Substituted diazenylanine as a fluorescent quencher and its use

Abstract

The present invention relates to substituted diazenylanilines of formula I, and their nucleotide conjugates, complexes and salts, which can potentially be used as fluorescence quenchers in chemical and biological sciences, such as cell imaging applications, diagnostic methods, fluorescent and non-fluorescent tags, pharmaceuticals, and other useful applications, as well as methods for preparing said novel compounds. More specifically, the present invention relates to 2,2'-((4-((2,5-disubstituted-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-2 / 3-substituted-phenyl)azanediyl)dialkanols, and to methods for preparing said compounds and their use as fluorescence quenchers in cell imaging applications, diagnostic methods, fluorescent and non-fluorescent tags, pharmaceuticals, and other useful applications.

Description

[Technical Field]

[0001] The present invention relates to fluorescent quenchers. In particular, the present invention relates to substituted diazenylanilines, and their nucleic acid conjugates, complexes, and salts, which can be used as fluorescent quenchers in chemical and biological sciences, such as in cell imaging applications, diagnostic methods, fluorescent tags, non-fluorescent tags, pharmaceuticals, and other useful applications. The present invention also relates to the synthesis of substituted diazenylanilines, and their nucleic acid conjugates, complexes, and salts. More specifically, the present invention relates to 2,2'-((4-((2,5-disubstituted-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-2 / 3-substituted phenyl)azandiyl)dialkanol, a method for producing the compound, and the use of the compound as a fluorescent quencher in cell imaging applications, diagnostic methods, fluorescent tags, non-fluorescent tags, pharmaceuticals, and other useful applications. [Background technology]

[0002] Cost-effective technological developments for detecting and quantifying chemical and biological substances have spurred widespread technological innovation in the fields of pharmaceuticals, diagnostics, and instruments. It has become one of the leading areas for identifying substances, from the micro level to peptides, proteins, nucleic acids, and other pharmaceutically important substances. These studies are intricately intertwined with our lives, as they play a crucial role in disease identification and can help detect and / or quantify biologically important entities.

[0003] Methods for identifying diagnostically useful analytes are based on a specific set of their inherent characteristics that bind to them in chemical and biological environments. To date, many binding methods exist, including antigen-antibody interactions, protein-enzyme interactions, nucleic acid modification systems (Northern blotting), and labeling techniques. Various labeling methods have been developed for the rapid and efficient labeling of oligonucleotides on the bench, and these methods are useful not only for creating small amounts of detection probes but also when mutation analysis is required.

[0004] Because the synthesis of fluorescent labels is controllable, labels detectable using fluorescence spectroscopy are of great interest. By simply introducing various groups, they can be derivatized, allowing for the creation of a wide variety of fluorescent labels corresponding to various types of moieties. Furthermore, these are readily available commercially. This method, based on the ability of fluorescent compounds to transfer energy absorbed from light to neighboring molecules, is being used in the development of homogeneous methods for nucleic acid detection.

[0005] To achieve robust, highly sensitive, and sufficiently specific real-time nucleic acid amplification assays, the use of appropriate fluorescent dye and quencher labeling pairs is essential. This includes the type of hybridization probe used in the assay and the number of targets to be detected.

[0006] In their excited states, fluorescent dyes may lose excitation energy in some cases, in addition to emitting photon energy. This fluorescence quenching can occur due to molecular motion (dynamic quenching), complex formation with other substances in the excited state (photoquenching), contact quenching (static quenching), or energy transfer to another molecule (fluorescence resonance energy transfer or FRET). Many nucleic acid fluorescence detection techniques use fluorescently labeled probes that function by quenching the fluorescence of an adjacent second fluorescent label, or use fluorescent-quencher pairs. Probes double-labeled with a reporter dye and a quencher dye monitor any biochemical events by measuring changes in their fluorescence. These events cause changes in the distance between the reporter and the quencher, and these events result in observed changes in fluorescence (Non-Patent Literature 1).

