Methods for producing fluorescent dyes

Abstract

The present disclosure relates, in general, to novel and easily accessible fluorescent compounds with improved fluorescence properties for expanding the multiplexing capabilities of fluorescence-based nucleic acid detection technologies, as well as to novel methods of producing the same. Moreover, conjugates, PCR probes and FRET pairs comprising these fluorescent compounds as well as methods for amplification and detection of a target nucleic acid utilizing these fluorescent compounds and methods of labeling are also provided.

Description

[0001] METHODS FOR PRODUCING FLUORESCENT DYES

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to novel fluorescent compounds with improved fluorescence quantum yield, extended lifetime, thermostable fluorescence, and superior quenching of background fluorescence in PCR probes for expanding the multiplexing capabilities of fluorescence-based nucleic acid detection technologies. The disclosure also relates to a novel method for producing the compounds disclosed herein as well as to conjugates, FRET pairs and kits comprising the compounds.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] The polymerase chain reaction (PCR) has become a ubiquitous tool of biomedical research, disease monitoring and diagnostics. Amplification of nucleic acid sequences by PCR is described in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188. PCR is now well known in the art and has been described extensively in the scientific literature. See PCR Applications, ((1999) Innis et al., eds., Academic Press, San Diego), PCR Strategies, ((1995) Innis et al., eds., Academic Press, San Diego); PCR Protocols, ((1990) Innis et al., eds., Academic Press, San Diego), and PCR Technology, ((1989) Erlich, ed., Stockton Press, New York). A "real-time" PCR assay is able to simultaneously amplify and detect and / or quantify the starting amount of the target sequence. The basic TaqMan® real-time PCR assay using the 5'-to-3 ' nuclease activity of the DNA polymerase is described in Holland et al., (1991) Proc. Natl. Acad. Sci. 88:7276-7280 and U.S. Patent No. 5,210,015. A real-time PCR without the nuclease activity (a nuclease-free assay) has been described in U.S. Patent Publication No. 20100143901A1. The use of fluorescent probes in realtime PCR is described in U.S. Patent No. 5,538,848.

[0006] A typical real-time PCR protocol with fluorescent probes involves the use of a labelled probe, specific for each target sequence. The probe is preferably labelled with one or more fluorescent moieties, which absorb and emit light at specific wavelengths. Upon hybridizing to the target sequence or its amplicon, the probe exhibits a detectable change in fluorescent emission as a result of probe hybridization or hydrolysis.

[0007] The major challenge of the real-time assay however remains the ability to analyze numerous targets in a single tube. In virtually every field of medicine and diagnostics, the number of loci of interest increases rapidly. For example, multiple loci must be analyzed in forensic DNA profiling, pathogenic microorganism detection, multi-locus genetic disease screening and multi-gene expression studies, to name a few. Commercial, fluorescence-based devices for automated polymerase chain reaction (PCR) can detect multiple targets in a single reaction vessel (multiplexing) by distinguishing light from differently colored fluorophores. The dyes are selected in a way to minimize their spectral overlap. Every fluorophore in the ensemble can be excited with light at or near the absorption maximum and the emitted light (fluorescence) is detected at or near the fluorescence maximum. By limiting the range of wavelengths (band) for excitation and emission with optical filters, individual fluorophores can be distinguished. The specific combination of an excitation band and a simultaneously detected emission band defines an optical channel, each allowing for the identification of one PCR target.

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

[0009] SUMMARY OF THE DISCLOSURE

[0010] The present disclosure is directed to dyes having improved fluorescence quantum yield, extended lifetime and thermostable fluorescence. Herein, it has been determined that the incorporation of one aryl group into the dyes of the present disclosure allows for tuning of their spectroscopic properties, such as their excitation and emission wavelengths. Generally, the effects of specific asymmetric modifications on the properties of such dyes cannot be reasonably predicted. By choosing suitable linker moieties the dyes of the present disclosure may be converted to their respective activated derivatives, such as their respective NHS-esters; or modified to incorporate a functional group capable of participating in a reaction for bio-molecular labeling. Additionally, the introduction of a linker allows the synthesis of phosphoramidites, such as those that are compatible with solid-phase synthesis of nucleic acids and phosphoramidite chemistry.

[0011] Further, a novel method of producing the fluorescent compound disclosed herein with exceptional yields, as well as without the need of using certain substances that may be of concern for human health or environment. These and other benefits are described further herein.

[0012] Embodiments of the disclosure include the following items:

[0013] [1] A fluorescent compound having a chemical structure according to Formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof: (I), wherein

[0014] A is an aryl;

[0015] R1to R7independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;

[0016] Y1and Y2independently are C(Rb)2, N(RC), S, O, or Se, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1, and each Rcindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;

[0017] R8is -OH, -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety; and

[0018] [X]’ is a counter anion, with the proviso that when any one of R1to R8has a negative charge, [X]’ is not present.

[0019] [2] The compound of [1], wherein A is a phenylene group.

[0020] [3] The compound of [1] or [2], wherein the compound has the structure of Formula (la): wherein A1to A4independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl.

[0021] [4] The compound of [3], wherein A1to A4independently are H, -OH, -OMe, azide, alkyne, - Br, or sulfonate.

[0022] [5] The compound of any one of the preceding items, wherein the compound has the structure of Formula (lb):

[0023] [6] The compound of any one of the preceding items, wherein each of R1to R7are H.

[0024] [7] The compound of any one of the preceding items, wherein each of Y1and Y2is C(CH3)2.

[0025] [8] The compound of any one of the preceding items, wherein the compound has the structure of Formula (Ic): wherein A1to A4independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl.

[0026] [9] The compound of any one of the preceding items, wherein the compound has the structure of Formula (Id):

[0027]

[0010] The compound of any one of the preceding items, wherein R8is -OH or NHRd.

[0028]

[0011] The compound of any one of the preceding items, wherein linker L comprises or consists of a saturated or unsaturated, unsubstituted or substituted hydrocarbon, optionally comprising one or more heteroatoms, such as P, O, N or S.

[0029]

[0012] The compound of any one of the preceding items, wherein linker L has as a backbone a straight- or branched-chain unsubstituted or substituted Ci to C20 alkyl chain, Ci to C20 alkenyl chain or a 1 to 75 atom chain consisting of carbon atoms, substituted carbon atoms and / or one or more atoms selected from P, O, N and S, wherein the backbone optionally comprises one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

[0030]

[0013] The compound of any one of the preceding items, wherein linker L has the structure of Formula (LI): wherein a and b are integers each independently ranging from 1 to 20; Q is a bond, -O, -S, -NH or -N(CH3); Rhand R1are independently H, a C1-C4 alkyl group, -F, -Cl, -NH or -N(CH3) or together are =0; and A and B are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon having between 1 and 40 carbon atoms, and optionally having one or more P, O, N, or S heteroatoms.

[0031]

[0014] The compound of any one of the preceding items, wherein linker L comprises a conjugable moiety.

[0032]

[0015] The compound of any one of the preceding items, wherein the conjugable moiety is selected from the group consisting of an active ester group, such as N-hydroxy succinimide (NHS) ester or sulfo-NHS ester, pentafluorophenyl ester, cabonylimidazole ester, quadratic acid esters, a hydroxybenzotriazole (HOBt) ester, or l-hydroxy-7-azabenzotriazole (HO At) ester, a sulfonylchloride unit, an acyl halide, a moiety comprising a phosphorami di te group or a moiety comprising a group capable of participating in a click chemistry reaction.

[0033]

[0016] The compound of

[0015] wherein the group capable of participating in the click chemistry reaction is selected from the group consisting of a bicyclo[6.1.0]nonyne group ("BCN"), dibenzocyclooctyne ("DBCO"), alkene, trans-cyclooctene ("TCO"), maleimide, an aldehyde, a ketone, an azide, an alkyne, a tetrazine, a thiol, a 1,3 -nitrone, a hydrazine, and a hydroxylamine.

[0034]

[0017] The compound of any one of the preceding items, wherein R8is selected from:

[0035] -OH, -OCH3,

[0036] wherein Reis -OH or a conjugable moiety, and Rfis H or a protecting group.

[0037]

[0018] The compound of any one of the preceding items having a structure selected from the following structures:

[0038]

[0019] The compound of any one of the preceding items, wherein [X]’ is selected from the group consisting of chloride, bromide, iodide, sulfate, benzene sulfonate, p-toluenesulfonate, p- bromobenzenesulfonate, methanesulfonate, trifluoromethanesulfonate, phosphate, perchlorate, tetrafluoroborate, hexafluorophosphate, tetraphenylboride, nitrate, and anions of aromatic or aliphatic carboxylic acids.

[0039]

[0020] The compound of any one of the preceding items, wherein the compound has improved fluorescence quantum yield, extended lifetime, and / or thermostable fluorescence, and / or superior quenching of background fluorescence in PCR probes compared to Cy5.5.

[0021] A method for producing a fluorescent compound having the chemical structure according to Formula (Ila) or (lib), or a stereoisomer or pharmaceutically acceptable salt thereof: wherein R1to R7and R11independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;

[0040] Y1and Y2independently are C(Rb)2, N(RC), S, O, or Se, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1, and each Rcindependently is H, deuterium, unsubstituted alkyl, or usubstituted heteroalkyl;

[0041] R8is -OH, -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety;

[0042] R9and R10independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl, or represent attachment points of an aryl, such as aryl A, for example phenylene;

[0043] [X]’ is a counter anion, with the proviso that when any one of R1to R11has a negative charge, [X]’ is not present; wherein the method comprises a first conversion:

[0044]

[0045]

[0023] The method of

[0022] , wherein substep (a) is conducted in the presence of ZnCh and H2SO4, or in the presence of BF3.

[0024] The method of

[0022] or

[0023] , wherein substep (a) is conducted in the presence of ZnCh and H2SO4, wherein optionally substep (a) is conducted first in the presence of ZnCh without H2SO4, followed by addition of H2SO4.

[0046]

[0025] The method of any one of

[0021] -

[0024] further comprising a preceding conversion before the first conversion:

[0047]

[0026] The method of

[0025] , wherein the preceding conversion is conducted in EtOAc, and / or wherein the product of the preceding conversion is purified on neutral alumina or by precipitation.

[0048]

[0027] The method of any one of

[0021] -

[0026] , wherein each of R1to R7and R11are H.

[0049]

[0028] The method of any one of

[0021] -

[0027] , wherein each of Y1and Y2is C(CH3)2.

[0050]

[0029] The method of any one of

[0021] -

[0028] , wherein R9and R10represent attachment points of an aryl, such as aryl A.

[0030] The method of any one of

[0021] -

[0029] , wherein R9and R10represent attachment points of phenylene.

[0051]

[0031] The method of any one of

[0021] -

[0030] for producing a fluorescent compound having the chemical structure according to Formula (Ila), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein the method comprises as the first conversion:

[0052]

[0053]

[0032] The method of any one of

[0021] -

[0031] for producing a fluorescent compound having the chemical structure according to any one of [l]-

[0020] , or a stereoisomer or pharmaceutically acceptable salt thereof, wherein the method comprises as the first conversion:

[0054]

[0033] The method of

[0032] , wherein the first conversion comprises the following substeps (a) and (b):

[0055]

[0034] The method of

[0032] or

[0033] , further comprising a preceding conversion before the first conversion:

[0056]

[0035] The method of any one of

[0021] -

[0034] , further comprising a second conversion after the first conversion: wherein R8is -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety.

[0057]

[0036] The method of

[0035] , wherein the second conversion comprises converting the carboxyl group to an active ester group, such as an NHS ester, optionally followed by reacting the active ester group with a linker L.

[0058]

[0037] The method of any one of

[0021] -

[0036] , further comprising conjugating a specific binding entity to the compound, optionally followed by removal of protecting groups.

[0059]

[0038] A conjugate comprising (i) a specific binding entity, and (ii) the compound of any one of [l]-

[0020] ,

[0060]

[0039] The conjugate of

[0038] , wherein the specific binding entity is coupled to the compound via Rd

[0061]

[0040] The conjugate of

[0038] or

[0039] , wherein the specific binding entity is an oligonucleotide, optionally comprising LNA, L-LNA, or PNA, or a protein, such as an antibody.

[0062]

[0041] The conjugate of any one of

[0038] -

[0040] , having the following structure of Formula (III): wherein Z is O, NH, ORdor NHRd, wherein Rdis a linker L.

[0063]

[0042] The conjugate of

[0041] having the structure of any one of Formulas (Illa) to (Illh): wherein Z is O, NH, ORdor NHRd, wherein Rdis a linker L, and wherein each Rgindependently represents H or a specific binding entity, with the proviso that at least one Rgrepresents a specific binding entity.

[0064]

[0043] A kit comprising (i) a first conjugate comprising a first oligonucleotide coupled to the compound of any one of [l]-

[0020] ; and (ii) a second conjugate comprising a second oligonucleotide coupled to a quencher, wherein optionally Oligonucleotide 1 and / or 2 comprises LNA, L-LNA, or PNA.

[0065]

[0044] A conjugate having Formula (V):

[0066] (Oligonucleotide 1) - [linker L - (Dye)] - [(Oligonucleotide 2)(Q 1)] (V), wherein

[0067] Oligonucleotide 1 and 2 are different from each other and are independently between about 5 and about 30 nucleotides in length;

[0068] Dye comprises the compound of any one of

[0001] -

[0020] , coupled to Oligonucleotide via linker L; and

[0069] QI is a quencher.

[0070]

[0045] The conjugate of

[0044] , wherein the Dye comprises a compound having the following structure:

[0071]

[0046] The conjugate of

[0044] or

[0045] , wherein the Dye comprises a compound having the following structure:

[0072]

[0047] The conjugate of any one of

[0044] -

[0046] , wherein at least one of Oligonucleotide 1 and / or 2 comprises LNA, L-LNA, or PNA.

[0073]

[0048] The conjugate of any one of

[0044] -

[0047] , wherein linker L is a substituted or unsubstituted aliphatic, heteroaliphatic, aromatic, or heteroaromatic group having between about 5 and about 15 carbon atoms.

[0074]

[0049] A kit comprising: (i) the conjugate of any one of

[0044] -

[0048] ; and (ii) a conjugate having Formula (VI):

[0075] [Oligonucleotide 3] - [Q2] (VI), wherein Oligonucleotide 3 is between about 5 and about 30 nucleotides in length, wherein Oligonucleotide 3 optionally comprises LNA, L-LNA, or PNA; and

[0076] Q2 is a quencher.

[0077]

[0050] The kit of

[0049] , wherein Oligonucleotide 3 is at least partially complementary to Oligonucleotide 1.

