; or may mean 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 a binding event, such as the binding of a hapten to an anti-hapten antibody or a guest molecule associated with a supramolecular host molecule.

[0203] As used herein, the term "secondary antibody" refers herein to an antibody that specifically binds to a primary antibody, thereby forming a bridge between the primary antibody and subsequent reagents, if any, such as labels, enzymes, etc. The secondary antibody is typically the second antibody used in immunohistochemical procedures.

[0204] 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 that are characterized by binding to each other to the substantial exclusion of binding to other molecules (e.g., the binding constant of the specific binding pair may be at least 10 greater than the binding constant of either member of the binding pair for other molecules in the biological sample). 3 M -1 , 10 4 M-1 or 10 5 M -1 Specific examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidin (such as streptavidin), and protein A). Specific binding moieties may also include molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0205] As used herein, the term "Stokes shift" refers to the difference (expressed in wavelength or frequency units) between the positions of the band maxima of the absorption and emission spectra (fluorescence and Raman are two examples) of the same electronic transition. 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, thereby losing its energy.

[0206] It is believed that the vast majority of small molecule fluorophores exhibit Stokes shifts of the order of 10-25 nm. Fluorophores with significantly larger Stokes shifts are broadly referred to as "large Stokes shift" (LSS) dyes, "high Stokes shift" dyes or "super Stokes (MegaStokes)" dyes. Two photophysical mechanisms are discussed in the literature to explain the occurrence of large Stokes shifts. The molecular geometry mechanism is based on the conformational relaxation of the fluorophore in the excited state and the rearrangement of the surrounding solvent dipoles caused thereby. The Stokes shift increases with the increase of the difference between the (balanced) molecular geometry and dipole moment of the ground state and the excited state. The large Stokes shift fluorescence of the electronic mechanism is attributed to the intramolecular charge transfer (ICT) of the excited state. A common problem of small Stokes shift fluorophores is the internal quenching of fluorescence. This self-quenching is caused by the spectral overlap of excitation and emission, and is particularly prevalent at high fluorophore concentrations. LSS dyes have better separated spectral bands, which minimizes the reabsorption of photons. There is a non-zero probability of excitation of the fluorophore outside its main excitation peak. Therefore, fluorescence from one dye inevitably leads to the total light detected in multiple emission channels. This spectral "crosstalk" or "bleed-through" can be compensated to a certain extent by using a predetermined correction factor for calculation. In addition, the scattering of the excitation light increases the background fluorescence of the adjacent channel. LSS dyes allow the reduction or even avoidance of crosstalk and scattering from other fluorophores. LSS dyes are particularly useful in experimental environments where many fluorophores produce strong background signals. The large spectral separation of LSS dyes allows more effective filtering of the excitation light, thereby enhancing the sensitivity of target detection. LSS dyes can obtain fluorescence data from optical channels that were previously unavailable. Promoted by wide spectral separation, and when used in combination with standard fluorophores, LSS dyes allow the increase of the multiplex capability of fluorescent PCR devices. In this way, LSS markers allow additional channels to be performed on established four to six-color instruments. In principle, 21 channels can be composed of filter combinations of six-color instruments. However, in practical applications, the number of channels is limited by the commercial availability of LSS dyes with sufficiently large Stokes shifts. Nine additional channels can be achieved based on the Stokes shift of 150 nm for currently commercially available LSS dyes. Light grey highlights the channels of standard dyes, while dark grey indicates channels for suitable LSS dyes that are not currently available. In contrast, resonant electron transfer (RET) probes produce large "virtual" Stokes shifts and can also be used to obtain these channels.

[0207] Whenever a group or moiety is described as "substituted" or "optionally substituted", the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as "substituted or unsubstituted", if substituted, the one or more substituents may be selected from one or more of the indicated substituents. If no substituents are indicated, it means that the indicated "optionally substituted" or "substituted" group may be substituted with one or more groups individually and independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclic, aralkyl, heteroaralkyl, (heteroalicyclic)alkyl, hydroxy, protected hydroxy, alkoxy, aryloxy, acyl, thiol, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamoyl alkyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxyl, protected C-carboxyl, O-carboxyl, isothiocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, ether, amino (e.g., monosubstituted amino or disubstituted amino) and protected derivatives thereof. Any of the above groups may include one or more heteroatoms, including O, N or S. For example, when a portion is substituted with an alkyl group, the alkyl group may include a heteroatom selected from O, N or S (e.g., -(CH 2 -CH 2 -O-CH 2 -CH 2 )-).

[0208] As used herein, the term "thiol-reactive group" refers to an agent or group that can form a covalent bond with a thiol group or another molecule.

[0209] Overview

[0210] The present disclosure relates to dyes, such as dyes with large Stokes shifts. The present disclosure also relates to conjugates and probes comprising one or more of the disclosed dyes. The present invention 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 disclosed herein can be used with any fluorescence-based PCR platform with a compatible filter. Conjugates comprising or derived from one of the dyes disclosed herein are also compatible with PCR using TAGS (temperature assisted signal generation) technology, as long as the dye exhibits stable fluorescence at temperatures up to 100°C (see U.S. Patent Nos. 11,028,433, 11,034,997, and 11,345,958; and U.S. Patent Publication No. 2021 / 0269857, the disclosures of which are incorporated herein by reference in their entirety.

[0211] A common problem with fluorophores with "small" Stokes shifts is internal quenching of fluorescence. This self-quenching is caused by spectral overlap of excitation and emission and is particularly prevalent at high fluorophore concentrations. "Large" Stokes shift dyes (such as those disclosed herein) generally have better separated spectral bands, which minimizes reabsorption of photons.

[0212] It is believed that there is a non-zero probability of excitation of a fluorophore outside its main excitation peak. Therefore, the fluorescence from the dye inevitably leads to the total light detected in multiple emission channels. This spectral "crosstalk" or "bleed-through" can be compensated to a certain extent by using a predetermined correction factor for calculation. In addition, the scattering of the excitation light increases the background fluorescence of the adjacent channels. "Large" Stokes shift dyes (such as those disclosed in the present invention) allow to reduce crosstalk and scattering from other fluorophores. "Large" Stokes shift dyes (such as those disclosed in the present invention) are particularly useful in experimental environments where many fluorophores produce strong background signals. The large spectral separation of "large" Stokes shift dyes (such as those disclosed in the present invention) is considered to allow more effective filtering of the excitation light, thereby enhancing the sensitivity of target detection.

[0213] "Large" Stokes shift dyes (such as those disclosed herein) also provide access to fluorescence data from previously inaccessible optical channels. Due to their broad spectral separation and when used in combination with standard fluorophores, "large" Stokes shift dyes (such as those disclosed herein) allow the multiplexing capability of fluorescent PCR devices to be increased by adding more channels to established four to six-color instruments. In principle, 21 channels can be provided by the filter combination of a six-color instrument. However, in practice, the number of channels is limited by the commercial availability of dyes with suitable spectral properties and sufficiently large Stokes shifts.

[0214] Dye or dye precursor

[0215] The present disclosure provides compounds having formula (I):

[0216]

[0217] in

[0218] R 1 is H or a protecting group;

[0219] R 2 For -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 -(carboxyl reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a group capable of participating in a "click chemistry" reaction. 1 -C 8 a branched or unbranched alkyl, a branched or unbranched heteroalkyl, or a cycloalkyl group; and

[0220] [X] - is the counter anion, provided that when R 2 When it has a negative charge, [X]- does not exist.

[0221] In some embodiments, [X] is chloride, bromide, iodide, sulfate, benzenesulfonate, p-toluenesulfonate, p-bromobenzenesulfonate, methanesulfonate, trifluoromethanesulfonate, phosphate, perchlorate, tetrafluoroborate, hexafluorophosphate, tetraphenylborate, nitrate, and an anion of an aromatic or aliphatic carboxylic acid. In some embodiments, R 1 is H. In other embodiments, R 1 is 9-fluorenylmethylcarbamate, tert-butylcarbamate, benzylcarbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, monomethoxytrityl (MMT), DMS and p-toluenesulfonamide. In some embodiments, R 2 The first carbon atom of is a primary carbon atom. 2 The first carbon atom of is a secondary carbon atom. In yet other embodiments, R 2 The first carbon atom of is a tertiary carbon atom. 2 In other embodiments, R 2 In other embodiments, R 2 In some embodiments, R 2 includes heteroalkyl groups having heteroatoms selected from O, N, or S. In some embodiments, R 2Including heteroalkyl groups with O heteroatoms. In some embodiments, the group capable of participating in the "click chemistry reaction" is selected from bicyclo [6.1.0] nonyne) group ("BCN"), dibenzocyclooctyne ("DBCO"), olefins, trans-cyclooctene ("TCO"), maleimide, aldehyde, ketone, azide, tetrazine, thiol, 1,3-nitrone, hydrazine and hydroxylamine. In some embodiments, R 1 is H; and R 2 It is one of BCN, DBCO, TCO, azide, tetrazine or maleimide. In some embodiments, the compound of formula (I) terminates with one of thiol-reactive groups, amine-reactive groups or carboxyl-reactive groups. 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 may include maleimide. In some embodiments, the thiol-reactive group may include haloacetyl. In some embodiments, the thiol-reactive group may include aziridine. In some embodiments, the thiol-reactive group may include acryloyl. In some embodiments, the thiol-reactive group may include arylating agent. In some embodiments, the thiol-reactive group may include vinyl sulfone. In some embodiments, the thiol-reactive group may include pyridyl disulfide. In some embodiments, the thiol-reactive group may include TNB-thiol. In some embodiments, the thiol reactive group may comprise a 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-dicarboximide), isothiocyanates, acyl azides, sulfonyl chlorides, sulfodichlorophenols, pentafluorophenols, tetrafluorophenols, 4-sulfo-2,3,5,6-tetrafluorophenyls, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, fluorophenol esters, sulfochlorophenols, uranium, carbodiimides, phthalimides, benzotriazoles, amide esters, anhydrides, etc. In some embodiments, the carbonyl reactive group is hydrazine, hydrazine derivatives, or amines. In some embodiments, R 2 For 2 -Maleimide-substituted C 1 -C 8 In some embodiments, R 2 For 2 -Maleimide-substituted C 1 -C 6 In some embodiments, R 2 For2 -Maleimide-substituted C 1 -C 4 In some embodiments, R 2 It is a classic-C 2 -CO 2 -Maleimide-substituted C 1 -C 8 In some embodiments, R 2 It is a classic-C 2 -CO 2 -Maleimide-substituted C 1 -C 6 In some embodiments, R 2 It is a classic-C 2 -CO 2 -Maleimide-substituted C 1 -C 4 In some embodiments, R 2 It is through-CO 2 -NHS ester substituted 1 -C 8 In some embodiments, R 2 It is through-CO 2 -NHS ester substituted 1-C 6 In some embodiments, R 2 It is through-CO 2 -NHS ester substituted 1 -C 4 In some embodiments, R 2 It is a classic-C 2 -CO 2 -NHS ester substituted C 1 -C 8 In some embodiments, R 2 It is a classic-C 2 -CO 2 -NHS ester substituted C 1 -C 6 In some embodiments, R 2 It is a classic-C 2-CO 2 -NHS ester substituted C 1 -C 4 In some embodiments, R 2 It is through-CO 2 -Hydrazine substituted C 1 -C 8 In some embodiments, R 2 For 2 -Hydrazine substituted C 1 -C 6 In some embodiments, R 2 For 2 -Hydrazine substituted C 1 -C 4 In some embodiments, R 2 It is a classic-C 2 -CO 2 -Hydrazine substituted C 1 -C 8 In some embodiments, R 2 It is a classic-C 2 -CO 2 -Hydrazine substituted C 1 -C 6 In some embodiments, R 2 It is a classic-C 2 -CO 2 -Hydrazine substituted C 1 -C 4 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D or one or more of the groups capable of participating in a "click chemistry" reaction 1 -C 8 In some embodiments, R 2 For the classics-M e , -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C8 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 8 In some embodiments, R 2 C substituted by BCN, DBCO or TCO 1 -C 6 In some embodiments, R 2 For -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 -(carboxyl reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a group capable of participating in a "click chemistry" reaction. 1 -C 6 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D or one or more of the groups capable of participating in a "click chemistry" reaction 1 -C 6 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 6 In some embodiments, R 2 For -Me, -Et, -CO 2 -C substituted with one or more of -OH, -D or halogen 1 -C 6 In some embodiments, R 2 C substituted by BCN, DBCO or TCO 1 -C 6 In some embodiments, R 2 For -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 -(carboxyl reactive group), -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or a group capable of participating in a "click chemistry" reaction. 1 -C 4 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D or one or more of the groups capable of participating in a "click chemistry" reaction 1 -C 4 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 4 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, -D or halogen 1 -C 4 In some embodiments, R 2 C substituted by BCN, DBCO or TCO1 -C 4 In some embodiments, R 2 Selected from:

[0222]

[0223] Examples of compounds of formula (I) include, but are not limited to, the following:

[0224]

[0225] The dye is

[0226]

[0227] In some embodiments, the compound of formula (I) comprises: as well as

[0228] In some embodiments, the compound of Formula (I) has the structure of Formula (IA):

[0229]

[0230] Where R 2 and [X] is as defined above.

