Dicationic fluorescent dyes

Abstract

The present invention provides water-soluble monocationic fluorescent dyes and water-soluble dicationic fluorescent dyes that exhibit stable fluorescence at high temperatures. The present invention also provides methods for producing the fluorescent dyes and methods for using these dyes in biological assays such as multiplex qPCR and tissue staining.

Description

[Technical Field]

[0001] The present invention relates generally to fluorescent compounds. In particular, the present invention relates to rhodamine-based fluorescent dyes and methods for synthesizing and using these dyes. [Background technology]

[0002] Fluorescent dyes with tailored properties are important for virtually any analytical method that relies on the detection of fluorescence. In the life sciences, applications of fluorescent dyes range from real-time polymerase chain reaction (PCR) assays to fluorescence imaging techniques (including, but not limited to, fluorescence microscopy, single-molecule imaging, super-resolution microscopy, and labeling and tracking experiments in cells).

[0003] The latest advances in analytical PCR are directed toward multiplexing, i.e., the simultaneous detection of multiple targets in an analytical sample. TAGS (Temperature-Assisted Signal Generation, disclosed in U.S. Patent Application Publication No. 2018 / 0073064, incorporated herein by reference in its entirety) PCR is a multiplexing technique that enables the measurement of multiple individual targets in each fluorescent channel by collecting fluorescent data at various temperatures during thermal cycling. Consequently, TAGS multiplexing using two or three temperature channels can double or triple the number of resolvable targets per optical channel. In principle, this technology can be deployed in any quantitative PCR (qPCR) instrument capable of collecting more than one fluorescent read per PCR cycle.

[0004] For any application relying on fluorescence detection, PCR using TAGS technology requires bright dyes. The brightness of a fluorophore is defined as the product of its extinction coefficient and quantum yield. The extinction coefficient determines the efficiency of a fluorophore in absorbing excitation light, while quantum yield is a measure of the number of photons emitted by a fluorophore per excitation event. A common problem is that the overall brightness of fluorophores tends to decrease significantly as one moves toward the red end of the electromagnetic spectrum. As a result, fluorophores in the near-infrared (NIR) spectral range (excitation and emission between 650 and 900 nm) exhibit reduced brightness, thereby reducing the overall sensitivity of fluorescence-based applications.

[0005] In reviewing PCR using TAGS technology, another requirement for fluorescent dyes is temperature-stable fluorescence emission. In particular, cyanine dyes such as Cy5, Cy5.5, or Cy7 exhibit a significant decrease in brightness at high temperatures, which is an inherent feature of their polymethine backbone with its conformationally flexible chain of conjugated double bonds.

[0006] Another requirement is to minimize background fluorescence from quenched probes, i.e., before polymerase cleavage and quenching. Background fluorescence is proportional to the number of PCR probes, resulting in an overall reduction in measurable signal gain per target. Thus, the accumulation of residual fluorescence depends on the efficiency with which each fluorophore is quenched, which is a function not only of molecular distance but also of the nature of the fluorophore-quencher pair.

[0007] The fluorescent dyes discussed in this disclosure address these key issues by introducing two structural changes to the basic general rhodamine structure: oxygen was substituted with silicon or germanium, while the aryl group was replaced with an imidazole or imidazolium heterocycle. The combined effect of both modifications significantly red-shifted rhodamine, which is smaller in size than typical fluorophores in this wavelength range.

[0008] Water solubility of fluorescent dyes is a prerequisite for any application, as water is the primary solvent and is often achieved by decorating the chromophore with negatively charged functional groups such as carboxylates or sulfates. The introduction of a second positive charge via an imidazolium moiety and miniaturization results in chromophores with very high water solubility. Dye size, charge, and solubility are also important parameters in the context of fluorescence microscopy, significantly affecting tissue, cell, or organelle penetration. For example, hydrophobicity can also lead to misleading fluorescent signals due to aggregation or strong interactions between the target and lipid bilayers. The mono- and dicationic dyes in this disclosure expand the number of available fluorophore scaffolds in the far-red to NIR spectral range. Their fluorescent thermal stability, improved background fluorescence, and high water solubility make these dyes useful for multiplex PCR and fluorescence microscopy. Summary of the Invention

[0009] In particular, the present invention relates to novel rhodamine-based fluorescent dyes and methods for synthesizing and using these dyes.

[0010] In one aspect, the present invention provides a compound of formula A: [ka] (In the formula, X is Si, Ge, or C; Y is Me, Et, iPr, or phenyl; Z is [ka] Any one of the following; W is [ka] Any one of the following; R1 and R2 are [ka] Any combination of; L is H, SO3H, CONH2, CO2H, N3, or any moiety that undergoes click chemistry functionality. The present invention provides a fluorescent dye.

[0011] In another aspect, the present invention provides a compound of formula B: [ka] (In the formula, X is Si, Ge, or C; Y is Me, Et, iPr, or Ph; Z is [ka] Any one of the following; R1 and R2 are [ka] Any combination of; L is H, SO3H, CONH2, CO2H, N3, or any moiety that undergoes click chemistry functionality; The present invention provides a fluorescent dye.

[0012] In one embodiment, Z is [ka] It is one of the following.

[0013] In another embodiment, Z is [ka] is.

[0014] In another aspect, the present invention provides a compound having the following structure: [ka] The present invention provides a fluorescent dye selected from the group consisting of:

[0015] In another aspect, the present invention provides a method for preparing the fluorescent dye of the present invention. Herein, the method for preparing the fluorescent dye may include the steps described in Examples 4, 5, 6, 7, or 8. In yet another aspect, the present invention provides a use of the fluorescent dye of the present invention. In one embodiment, the fluorescent dye of the present invention is used for detecting one or more target nucleic acids in a sample by PCR assay, particularly a multiplex PCR assay. In another embodiment, the detection of target nucleic acids in a PCR assay using the fluorescent dye of the present invention can be performed at high temperatures (e.g., 65°C or higher). In one embodiment, the fluorescent dye of the present invention is used for detecting one or more target proteins in a tissue sample by immunohistochemical assay. In another embodiment, the detection of target proteins in an immunohistochemical assay uses a primary or secondary antibody labeled with a fluorescent dye of the present invention. [Brief explanation of the drawings]

[0016] [Figure 1] The upper panel shows the fluorescence spectra for two representative fluorescent dyes, No. 29 and No. 30, and a commercially available reference dye. The lower panel shows the temperature dependence of fluorescence for the same series of dyes. These results demonstrate that fluorescent dyes with azetidine modifications have improved thermal stability of fluorescence. [Figure 2A] UPLC chromatogram after labeling of a DNA probe with a fluorescent dye using strain-promoted azide-alkyne cycloaddition (click chemistry). The reaction between the DBCO and azide moieties produces triazole regioisomers, which are separated as double peaks in the chromatogram. [Figure 2B] FIG. 2B shows the absorption spectrum of the DNA-dye conjugate extracted from the UPLC chromatogram peak shown in FIG. 2A. [Figure 3]Amplification curves for qPCR amplification using DNA probes labeled with Dye No. 36 compared to a commercially available reference dye are shown. Raw fluorescent signal divided by total gain (RFI). Results: Dye No. 36 is compatible with qPCR and demonstrates improved quenching with BHQ-2 compared to commercially available dyes in qPCR assays. [Figure 4] Light microscope images of immunohistochemically stained tonsillar tissue sections are shown at 10x, 20x, and 40x magnification (left to right). Formalin-fixed, paraffin-embedded tissue sections were treated with CONFIRM™ anti-Ki-67 (30-9) rabbit monoclonal primary antibody (IgG), which is directed against the C-terminal portion of the Ki-67 antigen, a biomarker abundant on tonsillar tissue. Ki-67 is a nuclear protein expressed in proliferating cells. Ki-67 staining can be used to aid in the evaluation of proliferative activity in normal and neoplastic tissues. Dye Azide No. 36 (top row) and reference dye CF680R (bottom row) were used for staining at a concentration of 200 μM. Results: The correct cellular structures were stained without obvious background, demonstrating the compatibility of dye No. 36 with immunohistochemical tissue staining. [Figure 5] 1 shows a general reaction scheme for synthesizing fluorescent dyes according to the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0017] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Essentially any method and material similar to those described herein can be used in the practice or testing of this invention, but only exemplary methods and materials are described. For the purposes of this invention, the following terms are defined as follows:

[0018] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0019] As used herein, unless otherwise specified, the term "alkyl group" refers to a saturated, monovalent, unbranched or branched hydrocarbon chain. Examples of alkyl groups include, but are not limited to, C1-C6 alkyl groups such as methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, and hexyl, as well as longer alkyl groups such as heptyl and octyl. An alkyl group can be unsubstituted or substituted with one or two suitable substituents.

[0020] As used herein, unless otherwise specified, the chemical symbols "Si" and "Ge" refer to silicon and germanium, respectively. Also, as used herein, "Me" refers to methyl, "Et" refers to ethyl, "iPr" refers to isopropyl, and "Ph" refers to phenyl.

[0021] As used herein, unless otherwise stated, the term -O-alkyl (or alkyl-O-) means an "alkoxy group," where alkyl is as defined above. An alkoxy group can be unsubstituted or substituted with one or more suitable substituents. The alkyl chain of an alkoxy group can be, for example, 1 to 6 carbon atoms in length.

[0022] As used herein, unless otherwise specified, the term "metal" refers to Li + , Na + , Ca 2+ , or Mg 2+ "Iron" refers to a Group I or II metal, including but not limited to:

[0023] As used herein, unless otherwise specified, the terms "linking group" and "linker" are used interchangeably and refer to the portion of a detectable label that can covalently link a base to a label, for example, forming a "bond" that links a nucleoside, nucleotide, or nucleic acid to the label. Examples of linkers include, but are not limited to, O, S, or NH. Optionally, the linking group or linker is a covalent bond (i.e., the label is covalently bonded to the base). As used herein, unless otherwise specified, the term "counterion" refers to a stable, synthetically available ion.

[0024] Examples of counterions include, but are not limited to, chloride, bromide, iodide, sulfate, benzenesulfonate, p-toluenesulfonate, p-bromobenzenesulfonate, methanesulfonate, trifluoromethanesulfonate, phosphate, perchlorate, tetrafluoroborate, hexafluorophosphate, tetraphenylborate, nitrate, and anions of aromatic or aliphatic carboxylic acids.