[0078]

[0051] A probe having the following Formula (VII):

[0079] [Dye 1] - [L]c- [5 ' - Oligonucleotide - 3'] - [L]c- [Dye 2] (VII), wherein one of Dye 1 or Dye 2 is the compound of any one of [l]-

[0020] , coupled via R8to L, when present, or to the Oligonucleotide, when L is not present; and the other one of Dye 1 or Dye 2 is a quencher; the oligonucleotide is between about 5 and about 60 nucleotides in length; each L is independently a Linker L; and c is 0 or 1.

[0080]

[0052] The probe of

[0051] , wherein one of Dye 1 or Dye 2 comprises a compound having the following structure:

[0081]

[0053] The probe of

[0051] or

[0052] , wherein one of Dye 1 or Dye 2 comprises a compound having the following structure:

[0082]

[0083]

[0054] A method for amplification and detection of a target nucleic acid in a sample comprising the steps of:

[0084] (a) contacting the sample containing the target nucleic acid in a single reaction vessel with

[0085] (i) one pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to opposite strands of a subsequence of the target nucleic acid;

[0086] (ii) an oligonucleotide probe that comprises an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence non-complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid that is bounded by the pair of oligonucleotide primers, wherein the probe further comprises an interactive dual label comprising a fluorescent compound of any one of [l]-

[0020] , located on the tag portion and a first quencher moiety located on the annealing portion and wherein the fluorescent compound is separated from the first quencher moiety by a nuclease susceptible cleavage site; and wherein prior to step (b), the tag portion is reversibly bound in a temperature-dependent manner to a quenching oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe and binds to the tag portion by hybridization, wherein the quenching oligonucleotide comprises at least a second quencher moiety capable of quenching the fluorescent compound on the tag portion when the quenching oligonucleotide is bound to the tag portion; (b) following step (a), amplifying the target nucleic acid by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5' to 3' nuclease activity such that during an extension step of each PCR cycle, the nuclease activity of the polymerase allows cleavage and separation of the tag portion from the first quencher moiety on the annealing portion of the probe;

[0087] (c) measuring one or more signals from the fluorescent compound at a first temperature at which the quenching oligonucleotide is bound to the tag portion;

[0088] (d) measuring one or more signals from the fluorescent compound at a second temperature, which is higher than the first temperature, at which the quenching oligonucleotide is not bound to the tag portion;

[0089] (e) obtaining a calculated signal value by subtracting a median or average of the one or more signals detected at the first temperature from a median or average of the one or more signals detected at the second temperature; whereby a calculated signal value that is higher than a threshold signal value allows determination of the presence of the target nucleic acid.

[0090]

[0055] The method of

[0054] , wherein the one or more nucleotide modifications is selected from the group consisting of Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), Bridged Nucleic Acid (BNA), 2'-0 alkyl substitution, L-enantiomeric nucleotide, and combinations thereof.

[0091]

[0056] A FRET pair comprising a first member having Formula (Villa) and a second member having Formula (Vlllb):

[0092] [Dye 1] - [L]c- [51- Oligonucleotide 1 - 3'] (Villa),

[0093] [5' - Oligonucleotide 2 - 3'] - [L]c- [Dye 2] (Vlllb), wherein one of Dye 1 or Dye 2 comprises the compound of any one of [l]-

[0020] , coupled via R8to L, when present, or to the Oligonucleotide, when L is not present; the other one of Dye 1 or Dye 2 is a Quencher; each L is independently a Linker L; c is 0 or 1; and

[0094] Oligonucleotide 1 and Oligonucleotide 2 are different from each other, optionally are between about 5 und about 30 nucleotides in length, and / or optionally comprise LNA, L- LNA, or PNA.

[0095]

[0057] The FRET pair of

[0056] , wherein one of Dye 1 or Dye 2 comprises a compound having the following structure.

[0058] The FRET pair of

[0056] or

[0057] , wherein one of Dye 1 or Dye 2 comprises a compound having the following structure:

[0096] In certain aspects a fluorescent compound is provided that may be applied in the method of producing and in the method of amplifying and detecting described in detail above, as well as may be used in a conjugate, a probe, a FRET pair or a kit as described in detail above, where said fluorescent compound has a chemical structure according to Formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof: wherein

[0097] A is an aryl;

[0098] R1to R7independently are H, sulfonate, deuterium, unsubstituted alkyl, or unsubstituted alkyl sulfonate;

[0099] Y1and Y2independently are C(Rb)2, S or O, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1;

[0100] R8is -OH, -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety; and

[0101] [X]’ is a counter anion, with the proviso that when any one of R1to R8has a negative charge, [X]’ is not present.

[0102] In certain embodiments, R1to R5independently are H, sulfonate, deuterium, unsubstituted alkyl, or unsubstituted alkyl sulfonate, while R6to R7independently are H, deuterium, or unsubstituted alkyl. In certain embodiments, R1to R5independently are H, sulfonate, deuterium, unsubstituted alkyl, or unsubstituted alkyl sulfonate, while R6to R7independently are H or deuterium. In certain embodiments, Y1and Y2independently are C(Rb)2, S or O, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1 and wherein the unsubstituted alkyl is methyl or ethyl. In certain embodiments, Y1and Y2independently are C(Rb)2 or O, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1. In certain embodiments, Y1and Y2independently are C(Rb)2 or O, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1 and wherein the unsubstituted alkyl is methyl or ethyl. In certain embodiments, Y1and Y2independently are C(Rb)2, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, - (OCH2CH2)XOH where x is an integer > 1. In certain embodiments, Y1and Y2independently are C(Rb)2, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1 and wherein the unsubstituted alkyl is methyl or ethyl. In certain embodiments, R8is -OH, -ORdor -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety BRIEF DESCRIPTION OF THE FIGURES

[0103] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements.

[0104] Fig- 1 shows the fluorescence excitation and emission spectra for Cy5.25B carboxylic acid (left panel) and Cy5.5 carboxylic acid (right panel).

[0105] Fig- 2 shows the temperature dependence of fluorescence emission for Cy5.25B carboxylic acid (solid line) in comparison to Cy5.5 carboxylic acid (dashed line). Each sample was excited at the excitation maximum and the fluorescence at the emission maximum was plotted as a function of temperature. For both dyes the fluorescence signal at 25 °C was normalized to 100%.

[0106] Fig- 3 shows PCR growth curves at three different temperatures for TaqMan DNA probes that were labelled with either Cy5.25B dye (A) or Cy5.5 dye (B). At each PCR cycle the fluorescence signal was recorded at 58 °C (solid lines), 80 °C (dashed lines), and 91 °C (dotted lines). In the left panels, the raw fluorescence plotted, whereas in the right panels the signal was divided by the background fluorescence before probe cleavage (RFI).

[0107] DETAILED DESCRIPTION

[0108] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0109] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0110] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of' or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0111] As used herein, the terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Likewise, 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. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0112] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. 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, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0113] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0114] As used herein, the symbol " intersecting a bond refers to a location in which a moiety is bonded to another moiety. In the structures shown herein, when not all natural valences of an atom are filled by named groups, it should be understood that the unfilled valences are filled by hydrogen.

[0115] As used herein, an alkyl is a hydrocarbon group having a saturated carbon chain. The chain may be branched, unbranched, or cyclic. 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, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl group substituted cycloalkyl groups, and cycloalkyl group substituted alkyl groups. In some embodiments, an alkyl is a Ci-Cio alkyl, C1-C5 alkyl, C1-C3 alkyl, methyl, ethyl, C3 alkyl, C4 alkyl, Cs alkyl, Ce alkyl, C7 alkyl, Cs alkyl, C9 alkyl, or C10 alkyl. In certain embodiments, a straight chain or branched chain alkyl has 8 or fewer carbon atoms in its backbone (e.g., Ci-Cs for straight chain, Ci-Cs for branched chain). Unless otherwise specified, the term alkyl encompasses substituted and unsubstituted alkyl, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0116] As used herein, a heteroalkyl is an alkyl containing at least one heteroatom, such as N, O, S, P, or S(O)n (where n is 1 or 2), replacing the one or more carbons of the hydrocarbon backbone. Unless otherwise specified, the term heteroalkyl encompasses substituted and unsubstituted heteroalkyl. In some embodiments, a heteroalkyl may have a chain length, including carbon atoms and heteroatoms, of from 1-10, 1-5, or 1-3, such as a chain length of 2, 3, 4, 5, 6, 7, 8, 9, or 10. A (hetero)alkyl indicates either an alkyl or a heteroalkyl.

[0117] As used herein, an aryl is a monovalent aromatic carbocyclic group of, for example, from 6 to 15 carbon atoms, having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., quinoline, indole, benzodioxole, and the like), provided that the point of attachment is through an atom of an aromatic portion of the aryl group and the aromatic portion at the point of attachment contains only carbons in the aromatic ring. If any aromatic ring portion contains a heteroatom, the group is a heteroaryl and not an aryl. Aryl groups can be monocyclic, bicyclic, tricyclic or tetracyclic, for example, in the context of the disclosure typically monocylic. Unless otherwise specified, the term aryl encompasses substituted and unsubstituted aryl. In the context of aryl A, it is noted that two carbon atoms of A are comprised as attachment points to the remaining structure in Formula (I). As used herein, a heteroaryl is an aryl having at least one heteroatom, i.e., one or more carbon atoms in the ring has been replaced with an atom having at least one lone pair of electrons, typically nitrogen, oxygen, phosphorus, silicon, or sulfur. Unless otherwise specified, the term heteroaryl encompasses substituted and unsubstituted heteroaryl. A (hetero)aryl indicates an aryl or a heteroaryl.

[0118] As used herein, the term "antibody" refers to immunoglobulins or immunoglobulin-like molecules, including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, (e.g., in mammals such as humans, goats, rabbits and mice) and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of 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 an epitope of an antigen. Antibodies may be composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins and the variants and portions of them well known in the art.

[0119] As used herein, the term "primary antibody" refers to an antibody, which binds specifically to the target protein antigen in a tissue sample. A primary antibody is generally the first antibody used in an immunohistochemical procedure.

[0120] As used herein, the term "secondary antibody" herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent (e.g., a label, an enzyme, etc.), if any. The secondary antibody is generally the second antibody used in an immunohistochemical procedure.

[0121] As used herein, "Ca to Cb" in which "a" and "b" are integers refer 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 heteroalicyclyl group. That is, the alkyl, alkenyl, alkynyl, ring of the cycloalkyl, ring of the cycloalkenyl, ring of the cycloalkynyl, ring of the aryl, ring of the heteroaryl or ring of the heteroalicyclyl can contain from "a" to "b", inclusive, carbon atoms. Thus, for example, a "Ci to C4 alkyl" group refers to all alkyl groups having from 1 to 4 carbons, that is, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH2CH(CH3) and C(CH3)3. As used herein, the term "click chemistry" refers to a chemical philosophy, independently defined by the groups of Sharpless and Meldal that describes chemistry tailored to generate substances quickly and reliably by joining small units together. "Click chemistry" has been applied to a collection of reliable and self-directed organic reactions (Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004-2021). For example, the identification of the copper catalyzed azide-alkyne [3+2] cycloaddition as a reliable molecular connection in water (Rostovtsev, V. V.; et al. Angew. Chem. Int. Ed. 2002, 41, 2596-2599) has been used to augment several types of investigations of biomolecular interactions (Wang, Q.; et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Speers, A. E.; et al. J. Am. Chem. Soc. 2003, 125, 4686-4687; Link, A. J.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 11164-11165; Deiters, A.; et al. J. Am. Chem. Soc. 2003, 125, 11782-11783). In addition, applications to organic synthesis (Lee, L. V.; et al. J. Am. Chem. Soc. 2003, 125, 9588-9589), drug discovery (Kolb, H. C.; Sharpless, K. B. Drug Disc. Today 2003, 8, 1128-1137; Lewis, W. G.; et al. Angew. Chem. Int. Ed. 2002, 41, 1053-1057), and the functionalization of surfaces (Meng, J.-C.; et al. Angew. Chem. Int. Ed. 2004, 43, 1255-1260; Fazio, F.; et al. J. Am. Chem. Soc. 2002, 124, 14397-14402; Collman, J. P.; et al. Langmuir 2004, 20(4), 1051-1053; Lummerstorfer, T.; Hoffmann, H. J. Phys. Chem. B 2004, 108, 13, 3963-3966) have also appeared. Generally, click chemistry encourages reactions that have modular applications that are wide in scope, that have a high chemical yield, that generate inoffensive byproducts, that are chemospecific, that require simple reaction conditions, that use readily available starting materials and reagents, that are solvent free or use benign solvents (such as water), that lead to easy product isolation, that have a large thermodynamic driving force to favor a reaction with a single reaction product, and that have a high atom economy. While certain of the general criteria can be subjective in nature, and not all criteria need to be met.

[0122] As used herein, the term "conjugate" refers to two or more molecules or moieties (including macromolecules or supra-molecular molecules) that are covalently linked into a larger construct. In some embodiments, a conjugate includes one or more biomolecules (such as oligonucleotides, polynucleotides, peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or more other molecules moieties.

[0123] As used herein, the terms "couple," "coupled," or "coupling" refers to the linking (e.g. covalently linking) of one molecule or atom to another molecule or atom.

[0124] As used herein, the term "derivative" is used in accordance with its plain ordinary meaning within chemistry and biology and refers to a chemical compound that is structurally related to another compound (i.e., a so-called "reference" compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.

[0125] As used herein, the term "heteroatom" is meant to include e.g. boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si). As noted herein, in some embodiments, a "heterocyclic ring" may comprise one or more heteroatoms. In other embodiments, an aliphatic group may comprise or be substituted by one or more heteroatoms.