[0007] Real-time nucleic acid amplification assays have a superior ability to obtain better qualitative and quantitative results. Furthermore, these assays can be performed in sealed tubes, thus avoiding contamination. Fluorescent nucleic acid hybridization probes contain various pairs of coordinating fluorescent dyes and quenchers. Some methods are based on pairs of complementary oligodeoxyribonucleotides, one of which remains as a probe for a single-stranded target sequence. The 5' end of one oligodeoxyribonucleotide is labeled with a donor fluorescent dye, and the 3' end of the other oligodeoxyribonucleotide is labeled with an acceptor fluorescent dye (Non-Patent Literature 2).

[0008] One of the key applications of probes containing reporter-quencher molecule pairs is their use in nucleic acid amplification reactions, such as polymerase chain reaction (PCR), to detect the presence and amplification of target nucleic acid sequences (Non-Patent Literature 3).

[0009] In the TaqMan assay, energy transfer efficiency decreases inversely with the sixth power of the distance between the reporter and the quencher; therefore, for the assay, it is preferable to position the donor and quencher at the 3'- and 5'-terminuses of the probe. Consequently, if the quencher is not close enough to the reporter to achieve the most efficient quenching, background luminescence originating from the probe may become very high (Non-Patent Literature 4).

[0010] Linear paired fluorescent-quencher probes are a standard tool for real-time PCR and are ideal as industrial marker standards for gene quantification in a wide range of applications due to their strong signal-to-noise ratio, low cost, and compatibility with different PCR techniques. Black hole quencher dyes BHQ0, BHQ1, BHQ2, and BHQ3 are used for quenching across the entire visible spectrum. FAM and BHQ dyes have demonstrated top-class performance, as reviewed in various scientific reports. TaqMan probes are used for quantitative real-time PCR analysis of gene expression, employing PCR primers as well as TaqMan probes with dye labeling (FAM) at the 5' end and minor groove binder (MGB) and non-fluorescent quencher (NFQ) / dark quencher at the 3' end. BHQ-1 is used to quench green and yellow dyes such as FAM, TET, and HEX. BHQ-2 and BHQ-3 have been reported to be used to quench orange or red pigments such as TAMRA, Texas Red, and Cy 5. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Chem. Commun., 2010, 46, 8154-8156 [Non-Patent Document 2] Nucleic Acids Research, 2002, Vol.30 No.21 e122 [Non-Patent Document 3] Acc. Chem. Res. 2011, 44, 2, 83-90 [Non-Patent Document 4] Nucleic Acids Res. 2011, 39, e112 [Overview of the Initiative]

[0012] (Purpose of the present invention) The main object of the present invention is to provide substituted diazenylanilines, as well as nucleic acid conjugates, complexes, and salts thereof, which can potentially be used as fluorescent quenchers in chemical and biological sciences, such as in cell imaging applications, diagnostic methods, fluorescent tags, non-fluorescent tags, pharmaceuticals, and other useful applications.

[0013] Another object of the present invention is to provide substituted diazenylanilines, as well as methods for producing nucleic acid conjugates, complexes, and salts thereof.

[0014] A further object of the present invention is to use substituted diazenylanilines, as well as their nucleic acid conjugates, complexes, and salts, in chemical and biological sciences, such as in cell imaging applications, diagnostic methods, fluorescent tags, non-fluorescent tags, pharmaceuticals, and other useful applications.

[0015] (Summary of the invention) Accordingly, the present invention relates to substituted diazenylanilines that can be potentially used as fluorescent quenchers in chemical and biological sciences such as cell imaging applications, diagnostic methods, fluorescent tags, non-fluorescent tags, pharmaceuticals and other useful applications, their synthesis and study of their fluorescent quenching properties, their nucleic acid conjugates, complexes and salts, and methods for producing the novel compounds.