[0025] The terms "click chemistry," "click reaction," and "click chemistry reaction" are used interchangeably and refer to the [3 + 2] cycloaddition between an alkyne and an azide, a reaction that enabled remarkable selectivity in conjugation reactions of biological samples, as first described by Rostovtsev et al., Angew. Chem. Int. Ed., 2002, 41, 2596-2599, incorporated herein by reference in its entirety. While early click chemistry reactions utilized copper as a catalyst, copper-free click chemistry reactions are preferred in nucleic acid research, and the use of dibenzocyclooctyne (DBCO) derivatives is described in WO 2009 / 067663, incorporated herein by reference in its entirety. Reagents such as DBCO-deoxythymidine (DBCO-dT) and DBCO-serinol are available as phosphoramidites for oligonucleotide synthesis from Glen Research (Sterling, VA).

[0026] Due to their spectral properties (absorption maxima in the region above about 650 nm and emission maxima above 670 nm), the compounds according to the present invention provide molecules that are highly suitable as dyes, particularly as fluorescent dyes. An absorption maxima at about 700 nm is particularly preferred. At the same time, the spectral properties of the molecule can be varied by varying the identity, number, and position of the residues Z, W, R1, and R2. In this way, fluorescent dyes with most absorption and emission maxima above 650 nm can be produced. Therefore, the subject of the present invention is also the use of the rhodamine derivatives according to the present invention as fluorescent dyes or laser dyes.

[0027] In the compounds of the present invention of general formula A or B, at least one of the residues R1 or R2 is preferably present in the form of an activating group suitable for coupling or a group that can be activated for coupling. Such an activating group is derived, in particular, from a carboxylic acid group or a sulfonic acid group that can be activated, and can be, for example, an acid ester, an acid anhydride, an acid halide, preferably a bromide, in particular a chloride, or an N-hydroxysuccinimide ester, or an ω-alkyl halide. Furthermore, such an activating group can also be, for example, a phosphoramidite.

[0028] To prepare conjugates containing the rhodamine derivatives according to the present invention, activated derivatives suitable for labeling biomolecules or other analytical reagents can be synthesized. The preparation of activated derivatives requires at least one activatable group, where activation is carried out according to standard protocols known to those skilled in the art. Depending on the subsequent application, various reactive groups can be introduced. Phosphoramidites and H-phosphonates can be derived, for example, from hydroxyl groups. Thus, the preparation of rhodamine phosphoramidites and H-phosphonates, respectively, is generally carried out according to previously known protocols (Methods in Mol. Biol. Vol. 20, "Protocols for Oligonucleotides and Analogs, Synthesis and Properties", S. Agrawal Hrsg., Humana Press, Totowa, NJ).

[0029] However, N-hydroxysuccinimide (NHS) esters are generally derived from carboxyl groups, maleimides, from amino groups (PY Reddy, Synthesis (1998) 999), or by elongation of activated carboxylic acids with the corresponding ω-aminoalkylmaleimides.

[0030] The preparation of NHS-esters is preferably carried out by This is carried out according to the method disclosed in EP 0 543 333, in which a free carboxylic acid is mixed with NHS in the presence of a condensation reagent, such as DCC or MEI. The preparation of rhodamine isothiocyanate is preferably carried out by reacting an amino group with thiophosgene (Advanced Organic Chemistry, McGraw Hill, 2nd ed., p. 383, 1997).

[0031] The subject of the present invention is therefore also activated derivatives of the rhodamines according to the invention, the reactive groups of which are preferably phosphoramidites, N-hydroxysuccinimide (NHS) esters, maleimidoalkylamides, H-phosphonates or isothiocyanates or ω-alkyl halides.

[0032] Another subject of the present invention is a conjugate that can be obtained by combining the rhodamine compound according to the present invention or its activated derivative.Therefore, such a conjugate is composed of at least two components, one component being the rhodamine derivative according to the present invention.Starting from the activated derivative, the production of the conjugate is carried out according to standard protocols.Appropriate conjugation methods to be applied are known to those skilled in the art.

[0033] The conjugates according to the invention can be used for analytical purposes as soon as the second component of the conjugate is capable of binding to the binding partner to be analyzed and the assembled complex can be identified by detection of the fluorescence emitted by the assembled complex after excitation with light of an appropriate absorption wavelength.

[0034] Due to absorption in near infrared, the compounds according to the present invention are also suitable for in vivo use.For this purpose, the water-soluble derivatives of the dyes according to the present invention and their conjugates are applied together with biomolecules.In vivo measurement is carried out by measuring fluorescence or absorption.

[0035] The use of the conjugate according to the present invention is particularly suitable for diagnostic analysis or the analysis of medical or biological substances.Therefore, the subject of the present invention is particularly conjugates that can interact with biomolecules.These are generally conjugates that also contain one or several biomolecules as additional components.

[0036] These biomolecules contained in conjugates can be, for example, single-stranded or double-stranded nucleic acids such as DNA, RNA or triplex structures, or nucleic acid analogs such as PNA, oligonucleotides and oligonucleotide derivatives, as well as single nucleotides, nucleotide derivatives, nucleotide analogs or nucleoside triphosphates.The labeling of such molecules is preferably carried out at 5'-position by NHS ester or phosphoramidite, but is preferably carried out at 3'-position via dye-substituted carrier materials such as CPG.The labeling of other positions, such as nucleic acid bases, is also preferably carried out by NHS ester.

[0037] For labeling of proteins, protein complexes, antibodies or amino acids, conjugation is preferably carried out by NHS ester, m-maleimide or isothiocyanate or ω-alkylhalogenide. Examples of further conjugate components are vitamins, steroid hormones, lipid molecules, and haptens. In addition, more complex biological structures such as membrane fractions or whole cells can also be labeled.

[0038] A particular embodiment of the conjugate according to the present invention is an oligonucleotide conjugated with a rhodamine derivative according to the present invention. Such labeled oligonucleotides can be used for previously known methods of detecting and analyzing nucleic acids, for example, by in situ hybridization (Meyne and Myzis, Methods Mol. Biol. 33, 63-74, 1994), or as primers in various sequencing methods (Sheealy et al., Anal. Chem. 67, 247-251, 1995).

[0039] In addition, the rhodamine dye-labeled nucleic acid ribonucleoside triphosphates and deoxyribonucleoside triphosphates of the present invention can be incorporated into nucleic acids as substrates for polymerases by various enzymatic reactions. In the case of DNA, this can be achieved, for example, by applying the nick translation method (Rigby et al., J. Mol. Biol. 113, p. 237, 1977) or by "random prime labeling" (Feinberg and Vogelstein, Anal. Biochem. 137, p. 266, 1984) using DNA polymerase. In the case of RNA, this can be achieved, for example, by transcription using T3, T7, or SP6 RNA polymerase. Another method of nucleic acid labeling is possible by the so-called 3'-tailing reaction using terminal transferase.

[0040] Further subjects of the present invention are therefore also the use of the conjugates according to the invention for labeling nucleic acids by chemical or enzymatic methods, as well as the use of hybridization probes labeled according to the invention for the detection and analysis of nucleic acids.

[0041] For analytical assays, the rhodamine derivatives according to the present invention are first excited with light of a suitable wavelength, such as a laser, a laser diode, or an LED. Depending on the analyte, fluorescence detection is carried out by a measurement method known to those skilled in the art. These include, for example, fluorescence microscopy for in situ methods or the detection of emitted radiation by a suitable photodiode.

[0042] Therefore, a further subject of the present invention is the use of dye conjugates for immunohistochemical staining of paraffin tissue sections. Immunohistochemistry allows the visualization of antigens through the sequential application, with washing steps in between, of a specific antibody that binds to the antigen (primary antibody), a secondary antibody that binds to the primary antibody (linked antibody), an enzyme complex, and a chromogenic substrate. Enzymatic activation of the chromogen results in a visible reaction product at the antigen site.

[0043] In addition to direct excitation of the dyes according to the present invention using radiant energy of an appropriate wavelength, excitation can also be achieved by so-called fluorescence resonance energy transfer (also known as Förster resonance energy transfer). This involves exciting a second fluorescent dye with light of an appropriate wavelength. Due to the local proximity of the two dyes, non-radiative energy transfer to the rhodamine derivative according to the present invention follows (Van der Meer et al., Resonance Energy Transfer, VCH, 1994). Detection of light emitted by this molecule at a specific wavelength can be preferably used for quantitation of the analyte. Therefore, the subject of the present invention is also the use of the rhodamine derivatives according to the present invention or corresponding conjugates as components of a fluorescence resonance energy transfer system.

[0044] The compounds according to the present invention are used as resonance energy donors or acceptors.The preferred resonance energy donors of all compounds according to the present invention are fluorescent dye conjugates suitable for spectral analysis.Therefore, the subject of the present invention is also the use of the rhodamine derivatives according to the present invention or corresponding conjugates together with suitable fluorescent resonance donor conjugates as fluorescent resonance energy acceptors in fluorescent resonance energy transfer systems.

[0045] For quantitative detection of nucleic acid, hybridization probes such as oligonucleotides are suitable that are labeled with fluorescent dyes, which can be detected by the principle of fluorescence resonance electron transfer (FRET).For oligonucleotides, the 5' terminal position can be labeled with one dye component of FRET system, and the 3' terminal position can be labeled with the remaining dye component of FRET system.In this case, oligonucleotides can also be labeled within the sequence.

[0046] Such oligonucleotides labeled with two dyes are used during nucleic acid amplification to detect the resulting product, and the emission of the fluorescence resonance energy acceptor is detected. When the oligonucleotide is not bound to the target, the fluorescence of the donor cannot be measured due to non-radiative energy transfer. However, when the oligonucleotide binds to the target DNA, the two dye components are locally separated due to the exonuclease activity of the DNA polymerase used, so that some fluorescence of the FRET donor becomes measurable (U.S. Patent No. 5,210,015).