[0126] As used herein, the term "oligonucleotide" refers to linear oligomers of natural or modified nucleosidic monomers linked by phosphodiester bonds or analogs thereof. Oligonucleotides include deoxyribonucleosides, ribonucleosides, anomeric forms thereof, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and the like, capable of specifically binding to a target nucleic acid. A “or modified nucleosidic monomer” or “modified nucleotide” in the context of an oligonucleotide refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced by a different nucleotide that provides a desired property to the oligonucleotide. Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, e.g., a t-butyl benzyl, a phosphate, a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7- propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, a C7- propargylamino-dA, a C7-propargylamino-dG, a 7-deaza-2-deoxyxanthosine, a pyrazolo- pyrimidine analog, a pseudo-dU, a nitro pyrrole, a nitro indole, 2’-0-methyl ribo-U, 2’-0-methyl ribo-C, an N4-ethyl-dC, an N6-methyl-dA, a 5-propynyl dU, a 5-propynyl dC, and the like. Many other modified nucleotides that can be substituted in the oligonucleotides are referred to herein or are otherwise known in the art. In certain embodiments, modified nucleotide substitutions modify melting temperatures (Tm) of the oligonucleotides relative to the melting temperatures of corresponding unmodified oligonucleotides. To further illustrate, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation or the like), increase the yield of an intended target amplicon, and / or the like in some embodiments. Examples of these types of nucleic acid modifications are described in, e.g., U.S. Patent No. 6,001,611. Other modified nucleotide substitutions may alter the stability of the oligonucleotide, or provide other desirable features. Usually, monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., at least 3, such as at least 5, to several tens of monomeric units, e.g., 40-60. For example, an oligonucleotide can have a length of at least 3, such as at least 5 or at least 10 nucleotides. For example, an oligonucleotide can have a length of 60 or less, such as 50 or less or 40 or less nucleotides. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'-3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T" denotes deoxythymidine, and "U" denotes the ribonucleoside, uridine, unless otherwise noted. Usually, oligonucleotides comprise the four natural deoxynucleotides; however, they may also comprise ribonucleotides or non-natural nucleotide analogs, as noted above. Non-natural nucleotides include, e.g. PNA, LNA, as well as L-DNA and L-RNA nucleotides. In some embodiments, an oligonucleotide can be an "LNA-modified oligonucleotide." An "LNA-modified oligonucleotide" refers to an oligonucleotide that is either fully or partially modified with one or more LNA monomers ("locked nucleic acid" monomers). Thus, an "LNA-modified oligonucleotide" may be composed entirely of LNA monomers, or an "LNA-modified oligonucleotide" may comprise one LNA monomer, two LNA monomers, etc. As used herein, the term "LNA monomer" refers to a class of conformationally restricted nucleotide analogs whose ribose ring is constrained by a (methylene) linkage between the 2'-oxygen and the 4'-carbon, i.e. such as nucleotides which comprise a covalent bridge between the 2' and 4' position (a 2' - 4' bridge). In some embodiments, LNA is applied in its beta-D-configuration which binds to naturally occurring nucleic acids with high affinity. alpha-L-LNA is also described and pairs also to naturally occurring nucleic acids with high affinity. In contrast to alpha-L-LNA, beta-L-LNA does not bind to naturally occurring nucleic acids but only to beta-L-configured nucleic acids like beta-L-DNA or beta-L-LNA. LNA monomers are further described within U.S. Pat. No. 6,268,490, U.S. Pat. No. 6,794,499, U.S. Pat. No. 7,034,133. The synthesis of locked nucleic acid derivatives is described in U.S. Patent No. 8,492,390. Yet other monomers are disclosed in PCT Publication Nos. WO98 / 39352, WO99 / 14226.

[0127] Where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g., single stranded DNA, RNA / DNA duplex, or the like, then selection of the appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill.

[0128] As used herein, the term "phosphoramidite" refers to a trivalent phosphorus group typically used in oligonucleotide synthesis. Detailed descriptions of the chemistry used to form oligonucleotides by the phosphoramidite method are provided in Caruthers et al., U.S. Pat. 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, pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (1991), each of which are incorporated by reference in their entirety. As used herein, the term "protecting group" refers to a moiety that when attached to a reactive group in a molecule reduces or prevents that reactivity. A "protected" molecule has one or more reactive groups (e.g., hydroxyl, amino, thiol, etc.) protected by protecting groups. Examples of protecting groups can be found in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York, 1999, Harrison and Harrison et al. Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996), and "Protection of Nucleosides for Oligonucleotide Synthesis," Current Protocols in Nucleic Acid Chemistry, ed. by Boyle, A. L., John Wiley & Sons, Inc., 2000, New York, N.Y., all of which are incorporated herein by reference in their entirety. A protecting group can be selected, for example, from dimethyl acetal, 1,3 -di oxolane, methyl ester, 2-cyanoethyl, 9-fluorenylmethyl carbamate, t- Butyl carbamate, benzyl carbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, monomethoxytrityl (MMT), DMS, DMT and -toluenesulfonamide.

[0129] As used herein, the terms "reactive group" or "reactive functional group" refer to a functional group that are capable of chemically associating with, interacting with, hybridizing with, hydrogen bonding with, 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 that a covalent linkage is formed between 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 to each other, hydrogen bond with each other, etc. In some embodiments, the "reaction" thus includes binding events, such as the binding of a hapten with an anti-hapten antibody, or a guest molecule associating with a supramolecular host molecule.

[0130] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind each other to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 103M'1greater, 104M'1greater or 105M'1greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). Particular examples of specific binding entities include oligonucleotides (such as oligonucleotides probes that can be designed to bind to specific target nucleic acid) or specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A). Specific binding moieties can also include the molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0131] Whenever a group or moiety is described as being "substituted" or "optionally substituted" that group may be unsubstituted or substituted with one or more of the indicated substituents. Likewise, when a group is described as being "substituted or unsubstituted" if substituted, the substituent(s) may be selected from one or more the indicated substituents. If no substituents are indicated, it is meant that the indicated "optionally substituted" or "substituted" group may be substituted with one or more group(s) individually and independently selected from, e.g., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C- carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, an amino, ether, amino (e.g. a mono-substituted amino group or a di -substituted amino group), and protected derivatives thereof. Any of the above groups may include one or more heteroatoms, including O, N, or S. For example, where a moiety is substituted with an alkyl group, that alkyl group may comprise a heteroatom selected from O, N, or S (e.g. -(CH2-CH2-O-CH2-CH2)-).

[0132] OVERVIEW

[0133] The present disclosure is directed to dyes, such as dyes having improved fluorescence quantum yield, extended lifetime, thermostable fluorescence, and superior quenching of background fluorescence in PCR probes, as well as methods for producing the same. The present disclosure is also directed to conjugates and probes including one or more of the disclosed dyes. The present disclosure also provides kits including one or more of the disclosed dyes; or one or more conjugates including one or more of the disclosed dyes. The dyes of the present disclosure may be used with any fluorescence-based PCR platform with compatible optical filters. Conjugates including one of the dyes disclosed herein are also compatible with PCR with TAGS (Temperature Activated Generation of Signal) technology (see United States Patent Nos. 11,028,433, 11,034,997, and 11,345,958; and in United States Patent Publication No. 2021 / 0269857, the disclosures of which are hereby incorporated by reference herein in their entireties).

[0134] DYES OR DYE PRECURSORS

[0135] The disclosure provides a fluorescent compound having a chemical structure according to Formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof: wherein

[0136] A is an aryl;

[0137] R1to R7independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;

[0138] Y1and Y2independently are C(Rb)2, N(RC), S, O, or Se, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1, and each Rcindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;

[0139] R8is -OH, -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein L optionally comprises a conjugable moiety; and

[0140] [X]’ is a counter anion, with the proviso that when any one of R1to R8has a negative charge, [X]’ is not present.

[0141] A represents an aryl. In some embodiments, A is a (substituted or unsubstituted) phenylene group. In some embodiments, the compound has the structure of Formula (la):

[0142] A1to A4independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl. In some embodiments, A1to A4independently are H, -OH, -OMe, azide, alkyne, -Br, or sulfonate. In some embodiments, each of A1to A4is H.

[0143] In some embodiments, the compound has the structure of Formula (lb): R1to R7independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl. In some embodiments, R1to R7independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, or - C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl. In some embodiments, each of R1to R7is H.

[0144] Y1and Y2independently are C(Rb)2, N(RC), S, O, or Se, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1, and each Rcindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl. In some embodiments, each of Rband Rcis -CH3. In some embodiments, each of Y1and Y2is C(CH3)2.

[0145] In some embodiments, the compound has the structure of Formula (Ic): (Ic), wherein A1to A4and R8are as described herein.

[0146] In some embodiments, the compound has the structure of Formula (Id):

[0147] R8is -OH, -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety. In some embodiments, R8is -OH, -OCH3 or -NHRd. Linker L is not particularly limited, and linkers used in conjugation chemistry are commonly known in the art. In some embodiments, the linker L comprises or consists of a saturated or unsaturated, unsubstituted or substituted hydrocarbon, optionally comprising one or more heteroatoms, such as P, O, N or S. In some embodiments, L may comprise carbonyl, amine, ester, ether, amide, imine, thione, or thiol groups. In some embodiments, linker L comprises 1 to 50 carbon atoms, such as 1 to 30, 1 to 20, or 1 to 10 carbon atoms. In some embodiments, linker L comprises 1 to 75 atoms, such as 1 to 60 atoms, 1 to 50, 1 to 40, or 1 to 30 atoms, not counting hydrogen atoms. In some embodiments, linker L has a molecular weight of lOOODa or less, such as 800Da or less.

[0148] In some embodiments, linker L has as a backbone a straight- or branched-chain unsubstituted or substituted Ci to C20 alkyl chain, Ci to C20 alkenyl chain or a 1 to 75 atom chain consisting of carbon atoms, substituted carbon atoms and / or one or more atoms selected from P, O, N and S, wherein the backbone optionally comprises one or more cyclic or heterocyclic aromatic or nonaromatic ring systems.

[0149] In some embodiments, linker L has the structure of Formula (LI): wherein a and b are integers each independently ranging from 1 to 20; Q is a bond, -O, -S, - NH or -N(CH3); Rhand R1are independently H, a C1-C4 alkyl group, -F, -Cl, -NH or -N(CH3) or together are =0; and A and B are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon having between 1 and 40 carbon atoms, and optionally having one or more P, O, N, or S heteroatoms, such as P, O, or N heteroatoms. Linker L according to Formula (LI) can be coupled to the oxygen or nitrogen of R8through A or B, for example. In some embodiments, Formula (LI) comprises 1 to 50 atoms, such as 1 to 40, or 1 to 30 atoms, not counting hydrogen atoms.

[0150] In some embodiments, a and b are integers ranging from 1 to 10. In some embodiments, a and b are integers ranging from 1 to 6. In other embodiments, a and b are integers ranging from 1 to 5. In some embodiments, a and b are both 1.

[0151] In some embodiments, A and B are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally having one or more P, O, N, or S heteroatoms, wherein A has between 1 and 12 carbon atoms, such as 1 to or 1 to 5, such as 5 carbon atoms, and B has between 1 and 40 carbon atoms, such as 10 to 40, such as 40 carbon atoms, and wherein Linker L according to Formula (LI) is coupled to the oxygen or nitrogen of R8through A. In some embodiments, A and B are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon having between 1 and 12 carbon atoms, and optionally having one or more P, O, N, or S heteroatoms. In some embodiments, L is obtainable from the following linkers (according to manufacturers’ instructions):

[0152] 5'-Amino-Modifier C6-TFA (GLEN RESEARCH CATALOG NO. 10-1916) Amino-Modifier C6 dT (GLEN RESEARCH CATALOG NO. 10-1039) Amino-Modifier C6 dC (10-1019)

[0153] Amino-Modifier C2 dT (Glen Research Catalog No. 10-1037) Amino-Modifier C6 dA (Glen Research Catalog No. 10-1089) N2-Amino-Modifier C6 dG (Glen Research Catalog No. 10-1529) Fmoc Amino-Modifier C6 dT (Glen Research Catalog No. 10-1536) 5'-Amino-Modifier 5 (Glen Research Catalog No. 10-1905) 5'-Amino-Modifier C6 (Glen Research Catalog No. 10-1906) 5'-DMS(O)MT-Amino-Modifier C6 (Glen Research Catalog No. 10-1907) 5'-Amino-Modifier C12 (Glen Research Catalog No. 10-1912) 5'-Amino-Modifier TEG CE-Phosphoramidite (Glen Research Catalog No. 10-1917) 5'-Amino-Modifier C3-TFA (Glen Research Catalog No. 10-1923) 5'-Amino-Modifier C6-PDA (Glen Research Catalog No. 10-1947) 5'-Amino-Modifier C12-PDA (Glen Research Catalog No. 10-1948) 5'-Amino-Modifier TEG PDA (Glen Research Catalog No. 10-1949)

[0154] Amino-Modifier Serinol Phosphorami dite (Glen Research Catalog No. 10-1997) PC Amino-Modifier Phosphoramidite (Glen Research Catalog No. 10-4906) 3'-Amino-Modifier C6 dC CPG (Glen Research Catalog No. 20-2019) 3'-Amino-Modifier C6 dT CPG (Glen Research Catalog No. 20-2039) 3'-PT-Amino-Modifier C3 CPG (Glen Research Catalog No. 20-2954) 3'-PT-Amino-Modifier C6 CPG (Glen Research Catalog No. 20-2956) 3'-Amino-Modifier C7 CPG 1000 (Glen Research Catalog No. 20-2958) 3'-Amino-Modifier Serinol CPG (Glen Research Catalog No. 20-2997) 3'-PT-Amino-Modifier C6 PS (Glen Research Catalog No. 26-2956) 5'-DBCO-TEG phosphoramidite (Glen Research Catalog No. 10-1941) DBCO-Serinol phosphoramidite (Glen Research Catalog No. 10-1998) DBCO-dT-CE phosphoramidite (Glen Research Catalog No. 10-1539) 5'-Bromohexyl Phosphoramidite (Glen Research Catalog No. 10-1946)

[0155] In some embodiments, the linker L comprises a conjugable moiety. For example, the conjugable moiety can comprise or consist of a group selected from an active ester group, such as N-hydroxy succinimide (NHS) ester or sulfo-NHS ester, pentafluorophenyl ester, cabonylimidazole ester, quadratic acid esters, a hydroxybenzotriazole (HOBt) ester, or l-hydroxy-7-azabenzotriazole (HO At) ester, a sulfonyl chloride unit, an acyl halide, a phosphoramidite group or a group capable of participating in a click chemistry reaction. An acyl halide in this context includes moieties comprising or consisting of -COX, wherein X is a halogen. A halogen includes, for example, fluoride, chloride, bromide and iodide.

[0156] In some embodiments, the group capable of participating in the click chemistry reaction is selected from the group consisting of a bicyclo[6.1.0]nonyne group ("BCN"), dibenzocyclooctyne ("DBCO"), alkene, trans-cyclooctene ("TCO"), maleimide, an aldehyde, a ketone, an azide, an alkyne, a tetrazine, a thiol, a 1,3 -nitrone, a hydrazine, and a hydroxylamine. In some embodiments, R8is selected from: wherein Reis -OH or a conjugable moiety, and Rfis H or a protecting group. In some embodiments, the compound of Formula (I) has a structure selected from the following structures:

[0157]

[0158] [X]’ is a counter anion, with the proviso that when any one of R1to R8has a negative charge, [X]’ is not present. In some embodiments, [X]’ is selected from the group consisting of chloride, bromide, iodide, sulfate, benzene sulfonate, p-toluenesulfonate, p-bromobenzenesulfonate, methanesulfonate, trifluoromethanesulfonate, phosphate, perchlorate, tetrafluoroborate, hexafluorophosphate, tetraphenylboride, nitrate, and anions of aromatic or aliphatic carboxylic acids. For example, [X]’ can be iodide.