[0231] In some embodiments, R 2 The first carbon atom of is a primary carbon atom. 2 The first carbon atom of is a secondary carbon atom. In yet other embodiments, R 2 The first carbon atom of is a tertiary carbon atom. 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 In some embodiments, R 2 In some embodiments, R 2 In some embodiments, R 2The compound of formula (IA) is substituted with a maleimide moiety. In some embodiments, the compound of formula (IA) is terminated with a thiol-reactive group, an amine-reactive group, or a carboxyl-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 amine reactive group is selected from NHS esters (e.g., NHS, sulfo-NHS, N-hydroxy-5-norbornene-2,3-dicarboximide), isothiocyanates, acyl azides, sulfonyl chlorides, sulfodichlorophenols, pentafluorophenols, tetrafluorophenols, 4-sulfo-2,3,5,6-tetrafluorophenyl, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, fluorophenol esters, sulfochlorophenols, uranium, carbodiimides, phthalimides, benzotriazoles, imidoesters, anhydrides, and the like. In some embodiments, the carbonyl reactive group is hydrazine, a hydrazine derivative, or an amine. In some embodiments, R 2 For 2 -Maleimide-substituted C 1 -C 8 In some embodiments, R 2 It is a classic-C 2 -CO 2 -Maleimide-substituted C 1 -C 8 In some embodiments, R 2 It is through-CO 2 -NHS ester substituted 1 -C 8 In some embodiments, R 2 It is a classic-C 2 -CO 2 -NHS ester substituted C 1 -C 8 In some embodiments, R 2 is a C substituted with a group capable of participating in a "click chemistry" reaction 1 -C 8 In some embodiments, the group capable of participating in the "click chemistry" reaction is BCN, DBCO or TCO. In some embodiments, R 2 For -Me, -Et, -CO 2 -, -OH, -phosphoramidite, -O-phosphoramidite, -D or one or more of the groups capable of participating in a "click chemistry" reaction 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, halogen or -D 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D or one or more of the groups capable of participating in a "click chemistry" reaction 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, halogen or -D 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 4 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction.1 -C 4 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, halogen or -D 1 -C 4 Branched or unbranched alkyl groups.

[0232] Examples of compounds of formula (IA) include, but are not limited to, those listed in Table 1a, Table 1b, and Table 1c:

[0233] Table 1a: Compounds of formula (IA) wherein R 2 It is primary carbon.

[0234]

[0235]

[0236] Table 1b: Compounds of formula (IA) wherein R 2 For secondary carbon,

[0237]

[0238] Table 1c: Compounds of formula (IA) wherein R 2 It is a tertiary carbon.

[0239]

[0240]

[0241] In some embodiments, the compound of Formula (I) has the structure of Formula (IB):

[0242]

[0243] in

[0244] "Protecting group" is 9-fluorenylmethylcarbamate, tert-butylcarbamate, benzylcarbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, monomethoxytrityl (MMT), DMS and p-toluenesulfonamide; and

[0245] R 2 and [X] is as defined herein.

[0246] In some embodiments, R 2 The first carbon atom of is a primary carbon atom. 2 The first carbon atom of is a secondary carbon atom. 2The first carbon atom of is a tertiary carbon atom. 2 It is through-CO 2 -NHS ester substituted 1 -C 8 In some embodiments, R 2 It is a classic-C 2 -CO 2 -NHS ester substituted C 1 -C 8 In some embodiments, R 2 For 2 -Maleimide-substituted C 1 -C 8 In some embodiments, R 2 It is a classic-C 2 -CO 2 -Maleimide-substituted C 1 -C 8 In some embodiments, R 2 is a C substituted with a group capable of participating in a "click chemistry" reaction 1 -C 8 In some embodiments, the group capable of participating in the "click chemistry" reaction is BCN, DBCO or TCO. In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D or one or more of the groups capable of participating in a "click chemistry" reaction 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, halogen or -D 1 -C 8In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D or one or more of the groups capable of participating in a "click chemistry" reaction 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, halogen or -D 1 -C 8 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 4 In some embodiments, R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 4 In some embodiments, R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, halogen or -D 1 -C 4 In some embodiments, R 2 It is -phosphoramidite or -O-phosphoramidite.

[0247] Non-limiting examples of compounds of formula (IB) are listed in Table 2a, Table 2b and Table 2c:

[0248] Table 2a: Compounds of formula (IA) wherein R 2 It is primary carbon.

[0249]

[0250]

[0251] Table 2b: Compounds of formula (IA) wherein R 2 It is secondary carbon.

[0252]

[0253] Table 2c: Compounds of formula (IA) wherein R 2 It is a tertiary carbon.

[0254]

[0255]

[0256] In some embodiments, R 1 is trifluoroacetamide and R 2 For -Me, -Et, -CO 2 - , -OH, -phosphoramidite, -O-phosphoramidite, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 8 In some embodiments, R 1 is trifluoroacetamide and R 2 For -Me, -Et, -CO 2 - , -OH, -D, halogen, or one or more of the groups capable of participating in a "click chemistry" reaction. 1 -C 8 In yet other embodiments, R 1 is trifluoroacetamide and R 2 For -Me, -Et, -CO 2 - C substituted with one or more of -OH, halogen or -D 1 -C 8 Branched or unbranched alkyl groups 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 Select from the group consisting of:

[0257]

[0258] In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 50 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 55 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 60 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 65 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 70 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 75 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 80 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 85 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 90 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 95 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (IB) has a Stokes shift of at least about 100 nm. In some embodiments, the compound of any one of formula (I), formula (IA), and formula (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.

[0259] Table 3; Properties of various conjugates of formula (IA).

[0260]

[0261] Table 3 above summarizes the spectral properties, including Stokes shift, of several compounds of formula (IA). The column "Amino Linker Reagent" corresponds to the primary amines that reacted with the R800 dye to obtain compounds R and 1a-1s of the present disclosure. The absorption and emission maxima and the resulting Stokes shifts are expressed in nanometers. The peak area of ​​the UPLC fluorescence peak was divided by the peak area of ​​the absorption peak to obtain an estimate of the brightness of the dye. Compound 1b was unstable and compound 1s did not show characteristics for the LSS dye (11 nm Stokes shift), so brightness data (nd) were not determined.

[0262] In some embodiments, dyes of any of Formula (I), Formula (IA), and Formula (IB) are thermostable. For example, Applicants surprisingly discovered that dyes of Formula (I), Formula (IA), and Formula (IB) exhibit thermostable fluorescence in the temperature range of about 25°C to about 100°C. This Fig.24 , which shows the change in fluorescence with temperature for Compound R, Compound 1i, Compound 1k, Compound 1j, and Compound 1n. No significant decrease in fluorescence was observed up to 100°C. The slightly more pronounced drift of Compound R and Compound 1n to higher fluorescence can be explained by their increased solubility at higher temperatures because they are more hydrophobic than carboxylic acid compounds 1i, Compound 1k, and Compound 1j. In some embodiments, the dye having any one of Formula (IA) is thermally stable in the temperature range of about 25°C to about 100°C. In some embodiments, Compounds 1a to 1s as described herein are thermally stable in the temperature range of about 25°C to about 100°C.

[0263] Conjugate

[0264] The present disclosure also provides conjugates comprising or derived from one or more compounds of formula (I), formula (IA) and formula (IB) and a specific binding entity. In some embodiments, one or more compounds of formula (I), formula (IA) and formula (IB) are directly coupled to the specific binding entity. In some embodiments, one or more compounds of formula (I), formula (IA) and formula (IB) are indirectly coupled to the specific binding entity. In some embodiments, the indirect coupling is carried out through one or more linkers.

[0265] In some embodiments, the conjugate comprises a compound derived from any one of Formula (I), Formula (IA), and Formula (IB) coupled directly or indirectly to a 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.

[0266] In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 60. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 55. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 50. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 45. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 40. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 35. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 30. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 25. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 20. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 15.

[0267] In some embodiments, the "specific binding entity" is an oligonucleotide, and the dye having 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 formula (I) is directly or indirectly coupled to the 3' end of the oligonucleotide.

[0268] In some embodiments, the conjugate comprising a compound derived from any one of Formula (I), Formula (IA), and Formula (IB) and a specific binding entity has the structure of Formula (II):

[0269]

[0270] In this article

[0271] R 1 is H or a protecting group;

[0272] R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 8 an alkyl, heteroalkyl or cycloalkyl group;

[0273] [Specific binding entity] is a protein or oligonucleotide;

[0274] Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and

[0275] a is 0, 1 or 2.

[0276] In embodiments where the specific binding entity is an oligonucleotide, the dye portion of the conjugate can be coupled to the 5' end or the 3' end of the oligonucleotide. In some embodiments, the oligonucleotide, whether coupled to the dye portion at the 5' end or the 3' end, comprises between about 5-mers and about 40-mers. In some embodiments, R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 6 In some embodiments, R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 4 Alkyl, heteroalkyl or cycloalkyl groups.

[0277] In some embodiments, Y may include a carbonyl, amine, ester, ether, amide, imide, thioketone or thiol group. In some embodiments, Y is a branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 between 2 and 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 between 2 and 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):

[0278]

[0279] 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 Independently H, C 1 -C 4 Alkyl, F, Cl or N(R c )(R d );R c and R d Independently CH 3 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.

[0280] 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 some 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, straight 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. It is independently a branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 4 carbon atoms and optionally having one or more O, N or S heteroatoms. In some embodiments, Y has a structure of formula (IIIB):

[0281]

[0282] in

[0283] d and e are each independently an integer in the range of 2 to 20;

[0284] Q is a bond, O, S or N (R c )(R d );

[0285] R c and R d Independently CH 3 or H; and

[0286] 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.

[0287] In some embodiments, Y has the structure of Formula (IIIC):

[0288]

[0289] Where R 3 and R 4 Each of is independently a bond or a group selected from carbonyl, amide, imide, ester, ether, -NH, -N-, thiol or thiol;

[0290] R 5 For key, C 1 -C 12 The alkyl or heteroalkyl groups include, and wherein R 5 can include a carbonyl, imide or thione;

[0291] R a and R b are independently H or methyl;

[0292] g and h are independently 0 or an integer ranging from 1 to 4;

[0293] i is 0, 1 or 2.