[0047] In another preferred embodiment, different dyes are located in two different hybridization probes that can hybridize in close proximity to the target nucleic acid. These probes can be, for example, two oligonucleotide probes that hybridize to the same strand of the target nucleic acid, with one dye at the 3'-terminal nucleotide of the first probe and the other at the 5'-terminal nucleotide of the second probe, so that the distance between the two is only a few nucleotides, i.e., between 0 and 30. When using fluorescein in combination with the rhodamine derivatives of the present invention, a distance of 0 to 15 nucleotides, particularly 1 to 5 nucleotides, and often only one nucleotide, has proven advantageous. Provided the nucleotide distance between the dye moieties is maintained, the probes can be used internally rather than conjugated with dyes at their ends. In the case of double-stranded target nucleic acid probes that bind to different strands of the target, a specific distance of 0 to 30 nucleotides between the two dye moieties can be used.

[0048] Therefore, a further subject of the present invention is the use of a conjugate according to the present invention, consisting of an oligonucleotide and a rhodamine derivative according to the present invention, for the analysis of nucleic acids, wherein a further conjugate is used, consisting of a second oligonucleotide and a further suitable fluorescent dye, preferably capable of fluorescence resonance energy transfer after excitation of the dye coupled to the second oligonucleotide. The combination of oligonucleotides is hereinafter referred to as a "FRET pair" and is indicated as such.

[0049] The use of such FRET pairs has proven to be particularly advantageous for detecting amplification products during or after polymerase chain reaction.Therefore, a further subject of the present invention is the use of the conjugate according to the present invention as a component of a FRET pair for detecting reaction products of nucleic acid amplification reactions.In a special test procedure, one of the two amplification primers can be simultaneously labeled with one of the two dyes used, thus providing one of the two components of FRET.

[0050] The use of an appropriate FRET pair for detecting amplification products allows so-called "real-time monitoring" of PCR reactions, and the data required for the generation of amplification products is determined according to the number of primed reaction cycles. This is generally achieved by the fact that the reaction conditions and temperature conditions during the annealing required for the amplification primers also allow the oligonucleotides of the FRET pair to hybridize to the target nucleic acid and emit a measurable fluorescent signal after appropriate excitation. The obtained data can be used to determine the amount of the target nucleic acid initially applied by quantitative analysis. Therefore, these embodiments are particularly important for quantitative RT-PCR experiments to quantify the RNA concentration of biological samples. Therefore, the present invention also relates to the use of the conjugates of the present invention as components of a FRET pair for detecting the reaction products of nucleic acid amplification reactions, where the reaction products are detected at each cycle. Furthermore, the present invention also relates to the use of the conjugates of the present invention as components of a FRET pair for quantifying the nucleic acid to be amplified.

[0051] In another embodiment, the detection of the amplification product is carried out after the completion of the amplification reaction, by continuously increasing the temperature during the fusion curve analysis after the hybridization of the FRET pair to the target nucleic acid to be detected. At the same time, the emitted fluorescence is measured according to the temperature. This allows the detection of sequences that hybridize with low stringency to the FRET pair used due to specific mismatches. The melting temperature measured in this way can be used to detect point mutations or other polymorphisms. Therefore, the subject of the present invention is also the use of the conjugate according to the present invention as a component of a FRET pair for determining the fusion curve, especially during the identification of polymorphisms and point mutations.

[0052] Such fluorescence resonance energy transfer processes can generally be used to detect molecule-molecule interactions, such as protein-protein interactions or antigen-antibody reactions. Particular advantages are achieved if the reaction is carried out under homogeneous conditions.

[0053] As counter ions, any cation suitable for charge neutralization and compatible with the anionic basic structure depending on the pH can be used.

[0054] The starting compound is preferably selected so that the synthesis product contains a group suitable for activation. Such a group is activated according to known methods to obtain a group suitable for subsequent coupling with a reactive group of a biologically active molecule to construct a biomolecule-dye conjugate. A linker can be inserted between the activated group and the biologically active molecule.

[0055] Linkers are used to change the distance between the dyes of the present invention and biomolecules. For example, aminocaproic acid can be inserted into the carboxylic acid for elongation. This can also involve changing the functionality, for example, by reacting the carboxylic acid with maleimidoalkylamine. Furthermore, the linker charge can affect the solubility of the dye. Amino acids with charge carriers, such as lysine or glutamic acid, are particularly suitable for this effect.

[0056] The compounds according to the invention provide novel compounds which, due to their spectroscopic properties (absorption maximum above 650 nm), are highly suitable as absorbing dyes suitable for coupling, in particular as fluorescent dyes for use with haptens, antibodies, protein conjugates, and for polynucleotide marking and latex dyeing (fluorescent latex).

[0057] The rhodamine derivative dyes according to the present invention are particularly suitable for incorporation into latex, since they are soluble in organic, water-immiscible solvents and can be incorporated into latex by a swelling procedure. Such fluorescent latexes, with diameters of about 50 nm to several microns, can be loaded with, for example, proteins, haptens, or nucleic acids by various coating methods. A dye mixture with at least one dye according to the present invention, so that resonance energy transfer can occur.

[0058] For example, conjugates of fluorescent dyes with proteins such as haptens or antibodies, such as theophylline, digoxin, T3, and T4, are suitable for use in diagnostic systems, particularly fluorescence immunoassays and fluorescence polarization immunoassays.

[0059] A further subject of the present invention is a procedure for detecting a first immunologically connectable substance, in which a conjugate of a compound according to the invention is used together with a second (immunologically) connectable substance, which may be similar or different with respect to the first substance, and the change in absorption, fluorescence or fluorescence polarization of the compound according to the invention, caused by an (immunological) binding reaction specific for the first substance, is analyzed and detected as a measure of the amount of substance contained in the sample.

[0060] A further subject of the present invention is the use of the conjugates according to the invention in immunoassays.

[0061] As used herein, a "biologically compatible" material is one that is not toxic as used and has no substantial deleterious effects on biological molecules.

[0062] As used herein, unless otherwise specified, the term "nucleobase" refers to adenine, cytidine, guanine, thymine, or uracil.

[0063] As used herein, unless otherwise specified, the term "nucleobase analog" refers to a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring capable of forming Watson-Crick hydrogen bonds with a complementary nucleobase or nucleobase analog. Preferably, the nucleobase analog is a purine, deazapurine, or pyrimidine. Exemplary nucleobase analogs include 7-deazaadenine, inosine, nebularine, nitropyrrole, nitroindole, 2-aminopurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, 5-propynylcytidine, isocytidine, isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil, and 0-thiouracil. 6 -methylguanine, N 6 -methyladenine, O 4 Examples of nucleobase analogs include, but are not limited to, 5,6-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole, ethenoadenine, etc. Further exemplary nucleobase analogs can be found in Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, FL, and the references cited therein, which are incorporated herein by reference.

[0064] As used herein, unless otherwise specified, the term "nucleoside" refers to a compound consisting of a nucleobase covalently linked to the C1' carbon of a substituted or unsubstituted ribose sugar. Exemplary substituted ribose sugars include those in which one or more of its carbon atoms, preferably one, and most preferably the 3' carbon atom, is substituted with one or more of the same or different -R, -OR, -NRR, or halogen groups, where each R is independently -H, (C1-C6) alkyl, or (C5-C 14)aryl. Particularly preferred ribose sugars are ribose, 2'-deoxyribose, 2',3'-dideoxyribose, 3'-haloribose, 3'-fluororibose, 3'-chlororibose, 3'-alkylribose, and the like. When the nucleobase is A or G, the ribose sugar is at the N-position of the nucleobase. 9 If the nucleobase is C, T, or U, the pentose sugar is attached to the N 1 (See, e.g., Kornberg and Baker, 1992, DNA Replication, 2nd ed., Freeman, San Francisco).

[0065] As used herein, unless otherwise specified, the term "nucleoside analog" refers to a nucleoside in which the nucleobase, the ribose sugar, or both, are replaced with their respective analogs. Exemplary nucleobase analogs are defined above. Exemplary ribose sugar analogs include, but are not limited to, substituted or unsubstituted furanoses having more or fewer than five ring atoms, such as erythrose and hexose, and substituted or unsubstituted 3- to 6-carbon acyclic sugars. Typical substituted furanoses and acyclic sugars are those in which one or more of the carbon atoms are substituted with one or more of the same or different -R, -OR, NRR, or halogen groups, where each R is independently H, (C1-C6) alkyl, or (C5-C 14 ) aryl.

[0066] As used herein, unless otherwise specified, the term "nucleotide" refers to a nucleotide in which one or more, typically one, of the ribose carbons has the following formula: [ka] [wherein a is an integer of 0 to 4. Preferably, a is 2, and the phosphate ester is attached to the 3' or 5' carbon of ribose, such as ribose 3'-triphosphate, 2'-deoxyribose 3-triphosphate, ribose 5'-triphosphate, 2'-deoxyribose 5'-triphosphate, 3'-halolibose 5'-triphosphate, 3'-alkylribose 5'-triphosphate, 2',3'-dideoxyribose 5'-triphosphate, etc.] It refers to a nucleoside substituted with a phosphate ester having the formula:

[0067] As used herein, unless otherwise specified, the term "nucleotide derivative" refers to a nucleotide in which the nucleobase, ribose sugar, and / or one or more phosphate esters are replaced with their respective analogs. Exemplary nucleobase and ribose sugar analogs are described above along with nucleoside analogs. Examples of phosphate ester analogs include, but are not limited to, alkyl phosphonates, methyl phosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphooselenoates, phosphorodiselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, phosphoramidates, boronophosphates, peptide nucleic acid (PNA) monomers, and the like, including any associated counterions, if present.

[0068] As used herein, unless otherwise specified, the term "protecting group" means a group that can be reversibly attached to a hydroxyl or amine moiety to render the hydroxyl or amine moiety unreactive during subsequent reactions and selectively cleaved to regenerate the hydroxyl or amine moiety after its protective purpose has been served. Examples of protecting groups are found in Greene, TW, Protective Groups in Organic Synthesis, 3rd Edition (1999), incorporated herein by reference. In one embodiment, the protecting group is stable in a basic reaction medium but can be cleaved by acid. Examples of base-stable, acid-labile protecting groups suitable for use in the present invention include, but are not limited to, ethers, such as methyl, methoxymethyl, methylthiomethyl, methoxyethoxymethyl, bis(2chloroethoxy)methyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, o-nitrobenzyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, 9-(9-phenyl-10-oxo)anthranyl, trimethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, and triisopropylsilyl; and esters, such as pivaloate, adamantoate, and 2,4,6-trimethylbenzoate.