[0159] It was discovered that the compounds of the present disclosure can provide excellent fluorescence quantum yield, extended lifetime and / or thermostable fluorescence. Thus, in some embodiments, the compound has improved fluorescence quantum yield, extended lifetime and / or thermostable fluorescence compared to a reference compound, such as Cy5.5.

[0160] In some embodiments, thermostable means that the compounds are capable of fluorescence emission at a temperature of up to 100 °C, such as up to 90 °C or up to 80 °C. In some embodiments, the compounds as described herein are thermostable over a temperature range of 25°C to l00°C.

[0161] DYE SYNTHESIS

[0162] The present disclosure provides methods of synthesizing fluorescent compounds of Formulas (Ila) (which encompasses the fluorescent compound of Formula (I)) and (lib), and derivatives and analogs thereof. Overview

[0163] The synthesis reported for rigidized penta- and heptamethine dyes is complex and the product is obtained only in small scale with low yield. See Michie et al., J. Am. Chem. Soc. 2017, 139, 12406-12409; WO 2019 / 040825 Al; and Eiring et al., Angew. Chem. Int. Ed. 2021, 60, 26685- 26693, the disclosures of which are incorporated herein by reference in their entirety. According to the present disclosure, the synthesis of rigidized penta- and heptamethine dyes can be significantly improved, scaled up and adapted for industrial production. The product of the multistep synthesis can now be obtained in gram scale. In addition, an important improvement for manufacturing purposes is the replacement of restricted compounds e.g. Substances of Very High Concern (SVHC).

[0164] Scheme 1 details the synthesis of the rigidized cyanine dye following previously reported literature (Michie et al., supra). Several changes have been introduced in the synthetic route, for example, use of halogenated solvent like dichloromethane (DCM) or chloroform could be reduced by at least partial replacement with more benign solvents compared to the art (Scheme 2). A bottle-neck reaction in the synthesis because of very low yield is the installation of the a-P unsaturated aldehyde via cross-metathesis.

[0165] One change introduced in this step includes the substitution of the protecting group for aldehyde of the coupling partner with less labile group (dimethyl acetal vs 1,3-dioxolane) that improved stability of the intermediate (see Scheme 2).

[0166] The key tetracyclization reaction was carried out initially using BBn in CH2CI2 under cryogenic temperature as described in the art (Scheme 1). Subsequently, BF3 (acetic acid complex) was used, which can be used in acetonitrile at room temperature (Scheme 2). When quenched, BBn and BF3 form B(OH)3 which is, unfortunately, a substance of very high concern and problematic for production. Other combinations of reagents were therefore tested and a combination of ZnCh and sulfuric acid in formic acid or in dichloromethane was found to work well at room temperature (Scheme 3).

[0167] In addition, it was shown that final purification can be performed using the reverse phase (RP) chromatography system (instead of using e.g. preparative HPLC) which may allow the production of larger batches.

[0168] As an example, the carboxylic acid of the dye obtained can then be used for the synthesis of a phosphoramidite reagent for oligo synthesis on solid support, for example (Scheme 4). For example, the dye can be coupled with a DMT-protected threoninol linker and then reacted with a phosphitylating reagent. Scheme 1: Old route (based on literature)

[0169] Scheme 2: New route (Gen II)

[0170] Scheme 3: Gen III of Cyclisation Step

[0171] Scheme 4: Phosphoramidite synthesis

[0172] Synthesis

[0173] The disclosure provides a method for producing a fluorescent compound having the chemical structure according to Formula (Ila) or (lib), or a stereoisomer or pharmaceutically acceptable salt thereof: wherein

[0174] R1to R7and R11independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;

[0175] Y1and Y2independently are C(Rb)2, N(RC), S, O, or Se, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1, and each Rcindependently is H, deuterium, unsubstituted alkyl, or usubstituted heteroalkyl;

[0176] R8is -OH, -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety;

[0177] R9and R10independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl, or represent attachment points of an aryl, such as aryl A;

[0178] [X]’ is a counter anion, with the proviso that when any one of R1to R11has a negative charge, [X]’ is not present; wherein the method comprises a first conversion:

[0179] The description of R1to R8, as well as Y1and Y2in the context of Formula (I) applies mutatis mutandis. The description of R1to R7applies equally to R11.

[0180] In some embodiments, R1to R7and R11independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl. In some embodiments, each of R1to R7and R11is H.

[0181] In some embodiments, each of Rband Rcis -CH3. In some embodiments, each of Y1and Y2is C(CH3)2.

[0182] In some embodiments, R9and R10independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl, or represent attachment points of an aryl, such as aryl A. In some embodiments, R9and R10represent attachment points of (substituted or unsubstituted) phenylene. The description of aryl A in the context of Formula (I) applies mutatis mutandis.

[0183] In some embodiments, the first conversion comprises the following substeps (a) and (b):

[0184]

[0185] In some embodiments, substep (a) is conducted in the presence of ZnCh and H2SO4, or in the presence of BF3. In some embodiments, substep (a) is conducted in the presence of ZnCh and H2SO4. In some embodiments, substep (a) is conducted first in the presence of ZnCh without H2SO4, followed by addition of H2SO4.

[0186] BF3 is typically present in a Lewis acid-Lewis base pair, such as BF3.OEt2, BF3.2AcOH, BF3.2H2O and BFs.MeCN. In some embodiments, BF3 is present as BF3.2AcOH.

[0187] In some embodiments, substep (a) is conducted at 4 to 30°C, such as 10 to 30°C, 15 to 25°C or 20 to 25°C.

[0188] In some embodiments, substep (a) is conducted for 1 to 20 hours, such as 2 to 20 hours, or 4 to 18 hours. When substep (a) is conducted in the presence of ZnCh and H2SO4, it is typically conducted for 3 to 5 hours, such as 3.5 to 4.5 hours, such as 4 hours.

[0189] In some embodiments, substep (a) is conducted in the presence of ZnCh and H2SO4 in formic acid, DCM, AcOEt, CEhCN, DMSO, THF, acetic acid, and mixtures thereof. In some embodiments, substep (a) is conducted in the presence of ZnCh and H2SO4 in formic acid and / or DCM.

[0190] In some embodiments, substep (a) is conducted in the presence of BF3 in CEhCN, DCM, AcOEt, DMSO, THF, acetic acid, diethyl ether, and mixtures thereof.

[0191] In some embodiments, substep (b) is conducted in the presence of MeOH and LiOH. In some embodiments, substep (b) is conducted first in the presence of MeOH without LiOH, followed by addition of LiOH.

[0192] In some embodiments, substep (b) is conducted in the presence of HC1, MeOH and LiOH. In some embodiments, substep (b) is conducted first in the presence of HC1 and MeOH without LiOH, followed by addition of LiOH.

[0193] In some embodiments, the method further comprises a preceding conversion before the first

[0194]

[0195] In some embodiments, the preceding conversion is conducted in the presence of a Ru- or Mo- containing olefin metathesis catalyst, such as a Grubbs catalyst or a Hovey da-Grubbs (HG) catalysts, such as a HGI or HGII catalyst, such as HGII catalyst M720 (dichlorofl, 3-bis(2, 4, 6- trimethylphenyl)-2-imidazolidinylidene](2-isopropoxyphenylmethylene)ruthenium(II)), having the structure:

[0196] Other suitable olefin metathesis (Schrock, Grubbs or Hovey da-Grubbs) catalysts include, for example, 2,6-diisopropylphenylimido-neophyliden-[(S)-(-)-BIPHEN]-molybdenum(VI), Ml 02 (benzylidenbis(tricyclohexylphosphin)dichlororuthenium, bis(tricyclohexylphosphin)benzyliden- ruthenium(IV)-dichloride), M204 (l,3-Bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichlor (phenylmethylen)(tricyclohexylphosphan)ruthenium, M700 (dichloro(2-isopropoxyphenylme- thylene) (tricyclohexylphosphine)ruthenium(II)), M710 (dichlorofl, 3-bis(2, 4, 6-trimethylphenyl)- 2-imidazolidinylidene][(2-isopropoxy)(5-trifluoroacetamido)benzylidene] ruthenium(II)), M730 (dichloro[l,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][(5-isobutoxycarbonylamino)-(2- isopropoxy )benzylidene]ruthenium(II)), M731 (dichlorofl, 3-bis(2,6-diisopropylphenyl) imidazolidin-2-ylidene][(5-isobutoxycarbonylamino)-(2-isopropoxy)benzylidene]ruthenium(II)), and M711 (fl,3-Bis(2,6-diisopropylphenyl)-2-imidazolidinylidene]dichloro[(2-isopropoxy)(5- trifluoroacetami do)b enzy li dene] ruthenium (II)) .

[0197] In some embodiments, the preceding conversion is conducted in EtOAc, DCM, CEECN, THF and mixtures thereof. In some embodiments, the preceding conversion is conducted in EtOAc.

[0198] In some embodiments, the preceding conversion is conducted at 4 to 30 °C, such as 10 to 30 °C, 15 to 25 °C or 20 to 25 °C.

[0199] In some embodiments, the product of the preceding conversion is purified on neutral alumina or by precipitation.

[0200] In some embodiments, the method is for producing a fluorescent compound having the chemical structure according to Formula (Ila), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein the method comprises as the first conversion:

[0201] In some embodiments, the method is for producing a fluorescent compound having the chemical structure according to Formula (I) (encompassing the Formula (IA), (IB), (IC) or (ID)), or a stereoisomer or pharmaceutically acceptable salt thereof, wherein the method comprises as the first conversion:

[0202] In some embodiments, the first conversion comprises the following substeps (a) and (b):

[0203] In some embodiments, further comprises a second conversion after the first conversion: wherein R8is -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety; for example, R8can be selected from: wherein Reis -OH or a conjugable moiety, and Rfis H or a protecting group. In some embodiments, the second conversion comprises converting the carboxyl group to an active ester group, such as an NHS ester.

[0204] In some embodiments, the second conversion comprises attachment of a linker L. For example, a linker L can be attached by converting the carboxyl group to an active ester group, followed by reacting the active ester group with a linker L. In some embodiments, the linker L is attached via an amide group (formed e.g. by the reaction with the active ester).

[0205] In some embodiments, the second conversion is conducted in the presence of 2- morpholinoethylisocyanide (MEI), CH3CN and / or N,N-diisopropylethylamine (DIPEA).

[0206] In some embodiments, the second conversion comprises the following substep:

[0207] For example, such a substep can be conducted in the presence of MEI (2- morpholinoethylisocyanide) and CH3CN. For example, such a substep can be conducted for a time of 1 -3h, such as 2h. For example, such a substep can be conducted at a temperature of 18-25 °C, such as 20-24 °C.

[0208] In some embodiments, the second conversion comprises the following substep:

[0209]

[0210] For example, such a substep can be conducted in the presence of N,N-diisopropylethylamine (DIPEA) and CH3CN. For example, such a substep can be conducted for a time of 1 -3h, such as 2h. For example, such a substep can be conducted at a temperature of 18-25 °C, such as 20-24 °C.

[0211] In some embodiments, the second conversion comprises the following substep: or

[0212]

[0213] For example, such a substep can be conducted in the presence of DIPEA and CH3CN. For example, such a substep can be conducted for a time of 0.5-2h, such as Ih. For example, such a substep can be conducted at a temperature of 18-25 °C, such as 20-24 °C. In some embodiments, in which R9and R10represent attachment points of an aryl A, the second conversion comprises the following substep:

[0214] In some embodiments, in which R9and R10represent attachment points of an aryl A, the second conversion comprises the following substep: In some embodiments, in which R9and R10represent attachment points of an aryl A, the second conversion comprises the following substep: In some embodiments, the method further comprises conjugating a specific binding entity to the compound, optionally followed by removal of protecting groups (when present).

[0215] If any protecting groups are present, e.g., in linker L, these may be removed at a suitable time during processing. For example, protecting groups (for example OH-protecting groups) may be removed after conjugating a specific binding entity to the compound. CONJUGATES

[0216] The present disclosure also provides conjugates comprising (i) a specific binding entity, and (ii) the fluorescent compound of Formula (I). In some embodiments, the one or more compounds having Formula (I) are directly coupled to the specific binding entity. In some embodiments, the one or more compounds having Formula (I) are indirectly coupled to the specific binding entity. In some embodiments, the indirect coupling is through one or more linkers. In some embodiments, the specific binding entity is coupled to the compound via Rd, optionally through a linker L.

[0217] In some embodiments, the conjugate has the following structure of Formula (III): wherein Z is O, NH, ORdor NHRd, wherein Rdis a linker L.

[0218] In some embodiments, the conjugate has the following structure of Formula (Illa): wherein Z is O, NH, ORdor NHRd, wherein Rdis a linker L.

[0219] In some embodiments, the conjugate has the following structure of Formula (Illb): wherein each Rgindependently represents H or a specific binding entity, with the proviso that at least one Rgrepresents a specific binding entity. In some embodiments, the conjugate has the following structure of Formula (IIIc): wherein each Rgindependently represents H or a specific binding entity, with the proviso that at least one Rgrepresents a specific binding entity.

[0220] In some embodiments, the conjugate has the following structure of Formula (Hid) or (Hie):

[0221] In some embodiments, the conjugate has the following structure of Formula (I I If), Illg or (Illh):

[0222]

[0223] In the structures of the fluorescent compound in the context of conjugates, probes or FRET pairs, the description of R1to R8, as well as Y1and Y2in the context of Formula (I) applies mutatis mutandis.

[0224] In some embodiments, the specific binding entity is an oligonucleotide, optionally comprising one or more modified nucleotides and / or optionally comprising one or more non-natural nucleotides, such as LNA, L-LNA, or PNA. In some embodiments, the specific binding entity is a protein, such as 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.

[0225] In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide comprises about 5 to about 60 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 55 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 50 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 45 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 40 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 35 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 30 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 25 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 20 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 15 nucleotides.

[0226] In some embodiments, the "Specific Binding Entity" is an oligonucleotide, and the dye having

[0227] Formula (I) is coupled directly or indirectly to a 5' end of the oligonucleotide. In some embodiments, the "Specific Binding Entity" is an oligonucleotide, the dye having Formula (I) is coupled directly or indirectly to a 3' end of the oligonucleotide.