[0294] In some embodiments, R a and R b Each is H. In some embodiments, Y is derived from:

[0295] 5′-Amino modifier C6-TFA (GLEN Research Catalog No. 10-1916)

[0296] Amino Modifier C6 dT (GLEN Research Catalog No. 10-1039)

[0297] Amino-L-threonine alcohol amide

[0298]

[0299] Amino modifier C6 dC (10-1019)

[0300] Amino Modifier C2 dT (Glen Research Cat. No. 10-1037)

[0301] Amino Modifier C6 dA (Glen Research Cat. No. 10-1089)

[0302] N2-Amino Modifier C6 dG (Glen Research Cat. No. 10-1529)

[0303] Fmoc amino modifier C6 dT (Glen Research Cat. No. 10-1536)

[0304] 5′-Amino Modifier 5 (Glen Research Cat. No. 10-1905)

[0305] 5′-Amino Modifier C6 (Glen Research Cat. No. 10-1906)

[0306] 5′-DMS(O)MT-amino modifier C6 (Glen Research Cat. No. 10-1907)

[0307] 5′-Amino Modifier C12 (Glen Research Cat. No. 10-1912)

[0308] 5′-Amino modifier TEG CE-phosphoramidite (Glen Research Cat. No. 10-1917)

[0309] 5′-Amino Modifier C3-TFA (Glen Research Cat. No. 10-1923)

[0310] 5′-Amino modifier C6-PDA (Glen Research Cat. No. 10-1947)

[0311] 5′-Amino modifier C12-PDA (Glen Research Cat. No. 10-1948)

[0312] 5′-Amino Modifier TEG PDA (Glen Research Cat. No. 10-1949)

[0313] Amino modifier serinol phosphoramidite (Glen Research catalog number 10-1997)

[0314] PC amino modifier phosphoramidite (Glen Research catalog number 10-4906)

[0315] 3′-Amino Modifier C6 dC CPG (Glen Research Cat. No. 20-2019)

[0316] 3′-Amino Modifier C6 dT CPG (Glen Research Cat. No. 20-2039)

[0317] 3′-PT-Amino Modifier C3 CPG (Glen Research Cat. No. 20-2954)

[0318] 3′-PT-Amino Modifier C6 CPG (Glen Research Cat. No. 20-2956)

[0319] 3′-Amino modifier C7 CPG 1000 (Glen Research Cat. No. 20-2958)

[0320] 3′-Amino modifier Serinol CPG (Glen Research Cat. No. 20-2997)

[0321] 3′-PT-Amino Modifier C6 PS (Glen Research Cat. No. 26-2956)

[0322] 5′-DBCO-TEG phosphoramidite (Glen Research Cat. No. 10-1941)

[0323] DBCO-serinol phosphoramidite (Glen Research Cat. No. 10-1998)

[0324] DBCO-dT-CE phosphoramidite (Glen Research Cat. No. 10-1539)

[0325] 5′-Bromohexylphosphoramidite (Glen Research Cat. No. 10-1946)

[0326] In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH_ substituted with one or more of C 1 -C 8 In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 6 In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; and R 3For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 4 In some embodiments, R 1 is H; the specific binding entity is an oligonucleotide; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and 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; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 6 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and 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; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 4 alkyl, heteroalkyl or cycloalkyl group, and Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S.

[0327] In some embodiments, the conjugate of Formula (II) has the structure of any one of Formula (IIA) and Formula (IIB):

[0328] [Dye]-[Y] a -[5′-oligonucleotide-3′] (IIA) or

[0329] [5′-oligonucleotide-3′]-[Y] a -[Dye] (IIB),

[0330] in

[0331] The dye is derived from any one of formula (I), formula (IA) or formula (IB);

[0332] Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and

[0333] a is 0, 1, or 2; and

[0334] The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer.

[0335] In some embodiments, dye is derived from any one of compound 1a to compound 1s (see Table 1a, Table 1c and Table 1c herein). In some embodiments, Y is a group having between 2 and about 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S, branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated. In some embodiments, Y is a group having between 2 and about 20 carbon atoms and optionally having one or more heteroatoms selected from O, N or S, branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated. In some embodiments, Y is a group having between 2 and about 10 carbon atoms and optionally having one or more heteroatoms selected from O, N or S, branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated. In some embodiments, Y has the structure of any one of formula (IIIA), formula (IIIB) and formula (IIIC) as described herein. In some embodiments, oligonucleotides include between about 5 aggressiveness and about 60 aggressiveness. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 55. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 50. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 45. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 40. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 35. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 30. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 25. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 20. In some embodiments, the oligonucleotide comprises a polymer between about 5 and about 15.

[0336] In some embodiments, the conjugate of Formula (II) has the structure of any one of Formula (IIC) and Formula (IID):

[0337]

[0338]

[0339] in

[0340] R 1 is H or a protecting group;

[0341] R 3 For -Me, -Et, -CO 2 -、-C 2-CO 2 -, -D or halogen substituted with one or more of C 1 -C 8 an alkyl, heteroalkyl or cycloalkyl group;

[0342] The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer;

[0343] Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is 0, 1 or 2.

[0344] In some embodiments, R 3 The first carbon atom of is a primary carbon atom. 3 The first carbon atom of is a secondary carbon atom. 3 The first carbon atom of is a tertiary carbon atom. 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 6 In some embodiments, R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 4In some embodiments, Y is a branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 between 2 and about 10 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In some embodiments, Y may comprise a carbonyl, amine, ester, ether, amide, imide, thione or thiol group. In some embodiments, Y has a structure of formula (IIIA):

[0345]

[0346] 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 Independently H, C 1 -C 4 Alkyl, F, Cl or N(R c )(R d );R c and R d Independently CH 3 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 heteroatoms of O, N or S. In some embodiments, d and e are integers in the range of 2 to 6.

[0347] In some embodiments, d and e are integers in the range of 2 to 10. In some 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, straight 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. It is independently a branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 4 carbon atoms and optionally having one or more O, N or S heteroatoms. In some embodiments, Y has a structure of formula (IIIB):

[0348]

[0349] in

[0350] d and e are each independently an integer in the range of 2 to 20;

[0351] Q is a bond, O, S or N (R c )(R d );

[0352] R c and R d Independently CH 3 or H; and

[0353] 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 heteroatoms of O, N or S. In some embodiments, Y has a structure of formula (IIIC):

[0354]

[0355] Where R 3 and R 4 Each of is independently a bond or a group selected from carbonyl, amide, imide, ester, ether, -NH, -N-, thiol or thiol;

[0356] R 5 For key, C 1 -C 12 The alkyl or heteroalkyl groups include, and wherein R 5 can include a carbonyl, imide or thione;

[0357] R a and R b are independently H or methyl;

[0358] g and h are independently 0 or an integer ranging from 1 to 4;

[0359] i is 0, 1 or 2.

[0360] In some embodiments, R 1 is H; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 In some embodiments, R 1 is H; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 6 In some embodiments, R 1 is H; and R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 4 In other embodiments, R 1 H; R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In other embodiments, R 1 H; R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 20 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In other embodiments, R1 H; R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 15 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In yet other embodiments, R 1 H; R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8 alkyl, heteroalkyl or cycloalkyl group; and Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 10 carbon atoms and optionally having one or more heteroatoms selected from O, N or S. In further embodiments, R 1 H; R 3 For -Me, -Et, -D, -C 2 -CO 2 -、-CO 2 - and -OH-substituted by one or more of C 1 -C 8Alkyl, heteroalkyl or cycloalkyl groups; and Y has the structure of any one of formula (IIIA), formula (IIIB) and formula (IIIC). In some embodiments, the oligonucleotide comprises between about 5 polymers and about 60 polymers. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises between about 5 and about 55 polymers. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises between about 5 and about 50 polymers. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises between about 5 and about 45 polymers. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises between about 5 and about 40 polymers. In some embodiments, the antisense oligonucleotide is single-stranded. In some embodiments, the oligonucleotide comprises between about 5 and about 35 polymers. In some embodiments, the oligonucleotide comprises between about 5 and about 30 polymers. In some embodiments, the oligonucleotide comprises between about 5 and about 25 polymers. In some embodiments, the oligonucleotide comprises between about 5 and about 20 polymers. In some embodiments, the oligonucleotide comprises between about 5 and about 15 polymers.

[0361] The present disclosure also relates to a conjugate comprising a compound of formula (I) and a hapten or enzyme (e.g., alkaline phosphatase; horseradish peroxidase). In some embodiments, the compound of formula (I) is directly coupled to the hapten or enzyme. In some embodiments, the compound of formula (I) is indirectly coupled to the hapten or enzyme. In some embodiments, the indirect coupling is carried out through one or more linkers. In some embodiments, the hapten is pyrazole (e.g., nitropyrazole); nitrophenyl compound; benzofuran; triterpene; urea (e.g., phenylurea); thiourea (e.g., phenylthiourea); rotenone or rotenone derivative; oxazole (e.g., oxazole sulfonamide); thiazole (e.g., thiazole sulfonamide); coumarin or coumarin derivative; or cyclolignan. In some embodiments, haptens include dinitrophenyl, biotin, digoxin and fluorescein and any derivatives or analogs thereof. Other haptens are described in U.S. Pat. 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.

[0362] Probe

[0363] The present disclosure also provides Probe, where The first dye of the probe is derived from any one of the compounds of Formula (I), Formula (IA) and Formula (IB), and wherein the second dye is a quencher. The probe can be used to As used herein, the term " "Probe" and "hydrolysis probe" can be understood interchangeably. In some embodiments, the first dye derived from the compound having formula (I) and the quencher are located near the end 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" can be understood in the broadest sense understood in the art. In addition, the terms "3' end" and "3' end" can be understood interchangeably as known in the art. In addition, it should be understood that the terms "3' end" and "3' end" used herein can refer to the 5' end of the nucleotide chain, but it is not excluded that at the 3' end, another molecular part (such as, for example, a fluorophore, a quencher, a binding part, etc.) is added to the 3' end of the probe.

[0364] The probe can hybridize to its target sequence. The composition of the probe may further comprise a pair of primers, such as a forward primer and a reverse primer. These primers are usually unlabeled. In addition, usually, the forward primer binds upstream of the band and the reverse primer binds downstream of the band, so that The probe binds to a sequence that is part of the strand being amplified. A PCR reaction, well known in the art, is performed. Thus, the target DNA is melted and then conditions are selected to enable the primers and probe to anneal to the target DNA. Subsequently, conditions are selected to enable a DNA polymerase to amplify the DNA strand between the primers. In the assay, the DNA polymerase typically has 5' to 3' exonuclease activity. Furthermore, the DNA polymerase may be Taq polymerase or a functional variant thereof. When the DNA polymerase reaches When the probe is attached, the 5' end is cut off. As a result, the compound having or derived from the orb of formula (I) or the quencher bound to the 5' terminal nucleotide is also cut off. In some embodiments, the compound having or derived from formula (I) is cleaved. Therefore, the compound having or derived from formula (I) and the quencher can diffuse in different directions. The spatial distance between the two can be significantly increased, and the fluorescence generated by the compound having or derived from formula (I) is significantly increased because it is no longer quenched by the dark quencher. In addition, real-time analysis can be performed. Determination. The determination can also be performed in a life-time PCR method. It can also be performed quantitatively in a qPCR reaction.