[0069] As used herein, unless otherwise specified, the term "salt" includes, but is not limited to, salts of acidic or basic groups that may be present in the compounds of the present invention. Compounds that are basic in nature can form a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare acceptable acid addition salts of such basic compounds include, but are not limited to, acids that form non-toxic acid addition salts, namely, sulfuric acid, citric acid, maleic acid, acetic acid, oxalic acid, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharinate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. The compounds of the present invention that contain an amino moiety can form acceptable salts with various amino acids in addition to the acids mentioned above. Compounds of the present invention that are acidic in nature can form base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

[0070] As used herein, unless otherwise specified, the term "solvate" refers to a compound of the present invention or a salt thereof that further contains a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile and non-toxic. The term solvate includes hydrates. Hydrates refer to a compound of the present invention or a salt thereof that further contains a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0071] As used herein, unless otherwise specified, the term "nucleoside or nucleotide" refers to a nucleoside and / or nucleotide, and / or mixtures thereof.

[0072] "Nucleoside analog" refers to a nucleoside in which the nucleobase, the ribose sugar, or both are replaced with the respective analog. Exemplary nucleobase analogs are defined above. Exemplary ribose sugar analogs include, but are not limited to, substituted or unsubstituted furanoses having more or fewer than five ring atoms, such as erythrose and hexose, and substituted or unsubstituted 3- to 6-carbon acyclic sugars. Typical substituted furanoses and acyclic sugars have one or more of the carbon atoms replaced with one or more of the same or different -R, -OR, NRR, or halogen groups, where each R is independently H, (C1-C6) alkyl, or (C5-C 14 ) aryl.

[0073] As used herein, unless otherwise specified, the terms "nucleotide derivative" and "nucleotide analog" are used interchangeably and refer to nucleotides in which the nucleobase, ribose sugar, and / or one or more phosphate esters are replaced with their respective analogs. Exemplary nucleobase and ribose sugar analogs are described above along with nucleoside analogs. Examples of phosphate ester analogs include, but are not limited to, alkyl phosphonates, methyl phosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphooselenoates, phosphorodiselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, phosphoramidates, boronophosphates, peptide nucleic acid (PNA) monomers, and the like, including any associated counterions, if present.

[0074] As used herein, unless otherwise specified, the term "nucleic acid" refers to a linear polymeric chain of nucleoside monomer units covalently linked to each other by phosphate internucleotide bonds. Unless otherwise specified, "nucleic acid" as used herein includes polymers of any length, including oligonucleotides, nucleic acids, and nucleic acids as the term is commonly used in the art. Thus, nucleic acids of the present invention can range in size from a few monomer units (e.g., 4-40) to hundreds, thousands, or even more monomer units. Such nucleic acids may also be described herein with respect to their function, such as primers or probes. Whenever a nucleic acid is represented by a sequence of letters, e.g., "ATGCCTG," it will be understood that the sequence is presented in the 5'→3' direction. Unless otherwise specified, nucleic acids whose sequences are described herein are 2'-deoxyribonucleic acids.

[0075] As used herein, unless otherwise specified, the term "nucleic acid analog" refers to a nucleic acid in which at least one nucleoside monomer unit is a "nucleoside analog" and / or at least one phosphate internucleotide linkage is a phosphate analog as defined above under "nucleotide analog." A preferred class of nucleic acid analogs is one in which the sugar and internucleotide linkage are replaced with uncharged neutral amides, such as morpholinocarbamates and peptide nucleic acids ("PNAs"). A preferred PNA is a PNA with an N-(2-aminoethyl)glycinamide backbone (see, e.g., Nielsen et al. (1991) Science, 254, 1497-1500).

[0076] As used herein, unless otherwise specified, the term "label" refers to a detectable molecule or atom covalently or noncovalently attached to a nucleoside or nucleotide, a nucleoside or nucleotide analog, a nucleic acid, a nucleic acid analog, or a terminator. In one embodiment, a nucleoside or nucleotide, a nucleoside or nucleotide analog, a nucleic acid, a nucleic acid analog, or a terminator has a detectable label covalently attached to a nucleobase. The term "label" may also refer to a molecule that modulates the detection of another detectable label, such as a quencher. As used herein, the term "detectable label" is intended to include not only molecules or labels that are "directly" detected (e.g., chromogens or fluorophores), but also moieties (e.g., biotin) that are "indirectly" detected by binding to a second, third, or more binding partners (e.g., avidin or streptavidin), one of which bears the "direct" label.

[0077] Other examples of labels include fluorescent compounds that become detectable by fluorescence when exposed to light of the appropriate wavelength and are detected and / or measured by microscopy or fluorometry. Commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, gamma-phthalaldehyde, and fluorescamine. Detectable labels include: 152 It may also be a fluorescent metal such as Eu or other labels of the lanthanide series that can be attached to oligonucleotides using metal chelating groups such as diethylenetriaminepentaacetic acid or ethylenediaminetetraacetic acid.

[0078] The label may be a chemiluminescent compound, the presence of which is detected by measuring the luminescence generated during the chemical reaction. Examples of useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. Similarly, oligonucleotides can be labeled with bioluminescent compounds and detected by measuring luminescence. In this case, catalytic proteins increase the efficiency of the chemiluminescent reaction. Examples of useful bioluminescent labeling compounds include luciferin, luciferase, and aequorin.

[0079] As used herein, unless otherwise specified, the term "reporter dye" refers to a compound that emits energy in the form of fluorescence when exposed to light. A "reporter dye chromophore" is the network of atoms in the reporter dye that, when exposed to light, emits a level of radiation detectable by conventional spectroscopic means.

[0080] As used herein, unless otherwise specified, the term "non-fluorescent" refers to a compound that, when exposed to radiation, does not emit radiation at levels detectable by conventional spectroscopic means.

[0081] As used herein, unless otherwise specified, the term "weakly fluorescent" refers to a compound that, when exposed to radiation, emits radiation at a low level that is detectable by conventional spectroscopic means.

[0082] As used herein, unless otherwise specified, the term "light" refers to electromagnetic energy having a wavelength that causes the reporter dye to fluoresce, where the wavelength may be in the range of 190 to 800 nm.

[0083] As used herein, unless otherwise specified, the term "specific" refers to a nucleic acid used in a reaction, e.g., a probe used in a hybridization reaction, a primer used in PCR, or a nucleic acid present in a composition, that hybridizes only with its intended target, but not with other nucleic acid molecules in a test sample under normal test circumstances.

[0084] As used herein, unless otherwise specified, the term "selective" refers to a nucleic acid used in a reaction, a post-transcriptional assay, a probe used in a hybridization reaction, a primer used in PCR, or a nucleic acid present in a pharmaceutical product that hybridizes to its intended target more frequently, more rapidly, or for a longer duration than other nucleic acids in a test sample under normal test circumstances.

[0085] As used herein, the term "hybridize under stringent conditions" refers to conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, or 75% or more) identical to each other typically remain hybridized to each other. Stringent conditions depend on the nature of the nucleic acids (e.g., length, GC content, etc.) and the method itself (hybridization, amplification, etc.). Such methods are known in the art and can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. In one embodiment, stringent hybridization conditions are hybridization in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.1×SSC, 0.2% SDS at about 68°C. In another embodiment, stringent hybridization conditions are hybridization in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65°C (i.e., one or more washes at 50°C, 55°C, 60°C, or 65°C). It is understood that the nucleic acids of the present invention do not include nucleic acid molecules that hybridize under these conditions only to nucleotide sequences consisting of only A or T nucleotides. For example, stringent hybridization of an oligonucleotide of approximately 15-40 bases to a complementary sequence in polymerase chain reaction (PCR) can be performed under the following conditions: a salt concentration of 50 mM KCl, a buffer concentration of 10 mM Tris-HCl, Mg 2+ A concentration of 1.5 mM, pH 7-7.5, and an annealing temperature of 55-60°C. Moderately stringent hybridization of oligonucleotides of approximately 15-40 bases to complementary sequences in polymerase chain reaction (PCR) can be performed under the following conditions: a salt concentration of 50 mM KCl, a buffer concentration of 10 mM Tris-HCl, Mg 2+Low stringency hybridization of oligonucleotides of approximately 15 to 40 bases to complementary sequences in polymerase chain reaction (PCR) can be performed under the following conditions: a salt concentration of 50 mM KCl, a buffer concentration of 10 mM Tris-HCl, Mg 2+ Concentration 1.5 mM, pH 7–7.5, and annealing temperature 37–47°C.

[0086] As used herein, unless otherwise specified, the term "stereoisomerically pure" refers to a composition that contains one stereoisomer of a compound and is substantially free of other stereoisomers of that compound. For example, a stereoisomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure composition of a compound having two chiral centers will be substantially free of other diastereoisomers of the compound. A typical stereoisomerically pure compound will contain more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers, more preferably more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, even more preferably more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, and most preferably more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound.

[0087] As used herein, unless otherwise specified, the term "substantially free" refers to a composition that contains one compound and no detectable or significant amounts of other compounds. A typical substantially free composition contains more than about 80% by weight of the desired compound and less than about 20% by weight of one or more other compounds, more preferably more than about 90% by weight of the desired compound and less than about 10% by weight of one or more other compounds, even more preferably more than about 95% by weight of the desired compound and less than about 5% by weight of one or more other compounds, and most preferably more than about 97% by weight of the desired compound and less than about 3% by weight of one or more compounds.

[0088] A method of nucleic acid amplification is the polymerase chain reaction (PCR), which is disclosed in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188, among other references. PCR typically uses two or more oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful for nucleic acid analysis include oligonucleotides that can act as initiation points for nucleic acid synthesis within the nucleic acid sequence of the target nucleic acid. Primers can be purified from restriction digests by conventional methods or produced synthetically. Primers can be single-stranded for maximum efficiency in amplification, but primers can also be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating. A "thermostable polymerase" is a polymerase enzyme that is thermostable, i.e., an enzyme that catalyzes the formation of primer extension products complementary to a template, and that does not irreversibly denature when exposed to elevated temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from, for example, Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, non-thermostable polymerases can also be used in PCR assays if the enzyme is supplemented.