[0016] Therefore, the present invention relates to general formula I: [ka] [wherein, R is independently selected from the group consisting of hydrogen and halogen; R1 and R2 are independently selected from the group consisting of hydrogen, substituted or unsubstituted (C1-C4) alkyl, and (C1-C6) alkoxy; R3 is selected from the group consisting of hydroxy, halogen, (C1-C6) alkoxy, substituted or unsubstituted (C1-C4) alkyl, thioalkyl (S C1-C6), methylamino, and dimethylamino; R4 is selected from the group consisting of hydrogen, hydroxy, halogen, (C1-C6) alkoxy, and substituted or unsubstituted (C1-C4) alkyl; m and n are selected from 0 to 3; Y1 and Y2 are independently selected from the group consisting of hydrogen, (C1-C6) alkyl, glycol, substituted or unsubstituted alkylaryl, oxoalkanoic acid, epoxy, N-hydroxysuccinimide ester, N-hydroxybenzotriazole ester, acid halide, acylimidazole, thioester, p-nitrophenyl ester, alkyl ester, mononucleotide unit, two or more mononucleotide units having or not having separate phosphate groups or polyphosphate groups linked by nucleoside groups] and its nucleic acid conjugates, complexes and salts are provided.

[0017] In a preferred embodiment of the present invention, the compound of formula I is selected from the following group: TIFF0007880419000002.tif212160

[0018] The present invention also provides a method for producing a compound of general formula I (wherein R, R1, R2, R3, R4, m, n, Y1 and Y2 are as defined above), and its nucleic acid conjugates, complexes and salts, comprising the following steps: [Chemical formula] a. A step of reacting a solution of unsubstituted or substituted 4-nitroaniline in HCl with a solution of sodium nitrite in distilled water at 0°C to form a diazonium salt, and then reacting it with substituted aniline to form the compound of formula S1; b. A step of reacting a substituted aniline mixture with a substituted alkyl halide in the presence of a base to form a compound of formula S2; c. A step of reacting the compound of formula S1 in HCl with a sodium nitrite solution to form a diazonium salt, and then reacting this with the compound of formula S-2 in the presence of NaOAc buffer to obtain a compound having general formula I; and d. The step of isolating the compound of general formula I from the reaction mixture and purifying it by washing with an organic solvent or by chromatography. The present invention provides a manufacturing method that includes the following:

[0019] In a preferred embodiment of the present invention, steps a to c of the above-described method are carried out for 1 minute to 3 days at a temperature range of 0°C to 100°C in the presence of an organic solvent selected from CH3CN, dimethyl sulfoxide, water, and tetrahydrofuran. In a preferred embodiment, the present invention is carried out in the following steps: [ka] a) A step of reacting compound S2 (Y1, Y2=OH, and m, n=1) in dried DCM with DMT-Cl in the presence of a base (DIPEA) under an inert atmosphere at room temperature to obtain compound S3; b) A step of reacting S3 with succinic anhydride and DMAP in an organic solvent for 12 to 48 hours to obtain compound S4; and c) A step of reacting the diazonium salt of S1 with S4 to obtain a compound having general formula I, This provides a method that includes [something].

[0020] The present invention also involves the following steps: [ka] a. A step in which the compound of formula S4 is coupled with the amine functional group of the solid support CPG beads of formula S5 to produce S6, and then the DMT group is deprotected to obtain S7; b. A step in which S7 is reacted with a nucleotide phosphoramidite to form oligonucleotide S8, and then 5'-modified with a hexynyl phosphoramidite to obtain product S9; c. A step of treating S9 with a base for cleaving oligonucleotides from a solid support to obtain S10; and d. A step of reacting S10 with a fluorescent dye azide to obtain an oligonucleotide probe of general formula S12. The present invention provides a method for producing a conjugate compound of general formula I (wherein Q is a compound of formula 1), including the compound Q.

[0021] The present invention provides compounds of general formula I that are useful for the analysis of nucleic acids (DNA, RNA), peptides, chemicals, pharmaceuticals, microorganisms, and other biological substances of diagnostic importance.

[0022] The present invention provides compounds of general formula I that are useful for developing diagnostic kits for detecting substances, hormones, pathogenic microorganisms and viruses, antibodies, and enzymes and nucleic acids, particularly those involved in disease conditions.