[0365] Using the probe disclosed herein Determination can be used for allelic differentiation, genotyping, bacterial identification determination, DNA quantitative and clinical samples in the determination of viral load, gene expression determination and the verification of microarray results.It can also be used for allelic differentiation, genotyping and bacterial identification analysis.Genotyping can be such as single nucleotide polymorphism (SNP) genotyping, and therefore includes determining the genotype at the interested locus place limited in the sample, wherein the locus is a mononucleotide.Or, genotyping can be copy number variation (CNV) genotyping.Copy number variation (CNV) is to find the DNA fragmentation of copy number (copy number of DNA sequence or its part) difference by comparing two or more genomes. As described above, sequences (and the loci of various SNPs and CNVs) can be obtained from databases such as the Database of Genomic Variants (DGV), the NCBI dbSNP database, the UCSC Genome Bioinformatics website, the Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER), the HapMap Project, the Sanger Institute Copy Number Variation Project, and the Human Structural Variation Project.

[0366] The present disclosure provides a probe having a structure of formula (IV), for example, Probe:

[0367] [Dye 1]-[Y] a -[5′-oligonucleotide-3′]-[Y] a -[Dye 2] (IV),

[0368] in

[0369] One of [Dye 1] or [Dye 2] is derived from any one of Formula (I), Formula (IA) and Formula (IB); and the other of Dye 1 or Dye 2 is a quencher;

[0370] The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer;

[0371] Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is 0, 1 or 2.

[0372] 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 the deep dark quenchers DDQ-I, DABCYL, Dark Quencher, Iowa FQ, Iowa RQ, Black Hole Series (BHQ-0, BHQ-1, BHQ-2, BHQ-3), QSY-7, DDQ-II, Iowa RQ, QSY-21, blackberry quencher (BBQ-650, available from LGC Biosearch); IDT double quencher (ZEN quencher; TAO quencher); Onyx quencher (available from MiliporeSigma) 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 of formula (IA). In other embodiments, the dye is derived from a compound of formula (IA) and wherein R 2 for

[0373]

[0374] Labeling probe

[0375] The present invention further provides a conjugate having the structure of formula (V):

[0376] [(oligo 1)(dye)]-linker-[(oligo 2)(Q1)](V),

[0377] in

[0378] Oligomer 1 and oligomer 2 are each different and are oligomers having between about 5-mers and about 30-mers;

[0379] The dye is derived from any of (I), (IA) or (IB);

[0380] Q1 is a quencher; and

[0381] The linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 40 carbon atoms.

[0382] In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 30 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 25 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 20 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 15 carbon atoms. In some embodiments, at least one of oligomer 1, oligomer 2 or the linker includes a nuclease sensitive cleavage site. In some embodiments, oligomer 1 and oligomer 2 may comprise DNA, L-DNA, RNA, L-RNA, LNA, L-LNA, PNA (peptide nucleic acid, as described in Nielsen et al., U.S. Pat. No. 5,539,082), BNA (bridged nucleic acid, such as 2',4'-BNA (NC) [2'-O,4'-C-aminomethylene bridged nucleic acid], as described in Rahman et al., J. Am. Chem. Soc. 2008; 130 (14): 4886-96), L-BNA, etc. (wherein "L-XXX" refers to the L enantiomer of the nucleic acid sugar unit) or any other known variations and modifications on the nucleotide base, sugar, or phosphodiester backbone. In some embodiments, one of oligomer 1 or oligomer 2 comprises L-DNA or consists entirely of L-DNA. In other embodiments, oligomer 1 comprises L-DNA or consists entirely of L-DNA. In other embodiments, oligomer 1 consists entirely of L-DNA. In some embodiments, the joint is an unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group with carbon atoms between about 5 and about 30. In other embodiments, the joint is an unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group with carbon atoms between about 5 and about 25. In other embodiments, the joint is an unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group with carbon atoms between about 5 and about 25. In some embodiments, the dye has a Stokes shift of at least about 60nm, at least about 70nm, at least about 80nm, at least about 90nm, at least about 100nm, etc. In some embodiments, the dye is derived from a compound with formula (IA). In other embodiments, the dye is derived from a compound with formula (IA), wherein R 2 for

[0383]

[0384] The present invention further provides an intermediate having the structure of formula (VI):

[0385] (Group capable of participating in click chemistry reaction)-(C 2 -C 8 )-O-[(oligonucleotide)(dye)]

[0386] (VI)

[0387] in

[0388] The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer; and

[0389] The dye is derived from any of (I), (IA) or (IB).

[0390] 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 for

[0391]

[0392] Reagent test kit

[0393] The present disclosure also provides a kit comprising at least two compounds having any one of formula (IA). The present disclosure also provides a kit comprising at least three compounds having any one of formula (IA). The present disclosure also provides a kit comprising at least four compounds having any one of formula (IA). The present disclosure also provides a kit comprising at least five compounds having any one of formula (IA). The present disclosure also provides a kit comprising at least six compounds having any one of formula (IA). The present disclosure also provides a kit comprising seven or more compounds having any one of formula (IA).

[0394] In some embodiments, at least one of the compounds of formula (IA) contained in any kit has a Stokes shift greater than about 50nm. In some embodiments, at least one of the compounds of formula (IA) contained in any kit has a shift greater than about 60nm. In some embodiments, at least one of the compounds of formula (IA) contained in any kit has a Stokes shift greater than about 70nm. In some embodiments, at least one of the compounds of formula (IA) contained in any kit has a Stokes shift greater than about 80nm. In some embodiments, at least one of the compounds of formula (IA) contained in any kit has a Stokes shift greater than about 90nm. In some embodiments, at least one of the compounds of formula (IA) contained in any kit has a Stokes shift greater than about 100nm. In some embodiments, at least one of the compounds of formula (IA) contained in any kit has a Stokes shift greater than about 110nm.

[0395] FRET Pair

[0396] The present disclosure also provides a kit including a FRET pair. FRET is a form of molecular energy transfer (MET), a process in which energy is transferred non-radioactively between a donor molecule and an acceptor molecule. FRET originates from the properties of certain compounds; when excited by exposure to light of a specific wavelength, they emit light at a different wavelength (i.e., they fluoresce). Such compounds are called fluorophores or fluorescent labels. In FRET, energy is transferred non-radioactively over a long distance (e.g., 10 angstroms to 100 angstroms) between a donor molecule (which may be a fluorophore) and an acceptor molecule (which may be a quencher or other fluorophore). The donor absorbs a photon and transfers the energy to the acceptor in a non-radioactive manner (Forster, 1949, Z. Naturforsch. A4: 321-327; Clegg, 1992, Enzyme Methods. 211: 353-388).

[0397] When two fluorophores with overlapping excitation and emission spectra are very close, the excitation of one fluorophore will cause it to emit light at a wavelength absorbed by the second fluorophore and these wavelengths stimulate the second fluorophore, causing it to emit fluorescence. In other words, the excited state energy of the first (donor) fluorophore is transferred to the adjacent second (acceptor) fluorophore by a resonance-induced dipole-dipole interaction. As a result, the lifetime of the donor molecule is shortened 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 is transferred to a non-fluorophore acceptor, the fluorescence of the donor is quenched, and the acceptor will not emit fluorescence subsequently. In this case, the acceptor acts as a quencher.

[0398] A pair of molecules that can participate in FRET is called a FRET pair. In order for energy transfer to occur, the donor and acceptor molecules must usually be very close (e.g., up to 70 angstroms to 100 angstroms) (Clegg, 1992, Enzyme Methods. 211: 353-388; Selvin, 1995, Enzyme Methods. 246: 300-334). As the distance between the donor and acceptor molecules increases, the efficiency of energy transfer decreases rapidly. In practice, this means that FRET occurs most efficiently up to a distance of about 70 angstroms.

[0399] In some embodiments of the present disclosure, the FRET pair comprises a first member comprising a dye derived from formula (I) directly or indirectly coupled to a first oligonucleotide; and a second member comprising a second oligonucleotide directly or indirectly coupled to a quencher. In some embodiments, the first member of the FRET pair comprises a conjugate having any one of formula (IIA), formula (IIB), formula (IIC), or formula (IID).

[0400] In some embodiments, the FRET pair comprises a first member having Formula (VIIA) and a second member having Formula (VIIB):

[0401] [Dye 1]-[Y] a -[5′-oligonucleotide 1-3′] (VIIA),

[0402] [5′-Oligonucleotide 2-3′]-[Y] a -[Dye 2] (VIIB),

[0403] in

[0404] One of Dye 1 or Dye 2 is derived from any one of Formula (I), Formula (IA) or Formula (IB);

[0405] The other of Dye 1 or Dye 2 is a quencher;

[0406] each Y is independently a branched or unbranched, straight chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S;

[0407] a is 0, 1, or 2; and

[0408] Oligonucleotide 1 and oligonucleotide 2 are different.

[0409] 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 for

[0410]

[0411]

[0412] Any quencher can be used in the compositions described herein without limitation, provided that it reduces the fluorescence intensity of the dye of formula (I) or a dye derived from formula (I) used. Commonly used quenchers for FRET include, but are not limited to, DeepDark Quencher DDQ-I, DABCYL, Dark quencher, Iowa FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa RQ, QSY-21 and Black Hole BHQ-3. Quenchers for use in the compositions provided herein can 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.).

[0413] In some embodiments, each Y is independently a branched or unbranched, straight or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 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 or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and about 15 carbon atoms and optionally having one or more heteroatoms selected from O, N or S.

[0414] In some embodiments, Oligonucleotide 1 and Oligonucleotide 2 include nucleotide modifications selected from locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA), 2'-O alkyl substitution, L-enantiomeric nucleotides, or combinations thereof. In some embodiments, the nucleotide modification includes LNA.

[0415] In some embodiments, the present disclosure provides a method for determining the genotype at a locus of interest in a sample containing genetic material, the method comprising the following steps: contacting the genetic material with a first probe having formula (VIIA) and a second probe having formula (VIIB); and detecting that one of the first and second probes binds 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 to the 3' end and a predetermined number of nucleotides (e.g., 4, 6, 8, 10, 12, 16, 20 nucleotides) comprising at least one DNA nucleotide and a predetermined number of locked nucleic acid nucleotides (e.g., at least five 2, 3, 4, 5, 6, 7, 8 locked nucleotides). In some embodiments, the nucleotides of the first probe include a first distinguishing position and the nucleotides of the second probe include a second distinguishing position, the second distinguishing position being located at the same nucleotide position in the second probe as the first distinguishing position in the first probe, the first distinguishing position including a nucleobase different from the second distinguishing position, wherein the nucleobases at other nucleotides of the first and second probes are the same.

[0416] Labeling probe

[0417] The present disclosure also provides a kit comprising (i) a conjugate having formula (V) and (ii) a conjugate having formula (VIII):

[0418] [(oligo 1)(dye)]-linker-[(oligo 2)(Q1)](V),

[0419] [Oligo 3]-[Q2](VIII),

[0420] in

[0421] The dye is derived from any of (I), (IA) or (IB);

[0422] Oligomer 1, oligomer 2, and oligomer 3 are each different and are oligomers having between about 5-mers and about 30-mers;

[0423] Q1 and Q2 are the same or different quenchers; and

[0424] The linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 40 carbon atoms.