[0089] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method of separating nucleic acid strands involves heating the nucleic acid until it is significantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration and the length and nucleotide composition of the nucleic acid being denatured, but typically range from about 90°C to about 105°C, depending on reaction characteristics such as temperature and nucleic acid length. Denaturation is typically carried out for about 5 seconds to 9 minutes. It may be preferable to use a short denaturation step to avoid prolonged exposure of each polymerase, such as Z05 DNA polymerase, to such high temperatures and thus risk loss of functional enzyme.

[0090] Once the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence on the target nucleic acid.

[0091] The annealing temperature can be about 35°C to about 70°C, or about 45°C to about 65°C, or about 50°C to about 60°C, or about 55°C to about 58°C. The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds; about 30 seconds to about 40 seconds). In this context, it can be advantageous to use different annealing temperatures to increase the comprehensiveness of each assay. This essentially means that at relatively low annealing temperatures, primers can bind to targets with single mismatches, thereby amplifying specific sequence variants. This can be desirable, for example, when a particular organism has known or unknown genetic variants that should also be detected. On the other hand, relatively high annealing temperatures have the advantage of exhibiting greater specificity, since as the temperature increases, the probability of primers binding to target sequences that do not exactly match decreases continuously. To take advantage of both phenomena, in some embodiments of the present invention, the above-mentioned method includes annealing at different temperatures, for example, first at a lower temperature and then at a higher temperature. For example, if the initial incubation is performed at 55°C for about 5 cycles, non-exactly matching target sequences may be (pre-) amplified. This can be followed by, for example, about 45 cycles at 58°C to obtain higher specificity throughout the majority of the experiment. In this way, potentially important genetic variants are not missed, while specificity remains relatively high.

[0092] The reaction mixture is then adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, generating a product complementary to the nucleic acid being analyzed. The temperature should be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., extension temperatures generally range from about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 65°C)). Extension times can range from about 10 seconds to about 5 minutes, or from about 15 seconds to 2 minutes, or from about 20 seconds to about 1 minute, or from about 25 seconds to about 35 seconds). The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing, and extension can be repeated as many times as necessary to generate the desired amount of amplification product corresponding to the target nucleic acid. The limiting factor of the reaction is the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) may be repeated at least once. For use in detection, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficiently for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0093] In PCR, the annealing and extension steps may be performed in the same step (one-step PCR), or as described above, in separate steps (two-step PCR). Performing annealing and extension together, i.e., under the same physical and chemical conditions, using a suitable enzyme, such as Z05 DNA polymerase, has the advantage of saving time for additional steps in each cycle and eliminating the need for additional temperature adjustment between annealing and extension. Thus, one-step PCR reduces the overall complexity of each assay.

[0094] In general, a reduction in the overall time for amplification may be preferable as it reduces the time to result and potentially leads to an earlier diagnosis.

[0095] Other nucleic acid amplification methods used include the ligase chain reaction (LCR; Wu DY and Wallace RB, Genomics 4 (1989) 560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193); and polymerase ligase chain reaction (Barany F., PCR Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO90 / 01069); Repair Chain Reaction (EP0439182A2), 3SR (Kwoh DY et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli JC et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO92 / 08808), and NASBA (US 5,130,238). Further examples include strand displacement amplification (SDA), transcription mediated amplification (TMA), and Qb amplification (for reviews, see, e.g., Whelen AC and Persing DH, Annu. Rev. Microbiol. 50 (1996), 349-373; Abramson RD and Myers TW, Curr. Opin. Biotechnol. 4 (1993), 41-47).

[0096] The terms "Cp value," "Ct value," "Cp," "Ct," or "crossing point of the amplification curve with the threshold" refer to a value that allows quantification of the input target nucleic acid. The Cp or Ct value can be measured according to the second derivative maximum method (Van Luu-The et al., "Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction," BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). In the second derivative method, Cp / Ct corresponds to the first peak of the second derivative curve. This peak corresponds to the beginning of the log-linear phase. The second derivative method calculates the second derivative value of the real-time fluorescence intensity curve, and only one value is obtained. The original Cp / Ct method is based on a locally defined, differentiable approximation of the intensity value, for example, by a polynomial function. Then, the third derivative is calculated. The Cp / Ct value is the minimum root of the third derivative. Cp / Ct can be determined using the fit point method, and Cp / Ct is determined by the intersection of the threshold line and the parallel line in the log-linear region (Van Luu-The et al., BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). The Cp / Ct value is provided by the Light Cycler instrument provided by Roche, calculated according to the second derivative maximum method.

[0097] The term "PCR efficiency" refers to a measure of amplification efficiency from cycle to cycle. PCR efficiency is calculated using the formula: % PCR efficiency = 10 (-傾き) -1) × 100, where the slope was calculated by linear regression with the log of copy number plotted on the y-axis and Cp plotted on the x-axis. PCR efficiency can be measured using perfectly matched or mismatched primer templates.

[0098] The terms "FRET" or "fluorescence resonance energy transfer" or "Förster resonance energy transfer" refer to the transfer of energy between at least two chromophores: a donor chromophore and an acceptor chromophore (called a quencher). Typically, the donor transfers energy to an acceptor when excited by light of a suitable wavelength. The acceptor typically re-emits the transferred energy in the form of light of a different wavelength. If the acceptor is a "dark" quencher, it dissipates the transferred energy in a form other than light. Whether a particular fluorophore acts as a donor or acceptor depends on the nature of the other components of the FRET pair. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ) (Biosearch Technologies, Inc., Novato, CA), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, IA), and BlackBerry® Quencher 650 (BBQ-650) (Berry & Assoc., Dexta, MI).

[0099] The above-described methods may be based on fluorescence resonance energy transfer (FRET) between a donor fluorescent moiety and an acceptor fluorescent moiety. A typical donor fluorescent moiety is fluorescein, and typical corresponding acceptor fluorescent moieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Detection typically involves exciting a sample with a wavelength absorbed by the donor fluorescent moiety and visualizing and / or measuring the wavelength emitted by the corresponding acceptor fluorescent moiety. In the methods of the present invention, detection can be followed by quantification of FRET. For example, detection can be performed after each cycle step. For example, detection can be performed in real time. Using commercially available real-time PCR instruments (e.g., LightCycler™ or TaqMan®), PCR amplification and detection of amplified products can be combined in a single closed cuvette, significantly reducing cycle times. Because detection occurs simultaneously with amplification, real-time PCR methods do not require manipulation of the amplified products, potentially reducing the risk of cross-contamination between amplified products. Real-time PCR significantly reduces turnaround time and is an attractive alternative to conventional PCR techniques in clinical laboratories.

[0100] The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714, and WO 97 / 46712. The LightCycler® instrument is a rapid thermal cycler combined with a microvolume fluorometer utilizing high-quality optics. This rapid thermal cycling technology uses thin glass cuvettes as reaction vessels. Heating and cooling of the reaction chamber is controlled by alternating between heated and ambient air. The low mass of air and the high surface area-to-volume ratio of the cuvette allow for very rapid temperature exchange within the thermal chamber.

[0101] TaqMan® technology utilizes a single-stranded hybridization probe labeled with two fluorescent moieties. When the first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the principle of FRET. The second fluorescent moiety is typically a quencher molecule. Typical fluorescent dyes used in this format include FAM, HEX, CY5, JA270, cyan, and Cy5.5, among others. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and is subsequently degraded during the extension phase by the 5'-3' exonuclease activity of Taq or another suitable polymerase known to those skilled in the art, such as mutant Z05 polymerase. As a result, the excited fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, fluorescence emission from the first fluorescent moiety can be detected upon excitation of the first fluorescent moiety in the absence of a quencher.

[0102] In both of the detection modes described above, the intensity of the emitted signal can be correlated with the number of original target nucleic acid molecules.

[0103] Recently, methods for using "tagged" TaqMan® probes to perform multiplex PCR assays have been described in U.S. Patent Application Publication Nos. 2018 / 0073056 and 2018 / 0073064, both of which are incorporated herein by reference in their entireties.

[0104] As an alternative to FRET, double-stranded DNA binding dyes, such as fluorescent DNA binding dyes (e.g., SYBRGREEN I® or SYBRGOLD® (Molecular Probes)), can be used to detect amplification products. Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Double-stranded DNA binding dyes, such as nucleic acid intercalating dyes, can also be used. When using double-stranded DNA binding dyes, a melting curve analysis is usually performed to confirm the presence of amplification products.

[0105] Molecular beacons in combination with FRET can also be used to detect the presence of amplification products using the real-time PCR method of the present invention. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows secondary structure formation (e.g., a hairpin). As a result of the formation of the secondary structure within the probe, both fluorescent moieties are spatially close together when the probe is in solution. After hybridization to the amplification product, the secondary structure of the probe is disrupted, separating the fluorescent moieties from each other so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0106] Thus, the method of the present invention is the above-described method using FRET, wherein the probe comprises a nucleic acid sequence that allows secondary structure formation, wherein the secondary structure formation brings the first and second fluorescent moieties into spatial proximity.

[0107] Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety.

[0108] Thus, in one embodiment, the donor and acceptor fluorescent moieties are within 5 nucleotides or less of each other on the probe. In a further embodiment, the acceptor fluorescent moiety is a quencher.

[0109] As noted above, in the TaqMan® format, during the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and is subsequently degraded in the extension step by the 5' to 3'-exonuclease activity of Taq or another suitable polymerase known to those of skill in the art, such as mutant Z05 polymerase. Thus, in one embodiment, in the above method, amplification uses a polymerase enzyme with 5' to 3'-exonuclease activity.

[0110] It is further advantageous to carefully select the length of the amplicon obtained as a result of the above-mentioned method. Generally, a relatively short amplicon increases the efficiency of the amplification reaction. Therefore, one aspect of the present invention is the above-mentioned method, wherein the amplified fragment comprises at most 450 bases, at most 300 bases, at most 200 bases, or at most 150 bases.

[0111] "Sequence" refers to the primary structure of nucleic acid, that is, the specific sequence of the single nucleic acid bases that each nucleic acid is composed of.It should be understood that the term "sequence" does not refer to a specific type of nucleic acid, such as RNA or DNA, but applies to both, as well as to other types of nucleic acid, such as PNA.When nucleic acid bases correspond to each other, particularly in the case of uracil (present in RNA) and thymine (present in DNA), these bases can be considered equivalent between RNA sequence and DNA sequence, as is well known to those skilled in the art.