[0228] In embodiments where the Specific Binding Entity is an oligonucleotide, the dye portion of the conjugate may be coupled to either a 5' end or a 3' end of the oligonucleotide. In some embodiments, the oligonucleotide, whether bound to the dye portion at a 5' end or a 3' end, comprises about 5 to about 40 nucleotides.

[0229] In some embodiments, the conjugate has the structure of any one of Formulas (IVa) or (IVb):

[0230] [Dye] - [L]c- [5' - Oligonucleotide - 3'] (IVa) or

[0231] [5' - Oligonucleotide - 3'] - [L]c- [Dye] (IVb), wherein

[0232] Dye is the fluorescent compound of Formula (I), coupled via R8to L, when present, or to the Oligonucleotide, when L is not present;

[0233] L is a linker L; and c is 0 or 1; and

[0234] Oligonucleotide is an oligonucleotide having about 5 to about 60 nucleotides.

[0235] In some embodiments, c is 1. Linker L can be as set forth herein. In some embodiments, L has the structure of Formulas (LI), as set forth herein.

[0236] In some embodiments, the oligonucleotide is single stranded. In some embodiments, the oligonucleotide comprises about 5 to about 55 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 50 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 45 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 40 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 35 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 30 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 25 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 20 nucleotides. In some embodiments, the oligonucleotide comprises about 5 to about 15 nucleotides.

[0237] The present disclosure is also directed to conjugates comprising a compound of Formula (I) and a hapten or an enzyme (e.g., alkaline phosphatase; horseradish peroxidase). In some embodiments, the compound having Formula (I) is directly coupled to the hapten or the enzyme. In some embodiments, the compound having Formula (I) is indirectly coupled to the hapten or the enzyme. In some embodiments, the indirect coupling is through one or more linkers. In some embodiments, the hapten is a pyrazole (e.g., nitropyrazoles); a nitrophenyl compounds; a benzofurazan; a triterpene; a ureas (e.g., phenyl ureas); a thiourea (e.g., phenyl thioureas); a rotenone or a rotenone derivative; an oxazole (e.g., oxazole sulfonamides); a thiazole (e.g., thiazole sulfonamides); a coumarin or a coumarin derivatives; or a cyclolignan. In some embodiments, the hapten is dinitrophenyl, biotin, digoxigenin, and fluorescein, and any derivatives or analogs thereof. Other haptens are described in United States Patent Nos. 8,846,320; 8,618,265; 7,695,929; 8,481,270; and 9,017,954, the disclosures of which are incorporated herein by reference in their entirety.

[0238] The disclosure also provides a kit comprising (i) a first conjugate comprising a first oligonucleotide coupled to the fluorescent compound of Formula (I); and (ii) a second conjugate comprising a second oligonucleotide coupled to a quencher. In some embodiments, Oligonucleotide 1 comprises DNA, L-DNA, LNA, L-LNA, or PNA. In some embodiments, Oligonucleotide 2 comprises DNA, L-DNA, LNA, L-LNA, or PNA. In some embodiments, Oligonucleotide 1 and 2 comprises DNA, L-DNA, LNA, L-LNA, or PNA.

[0239] The disclosure also provides a conjugate having Formula (V):

[0240] (Oligonucleotide 1) - [linker L - (Dye)] - [(Oligonucleotide 2)(Q 1)] (V), wherein

[0241] Oligonucleotide 1 and 2 are different from each other and are independently between about 5 and about 30 nucleotides in length;

[0242] Dye is the fluorescent compound of Formula (I), coupled to Oligonucleotide 1 via Linker L;

[0243] QI is a quencher; and

[0244] Linker L is as defined herein.

[0245] In some embodiments, the Dye in Formula (V) is a compound having the following structure:

[0246] The description of Ri to R? from the fluorescent compound as such applies.

[0247] In some embodiments, the Dye in Formula (V) is a compound having the following structure: In some embodiments, at least one of Oligonucleotide 1 and / or 2 comprises LNA, L-LNA, or PNA.

[0248] In some embodiments, at least one of Oligonucleotide 1, Oligonucleotide 2, or the linker L includes a nuclease susceptible cleavage site.

[0249] The disclosure also provides a kit comprising: (i) the conjugate of Formula (V); and (ii) a conjugate having Formula (VI):

[0250] [Oligonucleotide 3] - [Q2] (VI), wherein

[0251] Oligonucleotide 3 is between about 5 and about 30 nucleotides in length; and Q2 is a quencher.

[0252] In some embodiments, Oligonucleotide 3 comprises LNA, L-LNA, or PNA. In some embodiments, Oligonucleotide 3 is at least partially complementary to Oligonucleotide 1.

[0253] In some embodiments, Oligonucleotides 1 and / or 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, for example, 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. (where the "L-XXX" refers to the L-enantiomer of the sugar unit of the nucleic acids) or any other known variations and modifications on the nucleotide bases, sugars, or phosphodiester backbones. In some embodiments, one of Oligonucleotide 1 or Oligonucleotide 2 includes or consists of L-DNA. In some embodiments, Oligonucleotide 1 includes or consists of L-DNA. In some embodiments, Oligonucleotide 1 consists of L-DNA. In some embodiments, Oligonucleotide 2 includes or consists of L-DNA. In some embodiments, Oligonucleotide 2 consists of L-DNA. In some embodiments, QI and Q2 are the same. In other embodiments, QI and Q2 are different.

[0254] In some embodiments, the present disclosure provides a kit for detecting two or more target nucleic acid sequences in a sample comprising:

[0255] (a) two or more pairs of oligonucleotide primers with sequences that are complementary to each strand of the two or more target nucleic acid sequences;

[0256] (b) at least one oligonucleotide probe comprising two distinct portions:

[0257] (i) an annealing portion comprising a sequence at least partially complementary to one of the two or more target nucleic acid sequences and anneals within the one of the two or more target nucleic acid sequences, wherein the annealing portion comprises a first quencher moiety; and

[0258] (ii) a tag portion attached to the 5' terminus or to the 3' terminus of the annealing portion or attached via a linker between the 5' terminus and the 3' terminus of the annealing portion, and comprising a nucleotide sequence that is non-complementary to the one of the two or more target nucleic acid sequences, wherein the tag portion comprises a compound of Formula (I) and whose detectable signal is capable of being quenched by the first quencher moiety on the annealing portion, wherein the compound of Formula (I) is separated from the first quenching moiety by a nuclease susceptible cleavage site;

[0259] (c) at least one quenching oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe and hybridizes to the tag portion to form a duplex, wherein the quenching oligonucleotide comprises a second quencher moiety which quenches the detectable signal generated by the compound of Formula (I) on the tag portion when the quenching oligonucleotide is hybridized to the tag portion.

[0260] In some embodiments, the tag portion is attached to the 5' terminus of the annealing portion. In some embodiments, the tag portion is attached via a linker between the 5' terminus and the 3' terminus of the annealing portion. In some embodiments, the tag portion of the oligonucleotide probe or the quenching oligonucleotide or both the tag portion of the oligonucleotide probe and the quenching oligonucleotide contains one or more nucleotide modifications. In some embodiments, the one or more nucleotide modifications comprises a nucleotide modification selected from Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), Bridged Nucleic Acid (BNA), 2'-0 alkyl substitution, L-enantiomeric nucleotide, or combinations thereof. In some embodiments, the nucleotide modification comprises LNA. In some embodiments, the nucleotide modification comprises PNA. In some embodiments, the nucleotide modification comprises BNA. In some embodiments, the nucleotide modification comprises L-enantiomeric nucleotide. In some embodiments, the nucleotide modification comprises L-enantiomeric LNA (L-LNA). In some embodiments, the nucleotide modification comprises 2'-0 alkyl substitution. In some embodiments, the nucleotide modification comprises 2'-0 methyl substitution (2'-OMe).

[0261] The disclosure also provides a method for amplification and detection of a target nucleic acid in a sample comprising the steps of:

[0262] (a) contacting the sample containing the target nucleic acid in a single reaction vessel with

[0263] (i) one pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to opposite strands of a subsequence of the target nucleic acid;

[0264] (ii) an oligonucleotide probe that comprises an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence non-complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid that is bounded by the pair of oligonucleotide primers, wherein the probe further comprises an interactive dual label comprising a compound of Formula (I) located on the tag portion and a first quencher moiety located on the annealing portion and wherein the compound of Formula (I) is separated from the first quencher moiety by a nuclease susceptible cleavage site; and wherein prior to step (b), the tag portion is reversibly bound in a temperature-dependent manner to a quenching oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe and binds to the tag portion by hybridization, wherein the quenching oligonucleotide comprises at least a second quencher moiety capable of quenching the compound of Formula (I) on the tag portion when the quenching oligonucleotide is bound to the tag portion;

[0265] (b) following step (a), amplifying the target nucleic acid by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5' to 3' nuclease activity such that during an extension step of each PCR cycle, the nuclease activity of the polymerase allows cleavage and separation of the tag portion from the first quencher moiety on the annealing portion of the probe;

[0266] (c) measuring a suppressed signal from the compound of Formula (I) at a first temperature at which the quenching oligonucleotide is bound to the tag portion;

[0267] (d) increasing temperature to a second temperature at which the quenching oligonucleotide is not bound to the tag portion;

[0268] (e) measuring a temperature corrected signal from the compound of Formula (I) at the second temperature;

[0269] (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;

[0270] (g) repeating steps (b) through (f) through multiple PCR cycles;

[0271] (h) measuring the calculated signal values from the multiple PCR cycles to detect the presence of the target nucleic acid.

[0272] The disclosure also provides a method for amplification and detection of a target nucleic acid in a sample comprising the steps of

[0273] (a) contacting the sample containing the target nucleic acid in a single reaction vessel with

[0274] (i) one pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to opposite strands of a subsequence of the target nucleic acid;

[0275] (ii) an oligonucleotide probe that comprises an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence non-complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid that is bounded by the pair of oligonucleotide primers, wherein the probe further comprises an interactive dual label comprising a compound of Formula (I) located on the tag portion and a first quencher moiety located on the annealing portion and wherein the compound of Formula (I) is separated from the first quencher moiety by a nuclease susceptible cleavage site; and wherein prior to step (b), the tag portion is reversibly bound in a temperature-dependent manner to a quenching oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe and binds to the tag portion by hybridization, wherein the quenching oligonucleotide comprises at least a second quencher moiety capable of quenching the compound of Formula (I) on the tag portion when the quenching oligonucleotide is bound to the tag portion;

[0276] (b) following step (a), amplifying the target nucleic acid by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5' to 3' nuclease activity such that during an extension step of each PCR cycle, the nuclease activity of the polymerase allows cleavage and separation of the tag portion from the first quencher moiety on the annealing portion of the probe;

[0277] (c) measuring one or more signals from the compound of Formula (I) at a first temperature at which the quenching oligonucleotide is bound to the tag portion;

[0278] (d) measuring one or more signals from the compound of Formula (I) at a second temperature, which is higher than the first temperature, at which the quenching oligonucleotide is not bound to the tag portion;

[0279] (e) obtaining a calculated signal value by subtracting a median or average of the one or more signals detected at the first temperature from a median or average of the one or more signals detected at the second temperature; whereby a calculated signal value that is higher than a threshold signal value allows determination of the presence of the target nucleic acid.

[0280] In some embodiments, the PCR amplification of step (b) is allowed to reach an endpoint beyond the log phase of amplification. In some embodiments, the tag portion comprises a modification such that it is not capable of being extended by the nucleic acid polymerase. In some embodiments, the tag portion of the oligonucleotide probe or the quenching oligonucleotide or both the tag portion and the quenching oligonucleotide contain one or more nucleotide modifications. In some embodiments, the one or more nucleotide modifications is selected from the group consisting of Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), Bridged Nucleic Acid (BNA), 2'-0 alkyl substitution, L-enantiomeric nucleotide, and combinations thereof. Other methods of using TAGS probes are set forth within United States Patent Nos. 11,028,433, 11,034,997, and 11,345,958; and in United States Patent Publication No. 2021 / 0269857, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0281] Conjugates comprising a fluorescent compound and an oligonucleotide can be prepared, for example, by solid-phase DNA synthesis methods (see e.g. WO 2024 / 002924 for more details). The compound can be coupled to the 5’ end or the 3’ end of the oligonucleotide, for example.

[0282] PROBES

[0283] The present disclosure also provides (TaqMan®) probes, where a first dye of the probe is the fluorescent compound of Formula (I), and where a second dye is a quencher. Such probes may be used to conduct a TaqMan® assay, for example, as known in the art. As used herein, the terms "TaqMan® probe" and "hydrolysis probe" may be understood interchangeably. In some embodiments, the compound of Formula (I) and the quencher are located near the termini of the probe, and in some such embodiments, the compound having Formula (I) is located near the 5' terminus and the quencher is located near the 3' terminus. The term "3'-terminal" may be understood in the broadest sense as understood in the art. Further, the terms "3' terminus" and "3' end" may be understood interchangeably as known in the art. Also, it should be understood that the terms "5' terminus" and "5' end" as used herein may refer to the 5' end of the nucleotide strand but may not exclude that at the 3' end another molecular moiety (such as, e.g., a fluorophore, a quencher, a binding moiety or the like) is added to the 3' end of the probe.

[0284] The probe may hybridize to its target sequence. Further, a composition including a probe may further comprise a pair of primers, e.g., one forward and one reverse primer. These primers are generally unlabelled. Further, generally, the forward primer binds upstream, the reverse primer downstream of the band, such that the probe binds to a sequence that is a part of the strand that is amplified. A PCR reaction as well-known in the art is conducted. Thus, the target DNA is melted, then conditions are chosen that enable the annealing of the primers and the probe to the target DNA. Subsequently, conditions are chosen that enable the DNA polymerase to amplify the DNA strand between the primers. In the context of the TaqMan® assay, the DNA polymerase generally has a 5' to 3' exonuclease activity. Also, the DNA polymerase may be Taq polymerase or a functional variant thereof. When the DNA polymerase comes to the probe, the 5' end is cleaved off. Thereby, the compound of Formula (I), or quencher bound to the 5' terminal nucleotide(s) is also cleaved off. In some embodiments, the compound of Formula (I) is cleaved off. Consequently, the compound of Formula (I) and the quencher may diffuse in different directions. The spatial distance between both may be significantly increased and the fluorescence occurred by the compound of Formula (I) is significantly increased as it is not quenched by the dark quencher any longer. Also, the TaqMan® assay may be analyzed in real-time. The TaqMan® assay may also be conducted during a life-time PCR method. It may also be conducted quantitatively in a qPCR reaction.