[0425] In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 30 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 25 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 20 carbon atoms. In some embodiments, the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 15 carbon atoms. In some embodiments, one of oligomer 1 or oligomer 2 includes L-DNA or is completely composed of L-DNA. In other embodiments, oligomer 1 includes L-DNA or is completely composed of L-DNA. In other embodiments, oligomer 1 is completely composed of L-DNA. In other embodiments, oligomer 2 includes L-DNA or is completely composed of L-DNA. In other embodiments, oligomer 2 consists entirely of L-DNA. In some embodiments, Q1 and Q2 are identical. 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 for

[0426]

[0427] In some embodiments, the present disclosure provides a kit for detecting two or more target nucleic acid sequences in a sample, the kit comprising:

[0428] (a) two or more pairs of oligonucleotide primers having sequences complementary to each strand of two or more target nucleic acid sequences;

[0429] (b) at least one oligonucleotide probe comprising two distinct parts:

[0430] (i) an annealing moiety comprising a sequence that is at least partially complementary to one of the two or more target nucleic acid sequences and anneals within one of the two or more target nucleic acid sequences, wherein the annealing moiety comprises a first quencher moiety; and

[0431] (ii) a tag moiety attached to the 5′ end or the 3′ end of the annealing moiety or attached via a linker between the 5′ end and the 3′ end of the annealing moiety and comprising a nucleotide sequence that is not complementary to one of the two or more target nucleic acid sequences, wherein the tag moiety comprises a compound derived from formula (IA) and its detectable signal is capable of being quenched by a first quencher moiety on the annealing moiety, wherein the compound derived from formula (IA) is separated from the first quencher moiety by a nuclease-sensitive cleavage site;

[0432] (c) at least one quencher oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe and hybridizing with the tag portion to form a duplex, wherein the quencher oligonucleotide comprises a second quencher portion that quenches a detectable signal generated by the compound derived from Formula (IA) on the tag portion when the quencher oligonucleotide hybridizes with the tag portion.

[0433] In some embodiments, the label portion is attached to the 5' end of the annealing portion. In some embodiments, the label portion is attached via a joint between the 5' end and the 3' end of the annealing portion. In some embodiments, the label portion of the oligonucleotide probe or the quencher oligonucleotide or the label portion of the oligonucleotide probe and the quencher oligonucleotide both contain one or more nucleotide modifications. In some embodiments, 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-enantiomeric nucleotides or a combination 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-enantiomeric nucleotides. In some embodiments, the nucleotide modification includes L-enantiomeric 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).

[0434] A method for amplifying and detecting a target nucleic acid in a sample, the method comprising the following steps:

[0435] (a) contacting a sample containing a target nucleic acid in a single reaction vessel with

[0436] (i) a pair of oligonucleotide primers, each oligonucleotide primer being capable of hybridizing to opposite strands of a subsequence of a target nucleic acid;

[0437] (ii) an oligonucleotide probe comprising an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence that is not complementary to a target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence that is at least partially complementary to the target nucleic acid sequence and hybridizes to a region of a subsequence of the target nucleic acid defined by a pair of oligonucleotide primers, wherein the probe further comprises an interactive 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 wherein the compound having (or derived from) formula (IA) is separated from the first quencher portion by a nuclease-sensitive cleavage site; and

[0438] wherein prior to step (b), the tag moiety is reversibly bound in a temperature-dependent manner to a quencher oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag moiety of the oligonucleotide probe and bound to the tag moiety by hybridization, wherein the quencher oligonucleotide comprises at least a second quencher moiety capable of quenching a compound having (or derived from) formula (IA) on the tag moiety when the quencher oligonucleotide is bound to the tag moiety;

[0439] (b) after 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 can cleave and separate the tag portion from the first quencher portion on the annealed portion of the probe;

[0440] (c) measuring an inhibition signal from a compound having (or derived from) formula (IA) at a first temperature at which the quencher oligonucleotide is bound to the tag moiety;

[0441] (d) raising the temperature to a second temperature at which the quenching oligonucleotide does not bind to the tag moiety;

[0442] (e) measuring a temperature-corrected signal from a compound having (or derived from) formula (IA) at a second temperature;

[0443] (f) obtaining a calculated signal value by subtracting the suppressed signal detected at the first temperature from the temperature corrected signal detected at the second temperature;

[0444] (g) repeating steps (b) to (f) through multiple PCR cycles;

[0445] (h) Measuring the signal values ​​calculated from multiple PCR cycles to detect the presence of the target nucleic acid.

[0446] A method for amplifying and detecting a target nucleic acid in a sample, the method comprising the following steps:

[0447] (a) contacting a sample containing a target nucleic acid in a single reaction vessel with

[0448] (i) a pair of oligonucleotide primers, each oligonucleotide primer being capable of hybridizing to opposite strands of a subsequence of a target nucleic acid;

[0449] (ii) an oligonucleotide probe comprising an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence that is not complementary to a target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence that is at least partially complementary to the target nucleic acid sequence and hybridizes to a region of a subsequence of the target nucleic acid defined by a pair of oligonucleotide primers, wherein the probe further comprises an interactive 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 wherein the compound having (or derived from) formula (IA) is separated from the first quencher portion by a nuclease-sensitive cleavage site; and

[0450] wherein prior to step (b), the tag moiety is reversibly bound in a temperature-dependent manner to a quencher oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag moiety of the oligonucleotide probe and bound to the tag moiety by hybridization, wherein the quencher oligonucleotide comprises at least a second quencher moiety capable of quenching a compound having (or derived from) formula (IA) on the tag moiety when the quencher oligonucleotide is bound to the tag moiety;

[0451] (b) after 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 can cleave and separate the tag portion from the first quencher portion on the annealed portion of the probe;

[0452] (c) measuring one or more signals from the compound having (or derived from) formula (IA) at a first temperature, at which the quenching oligonucleotide is bound to the tag moiety;

[0453] (d) measuring one or more signals from the compound having (or derived from) formula (IA) at a second temperature, greater than the first temperature, at which the quenching oligonucleotide is not bound to the tag moiety;

[0454] (e) obtaining a calculated signal value by subtracting a median or average value of the one or more signals detected at the first temperature from a median or average value of the one or more signals detected at the second temperature;

[0455] A calculated signal value therein above a threshold signal value allows determining the presence of the target nucleic acid.

[0456] In some embodiments, the PCR of step (b) is allowed to amplify to an end point beyond the amplification logarithmic phase. In some embodiments, the tag portion comprises a modification that makes it impossible to be extended by a nucleic acid polymerase. In some embodiments, the tag portion of the oligonucleotide probe or the quencher oligonucleotide or both the tag portion and the quencher oligonucleotide contain one or more nucleotide modifications. In some embodiments, 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-enantiomeric nucleotides and combinations thereof.

[0457] Other methods using TAGS probes are described in U.S. Pat. Nos. 11,028,433, 11,034,997, and 11,345,958; and in U.S. Patent Publication No. 2021 / 0269857, the disclosures of which are incorporated herein by reference in their entireties.

[0458] synthesis

[0459] The present disclosure provides methods for synthesizing compounds of any one of Formula (I), Formula (IA) and Formula (IB) and derivatives and analogs thereof. The present disclosure also provides methods for synthesizing intermediates.

[0460] Overview

[0461] Where R 1 Compounds of formula (I) and formula (IB) with protecting groups allow stable amino protection to be formed under the conditions of solid phase synthesis using phosphoramidite chemistry in nucleic acid preparation. For example, the amino group can be protected as trifluoroacetamide (TFA, trifluoroacetyl protecting group) to give compounds 2a to 2s (see Tables 2a-2c), wherein R 1 For TFA protecting group. In some embodiments, TFA group can be cleaved during deprotection conditions common in solid phase synthesis of nucleic acid analogs, such as gaseous ammonia, ammonia or primary amine (methylamine, propylamine, tert-butylamine, etc.). Alternatively, amine can be protected as benzyl carbamate (benzyl chloroformate, Cbz or Z protecting group), or as 9-fluorenylmethylcarbamate (Fmoc protecting group).

[0462] Dye synthesis and accessibility

[0463] A single high-yield reaction from the inexpensive and commercially available Rhodamine 800 perchlorate dye can be used as the starting material together with a primary amine (fluorophore with a julolidine core structure, CAS No. [137993-41-0]).

[0464]

[0465] Compounds 1b, 1c, 1h-1j, 1k, 1o, 1r and 1s (see Tables 1a to 1c) can be used for biomolecule labeling, either by in situ activation of the carboxylic acid or by the corresponding NHS-esters. The NHS-esters are prepared by using trifluoroacetic anhydride (TFAA) and N-hydroxysuccinimide (NHS) in the presence of a base:

[0466]

[0467] The alcohol functional groups of compounds 2h, 2j, 2m and 2q (see Table 2a to Table 2c) can be directly converted into the corresponding phosphoramidites for 5′-modification of nucleic acids and nucleic acid analogs:

[0468]

[0469] The NHS esters of compounds 2b, 2c, 2h-1j, 2k, 2o, 2r and 2s (see Table 2a to Table 2c) can be converted into the corresponding phosphoramidites for internal modification of nucleic acids and nucleic acid analogs according to the following synthetic sequence:

[0470]

[0471] Compounds 1g and 2g (see Tables 1a and 2a) can be further used for copper-catalyzed click chemistry [Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition, CuAAC]. Compound 1g can be used for solution labeling of biomolecules, while compound 2g allows the introduction of alkyne groups during solid phase synthesis for on-column labeling.

[0472] result

[0473] Reference is made to a compound having formula (1A) (wherein R 2 The absorption and fluorescence maxima and brightness of compounds 1a to 1s were analyzed using butylamine. The results are set forth in Table 3 herein. Unexpectedly, it was found that direct binding to R 1 The configuration of the carbon of the amino group in has a profound effect on the fluorescence properties of the dye core. The dye absorption maximum changes by up to about 106 nm and the fluorescence changes by up to about 35 nm, corresponding to a Stokes shift of about 11 nm to about 92 nm.

[0474] A common problem with conventional fluorophores with "small" Stokes shifts is internal quenching of fluorescence. This self-quenching is caused by spectral overlap of excitation and emission and is especially prevalent at high fluorophore concentrations. LSS dyes (such as those described herein) generally have better separated spectral bands, which minimizes reabsorption of photons.

[0475] It is believed that there is a non-zero probability of excitation of a fluorophore outside its main excitation peak. Therefore, the fluorescence from the dye inevitably leads to the total light detected in multiple emission channels. This spectral "crosstalk" or "bleed-through" can be compensated to a certain extent by using a predetermined correction factor for calculation. In addition, the scattering of the excitation light increases the background fluorescence of adjacent channels. The dyes disclosed in the present invention (e.g., those with formula (IA)) allow to reduce or even eliminate crosstalk and scattering from other fluorophores. The dyes disclosed in the present invention (e.g., those with formula (IA)) are particularly useful in experimental environments where many fluorophores generate strong background signals. The large spectral separation of the dyes disclosed in the present invention (e.g., those with formula (IA)) allows to filter the excitation light more effectively, thereby enhancing the sensitivity of target detection (see, e.g., Figure 2 ).

[0476] It is also believed that the dyes of the present disclosure (e.g., those of formula (IA)) provide a way to acquire fluorescence data from previously inaccessible optical channels. Due to their broad spectrum separation and when used in combination with standard fluorophores, the dyes of the present disclosure allow for increasing the multiplexing capabilities of fluorescent PCR devices by adding more channels to established four to six color instruments (see Figure 2 ). In principle, 21 channels can be provided by the filter combination of a six-color instrument. In practice, however, the number of channels is limited by the commercial availability of filters with suitable spectral properties and sufficiently large Stokes shifts.

[0477] Activating the dye and coupling the activated dye to the DNA molecule

[0478] The present disclosure also provides methods of activating a compound of formula (I) and subsequently conjugating the activated compound to an oligomer. The following schematic diagram illustrates labeling of a DNA molecule with a DMT-MM solution.