[0112] Clinically relevant nucleic acids are often DNA, which can be derived from DNA viruses, such as Hepatitis B Virus (HBV) and Cytomegalovirus (CMV), or bacteria, such as Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). In such cases, it may be advantageous to use an internal control nucleic acid consisting of DNA to reflect the properties of the target nucleic acid. The terms "cell," "cell line," and "cell culture" can be used interchangeably, and all such designations include progeny. Thus, the term "transformant" or "transformed cell" includes the primary transformed cell and cultures derived from that cell, regardless of the number of transfers. Not all progeny may be precisely identical in DNA content due to deliberate or unintended mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of a transformant. Cells can be prokaryotic or eukaryotic.

[0113] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, a ribosome binding site, a positive and reverse regulatory element (U.S. Pat. No. 4,666,848, incorporated herein by reference), and optionally other sequences. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0114] The term "operably linked" refers to the positioning of a coding sequence such that the control sequences function to drive expression of the protein encoded by the coding sequence. Thus, a coding sequence "operably linked" to a control sequence refers to a configuration in which expression of the coding sequence is possible under the direction of the control sequences.

[0115] The terms "restriction endonucleases" and "restriction enzymes" refer to enzymes, typically of bacterial origin, that cut double-stranded DNA at or near a particular nucleotide sequence. Families of amino acid residues having similar side chains are defined herein. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine, glycine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0116] The term "reagent solution" refers to any solution containing at least one reagent required or used for PCR purposes. The most typical components are polymerase, nucleotides, primers, ions, magnesium, salts, pH buffers, nucleoside triphosphates (NTPs) or deoxyribonucleoside triphosphates (dNTPs), probes, fluorescent dyes (which may be bound to the probes), nucleic acid binders, and nucleic acid templates. Reagents may also be other polymerase reaction additives that affect the polymerase reaction or its monitoring.

[0117] The term "master mix" refers to a mixture of all or most of the components or factors necessary for PCR to occur, possibly excluding sample- and amplicon-specific templates and primers. Commercially available master mixes are usually concentrated solutions. A master mix may contain all reagents common to multiple samples, but may also be constructed for only one sample. The use of a master mix helps reduce pipetting errors and variability between samples due to differences in pipetting volume.

[0118] The term "thermostable polymerase" refers to an enzyme that is heat-stable, thermotolerant, and retains sufficient activity to subsequently carry out a primer extension reaction after being exposed to high temperatures for the time required to denature double-stranded nucleic acids. The heating conditions required for nucleic acid denaturation are well known in the art and are exemplified in U.S. Patent Nos. 4,965,188 and 4,889,818, which are incorporated herein by reference. As used herein, a thermostable polymerase is suitable for use in temperature cycling reactions such as PCR. Examples of thermostable nucleic acid polymerases include Thermus aquaticus (Taq) DNA polymerase, Thermus sp. Z05 polymerase, Thermus flavus polymerase, Thermotoga maritima polymerase, such as TMA-25 polymerase, TMA-30 polymerase, and Tth DNA polymerase.

[0119] A "polymerase with reverse transcriptase activity" refers to a nucleic acid polymerase that can synthesize DNA from an RNA template and can replicate single- or double-stranded DNA once the RNA has been reverse transcribed into single-stranded cDNA. In one embodiment of the present invention, the polymerase with reverse transcriptase activity is thermostable. In the amplification of RNA molecules by DNA polymerase, the first extension reaction is reverse transcription using an RNA template to generate a DNA strand. The second extension reaction using a DNA template generates a double-stranded DNA molecule. Thus, synthesis of a complementary DNA strand from the RNA template by DNA polymerase provides the starting material for amplification.

[0120] Thermostable DNA polymerases can be used in coupled, one-enzyme reverse transcription / amplification reactions. In this context, the term "homogeneous" refers to a two-step, single-addition reaction for reverse transcription and amplification of an RNA target. Homogeneous means that following the reverse transcription (RT) step, there is no need to open the reaction vessel or adjust the reaction components before the amplification step. In heterogeneous RT-PCR reactions, following reverse transcription and prior to amplification, one or more of the reaction components, such as amplification reagents, must be adjusted, added, or diluted, for example, and the reaction vessel must be opened or at least its contents must be manipulated. Both homogeneous and heterogeneous embodiments are contemplated by the scope of the present invention.

[0121] Reverse transcription is a key step in RT-PCR. For example, it is known in the art that RNA templates tend to form secondary structures that can hinder primer binding and / or cDNA strand elongation by the respective reverse transcriptases. Therefore, a relatively high temperature for the RT reaction is advantageous in terms of transcription efficiency. On the other hand, a high incubation temperature also provides higher specificity, i.e., the RT primers do not anneal to sequences that show mismatches with the expected sequence or sequences. In particular, in the case of multiple different target RNAs, transcribing and subsequently amplifying and detecting sequences with a single mismatch may also be desirable, for example, when there is a possibility that unknown or rare substrains or subspecies of organisms may be present in the fluid sample.

[0122] To obtain both of the benefits mentioned above, namely reduction of secondary structure and reverse transcription of templates with mismatches, the RT incubation can be carried out at more than one different temperature.

[0123] Therefore, one aspect of the present invention is a method as described above, wherein said incubation of the polymerase with reverse transcriptase activity is carried out at different temperatures from 30°C to 75°C, or from 45°C to 70°C, or from 55°C to 65°C.

[0124] A further important aspect of reverse transcription is that a long RT step can damage DNA templates that may be present in the fluid sample. If the fluid sample contains both RNA and DNA species, it is preferable to keep the duration of the RT step as short as possible, while at the same time ensuring the synthesis of sufficient amounts of cDNA for subsequent amplification and detection of any amplicons.

[0125] Thus, one aspect of the present invention is a method as described above, wherein the period for incubating the polymerase with reverse transcriptase activity is up to 30 minutes, 20 minutes, 15 minutes, 12.5 minutes, 10 minutes, 5 minutes, or 1 minute.

[0126] A further aspect of the present invention is the method as described above, wherein the polymerase having reverse transcriptase activity and comprising a mutation is selected from the group consisting of: a) CS5 DNA polymerase b) CS6 DNA polymerase c) Thermotoga maritima DNA polymerase d) Thermus aquaticus DNA polymerase e) Thermus thermophilus DNA polymerase f) Thermus flavus DNA polymerase g) Thermus filiformis DNA polymerase h) Thermus sp. sps17 DNA polymerase i) Thermus sp. Z05 DNA polymerase j) Thermotoga neapolitana DNA polymerase k) Thermosipho africanus DNA polymerase l) Thermus caldophilus DNA polymerase

[0127] In particular, enzymes carrying mutations in the polymerase domain are suited to these requirements, improving their reverse transcription efficiency in terms of faster extension rates. Thus, in the above methods, a polymerase with reverse transcriptase activity is a polymerase that comprises a mutation that improves the rate of nucleic acid extension and / or improves reverse transcriptase activity compared to the respective wild-type polymerase.

[0128] In one embodiment, in the above method, the polymerase with reverse transcriptase activity is a polymerase comprising a mutation that improves reverse transcriptase activity compared to the respective wild-type polymerase.

[0129] Polymerases carrying point mutations that make them particularly useful are disclosed in WO 2008 / 046612. In particular, the polymerases used contain at least the following in the polymerase domain: The mutant DNA polymerase may have a motif of TGRLSS-Xb7-Xb8-PNLQN; where Xb7 is an amino acid selected from S or T and Xb8 is an amino acid selected from G, T, R, K, or L, and the polymerase comprises 3' to 5' exonuclease activity and has improved nucleic acid extension rate and / or reverse transcription efficiency compared to a wild-type DNA polymerase, wherein Xb8 in the wild-type DNA polymerase is an amino acid selected from D, E, or N.

[0130] An example includes mutants of thermostable DNA polymerases derived from Thermus species Z05 (e.g., as described in U.S. Pat. No. 5,455,170), which contain mutations in the polymerase domain compared to the respective wild-type enzyme Z05. An embodiment of the methods of the present invention is a mutant Z05 DNA polymerase, in which the amino acid at position 580 is selected from the group consisting of G, T, R, K, and L.

[0131] For reverse transcription using a thermostable polymerase, Mn 2+ or Mg 2+ Divalent cations such as manganese chloride (MnCl), manganese acetate [Mn(OAc)], or manganese sulfate (MnSO), or magnesium chloride (MgCl), magnesium acetate [Mg(OAc)], or magnesium sulfate (MgSO), are typically included as salts. For example, when MnCl is included in a reaction containing 50 mM Tricine buffer, MnCl is generally present at a concentration of 0.5-7.0 mM; when 200 μM each of dGTP, dATP, dUTP, and dCTP is utilized, MnCl is generally present at 2.5-3.5 mM.

[0132] A "modified" thermostable polymerase refers to a polymerase in which at least one monomer differs from the reference sequence, for example, a natural or wild-type form of a polymerase, or another modified form of a polymerase. Exemplary modifications include insertion, deletion, and substitution of monomers. Modified polymerases also include chimeric polymerases that have distinguishable component sequences (e.g., structural or functional domains) derived from two or more parents. The definition of modified polymerase also includes definitions that consist of chemical modifications of the reference sequence. Examples of modified thermostable polymerases include G46E E678G CS5 DNA polymerase, G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G S671F CS5 DNA polymerase, G46E L329A D640G S671F E678G CS5 DNA polymerase, G46E E678G CS6 DNA polymerase, Z05 DNA polymerase, ΔZ05 polymerase, ΔZ05-Gold polymerase, ΔZ05R polymerase, E615G Taq DNA polymerase, E678G TMA-25 polymerase, and E678G TMA-30 polymerase.

[0133] The term "thermoactive polymerase" refers to an enzyme that is activated at the high temperatures (e.g., 55-80°C) required to ensure specific priming and primer extension.

[0134] The terms "peptide," "polypeptide," and "protein" are used interchangeably. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Amino acid sequences are written from the amino terminus to the carboxy terminus unless otherwise specified. Single-stranded nucleic acid sequences are written 5' to 3' unless otherwise specified. The top strand of a double-stranded nucleic acid sequence is written 5' to 3' unless otherwise specified, and the bottom strand is written 3' to 5' unless otherwise specified.