[0285] A TaqMan® assay using the probes of the present disclosure may be used for the discrimination of alleles, genotyping, bacterial identification assays, DNA quantification, and the determination of the viral load in clinical specimen, gene expression assays and verification of microarray results. It may also be used for the discrimination of alleles, genotyping, and bacterial identification assays. Genotyping may be single nucleotide polymorphisms (SNP) genotyping, for example, and therefore include the determination of a genotype at defined a locus of interest in a sample, wherein the locus is a single nucleotide. Alternatively, genotyping may be copy number variant (CNV) genotyping. A copy number variant (CNV) is a segment of DNA in which differences of copynumber (number of copies of a DNA sequence or portions thereof) have been found by comparison of two or more genomes. As discussed above, sequences (and loci of various SNPs and CNVs) may be obtained from databases such as The Database of Genomic Variants (DGV), the NCBI dbSNP database, the UCSC Genome Bioinformatics Site, 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.

[0286] The disclosure provides a probe having the following Formula (VII):

[0287] [Dye 1] - [L]c- [5 ' - Oligonucleotide - 3'] - [L]c- [Dye 2] (VII), wherein one of Dye 1 or Dye 2 is the compound of Formula (I), coupled via R8to L, when present, or to the Oligonucleotide, when L is not present; and the other one of Dye 1 or Dye 2 is a quencher; the oligonucleotide is between about 5 and about 60 nucleotides in length; each L is independently a Linker L; and c is 0 or 1.

[0288] In some embodiments, the oligonucleotide comprises LNA, L-LNA, or PNA.

[0289] In some embodiments, one of Dye 1 or Dye 2 is a compound having the following structure:

[0290]

[0291] In some embodiments, one of Dye 1 or Dye 2 is a compound having the following structure:

[0292] In some embodiments, the Quencher is a molecule which decreases the fluorescence intensity of the fluorescent compound of Formula (I). In some embodiments, the Quencher is selected from Deep Dark Quencher DDQ-I, DABCYL, Eclipse® Dark quencher, Iowa Black® FQ, Iowa Black® RQ, Black Hole Quencher® series (BHQ-0, BHQ-1, BHQ-2, BHQ-3), QSY-7, DDQ-II, Iowa Black® RQ, QSY-21, Black Berry Quencher (BBQ-650, available from LGC Biosearch); IDT double quencher (ZEN Quencher; TAO Quencher); Onyx Quencher (available from Millipore Sigma), and TAMRA quencher. FRET PAIRS

[0293] The present disclosure also provides kits comprising a FRET pair. FRET is a form of molecular energy transfer (MET), a process by which energy is passed non-radioactively between a donor molecule and an acceptor molecule. FRET arises from the properties of certain chemical compounds; when excited by exposure to particular wavelengths of light, they emit light (i.e., they fluoresce) at a different wavelength. Such compounds are termed fluorophores or fluorescent labels. In FRET, energy is passed non-radioactively over a long distance (e.g., 10-100 Angstroms) between a donor molecule, which may be a fluorophore, and an acceptor molecule, which may be a quencher or another fluorophore. The donor absorbs a photon and transfers this energy non- radioactively to the acceptor (Forster, 1949, Z. Naturforsch. A4:321-327; Clegg, 1992, Methods Enzymol. 211 :353-388).

[0294] When two fluorophores whose excitation and emission spectra overlap are in close proximity, excitation of one fluorophore will cause it to emit light at wavelengths that are absorbed by, and that stimulate, the second fluorophore, causing it in turn to fluoresce. In other words, the excited- state energy of the first (donor) fluorophore is transferred by a resonance induced dipole-dipole interaction to the neighboring second (acceptor) fluorophore. As a result, the lifetime of the donor molecule is decreased 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 without subsequent emission of fluorescence by the acceptor. In this case, the acceptor functions as a quencher.

[0295] Pairs of molecules that can engage in FRET are termed FRET pairs. In order for energy transfer to occur, the donor and acceptor molecules must typically be in close proximity (e.g., up to 70 to 100 Angstroms) (Clegg, 1992, Methods Enzymol. 211 :353-388; Selvin, 1995, Methods Enzymol. 246:300-334). The efficiency of energy transfer falls off rapidly with increased distance between the donor and acceptor molecules. Effectively, this means that FRET can most efficiently occur up to distances of about 70 Angstroms.

[0296] In some embodiments of the present disclosure, a FRET pair comprises a first member including a dye of Formula (I) coupled directly or indirectly to a first oligonucleotide; and a second member including a second oligonucleotide coupled directly or indirectly to a quencher.

[0297] The disclosure provides a FRET pair comprising a first member having Formula (Villa) and a second member having Formula (Vlllb):

[0298] [Dye 1] - [L]c- [5' - Oligonucleotide 1 - 3'] (Villa),

[0299] [5' - Oligonucleotide 2 - 3'] - [L]c- [Dye 2] (Vlllb), wherein one of Dye 1 or Dye 2 is the compound of Formula (I), coupled via R8to L, when present, or to the Oligonucleotide, when L is not present; the other one of Dye 1 or Dye 2 is a Quencher; each L is independently a Linker L; c is O or l; and

[0300] Oligonucleotide 1 and Oligonucleotide 2 are different from each other.

[0301] In some embodiments, Oligonucleotides 1 and 2 are between about 5 and about 30 nucleotides in length. In some embodiments, Oligonucleotide 1 and / or 2 comprises LNA, L-LNA, or PNA.

[0302] In some embodiments, one of Dye 1 or Dye 2 is a compound having the following structure.

[0303] In some embodiments, one of Dye 1 or Dye 2 is a compound having the following structure:

[0304]

[0305] Any quencher may be used without limitation in the compositions described herein provided that it decreases the fluorescence intensity of the dye of Formula (I) that is being used. Quenchers commonly used for FRET include, but are not limited to, Deep Dark Quencher DDQ-I, DAB CYL, Eclipse® Dark quencher, Iowa Black® FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black® RQ, QSY-21, and Black Hole Quencher® BHQ-3. Quenchers for use in the compositions provided herein may be obtained commercially, for example, from Eurogentec (Belgium), Epoch Biosciences (Bothell, Wash.), Biosearch Technologies (Novato Calif.), Integrated DNA Technologies (Coralville, Iowa) and Life Technologies (Carlsbad, Calif.).

[0306] In some embodiments, Oligonucleotide 1 and / or 2 includes a nucleotide modification selected from Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), Bridged Nucleic Acid (BNA), 2'-0 alkyl substitution, L-enantiomeric nucleotide, or combinations thereof. In some embodiments, the nucleotide modification comprises LNA.

[0307] In some embodiments, the present disclosure provides a method of determining a genotype at a locus of interest in a sample comprising genetic material, the method comprising the steps of: contacting the genetic material with a first probe having Formula (Villa) and a second probe having Formula (Vlllb); and detecting the binding of one of the first and second probe 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 a 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 comprising a first discriminating position and the nucleotides of the second probe comprising a second discriminating position at a same nucleotide location in the second probe as the first discriminating position in the first probe, the first discriminating position comprising a different nucleobase than the second discriminating position, wherein the nucleobases at the other nucleotides of the first and second probes being the same. EXAMPLES

[0308] The following examples are given to illustrate embodiments of the present disclosure as it is presently preferred to practice. It will be understood that the examples are illustrative, and that the disclosure is not considered as restricted except as indicated in the appended claims.

[0309] Abbreviations: Ac = Acetyl; CPG = Controlled pore glass; dATP = 2’ -Deoxy adenosine 5’- triphosphate; dCTP = 2’ -Deoxy cytidine 5 ’-triphosphate; dGTP = 2 ’-Deoxy guanosine 5’- triphosphate; DMSO = Dimethyl sulfoxide; EDTA = Ethylenediaminetetraacetic acid; ETT = 5- Ethylthio-lH-tetrazole; dUTP = 2 ’-Deoxyuridine 5 ’-triphosphate; MEI = 2- Morpholinoethylisocyanide; qPCR = Quantitative polymerase chain reaction; Pac = Phenoxy acetyl; Pac2O = phenoxyacetic anhydride; RFI = Relative fluorescence increase; RT = Room temperature; TEAA = Triethylammonium acetate.

[0310] Example 1: Methyl 2-[(2£)-l-but-3-enyl-2-[(2£',4£)-5-[3-[2-(l,3-dioxolan-2-yl)ethyl]-l,l- dimethyl-benzo[e]indol-3-ium-2-yl]penta-2,4-dienylidene]-3,3-dimethyl-indolin-5-yl]acetate triflate l-(But-3-en-l-yl)-5-(2-methoxy-2-oxoethyl)-2,3,3-trimethyl-3H-indol-l-ium triflate (3.5 g, 8.0 mmol) and 3-anilinoacraldehyde anil hydrochloride (2.08 g, 8.0 mmol) were suspended in acetic anhydride (140 mL). The mixture was heated for 1 h at 75 °C. Then the solution was cooled to 0 °C, 3-(2-(l,3-dioxolan-2-yl)ethyl)-l,l-dimethyl-2-methylene-2,3-dihydro-lH-benzo[e]indole (2.49 g, 8.0 mmol) and NEts (5.59 mL, 8.0 mmol) were added. The solution was stirred for 10 min at 0 °C then 30 min at rt. The reaction mixture was concentrated under reduced pressure. The crude mixture was purified by reverse phase chromatography (Cl 8, eluents: A: H2O + 0.05% TFA, B: CH3CN + 0.05% TFA, gradient: 45-100% B) to afford methyl 2-[(2E)-l-but-3-enyl-2-[(2E,4E)-5- [3-[2-(l,3-dioxolan-2-yl)ethyl]-l,l-dimethyl-benzo[e]indol-3-ium-2-yl]penta-2,4-dienylidene]- 3,3-dimethyl-indolin-5-yl]acetate triflate (5.1 g, 81% yield) as a blue solid. HPLC-MS (ESI) calculated for C41H47N2O4+ [M]+ 631.3, experimental 631.3 (m / z).

[0311] XH NMR (400 MHz, CD3OD): 5 (ppm) 8.34 (br t, J= 13.1 Hz, 1H), 8.12-8.28 (m, 2H), 7.94-8.06 (m, 2H), 7.55-7.69 (m, 2H), 7.49 (t, J= 7.5 Hz, 1H), 7.40 (s, 1H), 7.31 (d, J= 8.2 Hz, 1H), 7.22 (d, J= 8.3 Hz, 1H), 6.63 (br t, J= 12.3 Hz, 1H), 6.36 (br d, J= 13.9 Hz, 1H), 6.27 (br d, J= 13.7 Hz, 1H), 5.89 (ddt, J= 17.0, 10.0, 7.1 Hz, 1H), 4.95-5.15 (m, 3H), 4.37 (br t, J= 7.0 Hz, 2H), 4.16 (br t, J= 6.9 Hz, 2H), 3.76-4.02 (m, 4H), 3.72 (s, 2H), 3.70 (s, 3H), 2.58 (q, J = 6.9 Hz, 2H), 2.18- 2.33 (m, 2H), 1.99 (s, 6H), 1.72 (s, 6H).

[0312] 19F NMR (376 MHz, CD3OD) 5 (ppm) -80.1.

[0313] 13C NMR (101 MHz, CD3OD): 5 (ppm) 176.5, 174.3, 174.0, 154.9, 154.8, 142.9, 141.0, 135.4,

[0314] 135.3, 133.6, 132.7, 131.7, 131.2, 130.9, 129.5, 128.9, 126.8, 126.7, 126.3, 124.7, 123.6, 123.5,

[0315] 120.4, 119.0, 112.3, 112.0, 104.7, 103.1, 66.2, 52.7, 52.7, 50.5, 44.1, 41.3, 40.4, 33.1, 32.0, 28.2, 27.5.

[0316] Example 2: 2-((l£',3£)-5-((£)-l-((£')-4-(l,3-Dioxolan-2-yl)but-3-en-l-yl)-5-(2-methoxy-2- oxoethyl)-3,3-dimethylindolin-2-ylidene)penta-l,3-dien-l-yl)-3-(2-(l,3-dioxolan-2-yl)ethyl)- l,l-dimethyl-lEZ-benzo[e]indol-3-ium triflate

[0317] Methyl 2-[(2E)-l-but-3-enyl-2-[(2E,4E)-5-[3-[2-(l,3-dioxolan-2-yl)ethyl]- 1,1 -dimethyl- benzo[e]indol-3-ium-2-yl]penta-2,4-dienylidene]-3,3-dimethyl-indolin-5-yl]acetate triflate (10.0 g, 12.2 mmol) was dissolved in dry EtOAc (150 mL), the solution was degassed with Ar for 10 min. Then 2-vinyl-l,3-dioxolane (49.0 mL, 0.49 mol) and Hovey da-Grubbs Catalyst M720 (767 mg, 1.22 mmol) were added. The mixture was stirred for 2 h at rt under argon. Then the solution was poured into 2 L of a hexane / Et2O (1 : 1) mixture. The blue precipitate obtained was separated from the green solution and was redissolved in EtOAc (400 mL). The precipitation procedure was repeated. The blue solid was collected by filtration and dried under vacuum to afford 2-((lE,3E)-5-((E)-l-((E)-4-(l,3-dioxolan-2-yl)but-3-en-l-yl)-5-(2-methoxy-2-oxoethyl)- 3 ,3 -dimethylindolin-2-ylidene)penta- 1 ,3 -dien- 1 -y l)-3 -(2-(l ,3 -dioxolan-2-yl)ethyl)- 1 , 1 -dimethyl- lH-benzo[e]indol-3-ium Inflate (10.5 g, 96% yield, HPLC purity 67%, corrected yield 64%, main impurity residual starting material) which was used in the next step without purification.

[0318] HPLC-MS (ESI) calculated for C44HSIN2O6+[M]+703.4, experimental 703.4 (m / z).

[0319] 'H NMR (400 MHz, CD3OD ): 5 (ppm) 8.27-8.39 (m, 1H), 8.14-8.27 (m, 2H), 8.01 (d, J= 8.9 Hz, 1H), 7.99 (br d, J= 8.5 Hz, 1H), 7.55-7.70 (m, 2H), 7.43-7.54 (m, 1H), 7.40 (d, J= 1.6 Hz, 1H), 7.26-7.35 (m, 1H), 7.17-7.26 (m, 1H), 6.52-6.71 (m, 1H), 6.36 (br d, J= 13.9 Hz, 1H), 6.26 (br d, J= 13.6 Hz, 1H), 5.89-6.01 (m, 1H), 5.41-5.51 (m, 1H), 5.09 (d, J= 5.8 Hz, 1H), 5.01 (t, J= 3.8 Hz, 1H), 4.38 (br t, J= 7.0 Hz, 2H), 4.13-4.22 (m, 2H), 3.82-3.97 (m, 4H), 3.74-3.82 (m, 4H), 3.72 (s, 2H), 3.70 (s, 3H), 2.50-2.67 (m, 2H), 2.25 (td, J= 6.9, 3.9 Hz, 2H), 1.99 (s, 6H), 1.70-1.75 (m, 6H).