[0479]

[0480] The dye is Synthesis of TAGS probes

[0481] The present disclosure also provides methods for synthesizing TAGS probes, wherein the TAGS probes include compounds having formula (I) and being thermally stable up to about 100° C. The use of such TAGS probes is described herein and in U.S. Pat. Nos. 11,028,433, 11,034,997, and 11,345,958; and in U.S. Patent Publication No. 2021 / 0269857, the disclosure of which is incorporated herein by reference in its entirety.

[0482] In some embodiments, by first preparing a 5'-N 3-modified DNA to synthesize a probe (such as one having formula (V)):

[0483]

[0484] 5'-N 3 The -modified DNA is then coupled to an oligonucleotide comprising a quencher and a first reactive group, such as a reactive group capable of participating in a "click chemistry" reaction (e.g., DBCO). When reacted with an oligonucleotide comprising a quencher and a first reactive group, the 5'-N 3 The -modified DNA is then "clicked" into place to provide the probe of formula (V) shown.

[0485] The present disclosure also provides methods for directly coupling an oligonucleotide having a terminal amine group to a cyano moiety present at the mesogenic position of a dye core to provide any of compounds of formula (VIIA) or formula (VIIB) (see, e.g., 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 substituted oligonucleotide having one or more substituents (e.g., -CO 2 -、-C 2 -CO 2 -, -D or halogen) 1 -C 8 branched or unbranched alkyl, branched or unbranched heteroalkyl or cycloalkyl groups.

[0486]

[0487] in

[0488] Dye-CN represents a dye with a cyano moiety at the mesoposition of the dye core;

[0489] R 3 For -Me, -Et, -CO 2 -、-C 2 -CO 2 -, -D or halogen substituted with one or more of C 1 -C 8 an alkyl, heteroalkyl or cycloalkyl group; and

[0490] The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer.

[0491] 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] non-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 or Cyrene. In some embodiments, the reaction is carried out at a temperature range between about 20°C and about 70°C. In some embodiments, the reaction is carried out for a period of time between about 60 minutes and about 72 hours. In some embodiments, the dye is rhodamine. In some embodiments, the dye is Rhodamine 800.

[0492] Examples

[0493] The following examples are given to illustrate embodiments of the present disclosure as it is presently preferably practiced. It should be understood that the examples are illustrative and that the present disclosure should not be considered as being limited except as indicated in the appended claims.

[0494] abbreviation

[0495] 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 = ethylenediaminetetrakis Acetic acid; eq. = molar equivalent; EtOH = ethanol, EU = emission units; FAM = fluorescein; HAA = hexyl ammonium acetate buffer, HCl = hydrochloride; HEX = hexachlorofluorescein; LSS = large Stokes shift; MeCN = acetonitrile; nd = 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-performance liquid chromatography coupled to mass spectrometry.

[0496] General Materials and Methods

[0497] R800 perchlorate dye [137993-41-0] and 1-bicyclo[1.1.1]pentylamine hydrochloride were obtained from MilliporeSigma (Burlington, MA, USA). trans-4-Aminocyclohexane-carboxylic acid was obtained from TCI America (Portland, OR, USA). 9 -Butylamine was obtained from C / D / N Isotopes Inc. (Pointe-Claire, Quebec, Canada). 3-Aminobicyclo[1.1.1]pentane-1-carboxylic acid hydrochloride was obtained from AA Blocks Inc. (San Diego, California, USA). Bicyclo[2.2.2]octan-1-amine hydrochloride was obtained from 1Click Chemistry (Kendall Park, New Jersey, USA). 4-Aminobicyclo[2.2.2]octan-1-ol HCl and 4-aminobicyclo[2.2.2]octan-1-carboxylic acid were obtained from Absolute Chiral (San Diego, California, USA). Reagents and materials for chemical DNA synthesis were obtained from Glen Research (Sterling, Virginia, USA). TEAB buffer was obtained as a ready solution (1.0 M, pH 8.5) and was used without further dilution. TEAA and HAA buffers were prepared by diluting commercial stock solutions (Glen Research, Sterling, Virginia, USA) with water to a final concentration of 100 mM. Unless otherwise stated, all other reagents were purchased from MilliporeSigma (Burlington, MA, USA). Solvents used in chemical reactions were dried over activated molecular sieves from Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic solvents (HPLC grade) were obtained from MilliporeSigma (Burlington, MA, USA) or VWR (Radnor, PA, USA). Ultrapure water was obtained from Purification system (MilliporeSigma), resistivity at 25°C is at least 18.2 MΩ·cm.

[0498] Chemical reactions at Eppendorf C (Enfield, Connecticut, USA). Microwave-assisted reactions were performed using CEM (Matthews, NC, USA). SP microwave system was used, which was equipped with a focused single-mode reaction chamber (2.45 GHz) in thick-walled glass vials (2.0 mL or 10.0 mL). The reaction temperature was monitored with a built-in infrared temperature sensor and kept constant by automatic power control. The microwave-assisted reaction was stirred under active cooling using compressed air. An automated flash chromatography system ( R + Lumen) for flash column chromatography. Replacement of the dye counterion is achieved by standard ion exchange procedures such as ion exchange, SPE, liquid-liquid extraction or organic solvent precipitation.

[0499] DNA oligomers carrying 3′-modifications are preloaded with spacers C3, phosphate or BHQ-2 (Black Hole ) was synthesized on CPG of 5′-amino-2-nitropropene. A DNA sequence with primary amino modification was synthesized by solid phase DNA synthesis using amino-modifying agent phosphoramidite. For 5′ end modification, 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (5′-amino-C 6 For internal modification, 5'-dimethoxytrityl-5-[N_(trifluoroacetamidohexyl)-3-acrylimide]-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N_diisopropyl)]-phosphoramidite (amino-C 6 -dT). Due to sequence context, amino-C 6 -dT can also be used at the 5' end. DNA sequences with 5'-azido modifications were prepared by synthesizing 5'-bromo-modified DNA with bromohexyl phosphoramidite and subsequently converting the bromine to an azido group on a solid support. Br / N was carried out by adding sodium azide and sodium iodide in DMSO (100 mM each) to CPG and heating the mixture at 65°C for 1.0 h (quantitative reaction). 3 -Exchange. After DNA synthesis and on-column modification, DNA was cleaved, deprotected, desalted, and precipitated using standard methods. The latter two steps were performed to ensure removal of any trace amines. A rotary vacuum concentrator (SpeedVac, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used. TM ) or a freeze dryer (Labconco freeze dryer, ice capacity 4.5 L, collector temperature -105°C, Labconco Corp., Kansas City, MO, USA) to dry the aqueous solution of the DNA sequence.

[0500] UPLC analysis was performed using a diode array, fluorescence and mass spectrometer (ZSpray TM ) detector was used on a Waters I-class ACQUITY UPLC (Waters Corporation, Milford, Massachusetts, USA). 1.7 μm, 2.1×50 mm) with an appropriate gradient of TEAA buffer (100 mM, pH 7.0) to MeCN at a flow rate of 1.0 ml / min. The chromatograms of DNA were recorded at 260 nm and the absorption maximum of the corresponding dye or dye-labeled probe was recorded. Semi-preparative HPLC purification was performed on a 996 photodiode array detector and a Waters XBridge TM BEH C18 OBD Prep Column( 5 μm, 19.0×250 mm) at a flow rate of 10.0 ml / min. Before injection, the sample was passed through Filter through a syringe filter (0.22 μm). Use NanoDrop One C Absorption spectra were obtained with a spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and background correction was performed at 750 nm. Fluorescence spectra and thermal stability of fluorescence data were recorded using a Cary Eclipse fluorescence spectrophotometer with a temperature controller (Agilent Technologies, Santa Clara, CA, USA).

[0501] Example 1: General procedure for preparing compound R, compound 1a to compound 1s

[0502] R800 perchlorate dye (1.0 eq., 100 mM in DMSO, 10 μL) in DMSO was converted with primary amine (5.0 eq., 100 mM in DMSO) in the presence of base at 50°C in a reaction mixer. 6 The amine of the present invention is 1-butylamine, ethanolamine, 2-methoxyethylamine, 2-(2-aminoethoxy) ethanol, propargylamine, tert-butylamine and 2-amino-2-methyl-1-propanol, alkali is DIPEA (1.0eq.).For amine (glycine, β-alanine, L-Ala, L-leucine, L-valine, L-(+ / -)-3-aminobutyric acid, trans-4-aminocyclohexanecarboxylic acid, 4-aminobicyclo [2.2.2] octane-1-carboxylic acid) cesium carbonate (2.0eq.) with carboxylate functional group is used as alkali.For the amine obtained with hydrochloride form (1-bicyclo [1.1.1] amylamine hydrochloride, 3-aminobicyclo [1.1.1] pentane-1-carboxylic acid hydrochloride, bicyclo [2.2.2] pungent-1-amine hydrochloride), increase the amount of cesium carbonate with neutralization acid (5.0eq.).The identity of dye product has been confirmed by the mass spectrometry in the positive ion mode.

[0503] The spectral characteristics of dye products R, 1a to 1s were determined by UPLC-MS analysis. The reaction solution sample (0.5 μL) was diluted with MeCN (29.5 μL) and separated on a C18 stationary phase using a TEAA buffer mobile phase and a MeCN gradient (40% to 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 in which the maximum absorbance of the corresponding dye was about 0.5-1.0AU.

[0504] To determine the fluorescence per unit of absorption as a measure of dye brightness, an amount of sample was injected into the UPLC to give approximately 0.1 AU at the maximum absorbance of the corresponding dye. Fluorescence emission was measured by excitation at the absorption maximum (3D fluorescence mode, 1.0 PMT gain, 1.0 second time constant) and a 100 nm fluorescence emission window centered at the maximum emission. The peak area of ​​the fluorescence peak was divided by the peak area of ​​the absorption peak.

[0505] Results: The analytical data of the reaction of n-butylamine with R800 to generate compound R is shown in Figure 3A , Figure 3B and Figure 3C The detailed analytical data of Compound 1a to Compound 1s are shown in Figures 4 to 23 (A: chromatogram; B: excitation and emission spectra; C: mass spectra). The spectral data of Compound R and Compound 1a to Compound 1s are summarized in Table 1. Compound 1b was unstable and was not further analyzed ( Figure 6 Surprisingly, compound 1s is not a LSS dye and shows typical spectral features of “conventional” fluorescent dyes (~11 nm Stokes shift, Fig. 23B ). This result is in sharp contrast to compound 1g, which showed a Stokes shift of 81 nm ( Fig. 11B ). Taken together, these results suggest that the linker moiety has a profound effect on the spectral properties of the dye core.

[0506] Example 2: Synthesis of Compound 1j

[0507]

[0508] (+ / -)-3-aminobutyric acid (5.0eq., 151μmol) and cesium carbonate (2.0eq., 61μmol) are fully mixed in dry DMSO (909μL). Add R800 perchlorate dye (1.0eq., 30μmol, 33mM) and 50℃ of mixed suspensions, during which the color of the reaction mixture changes from dark blue to yellow-orange. Filter the reaction mixture with a rotary filter and dilute the filtrate with sodium iodide aqueous solution (0.5M). By centrifugation precipitation, dry under high vacuum, and purify with preparative HPLC. For DNA labeling, the acetate counter ion is exchanged for iodide.

[0509] Results: The analytical data in Figure 14 demonstrate the successful synthesis of compound 1j (A: chromatogram; B: excitation and emission spectra; C: mass spectra). Compound 1j was used for DNA labeling in Example 7 ( Fig. 27 ).