[0023] The present invention provides compounds of general formula I, which are useful in the manufacture of fluorescent probes, tags, markers, diagnostic agents, ion sensors, and pharmaceuticals for detecting / capturing ions in fluorescence-based imaging and / or analysis related to cells, body fluids, chemical mixtures, and / or other useful applications.

[0024] The present invention also provides compositions of a compound of general formula I with acetyl, azide, n-hydroxysuccinimide, oxoalkanoic acid, glycolate, thiol, amine, hydroxide, maleimide, tetrazine, phosphate, sodium salt, potassium salt, or phosphoramidite.

[0025] In preferred embodiments of the present invention, compounds of general formula I are useful for producing double-labeled probes and for single, double, and multiplex analysis of them in RT-PCR or other related detection systems.

[0026] In one embodiment of the present invention, the compound is useful as a fluorescent quencher in chemical and biological sciences.

[0027] In another embodiment of the present invention, the compound exhibits a broad quenching range between 450 nm and 700 nm.

[0028] Furthermore, the present invention provides compounds having general formula I that can potentially be used as fluorescent quenchers in chemical and biological sciences, such as for cell imaging applications, fluorescent and non-fluorescent tags, and other useful biological applications (e.g., the development of diagnostic kits). [Brief explanation of the drawing]

[0029] The following drawings form part of this specification and are included to further illustrate aspects of this disclosure. This disclosure may be better understood by referring to the drawings in conjunction with the detailed description of the specific embodiments shown herein: [Figure 1] Figure 1 shows the absorption spectra of newly synthesized quenching agent derivatives (1-9) according to embodiments of this disclosure. [Figure 2] Figure 2 shows a comparison of the absorption of BHQ-2 and CDRI-Q2 at a concentration of 20 μM in PCR buffer, according to an embodiment of the present disclosure. [Figure 3] Figure 3 shows the fluorescence quenching of 5-FAM in the presence of various concentrations of CDRI Q2(1) in PCR buffer, according to embodiments of the present disclosure. [Figure 4] Figure 4 shows the fluorescence quenching of CY3 in the presence of various concentrations of CDRI Q2(1) in PCR buffer, according to embodiments of the present disclosure. [Figure 5]Figure 5 shows the fluorescence quenching of 5-TAMRA in the presence of various concentrations of CDRI Q2(1) in PCR buffer, according to embodiments of the present disclosure. [Figure 6] Figure 6 shows the fluorescence quenching of CalFluor in the presence of various concentrations of CDRI Q2(1) in PCR buffer, according to embodiments of the present disclosure. [Figure 7] Figure 7 shows the fluorescence quenching of Texas Red in the presence of various concentrations of CDRI Q2(1) in PCR buffer, according to embodiments of the present disclosure. [Figure 8] Figure 8 shows the fluorescence quenching of Cy5 in the presence of various concentrations of CDRI Q2(1) in PCR buffer, according to embodiments of the present disclosure. [Figure 9] Figure 9 shows RT-PCR data with the cycle threshold (Ct) on the X-axis and RFU on the Y-axis, according to an embodiment of the present disclosure. [Figure 10] Figure 10 shows the detection of SARS-CoV-2 virus genes E and RdRp and the housekeeping gene RnaseP by multiplex RT-PCR using a positive control according to an embodiment of the present disclosure, where the data represent the cycle threshold (Ct) on the X axis and RFU on the Y axis. [Figure 11] Figure 11 shows the detection of SARS-CoV-2 viral genes E and RdRp and housekeeping gene RnaseP by multiplex RT-PCR using positive RNA samples from COVID-19 positive patients according to embodiments of the present disclosure; data represent cycle threshold (Ct) on the X axis and RFU on the Y axis.

[0030] (abbreviation) PCR (polymerase chain reaction) TDW (Triple-Distilled Water)

[0031] (Detailed description of the invention) Those skilled in the art will recognize that this disclosure is subject to modifications and alterations other than those specifically described. It should be understood that this disclosure includes all such modifications and alterations. This disclosure also encompasses all of the aforementioned processes, features, compositions, and compounds, individually or collectively, and any combination of one or more of the aforementioned processes or features.