[0320] 19F NMR (376 MHz, CD3OD) 5 (ppm) -80.1.

[0321] 13C NMR (101 MHz, CD3OD): 5 (ppm) 175.1, 172.8, 172.4, 153.4, 153.3, 141.4, 141.3, 139.5, 133.9, 133.7, 132.1, 131.2, 130.7, 130.5, 130.2, 129.7, 129.4, 128.0, 127.3, 125.3, 124.8, 123.1, 122.0, 110.8, 110.4, 103.2, 102.7, 101.6, 64.7, 64.4, 51.2, 51.1, 49.0, 42.4, 39.8, 38.8, 30.4, 29.7, 26.7, 25.9.

[0322] Example 3: (9aR,10aS,llaR)-17-(Carboxymethyl)-19,19,22,22-tetramethyl-8,9,9a,10a,ll, lla,12,13,19,22-decahydroindolo[2,l-a]naphtho[l",2":2',3']indolizino [8',7':5,6]pyrano[2,3- g]isoquinolin-7-ium trifluoroacetate (method 1)

[0323] 2-((lE,3E)-5-((E)-l-((E)-4-(l,3-Dioxolan-2-yl)but-3-en-l-yl)-5-(2-methoxy-2-oxoethyl)-3,3- dimethylindolin-2-ylidene)penta-l,3-dien-l-yl)-3-(2-(l,3-dioxolan-2-yl)ethyl)-l,l-dimethyl-lH- benzo[e]indol-3-ium triflate (10.0 g, 12.0 mmol) was dissolved in dry CH2CI2 under Ar. Then ZnC12 solution in 2-MeTHF (12.5 mL, 24 mmol; 1.9 M) was added. The reaction was stirred at 40 °C for 1 h. Then aq. H2SO4 (15.2 mL, 143 mmol, 50%) was added and the solution stirred at rt for 15 min. Dry MeOH (125 mL) was added and the solution heated at 60 °C for 1 h. The solution was concentrated to half volume under vacuum and poured into 2 L of water. The blue precipitate was separated from the solution and washed with water. The solid was dissolved in MeOH (200 mL) and LiOH (864 mg, 36 mmol) dissolved in 20 mL of water was added. The solution was stirred at rt for 2 h. The solution was neutralized until pH 7 with IM HC1 and concentrated under vacuum. The crude was purified by reverse phase chromatography (Cl 8, eluents: A: H2O + 0.05% TFA, B: CH3CN + 0.05% TFA, gradient: 20-80% B) to afford (2.7 g, 33% yield) as a blue solid. HPLC-MS (ESI) calculated for C39H39N2Ch+[M]+583.3, experimental 583.3 (m / z).

[0324] 'H NMR (400 MHz, CD3CN): 5 (ppm) 8.27 (d, J= 8.5 Hz, 1H), 7.94-8.10 (m, 2H), 7.87 (s, 1H), 7.70 (s, 1H), 7.65 (ddd, J= 8.5, 7.0, 1.3 Hz, 1H), 7.40-7.56 (m, 3H), 7.34 (dd, J= 8.2, 1.6 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 4.45-4.68 (m, 2H), 4.28-4.45 (m, 1H), 4.18-4.26 (m, 1H), 3.93 (td, J = 13.4, 4.3 Hz, 1H), 3.80 (td, J= 13.1, 4.1 Hz, 1H), 3.71 (s, 2H), 2.70-2.80 (m, 1H), 2.50-2.64 (m, 1H), 2.44 (dt, J = 11.6, 4.5 Hz, 1H), 2.26-2.38 (m, 1H), 2.01-2.06 (m, 1H), 2.00 (s, 3H), 1.99 (s, 3H), 1.75-1.82 (m, 1H), 1.73 (s, 3H), 1.73 (s, 3H), 1.41 (q, J= 11.8 Hz, 1H).

[0325] 19F NMR (376 MHz, CD3CN): 5 (ppm) -77.2.

[0326] 13C NMR (101 MHz, CD3CN): 5 (ppm) 173.1, 169.5, 168.2, 160.3, 143.3, 142.6, 142.1, 140.6, 140.3, 134.2, 133.0, 132.5, 131.2, 130.8, 130.6, 128.9, 128.5, 125.7, 124.3, 123.1, 115.6, 111.4, 111.1, 72.8, 70.9, 51.3, 49.9, 49.8, 44.0, 42.2, 40.8, 35.9, 31.7, 27.8, 27.7, 27.5, 27.2, 27.0.

[0327] Example 4: (9aR,10aS,llaR)-17-(Carboxymethyl)-19,19,22,22-tetramethyl-8,9,9a,10a,ll, lla,12,13,19,22-decahydroindolo[2,l-a]naphtho[l",2":2',3']indolizino [8',7':5,6]pyrano[2,3- g]isoquinolin-7-ium trifluoroacetate (method 2)

[0328] 2-((lE,3E)-5-((E)-l-((E)-4-(l,3-Dioxolan-2-yl)but-3-en-l-yl)-5-(2-methoxy-2-oxoethyl)-3,3- dimethylindolin-2-ylidene)penta-l,3-dien-l-yl)-3-(2-(l,3-dioxolan-2-yl)ethyl)-l,l-dimethyl-lH- benzo[e]indol-3-ium triflate (8.4 g, 10.3 mmol) was dissolved in dry CH3CN (180 mL) under Ar and then BF3 acetic acid complex (17.5 mL, 124 mmol) was added. The solution was stirred at rt for 24 h. The solution was quenched by addition of sat. aq. NaHCCh solution (100 mL) and extracted with EtOAc (3x 200 mL). The organic layers were combined, dried over Na2SO4, filtered and concentrated under vacuum. The residue was dissolved in MeOH (180 mL) and 1 M HC1 (20.6 mL, 20.6 mmol) was added. The solution was stirred at 60 °C for 6 h. After cooling to rt, LiOH (1.2 g, 51.5 mmol) dissolved in water (50 mL) was added. The solution was stirred at rt for 2 h. The solution was neutralized until pH 7 with 1 M HC1 and concentrated under vacuum. The crude was purified by reverse phase chromatography (Cl 8, eluents: A: H2O + 0.05% TFA, B: CH3CN + 0.05% TFA, gradient: 20-80% B) to afford (2.0 g, 27% yield) as a blue solid.

[0329] Analytical data was in agreement with example 3.

[0330] Example 5: ((9aR,10aS,llaR)-17-(Carboxymethyl)-19,19,22,22-tetramethyl-8,9,9a,10a,ll, lla,12,13,19,22-decahydroindolo[2,l-a]naphtho[l",2":2',3']indolizino [8',7':5,6]pyrano[2,3- g]isoquinolin-7-ium iodide)

[0331] A column was packed with resin for anion exchange (250 g, Dowex 1X2-100(0)). The resin was converted to the iodide form by washing it with aq. Nal (10%) solution (600 mL) and then conditioned with CH3CN / H2O 1 : 1. The sample (2.70 g, 3.9 mmol) was dissolved in CH3CN / H2O 1 :1 and applied dropwise to the top of the column. The compound was eluted with CH3CN / H2O 1 :1. The product containing fractions were collected and the solvent removed under vacuum to afford (2.66 g, 96% yield) as a blue solid.

[0332] HPLC-MS (ESI) calculated for C39H39N2O3+[M]+583.3, experimental 583.3 (m / z).

[0333] 'H NMR (400 MHz, CD3CN): 5 (ppm) 8.27 (d, J = 8.5 Hz, 1H), 8.03 (dd, J = 8.2, 2.4 Hz, 2H), 7.89 (s, 1H), 7.70 (s, 1H), 7.66 (ddd, J= 8.5, 7.0, 1.3 Hz, 1H), 7.46-7.56 (m, 2H), 7.43 (d, J= 1.3 Hz, 1H), 7.33 (dd, J= 8.2, 1.5 Hz, 1H), 7.18 (d, J= 8.2 Hz, 1H), 4.53-4.69 (m, 2H), 4.30-4.40 (m, 1H), 4.22 (dd, J= 13.9, 3.6 Hz, 1H), 3.94 (td, J = 13.5, 4.1 Hz, 1H), 3.80 (td, J= 13.1, 4.1 Hz, 1H), 3.71 (s, 2H), 2.70-2.81 (m, 1H), 2.56 (br dd, J= 16.7, 4.9 Hz, 1H), 2.45 (dt, J= 11.5, 4.6 Hz, 1H), 2.34 (dt, J = 13.3, 3.7 Hz, 1H), 2.01-2.08 (m, 1H), 2.00 (s, 3H), 2.00 (s, 3H), 1.75-1.81 (m, 1H), 1.74 (s, 6H), 1.41 (q, J= 11.8 Hz, 1H).

[0334] 13C NMR (101 MHz, CD3CN): 5 (ppm) 173.1, 169.6, 168.4, 143.5, 142.7, 142.3, 140.8, 140.5, 134.4, 133.0, 132.7, 131.4, 131.0, 130.7, 129.2, 129.0, 128.6, 125.8, 124.5, 123.3, 114.8, 112.2, 111.6, 111.3, 73.0, 71.0, 51.4, 50.0, 44.1, 42.4, 40.9, 36.1, 31.9, 28.0, 27.9, 27.7, 27.4, 27.4, 27.1. Example 6 : (9aR, 1 OaS, 11 aR)- 17-(2-((6-(((2R,3R)- l-(bis(4-Methoxy phenyl)(phenylmethoxy)- 3-hydroxybutan-2-yl)amino)-6-oxohexyl)amino) -2- oxoethyl) -19,19,22,22 - tetramethyl-8,9, 9a,10a,ll,lla,12,13,19,22-decahydroindolo [2,1-a] naphtha [1",2":2',3'] indolizino [8',7':5,6] pyrano [2,3-g] isoquinolin-7-ium iodide

[0335] (9aR, 1 OaS, 11 aR)- 17-(Carboxymethyl)- 19, 19,22,22-tetramethyl-,9,9a, 1 Oa, 11 , 11 a, 12, 13 , 19,22- decahydroindolo[2,l-a]naphtho[l",2":2',3']indolizino [8',7':5,6]pyrano[2,3-g]isoquinolin-7-ium iodide (2.5 g, 3.5 mmol) and N-hydroxysuccinimide (810 mg, 7.0 mmol) were dissolved in dry acetonitrile (75 mL) under Ar, then 2-morpholinoethylisocyanide (965 pL, 7.0 mmol) was added. The solution was stirred at rt for 2 h. Then DIPEA (1.8 mL, 10.5 mmol) and 6-amino-N-((2R,3R)- l-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxybutan-2-yl) hexanamide (2.19 g, 4.2 mmol) were added. The solution was stirred at rt for 1 h. The solution was concentrated to half volume and EtOAc was added. The precipitate obtained was collected by filtration and washed with EtOAc to afford (3.8 g, 89% yield) as a blue solid.

[0336] HPLC-MS (ESI) calculated for C?oH77N407+[M]+1085.6, experimental 1085.4 (m / z).

[0337] ‘H NMR (400 MHz, CDCh): 5 (ppm) 8.11 (d, J = 8.5 Hz, 1H), 7.90 (d, J= 8.7 Hz, 2H), 7.73 (s, 1H), 7.52-7.64 (m, 2H), 7.44 (t, J= 13 Hz, 1H), 7.31-7.41 (m, 4H), 7.20-7.31 (m, 6H), 7.14-7.18 (m, 1H), 7.01-7.06 (m, 2H), 6.79 (dd, J= 8.9, 1.3 Hz, 4H), 6.50 (br d, J= 8.7 Hz, 1H), 4.68 (br dd, = 11.5, 4.8 Hz, 1H), 4.56 (br dd, J= 11.1, 4.8 Hz, 1H), 4.25-4.37 (m, 1H), 4.10-4.19 (m, 1H), 3.96-4.07 (m, 1H), 3.89-3.95 (m, 1H), 3.78-3.87 (m, 1H), 3.77-3.75 (m, 1H), 3.74 (s, 6H), 3.67- 3.70 (m, 1H), 3.64 (s, 2H), 3.30-3.37 (m, 1H), 3.14-3.25 (m, 3H), 2.75-2.88 (m, 1H), 2.55-2.70 (m, 2H), 2.31-2.52 (m, 2H), 2.23 (t, = 7.47 Hz, 2H), 2.07-2.13 (m, 1H), 1.97-2.05 (m, 6H), 1.72- 1.78 (m, 6H), 1.39-1.69 (m, 6H), 1.29-1.39 (m, 2H), 1.12 (d, J= 6.4 Hz, 3H).

[0338] 13C NMR (101 MHz, CDCh): 5 (ppm) 173.6, 170.8, 168.8, 166.6, 158.4, 144.6, 142.5, 141.5, 140.3, 138.9, 135.8, 135.6, 134.3, 133.3, 131.7, 130.6, 130.0, 129.9, 129.8, 128.2, 128.1, 128.0, 127.8, 126.8, 124.9, 123.6, 122.1, 114.1, 113.1, 110.8, 110.3, 109.7, 86.2, 72.0, 70.0, 67.6, 64.4, 55.2, 54.2, 50.5, 49.4, 43.4, 43.2, 41.5, 39.1, 36.5, 34.9, 31.0, 28.6, 28.3, 27.9, 27.8, 27.6, 27.4, 26.9, 26.2, 25.1, 21.0, 20.1. Example 7: (9aR,10aS,llaR)-17-(2-((6-(((2R,3R)-l-(bis(4-methoxyphenyl)(phenyl)methoxy) -3-(((2-cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)butan-2-yl)amino) -6- oxohexyl) amino) -2-oxoethyl)-19,19,22,22-tetramethyl-8,9,9a,10a,ll,lla,12,13,19,22-decahydroindolo [2,l-a]naphtho[l",2":2',3']indolizino[8',7':5,6]pyrano[2,3-g]isoquinolin-7-ium iodide

[0339] (9aR,10aS,l laR) -17- (2-((6-(((2R,3R) -1- (bis(4-Methoxyphenyl)(phenyl)methoxy) -3- hydroxy- butan -2-yl)amino)-6-oxohexyl)amino)-2-oxoethyl)-19,19,22,22-tetramethyl-8,9,9a,10a,l 1,1 la, 12,13,19,22-decahydroindolo[2,l-a]naphtho[l",2":2',3']indolizino[8',7':5,6] pyrano [2,3-g] isoquinolin-7-ium iodide (1.3 g, 1.07 mmol) was dissolved in dry CH2CI2 (15 mL) under argon atmosphere. Then DIPEA (550 pL, 3.2 mmol) and 2-cyanoethyl N,N- diisopropylchlorophosphoramidite (228 pL, 1.07 mmol) were added. The solution was stirred at rt for 30 min. Then 10 mL of aq. NaHCCh (5%) solution was added. The organic phase was separated and dried over Na2SO4, filtered and concentrated. The residue was dissolved in a small amount of CH2CI2 and hexane was added. The supernatant was removed and the solid redissolved, procedure was repeated 4 times. The precipitate obtained was dried under vacuum, to afford (1.0 g, 70% yield) as a blue solid.