[0510] Example 3: Synthesis of Compound 1k

[0511]

[0512] Trans-4-aminocyclohexanecarboxylic acid (2.0 eq., 403 μmol) and cesium carbonate (2.0 eq., 403 μmol) were thoroughly mixed in dry DMSO (8.1 mL). R800 perchlorate dye (1.0 eq., 403 μmol) was added and the suspension was heated for 15 minutes. The reaction mixture was heated at 50°C in a microwave reactor during which time the color of the reaction mixture changed from dark blue to dark green. The reaction progress was monitored by UPLC-MS. Any undissolved solids were removed by centrifugation. The solution was purified by 0.22 μm Syringe filter is filtered and added dropwise in the sodium iodide (0.5M) aqueous solution of stirring. The mixture is vortexed and separated by centrifugation solid. Residue is dried under high vacuum. Remove any unreacted R800 starting material and decomposition products by precipitating dye from DCM at-20 ℃. Product is dried under high vacuum and lyophilized from MeCN to obtain the yellow-orange target compound.

[0513] Results: The analytical data in Figure 15 demonstrated the successful synthesis of compound 1k (A: chromatogram; B: excitation and emission spectra; C: mass spectrum).

[0514] Example 4: Synthesis of Compound 1q

[0515]

[0516] 4-aminobicyclo[2.2.2]octan-1-ol hydrochloride (2.0 eq., 403 μmol) and cesium carbonate (2.0 eq., 403 μmol) were thoroughly mixed in dry DMSO (8.1 mL). R800 perchlorate dye (1.0 eq., 403 μmol) was added and the suspension was heated for 10 minutes. The reaction mixture was heated at 50°C in a microwave reactor during which time the color of the reaction mixture changed from dark blue to brownish orange. The reaction progress was monitored by UPLC-MS. A 0.22 μm The syringe filter removes any undissolved solids, and the solution is added dropwise to a stirred solution of sodium iodide (2.0 g) in water (30 mL). The precipitate is vortexed and then separated by centrifugation. Any unreacted R800 starting material, decomposition products, water are removed by extracting the solid with anhydrous toluene (2 × 20 mL, or until the supernatant is no longer blue). The residue is dried under high vacuum. Excess salt is removed by dissolving the crude product in DCM and filtering out the colorless solid. DCM is evaporated under reduced pressure and the solid is redissolved in MeCN. The solution is frozen and lyophilized in liquid nitrogen to obtain a bright orange target compound with a 20% yield and 85% purity.

[0517] Results: The analytical data in Figure 21 demonstrated the successful synthesis of compound 1q (A: chromatogram; B: excitation and emission spectra; C: mass spectrum).

[0518] Example 5: Fluorescence thermal stability

[0519] A small amount of LSS dye sample 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 the corresponding LSS dye at the excitation maximum and recording the fluorescence at the emission maximum at a heating rate of 1°C / min from 25°C to 100°C.

[0520] Results: The fluorescence of compound R, compound 1i, compound 1k, compound 1j and compound 1n changed with temperature. Fig.24 The results show no significant decrease in fluorescence up to 100°C, demonstrating the thermal stability of the fluorescence of the LSS dyes disclosed herein. The slightly more pronounced shifts of Compound R and Compound 1n to higher fluorescence can be explained by their increased solubility at higher temperatures, as they are more hydrophobic compared to the carboxylic acid compounds 1i, 1k, and 1j.

[0521] Example 6: Direct labeling of DNA with R800 dye

[0522]

[0523] In order to make oligonucleotides dissolve in polar organic solvents, amino-modified DNA is converted into TEA salt via standard salt exchange method. Remove water by freeze drying, and the residue is redissolved in anhydrous DMSO. After adding DIPEA (1.0eq., 100nmol) and R800 perchlorate dye (5.0eq., 500nmol), DNA concentration is 1.0mM. The labeling reaction is carried out at RT for 67 hours. The analytical amount of the reaction solution is analyzed by UPLC-MS to determine the score of the labeled DNA (TEAA buffer, pH 7.0, 10% to 40% MeCN, 2.0 minutes). Excess dye is removed by EtOH precipitation, and the labeled DNA is purified by preparative HPLC using a biphasic gradient (10% to 25% MeCN, 5 minutes, then 25% to 45% MeCN, 20 minutes). After confirming the identity of the product by mass spectrometry, the labeled DNA is separated with a purity of >95% by standard procedures.

[0524] Results: The effectiveness of the direct labeling approach was demonstrated by the successful derivatization of a variety of amino-modified DNA probe sequences using R800. The analytical data of the labeling reaction of the probe sequence is Fig.25 The labeled DNA was used in Example 10. The analysis data of three 5′-azido-modified DNA sequences are shown in Fig.26A (chromatogram) and Fig.26B (absorption and mass spectrometry). These results demonstrate that site-specific labeling of DNA can be achieved without active ester or click chemistry functionalities. With this approach, the molecular configuration of the DNA amino-like body is incorporated and becomes an integral part of the dye structure.

[0525] Example 7: Labeling DNA with LSS dye carboxylic acid

[0526] After automated solid phase synthesis and standard workup procedures, analytical amounts of unpurified DNA were analyzed using UPLC-MS to determine the fraction of target sequence that was amino-modified. The total amount of DNA was determined using a spectrophotometer using the calculated extinction coefficient of the DNA sequence at 260 nm.

[0527] DNA precipitation: The unpurified DNA obtained in solid phase synthesis was desalted with water using standard methods (NAP-25 or SPE) and dried using a rotary vacuum concentrator. In a reaction tube, the DNA was redissolved in water (0.1 mL) with the aid of a warm water bath. Sodium iodide was added to a final concentration of 10.0 M. Anhydrous ethanol (200 degrees, 30.0 mL) was added and the tube was vortexed. The suspension was centrifuged at maximum speed (5.0 minutes) in a tabletop centrifuge. The supernatant was carefully decanted and discarded. Excess sodium iodide was removed by washing the precipitate with anhydrous ethanol. The precipitate was dried under high vacuum, redissolved in TEAB buffer (pH 8.5), and used immediately for labeling reactions.

[0528] Fluorescent dye carboxylic acid activation: In a reaction bottle, carboxylic acid dye (1.0 eq.) was dissolved in anhydrous DMSO (8 mM). DIPEA (2.0 eq.) was added and the solution was briefly mixed. In another glass vial, tetrafluoroborate of DMT-MM (2.0 eq.) was weighed, the dye carboxylic acid solution was added, and then vigorously mixed until all solids were dissolved.

[0529] The reaction was then shaken at RT for 15 min.

[0530] Labeling reaction: The activated dye solution (3.0 eq.) was quickly mixed with amino-modified DNA (1.0 eq. primary amine) and the labeling reaction was carried out in a reaction mixer at RT 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 injection into the UPLC (7.0 μL).

[0531] Purification: Labeled DNA was purified by reverse phase liquid chromatography with TEAA (0.1 M, pH 7.0) and MeCN. The combined product fractions were concentrated on a centrifugal vacuum concentrator and desalted by size exclusion chromatography. The purified DNA probe (0.1 mM) was lyophilized and then redissolved in TE buffer (10.0 mM Tris.HCl, 1.0 mM EDTA) for qPCR.

[0532] result: Fig. 27 The analytical data in demonstrate the successful labeling of amino-modified DNA with LSS dye 1j (A: chromatogram; B: excitation and emission spectra; C: mass spectra). Since compound 1j is a racemic mixture of enantiomers, the labeled DNA is a mixture of diastereomers and is separated as a double peak.

[0533] Example 8: Preparation of dye-labeled DNA probes using click chemistry

[0534] Dye-labeled DNA probes can also be prepared by strain-promoted azide-alkyne cycloaddition between DNA-bound DBCO or BCN and azide-modified dyes. DNA probes containing 5'-DBCO modification, internal BHQ-2 and 3'-C3 extension blocker were prepared by solid phase DNA synthesis and purification using standard methods. DNA (1.0 eq., 100 μM, 50 mM TEAA buffer, pH 7.0) and azidated dye (1.1 eq., 100 μM, 50 mM TEAA buffer, pH 7.0) were mixed and stored at 40°C for 2 hours in a shaker. Any excess dye was removed by ethanol precipitation. UPLC analysis showed quantitative labeling of DNA. The above protocol has been used to prepare several DNA conjugates with common fluorescent dyes. It is expected that LSS dyes with azido linkers will produce DNA-LSS dye conjugates in the same manner.

[0535] Example 9: Preparation of branched DNA probes using click chemistry

[0536] Branched DNA probes for thermal multiplexing were prepared by strain-promoted azide-alkyne cycloaddition between an oligonucleotide with 5'-BHQ-2 and internal DBCO modifications and a 5'-azido-modified oligonucleotide with a dye at the penultimate position (e.g., labeled DNA from Example 6). The two DNA sequences were mixed in a stoichiometric ratio of 1:1.1 in TEAA buffer (50 mM, pH 7.0) and stored at 40°C in a reaction mixer for 2 hours. UPLC analysis showed quantitative click reactions with some residual DNA excess that could be removed by HPLC purification. Example 10: PCR amplification using dye-labeled DNA probes

[0537] All qPCR components were prepared using nuclease-free water. A total volume of 50 μL of reaction mixture was prepared by mixing three components called master mix (20 μL), buffer mix (20 μL), and dNTP mix (10 μL). The master mix contained Tricine buffer (pH 8.2), manganese acetate, potassium acetate, glycerol, DMSO, detergent, target DNA (5000 copies / reaction), polymerase aptamer, forward and reverse primer DNA and polymerase, and Probe. dNTP mix contained dATP, dCTP, dGTP (2.0 mM each) 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 probe is a DNA sequence prepared in Example 6 with a large Stokes shift (LSS) dye and a BHQ-2 quencher ( Fig.25For comparison, another qPCR assay contained the same sequence labeled with Cy5.5. The plate was sealed and used They were subjected to amplification cycles using the 480 system (Fritz Hofmeyer-La Roche GmbH, Basel, Switzerland). Growth curves were analyzed based on fluorescence data collected in appropriate combinations of excitation and emission channels.

[0538] result: Fig.28 The results for the analysis of the chromatin stained with LSS and Cy5.5 are shown. PCR growth curve of the probe, such as Figure 2 As shown, these curves were measured in their respective optical channels. Specifically, the fluorescence of the LSS-labeled probe was detected in channel RLS 1 (435 nm excitation and 580 nm emission), while the fluorescence of the Cy5.5-labeled probe was detected in the Cy5.5 channel (580 nm excitation and 700 nm emission). The two growth curves are superimposed on Fig.28 The LSS dye signal showed a higher fluorescence signal than the traditional Cy5.5 dye. This experiment proved that LSS dye can be used as General applicability and compatibility of bright reporters in PCR, and it is expected that other LSS dye variants in the present disclosure will generate qPCR signals in the same manner.

[0539] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. If necessary, various aspects of the embodiments may be modified to provide yet further embodiments using concepts from the various patents, applications, and publications.

[0540] Although the present disclosure has been described with reference to some illustrative embodiments, it should be understood that those skilled in the art can design many other modifications and embodiments within the spirit and scope of the principles of the present disclosure. More specifically, without violating the spirit of the present disclosure, within the scope of the above disclosure, the drawings and the appended claims, the components and / or arrangements of the subject combination arrangement may be reasonably varied and modified. In addition to the variations and modifications in the components and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

1. A compound having formula (I): in R 1 is H or a protecting group; R 2 is a C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl, or cycloalkyl group substituted with one or more of –Me, –Et, –CO2-, –CO2-(thiol reactive group), –CO2-(amine reactive group), –CO2-(carboxyl reactive group), –C2-CO2-, –C2-CO2-(thiol reactive group), –C2-CO2-(amine reactive group), –C2-CO2-(carboxyl reactive group), –OH, –phosphoramidite, –O-phosphoramidite, –D, halogen, or a group capable of participating in a “click chemistry” reaction; and [X] - is the counter anion, provided that when R 2 When it has a negative charge, [X] - Does not exist.