[0135] Although the foregoing invention has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all of the compositions and methods described above may 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 was individually indicated to be incorporated by reference for all purposes.

[0136] The chemical structures of the compounds disclosed in this application are numbered and shown as follows:

[0137] [Table 1]

[0138] [Table 2]

[0139] [Table 3]

[0140] [Table 4]

[0141] [Table 5] [Example]

[0142] The following examples are presented to illustrate embodiments of the invention that are presently preferred to be practiced, it being understood that the examples are illustrative and that the invention is not to be considered as limited except as set forth in the appended claims.

[0143] Abbreviations: abs. = anhydrous, AcOH = acetic acid, aq = aqueous, dATP = 2'-deoxyadenosine 5'-triphosphate, dCTP = 2'-deoxycytidine 5'-triphosphate, dGTP = 2'-deoxyguanosine 5'-triphosphate, DBCO = dibenzocyclooctyne modified; DCM = dichloromethane, DMF = N,N'-dimethylformamide, DMSO = dimethyl sulfoxide, dUTP = 2'-deoxyuridine 5'-triphosphate, eq. = molar equivalent, EtO = diethyl ether, EtOAc = ethyl acetate, MeCN = acetonitrile, MeOH = methanol, qPCR = real-time polymerase chain reaction, RT = room temperature, SPE = solid phase extraction, TBAF = tetrabutylammonium fluoride, THF = tetrahydrofuran, TLC = thin layer chromatography, UPLC-MS = ultra-performance liquid chromatography coupled to a mass spectrometer.

[0144] General Materials and Methods Reagents were obtained from MilliporeSigma (Burlington, MA, USA) unless otherwise noted. tert.-Butyllithium solution (1.5 M in n-pentane) was obtained from AlfaAesar (Haverill, MA, USA). 1-(3-Bromophenyl)azetidine and methyl 1-butyl-1H-imidazole-2-carboxylate were obtained from ChemShuttle (Hayward, CA, USA). Methyl 1-methyl-1H-imidazole-2-carboxylate was obtained from AA Blocks LLC (San Diego, CA, USA). Methyl 1,4,5-trimethyl-1H-imidazole-2-carboxylate was obtained from ChemSpace (Monmouth Junction, NJ, USA). 2-(Trimethylsilyl)phenyl trifluoromethanesulfonate was obtained from TCI America (Portland, OR, USA). 1-Azido-2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethane (bromo-PEG-azide) was obtained from Broad Pharm (San Diego, CA, USA). Reagents and materials for chemical DNA synthesis were obtained from Glen Research (Sterling, VA, USA).

[0145] All synthetic transformations were carried out using flame-dried glassware under a dry argon atmosphere unless otherwise noted. Solvents for chemical reactions were obtained from Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA) and stored over activated molecular sieves. Chromatographic solvents (HPLC grade) were obtained from MilliporeSigma or VWR (Radnor, PA, USA) and used without further purification. Microwave-assisted reactions were performed using a Discover® SP microwave system from CEM (Matthews, NC, USA) equipped with a focused single-mode reaction chamber (2.45 GHz) in a heavy-walled glass vial (2.0 mL or 10.0 mL). The reaction temperature was monitored with a built-in IR temperature sensor and maintained constant by automatic power control. Microwave-assisted reactions were stirred under active cooling with compressed air. Flash column chromatography was performed using an automated flash chromatography system (CombiFlash® Rf) from Teledyne-Isco (Lincoln, NE, USA). +UPLC analysis was performed using a Waters I-class ACQUITY UPLC (Waters Corporation, Milford, MA, USA) equipped with diode array, fluorescence, and mass spectrometry (ZSpray™) detectors. A Waters Oligonucleotide BEH C18 column (130 Å, 1.7 μm, 2.1 × 50 mm) was used with an appropriate gradient of TEAA buffer (100 mM, pH 7.0) or HO (0.1% formic acid) against MeCN at 1.0 ml / min. Semi-preparative HPLC purification was performed on a Waters 600 HPLC using a 996 photodiode array detector and a Waters XBridge™ BEH C18 OBD Prep column (130 Å, 5 μm, 19.0 × 250 mm) at 10.0 ml / min. Samples were filtered through a Teflon syringe filter (0.20 μm) before injection. Absorption spectra were obtained using an Agilent 8453 UV-visible spectrophotometer (Santa Clara, CA, USA). Fluorescence spectra were recorded on an Agilent Cary Eclipse fluorescence spectrophotometer.

[0146] Depending on the respective workup or purification method, compounds bearing one or more positive charges (24–36) were obtained as acetate, chloride, bromide, or iodide salts. Counterion replacement was achieved by standard ion-exchange procedures using appropriate ion-exchange resins, liquid-liquid extraction, or precipitation from organic solvents.

[0147] Example 1: Reaction Scheme General Procedure

[0148] A general reaction scheme for preparing fluorescent dyes according to the present disclosure is provided in Figure 5. As shown in the figure, the method for preparing fluorescent dyes includes a dye core-forming step. Here, a double halogen-metal exchange of dihalide (1) with the corresponding dilithium compound using an organolithium reagent (e.g., tert-butyllithium) is performed. The intermediate is then reacted with an alkyl ester, such as the methyl ester of 1-alkyl- or 1-aryl-1H-imidazole-2-carboxylate (2) ("in situ"). Aromatization is then carried out by removing water from the hydroxyl intermediate with the addition of a Brønsted acid, such as acetic acid ("one-pot reaction"), to yield compound (3). The dye core-forming reaction is followed by quaternization of the imidazole to an imidazolium compound. In some embodiments, quaternization of imidazole (3) to an imidazolium compound is carried out by nucleophilic substitution with an alkyl halide, alkyl tosylate, alkyl triflate, or alkyl mesylate to yield alkylated compound (4). In another embodiment, the quaternization of imidazole 3 to an imidazolium compound is carried out using a cyclic lactone, such as beta-propiolactone, in the presence of a Lewis acid, such as aluminum tribromide, to accept carboxylic acid 5. In yet another embodiment, the quaternization of the 3-imidazole nitrogen 3 to an imidazolium compound is carried out using an aryline generated in situ from the 2-(trimethylsilyl)phenyl trifluoromethanesulfonate reagent and a fluoride donor (e.g., tetrabutylammonium fluoride or tetrabutylammonium difluorotriphenylsilicate) to afford arylated compound 6.

[0149] Example 2: General procedure for the synthesis of compounds 1-10 [ka] X is Si, Ge; Y is Me, iPr, Ph; Z is NMe2, NC3H6.

[0150] Compounds 1-10 [3,3'-(dimethylsilanediyl)bis(N,N-dimethylaniline) 1, bis(3-(azetidin-1-yl)phenyl)dimethylsilane 2,3,3'-(diisopropylsilanediyl)bis(N,N-dimethylaniline) 3, bis(3-(azetidin-1-yl)phenyl)diisopropylsilane 4,3,3'-(diphenylsilanediyl)bis(N,N-dimethylaniline) 5, bis(3-(azetidin-1-yl)phenyl)diphenylsilane 6,3,3'-(dimethylgermanediyl)bis(N,N-dimethylaniline) 7, bis(3 3-(azetidin-1-yl)phenyl)dimethylgermane 8,3,3'-(diphenylgermanediyl)bis(N,N-dimethylaniline) 9, bis(3-(azetidin-1-yl)phenyl)diphenylgermane 10) were synthesized starting from 3-bromo-N,N-dimethylaniline or 1-(3-bromophenyl)azetidine and a silane or germane reagent selected from dichlorodimethylsilane, dichlorodiisopropylsilane, dichlorodiphenylsilane, dimethylgermanium dichloride, or diphenylgermanium dichloride, respectively.

[0151] A solution of 3-bromo-N,N-dimethylaniline or 1-(3-bromophenyl)azetidine (2.4 equiv.) in anhydrous THF (17.0 mL) was cooled to -78 °C, and then n-butyllithium solution (2.4 equiv., 2.5 M in hexane) was slowly added dropwise. The reaction was stirred at -78 °C for 0.5 h. At the same temperature, a solution of silane or germane (1.0 equiv., 4.0 mmol), respectively, in THF (2.2 mL) was added. The cooling bath was then removed, and the reaction mixture was stirred at room temperature for 3 h or until complete conversion of the starting material was observed by TLC analysis (silica, EtOAc / hexane). The reaction was quenched with saturated aqueous NH4Cl (5.0 mL), diluted with HO (20 mL), and extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous MgSO4, and filtered. The solvent was then evaporated, and the residue was dried under high vacuum. The crude product was purified by silica flash chromatography (silica, 0-10% EtOAc in hexanes) to give the title compound as a colorless oil. The identity of the product was confirmed by NMR and UPLC-MS analysis (C 18 , HO, 0.1% formic acid, 50–100% MeCN in 2.0 min).

[0152] Example 3: General procedure for the synthesis of compounds 11-20 [ka] X is Si, Ge; Y is Me, iPr, Ph; Z is NMe2, NC3H6.

[0153] Compounds 11-20 [3,3'-(dimethylsilanediyl)bis(4-bromo-N,N-dimethylaniline)] 11, bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylsilane 12, 3,3'-(diisopropylsilanediyl)bis(4-bromo-N,N-dimethylaniline) 13, bis(5-(azetidin-1-yl)-2-bromophenyl)diisopropylsilane 14, 3,3'-(diphenylsilanediyl)bis(4-bromo-N,N-dimethylaniline) 15, bis (5-(azetidin-1-yl)-2-bromophenyl)diphenylsilane 16,3,3'-(dimethylgermanediyl)bis(4-bromo-N,N-dimethyl-aniline) 17, bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylgermane 18,3,3'-(diphenylgermanediyl)bis(4-bromo-N,N-dimethylaniline) 19, bis(5-(azetidin-1-yl)-2-bromophenyl)diphenylgermane 20) were synthesized from compounds 1–10.