[0340] HPLC-MS (ESI) calculated for C79H94N6O8P [M]+1285.7, experimental 1285.5 (m / z).

[0341] 'H NMR (400 MHz, CDCI3): 5 (ppm) 8.14 (br d, J= 8.9 Hz, 1H), 7.92 (d, J= 8.5 Hz, 2H), 7.85 (s, 1H), 7.67 (s, 1H), 7.57-7.65 (m, 1H), 7.51-7.56 (m, 1H), 7.47 (t, J= 7.2 Hz, 1H), 7.33-7.42 (m, 3H), 7.22-7.33 (m, 6H), 7.16-7.22 (m, 1H), 7.05 (d, J= 8.2 Hz, 1H), 6.73-6.87 (m, 4H), 6.54-6.59 (m, 1H), 5.89 (br d, J= 92 Hz, 0.6H), 5.70 (br d, J= 9.2 Hz, 0.4H), 4.73 (dd, J= 11.3, 5.1 Hz, 1H), 4.57-4.62 (m, 1H), 4.26-4.38 (m, 1H), 4.13-4.25 (m, 1H), 4.03-4.10 (m, 1H), 3.85-3.98 (m, 1H), 3.79-3.81 (m, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.64-3.73 (m, 1H), 3.62 (s, 2H), 3.44-3.56 (m, 4H), 3.12-3.24 (m, 4H), 3.05-3.09 (m, 1H), 2.83-2.91 (m, 1H), 2.56-2.70 (m, 1H), 2.36-2.53 (m, 2H), 2.15-2.24 (m, 1H), 2.11-2.15 (m, 1H), 2.09 (s, 3H), 2.07 (s, 3H), 1.81 (dd, J = 6.5, 1.7 Hz, 6H), 1.65-1.77 (m, 1H), 1.46-1.57 (m, 4H), 1.20-1.37 (m, 6H), 1.09-1.20 (m, 12H), 0.97 (d, J = 6.8 Hz, 3H).

[0342] 31P NMR (162 MHz, CDCh): 5 (ppm) 148.0, 147.6. Example 8: Fluorescence spectra and temperature dependence of fluorescence emission

[0343] Fluorescence spectra and temperature dependence of fluorescence emission for Cy5.5 (GE Healthcare, Chicago, IL, U.S.A.) and Cy5.25B carboxylic acid (reflected in Formula Id, wherein R8= OH; cf. Example 5) were obtained with a Cary Eclipse fluorescence spectrophotometer with temperature controller (Agilent Technologies, Santa Clara, CA, U.S.A.). Stock solutions of cyanine dye carboxylic acids in DMSO were diluted with TEAA buffer (0.1 M, pH 7.0) to a DMSO concentration of 1.0%. TEAA buffer was prepared from diluting commercially available stock solution (Glen Research, Sterling, VA, U.S.A.) with ultrapure water to a final concentration of 100 mM. Ultrapure water was obtained from a Milli-Q® purification system (MilliporeSigma) with a resistivity of at least 18.2 MQ cm at 25 °C.

[0344] The fluorescence emission was acquired with excitation and emission slit widths of 2.5 nm and 5.0 nm, respectively. The temperature dependence of the dyes was recorded by exciting the dye at the excitation maximum and recording the fluorescence at the emission maximum from 25°C to 100°C at a heating rate of 3.0°C / min.

[0345] Results: The fluorescence excitation and emission spectra for Cy5.25B and Cy5.5 are shown in Fig. 1. Both cyanine dyes exhibit very similar spectral features and excitation and emission maxima that align very well with the filter sets of the Roche LightCycler® 480 and cobas® x800 systems. The fluorescence emission as a function of temperature for Cy5.25B and Cy5.5 are shown in Fig. 2, demonstrating a significantly more thermostable fluorescence for the rigidized dye Cy5.25B compared to Cy5.5.

[0346] Example 9: PCR amplification with dye labelled DNA probes

[0347] Oligonucleotides were synthesized at 1.0 pmol scale with a MerMade 12 oligonucleotide synthesizer (LGC Biosearch Technologies, Teddington, UK) using standard methods with reagents from Glen Research (Sterling, VA, U.S.A.). Oligonucleotide synthesis was performed with phosphoramidites that are compatible with “UltraMild” deprotection (Pac-dA, Ac-dC, and iPr-Pac-dG), ETT activator, Pac2O Cap A, and CPG that was preloaded with spacer C3 or BHQ- 2 (Black Hole Quencher®). The dye amidites were used in anhydrous acetonitrile (0.1 M) over molecular sieves (3 A). The coupling time for Cy5.5 and Cy5.25B phosphoramidites was 10 min.. Following synthesis the oligonucleotides were cleaved and deprotected by treatment of the CPG with K2CO3 / MeOH (50 mM) for 4-12 h. The CPG was filtered off and the oligonucleotide solution was diluted with TEAA buffer (2.0 M) for purification and desalting with standard methods. Aqueous solutions of purified DNA were dried with a rotary vacuum concentrator (SpeedVacTM, Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) or lyophilizer (Labconco freeze dryer with 4.5 L ice capacity and -105°C collector temperature; Labconco Corp., Kansas City, MO, U.S.A.), and redissolved in storage buffer containing tricine and EDTA.

[0348] All qPCR components were prepared with nuclease-free water. Reaction mixtures with a total volume of 50 pL were prepared by combining master mixture (20 pL), and dNTP mixture (10 pL). The master mixture contained tricine buffer (pH 8.2), manganese acetate, potassium acetate, glycerol, DMSO, detergent, target DNA (5000 copies / reaction), polymerase aptamer, DNA probe, forward and reverse primer DNA, and polymerase enzyme. The dNTP mixture contained dATP, dCTP, dGTP (2.0 mM each), and dUTP (4.0 mM). Each qPCR was prepared as triplicates in the wells of a 96-well plate. The plate was sealed and subjected to amplification cycles with a LightCycler® 480 System (F. Hoffmann-La Roche, Basel, Switzerland). At each PCR cycle the raw fluorescence was recorded at three temperatures (58, 80, and 91 °C), giving three PCR curves that were plotted individually. The fluorescence in RFI units was obtained by dividing the signal by background fluorescence, which is the fluorescence before DNA probe cleavage. For this purpose the average fluorescence signal during the first five PCR cycles was used.

[0349] Results: This experiment demonstrates the compatibility of the Cy5.25B dye as a reporter in TaqMan PCR and multiplexing PCR with TAGS® technology, see EP 3 512 960 for details. The growth curves demonstrate superior quenching of background fluorescence, higher thermostability of fluorescence, and brighter fluorescence signal in all thermal channels for Cy5.25B compared to Cy5.5 (Fig. 3).

[0350] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually indicated to be incorporated by reference for all purposes.

Claims

CLAIMS1. A fluorescent compound having a chemical structure according to Formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof:whereinA is an aryl;R1to R7independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;Y1and Y2independently are C(Rb)2, N(RC), S, O, or Se, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1, and each Rcindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;R8is -OH, -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety; and[X]’ is a counter anion, with the proviso that when any one of R1to R8has a negative charge, [X]’ is not present.

2. The compound of claim 1, wherein the compound has the structure of Formula (la):wherein A1to A4independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl.

3. The compound of claim 1 or 2, wherein the compound has the structure of Formula (lb):

4. The compound of any one of the preceding claims, wherein each of R1to R7are H, and / or each of Y1and Y2is C(CHs)2.

5. The compound of any one of the preceding claims, wherein R8is selected from:wherein Reis -OH or a conjugable moiety, and Rfis H or a protecting group.

6. A method for producing a fluorescent compound having the chemical structure according to Formula (Ila) or (lib), or a stereoisomer or pharmaceutically acceptable salt thereof:wherein R1to R7and R11independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a(hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl;Y1and Y2independently are C(Rb)2, N(RC), S, O, or Se, wherein each Rbindependently is unsubstituted alkyl, H, deuterium, -(OCH2CH2)xOH where x is an integer > 1, and each Rcindependently is H, deuterium, unsubstituted alkyl, or usubstituted heteroalkyl;R8is -OH, -ORd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety;R9and R10independently are H, sulfonate, -N(Ra)2, deuterium, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkyl sulfonate, unsubstituted aminoalkyl, a (hetero)aryl, or -C(O)ORa, where each Raindependently is H, deuterium, unsubstituted alkyl, or unsubstituted heteroalkyl, or represent attachment points of an aryl, such as aryl A, for example phenylene;[X]’ is a counter anion, with the proviso that when any one of R1to R11has a negative charge, [X]’ is not present; wherein the method comprises a first conversion:

7. The method of claim 6, wherein the first conversion comprises the following substeps (a) and (b):

8. The method of claim 7, wherein substep (a) is conducted in the presence of ZnCh and H2SO4, or in the presence of BF 3.

9. The method of any one of claims 6 to 8 further comprising a preceding conversion before the first conversion:

10. The method of any one of claims 6 to 9 for producing a fluorescent compound having the chemical structure according to any one of claims 1 to 5, or a stereoisomer or pharmaceutically acceptable salt thereof, wherein the method comprises as the first conversion:

11. The method of any one of claims 6 to 10 further comprising a second conversion after the first conversion:orwherein R8is -0Rd, -NH2 or -NHRd, wherein Rdis a conjugable moiety, or a linker L, wherein linker L optionally comprises a conjugable moiety.

12. The method of any one of claims 6 to 11 further comprising conjugating a specific binding entity to the compound, optionally followed by removal of protecting groups.

13. A conjugate comprising (i) a specific binding entity, and (ii) the compound of any one of claims 1 to 5, wherein the conjugate optionally has the following structure of Formula (III):wherein Z is O, NH, ORdor NHRd, wherein Rdis a linker L.

14. A kit comprising (i) a first conjugate comprising a first oligonucleotide coupled to the compound of any one of claims 1 to 5; and (ii) a second conjugate comprising a second oligonucleotide coupled to a quencher, wherein optionally Oligonucleotide 1 and / or 2 comprises LNA, L-LNA, or PNA.

15. A conjugate having Formula (V):(Oligonucleotide 1) - [linker L - (Dye)] - [(Oligonucleotide 2)(Q 1)] (V), whereinOligonucleotide 1 and 2 are different from each other and are independently between about 5 and about 30 nucleotides in length;Dye comprises the compound of any one of claims 1 to 5, coupled to Oligonucleotide via linker L; andQI is a quencher.

16. A kit comprising: (i) the conjugate of claim 15; and (ii) a conjugate having Formula (VI):[Oligonucleotide 3] - [Q2] (VI),whereinOligonucleotide 3 is between about 5 and about 30 nucleotides in length, wherein Oligonucleotide 3 optionally comprises LNA, L-LNA, or PNA; andQ2 is a quencher.

17. A probe having the following Formula (VII):[Dye 1] - [L]c- [5 ' - Oligonucleotide - 3'] - [L]c- [Dye 2] (VII), wherein one of Dye 1 or Dye 2 is the compound of any one of claims 1 to 5, coupled via R8to L, when present, or to the Oligonucleotide, when L is not present; and the other one of Dye 1 or Dye 2 is a quencher; the oligonucleotide is between about 5 and about 60 nucleotides in length; each L is independently a Linker L; and c is 0 or 1.

18. A method for amplification and detection of a target nucleic acid in a sample comprising the steps of:(a) contacting the sample containing the target nucleic acid in a single reaction vessel with(i) one pair of oligonucleotide primers, each oligonucleotide primer capable of hybridizing to opposite strands of a subsequence of the target nucleic acid;(ii) an oligonucleotide probe that comprises an annealing portion and a tag portion, wherein the tag portion comprises a nucleotide sequence non-complementary to the target nucleic acid sequence, wherein the annealing portion comprises a nucleotide sequence at least partially complementary to the target nucleic acid sequence and hybridizes to a region of the subsequence of the target nucleic acid that is bounded by the pair of oligonucleotide primers, wherein the probe further comprises an interactive dual label comprising a fluorescent compound of any one of claims 1 to 5, located on the tag portion and a first quencher moiety located on the annealing portion and wherein the fluorescent compound is separated from the first quencher moiety by a nuclease susceptible cleavage site; and wherein prior to step (b), the tag portion is reversibly bound in a temperature-dependent manner to a quenching oligonucleotide comprising a nucleotide sequence at least partially complementary to the tag portion of the oligonucleotide probe and binds to the tag portion by hybridization, wherein the quenching oligonucleotide comprises at least a secondquencher moiety capable of quenching the fluorescent compound on the tag portion when the quenching oligonucleotide is bound to the tag portion;(b) following step (a), amplifying the target nucleic acid by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5' to 3' nuclease activity such that during an extension step of each PCR cycle, the nuclease activity of the polymerase allows cleavage and separation of the tag portion from the first quencher moiety on the annealing portion of the probe;(c) measuring one or more signals from the fluorescent compound at a first temperature at which the quenching oligonucleotide is bound to the tag portion;(d) measuring one or more signals from the fluorescent compound at a second temperature, which is higher than the first temperature, at which the quenching oligonucleotide is not bound to the tag portion;(e) obtaining a calculated signal value by subtracting a median or average of the one or more signals detected at the first temperature from a median or average of the one or more signals detected at the second temperature; whereby a calculated signal value that is higher than a threshold signal value allows determination of the presence of the target nucleic acid.

19. A FRET pair comprising a first member having Formula (Villa) and a second member having Formula (Vlllb):[Dye 1] - [L]c- [51- Oligonucleotide 1 - 3'] (Villa),[5' - Oligonucleotide 2 - 3'] - [L]c- [Dye 2] (Vlllb), wherein one of Dye 1 or Dye 2 comprises the compound of any one of claims 1 to 5, coupled via R8to L, when present, or to the Oligonucleotide, when L is not present; the other one of Dye 1 or Dye 2 is a Quencher; each L is independently a Linker L; c is 0 or 1; andOligonucleotide 1 and Oligonucleotide 2 are different from each other, optionally are between about 5 und about 30 nucleotides in length, and / or optionally comprise LNA, L- LNA, or PNA.

Images