2. The compound of claim 1, wherein the thiol-reactive group is selected from the group consisting of haloacetyl, maleimide, iodoacetamide, aziridine, acryloyl, arylating agent, vinyl sulfone, methanethiosulfonate, pyridyl disulfide and TNB-thiol.

3. The compound of claim 1, wherein 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, glyoxals, epoxides, ethylene oxides, carbonates, aryl halides, fluorophenol esters, sulfochlorophenols, carbodiimides, phthalimides, benzotriazoles, imidoesters, and anhydrides.

4. The compound of claim 1, wherein the carbonyl-reactive group is selected from the group consisting of hydrazine, hydrazine derivatives and amines.

5. A compound according to any one of claims 1 to 4, wherein R 2 Selected from the group consisting of: C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl, or cycloalkyl group substituted with –CO2-maleimide or –C2-CO2-maleimide; C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl, or cycloalkyl group substituted with –CO2-NHS ester or –C2-CO2-NHS ester; and C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl, or cycloalkyl group substituted with –CO2-hydrazine or –C2-CO2-hydrazine.

6. The compound according to claim 1, wherein the group capable of participating in a "click chemistry reaction" is selected from the group consisting of: a bicyclo[6.1.0]nonyne) group ("BCN"), a dibenzocyclooctyne ("DBCO"), an olefin, a trans-cyclooctene ("TCO"), a maleimide, an aldehyde, a ketone, an azide, a tetrazine, a thiol, a 1,3-nitrone, a hydrazine and a hydroxylamine.

7. The compound according to claim 6, wherein R 2 is a C1-C6 branched or unbranched alkyl group substituted by BCN, DBCO or TCO.

8. The compound according to claim 1, wherein R 2 For –Me, –Et, –CO2 - A C1-C6 branched or unbranched alkyl group substituted with one or more of -OH, -D or halogen; or Where R 2 Select from the group consisting of:

9. The compound according to claim 1, wherein R 1 is a protecting group; and wherein R 2 Includes –phosphoramidites or –O-phosphoramidites.

10. A compound selected from the group consisting of: The dye is And where [X] - as counter anion.

11. The compound according to claim 10, wherein [X] - Selected from the group consisting of chloride, bromide, iodide, sulfate, benzenesulfonate, p-toluenesulfonate, p-bromobenzenesulfonate, methanesulfonate, trifluoromethanesulfonate, phosphate, perchlorate, tetrafluoroborate, hexafluorophosphate, tetraphenylborate, nitrate, and anions of aromatic or aliphatic carboxylic acids.

12. A compound selected from the group consisting of: Where [X] - as counter anion.

13. A compound having formula (IA): Where R 2 For –Me, –Et, –CO2 - , –CO2– (thiol reactive group), –CO2– (amine reactive group), –CO2– (carboxyl reactive group), –C2–CO2 - a C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl, or cycloalkyl group substituted with one or more of: –C2–CO2– (thiol reactive group), C2–CO2– (amine reactive group), –C2–CO2– (carboxyl reactive group), –OH, phosphoramidite, –O-phosphoramidite, –D, halogen, or a group capable of participating in a “click chemistry” reaction; and [X] - is the counter anion, provided that when R 2 When it has a negative charge, [X] - Does not exist.

14. The compound according to claim 13, wherein R 2 is selected from the group consisting of a C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl, or cycloalkyl group substituted with –CO2-maleimide or –C2-CO2-maleimide; a C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl, or cycloalkyl group substituted with –CO2-NHS ester or –C2-CO2-NHS ester; and a C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl, or cycloalkyl group substituted with –CO2-hydrazine or –C2-CO2-hydrazine; or Where R 2 is a C1-C8 branched or unbranched alkyl group substituted with a group capable of participating in a "click chemistry" reaction; or Where R 2 For –Me, –Et, –CO2 - A C1-C8 branched or unbranched alkyl group substituted with one or more of -OH, -OH, halogen or -D; or Where R 2 is a –phosphoramidite or an –O-phosphoramidite; or Where R 2 Select from the group consisting of:

15. The compound of any one of claims 13 to 14, wherein the compound has a Stokes shift of at least about 70 nm.

16. The compound of any one of claims 13 to 15, wherein the compound is thermally stable in a temperature range of about 25°C to about 100°C.

17. A conjugate comprising (i) a specific binding entity and (ii) a dye moiety derived from a compound according to any one of claims 1 to 16.

18. A conjugate having formula (II): in R 1 is H or a protecting group; R 3 is a C1-C8 alkyl, heteroalkyl or cycloalkyl group substituted by one or more of -Me, -Et, -CO2-, -C2-CO2-, -D or halogen; The "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 between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is 0, 1 or 2.

19. The conjugate according to claim 18, wherein Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and about 25 carbon atoms and optionally having one or more heteroatoms selected from O, N or S.

20. The conjugate according to any one of claims 18 to 19, wherein Y has the structure of formula (IIIC): Where R 3 and R 4 Each of is independently a bond or a group selected from carbonyl, amide, imide, ester, ether, -NH, -N-, thiol or thiol; R 5 is a bond, C1–C 12 The alkyl or heteroalkyl groups include, and wherein R 5 can include a carbonyl, imide or thione; R a and R b are independently H or methyl; g and h are independently 0 or an integer ranging from 1 to 4; i is 0, 1 or 2.

21. A conjugate having any one of formula (IIC) or (IID): in R 1 is H or a protecting group; R 3 is a C1-C8 branched or unbranched alkyl, branched or unbranched heteroalkyl or cycloalkyl group substituted by one or more of -Me, -Et, -CO2-, -C2-CO2-, -D or halogen; The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer; Y is a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is 0, 1 or 2.

22. The conjugate according to claim 21, wherein R 3 The first carbon atom of is a primary carbon atom; or Where R 3 The first carbon atom of is a secondary carbon atom; or Where R 3 The first carbon atom is a tertiary carbon atom.

23. The conjugate according to claim 21, wherein R 1 is H; and R 3 is a C1-C8 alkyl, heteroalkyl or cycloalkyl group substituted by one or more of —Me, —Et, —D, —C2—CO2—, —CO2— and —OH—.

24. The conjugate according to claim 23, wherein Y is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 30 carbon atoms and optionally having one or more heteroatoms selected from O, N or S.

25. A kit comprising: (i) a first conjugate comprising a first oligonucleotide coupled to a dye moiety derived from a compound according to any one of claims 1 to 16; and (ii) a second conjugate comprising an oligonucleotide coupled to a quencher.

26. A probe having formula (IV): [Dye 1]–[Y] a –[5'–oligonucleotide–3']–[Y] a –[Dye 2](IV), where One of [Dye 1] or [Dye 2] is derived from a compound according to any one of claims 1 to 16; and the other of Dye 1 or Dye 2 is a quencher; The oligonucleotide is an oligonucleotide having between about 5 and about 60 mer; each Y is independently a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic or aromatic group having between 2 and about 40 carbon atoms and optionally having one or more heteroatoms selected from O, N or S; and a is 0, 1 or 2.

27. The probe according to claim 26, wherein the one of [Dye 1] or [Dye 2] is derived from a compound having formula (IA): Where R 2 For –Me, –Et, –CO2 - A C1-C8 branched or unbranched alkyl group substituted by one or more of -OH, -D or halogen.

28. The probe according to claim 26, wherein R 2 Selected from:

29. A conjugate having formula (V): [(oligo 1)(dye)]–linker–[(oligo 2)(Q1)](V), in Oligo 1 and oligo 2 are each different and are oligonucleotides having between about 5-mers and about 30-mers; The dye is derived from a compound according to any one of claims 1 to 21; Q1 is a quencher; and The linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 30 carbon atoms.

30. The conjugate of claim 29, wherein the dye is derived from a compound having formula (IA): Where R 2 For –Me, –Et, –CO2 - A C1-C8 branched or unbranched alkyl group substituted by one or more of -OH, -D or halogen.

31. The conjugate according to claim 29, wherein R 2 Selected from:

32. The conjugate of any one of claims 29 to 31, wherein the dye has a Stokes shift of at least about 70 nm.

33. The conjugate according to any one of claims 29 to 32, wherein at least one of Oligo 1 and Oligo 2 comprises LNA, L-LNA or PNA.

34. The conjugate of any one of claims 29 to 33, wherein the linker is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic or heteroaromatic group having between about 5 and about 15 carbon atoms.

35. A kit comprising: (i) the conjugate according to any one of claims 29 to 34; and (ii) a compound having the formula (VIII): [Oligo 3]–[Q2](VIII), in Oligo 3 is an oligonucleotide having between 5 and 30 mers; and Q2 is a quencher.

36. 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), in One of Dye 1 or Dye 2 is derived from a compound according to any one of claims 1 to 33; 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 between 2 and 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.

37. The FRET pair of claim 36, wherein said one of Dye 1 or Dye 2 is derived from a compound having formula (IA): Where R 2 For –Me, –Et, –CO2 - A C1-C8 branched or unbranched alkyl group substituted with one or more of -OH, -D or halogen; or Where R 2 For –Me, –Et, –CO2 - A C1-C6 branched or unbranched alkyl group substituted with one or more of -OH, -D or halogen; or Where R 2 Select from the group consisting of:

38. A method for amplifying and detecting a target nucleic acid in a sample, the method comprising the steps of: (a) contacting the sample containing the target nucleic acid with the following in a single reaction vessel: (i) a pair of oligonucleotide primers, each oligonucleotide primer being capable of hybridizing to opposite strands of a 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 that is not complementary to a target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence that is at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid defined by the pair of oligonucleotide primers, wherein the probe further comprises an interactive dual label comprising a dye derived from a compound according to any one of claims 13 to 21 located on the tag portion and a first quencher portion located on the annealing portion and wherein the dye is separated from the first quencher portion by a nuclease-sensitive cleavage site; and wherein prior to step (b), the tag portion is reversibly bound in a temperature-dependent manner to a quencher oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe and bound to the tag portion by hybridization, wherein the quencher oligonucleotide comprises at least a second quencher moiety capable of quenching the dye on the tag portion when the quencher oligonucleotide is bound to the tag portion; (b) after 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 allows 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 quencher oligonucleotide is bound to the tag moiety; (d) measuring one or more signals from the dye at a second temperature higher than the first temperature, at which the quencher oligonucleotide does not bind to the tag moiety; (e) obtaining a calculated signal value by subtracting a median or average value of the one or more signals detected at the first temperature from a median or average value of the one or more signals detected at the second temperature; A calculated signal value therein above a threshold signal value allows determining the presence of the target nucleic acid.

39. The method of claim 38, wherein the one or more nucleotide modifications are selected from the group consisting of locked nucleic acids (LNA), peptide nucleic acids (PNA), bridged nucleic acids (BNA), 2'-O alkyl substitutions, L-enantiomeric nucleotides, and combinations thereof.

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

41. The method of claim 40, wherein 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]non-5-ene (DBN), 1,1,3,3-tetramethylguanidine (TMG) and 2,2,6,6-tetramethylpiperidine; and wherein the solvent is selected from the group consisting of: dimethyl sulfoxide (DMSO), sulfolane, N-butylpyrrolidone, γ-valerolactone, δ-valerolactone, N-methylpyrrolidone, N,N-dimethylformamide, sulfolane and dihydro-levulinone.

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