[0154] Aniline or azetidine (1.0 equiv., 3.0 mmol) was dissolved in anhydrous DMF (18.9 mL) and cooled in an ice bath. While stirring, N-bromosuccinimide (2.02 equiv.) was added over 0.5 h. The reaction was stirred at room temperature for 3 h or until complete conversion of the starting material was observed by TLC analysis (silica, EtOAc / hexanes). The mixture was diluted with DCM and extracted twice with half-saturated aqueous NaHCO3. The organic layer was washed with saturated aqueous NaCl, dried over MgSO4, and filtered. The solvent was then evaporated, and the residue was dried under high vacuum. Pure products were obtained by flash chromatography (silica, 0–10% EtOAc in hexanes) or recrystallization. The identity of the products was confirmed by NMR and UPLC-MS analysis (C). 18 , HO, 0.1% formic acid, 50–100% MeCN in 2.0 min).

[0155] Example 4: General procedure for the synthesis of compounds 24-28 [ka] X is Si, Ge; Y is Me, iPr, Ph; Z is NMe2, NC3H6; R is Me, Bu.

[0156] Dyes 24–28 were synthesized from compounds 11, 12, and 20 and their respective imidazole methyl esters 21–23 [methyl 1-methyl-1H-imidazole-2-carboxylate 21, methyl 1,4,5-trimethyl-1H-imidazole-2-carboxylate 22, and methyl 1-butyl-1H-imidazole-2-carboxylate 23].

[0157] A solution of each dibromide (1.0 equiv., 450 μmol) in anhydrous THF (5.75 mL) was cooled to −78°C. tert.-Butyllithium solution (4.4 equiv., 1.5 M in n-pentane) was added dropwise, and the reaction was stirred at −78°C for 30 minutes. The reaction temperature was raised to −20°C, followed by the slow addition of a solution of each imidazole methyl ester (2.2 equiv.) in anhydrous THF (5.75 mL) over 0.5 hours. The mixture was stirred at room temperature for 18 hours. The pH of the reaction mixture was set to 4.0 with glacial acetic acid, and the solution was stirred at room temperature for 4 hours, during which time an intense blue coloration developed. The reaction mixture was extracted with saturated aqueous KBr and DCM. The combined organic layers were washed with HO, dried over NaSO, and filtered. The solvent was evaporated under reduced pressure. The residue was redissolved in a minimum amount of DCM and added to a stirred solution of EtO. A blue precipitate formed, which was isolated and washed by centrifugation. The yellow supernatant was discarded. The dark blue pigment was dried under high vacuum and purified by semi-preparative HPLC (C 18 , TEAA / MeCN).

[0158] Example 5: General procedure for the synthesis of compounds 29 and 30 [ka] Z is NMe2, NC3H6.

[0159] Imidazolium dyes 29 and 30 were synthesized from dyes 24 and 25, respectively.

[0160] A solution of the imidazole dye (1.0 equiv., 17 μmol) and methyl iodide (2.0 equiv.) in MeCN was heated at 80 °C for 6 h by microwave irradiation. The reaction mixture was added to stirring EtO. The precipitate was isolated by centrifugation and the supernatant was discarded. Finally, the crude product was purified by semi-preparative HPLC (C 18 The fluorescence spectrum and temperature dependence of fluorescence are shown in Figure 1.

[0161] Example 6: General procedure for the synthesis of dye 31 [ka]

[0162] A solution of the imidazole dye (1.0 equiv., 17 μmol, final concentration 30 mM) and 2-(trimethylsilyl)phenyl trifluoromethanesulfonate (5.0 equiv.) in anhydrous MeCN was prepared, and the reaction was initiated by the final addition of anhydrous TBAF (1.0 M in THF, 5.0 equiv.). The stirred solution was heated at 50 °C for 1.0 h using microwave irradiation. UPLC-MS analysis (30–60% MeCN in 2 min) confirmed the reaction was complete. The mixture was poured into stirred EtO, to which solid NaI (2.0 equiv.) was added. A precipitate formed and was isolated by centrifugation. The supernatant was discarded, and the residue was dissolved in HO. Insoluble by-products were removed using a Teflon syringe filter, and the imidazolium dye was purified by semi-preparative HPLC (C). 18 , TEAA, 30-60% MeCN) from the supernatant.

[0163] Example 7: General procedure for synthesizing dyes 32-35 [ka] X is Si or Ge; R1 is Me or Bu; and R2 is H or Me.

[0164] A solution of imidazole dye (1.0 equiv., 20 μmol, final concentration 78 mM) and β-propiolactone (10.0 equiv.) in anhydrous MeCN was prepared, and AlBr was added last to initiate the reaction. The solution was stirred at room temperature for 1.0 h, during which time some precipitate formed. UPLC-MS analysis (30–60% MeCN in 2 min) confirmed the reaction was complete. The mixture was poured into stirred EtO. A precipitate formed and was isolated by centrifugation. The residue was washed with EtO, dried under high vacuum, and redissolved in MeCN (400 μL). TEAA buffer (3.6 mL) was added, and insoluble byproducts were removed with a Teflon syringe filter. The imidazolium dye was purified by semi-preparative HPLC (C). 18 , TEAA, 30-60% MeCN) from the supernatant.

[0165] Example 8: General procedure for the synthesis of dye 36 [ka]

[0166] A solution of imidazole dye (1.0 equiv., 17 μmol, final concentration 50 mM) and bromo-PEG-azide (2.0 equiv.) and NaI (10.0 equiv.) in anhydrous MeCN was heated by microwave irradiation at 50 °C for 12 h. The product was precipitated from EtO and analyzed by semi-preparative HPLC (C 18 The purified product was desalted by SPE.

[0167] Example 9: Preparation of dye-labeled DNA probes Dye-labeled DNA probes were prepared by strain-promoted azide-alkyne cycloaddition between DNA-bound DBCO and azide-modified dyes. 30-mer DNA probes containing a 3'-C3 extension blocker, internal BHQ-2, and 5'-DBCO dT modifications were prepared by solid-phase DNA synthesis and purified using standard methods. DNA (1.0 eq., 100 μM, 50 mM TEAA) and dye azide No. 36 (1.1 eq., 100 μM, 50 mM TEAA) were mixed and incubated at room temperature on a shaker for 2 h. A DNA conjugate containing a commercially available reference dye (azide-modified) was prepared in a similar manner. Excess dye was completely removed by ethanol precipitation using standard procedures. UPLC analysis confirmed quantitative DNA labeling (Figure 2A and Figure 2B).

[0168] Example 10: PCR amplification using dye-labeled DNA probes All qPCR components were prepared using nuclease-free HO. A reaction mixture with a total volume of 50 μL was prepared by mixing three components, referred to as the master mix (20 μL), buffer mix (20 μL), and dNTP mix (10 μL). The master mix contained Tricine buffer (pH 8.2), manganese acetate, potassium acetate, glycerol, DMSO, surfactant, target DNA (5000 copies / reaction), polymerase aptamer, labeled DNA probe, forward and reverse primer DNA, and polymerase enzyme. The dNTP mix contained dATP, dCTP, dGTP (2.0 mM each), and dUTP (4.0 mM). Each qPCR run was prepared in triplicate in wells of a 96-well plate. The plate was sealed and subjected to amplification cycling using a LightCycler® 480 system (Fritz Hoffmann-La Roche, Basel, Switzerland). Growth curves were analyzed from fluorescence data collected in the Cy5.5 channel and are shown in Figure 3.

[0169] Example 11: Immunohistochemical tissue staining Dye azide No. 36 and CF680R were used to stain tonsillar tissue sections. Formalin-fixed, paraffin-embedded tissue sections were treated with CONFIRM™ anti-Ki-67(30-9) Rabbit Monoclonal Primary Antibody (Ventana Medical Systems, Inc., Tucson, Arizona, USA) and labeled with each dye azide at a concentration of 200 μM. Light microscope images at 10x, 20x, and 40x magnification are shown in Figure 4.

Claims

1. Formula A or B 【Chemistry 1】 a fluorescent dye of the formula: (In the formula, X is Si, Ge, or C; Y is Me, Et, iPr, or Ph; Z is 【Chemistry 2】 any one of: W is 【Transformation 3】 any one of: R 1 and R 2 teeth, 【Chemistry 4】 any combination of L is H, CONH 2 , CO 2 H, N 3 (which is Fluorescent dye.

2. Z is, 【Transformation 5】 The fluorescent dye according to claim 1 ,

3. The dye is 【Transformation 6】 3. The fluorescent dye according to claim 1, selected from the group consisting of:

4. A method for preparing the fluorescent dye according to any one of claims 1 to 3, comprising the steps of: a) performing a double halogen-metal exchange of the dihalide to the corresponding dilithium compound using an organolithium reagent; b) reacting the dilithium compound with an alkyl ester to form a hydroxyl intermediate; c) adding a Bronsted acid to the hydroxyl intermediate to effect aromatization with elimination of water to form an imidazole; and d) quaternizing the imidazole to form an imidazolium A method comprising:

5. 5. The method of claim 4, wherein the quaternization is carried out by nucleophilic substitution with an alkyl halide, alkyl tosylate, alkyl triflate or alkyl mesylate.

6. 5. The method of claim 4, wherein the quaternization is carried out using a cyclic lactone in the presence of a Lewis acid.

7. 5. The method of claim 4, wherein the quaternization is carried out using an aline generated in situ from the 2-(trimethylsilyl)phenyl trifluoromethanesulfonate reagent and a fluoride donor.

8. 1. A method for detecting the presence of one or more target nucleic acids in a sample in a polymerase chain reaction (PCR) assay, comprising: - contacting the sample with an oligonucleotide probe labeled with at least one fluorescent dye according to any one of claims 1 to 3; and - measuring a fluorescent signal generated from said labeled oligonucleotide, whereby said detection of said fluorescent signal indicates the presence of said one or more target nucleic acids in said sample. By, method.

9. 9. The method of claim 8, wherein the PCR assay is a multiplex PCR assay.

10. 10. The method of claim 8 or 9, wherein measuring the fluorescent signal is carried out at a temperature of 65°C or higher.

11. 1. A method for detecting the presence of one or more target proteins in a tissue sample in an immunohistochemical assay, comprising: - contacting the tissue sample with at least one fluorescent dye-labeled binding member according to any one of claims 1 to 3, which interacts directly or indirectly with the target protein; and - visualizing a fluorescent signal generated in said tissue sample, whereby said detection of said fluorescent signal indicates the presence of said one or more target proteins in said tissue sample. By, method.

12. 12. The method of claim 11, wherein the binding member is a primary antibody that binds to the one or more target proteins or a secondary antibody that binds to the primary antibody.

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