[0032] The various aspects of the present invention can be better understood and appreciated by being described in detail in relation to certain preferred embodiments and optional embodiments.

[0033] For convenience, before further description of this disclosure, certain terms and examples used herein are given here. These definitions should be read in light of the rest of this disclosure and understood by those skilled in the art. The terms used herein have meanings that are recognized and known to those skilled in the art, but for convenience and completeness, certain terms and their meanings are given below.

[0034] (definition) The articles "a," "an," and "the" are used to indicate that the grammatical object of the article is one or more (i.e., at least one).

[0035] The terms "includes" and "contains" are used in an inclusive and open sense, meaning that additional elements may be included. The terms are not intended to be interpreted as "consisting of only."

[0036] Throughout this specification, unless otherwise specified in the context, the word “includes,” and variations such as “includes” and “contains,” are understood to mean including the element or process, or group of elements and processes, described, but not to mean excluding other elements or processes, or groups of elements or processes.

[0037] Accordingly, the present invention relates to substituted diazenylanilines that can be potentially used as fluorescent quenchers in chemical and biological sciences, such as for cell imaging applications, diagnostic methods, fluorescent tags, non-fluorescent tags, pharmaceuticals and other useful applications, as well as the synthesis and study of the fluorescent quenching properties of their nucleotide conjugates, complexes and salts, and methods for producing the novel compounds.

[0038] The term "quenching probe" refers to a quenching agent that can be used to quench and / or reduce fluorescence emission in various ultraviolet-visible regions in response to a specific analyte / substance.

[0039] This invention relates to formula I: [ka] [In the formula, R is selected from the group consisting of hydrogen and halogens; R1 and R2 are independently selected from the group consisting of hydrogen, substituted or unsubstituted (C1-C4) alkyl and (C1-C6) alkoxy; R3 is selected from the group consisting of hydroxy, halogen, (C1-C6) alkoxy, substituted or unsubstituted (C1-C4) alkyl, thioalkyl (SC1-C6), methylamino, and dimethylamino; R4 is selected from the group consisting of hydrogen, hydroxyl, halogen, (C1-C6) alkoxy and substituted or unsubstituted (C1-C4) alkyl; m and n are numbers independently selected from 0 to 3; Y1 and Y2 are independently selected from the group consisting of hydrogen, (C1-C6) alkyl, glycol, substituted or unsubstituted alkylaryl, oxoalkanoic acid, epoxy, N-hydroxysuccinimide ester, N-hydroxybenzotriazole ester, acid halide, acylimidazole, thioester, p-nitrophenyl ester, alkyl ester, phosphoramidite, mononucleotide units, and two or more mononucleotide units having or not having separate phosphate groups or polyphosphate groups linked by nucleoside groups. The compound is provided.

[0040] The following is a list of representative substituted diazenylanine compounds: TIFF0007880419000007.tif212160

[0041] Formula I (where R, R1, R2, R 3、 Scheme I shows a method for producing the compound (where R4, m, n, Y1, and Y2 are as defined above). [ka]

[0042] This method includes the following steps: a. A step of reacting a solution of unsubstituted or substituted 4-nitroaniline in HCl with a solution of sodium nitrite in distilled water at 0°C to form a diazonium salt, and then reacting it with substituted aniline to form a compound having general formula S1; b. A step of reacting a substituted aniline mixture with a substituted alkyl halide in the presence of a base to form a compound having the general formula S2; c. A step of reacting a compound of general formula S1 in HCl with a sodium nitrite solution to form a diazonium salt, and then reacting this with a compound of general formula S-2 in the presence of NaOAc buffer to obtain a compound having general formula I; and d. The process of isolating the compound of general formula I from the reaction mixture and purifying it by washing with an organic solvent or by chromatography.

[0043] The reaction is carried out in common organic solvents, particularly CH3CN, dimethyl sulfoxide, water, and tetrahydrofuran, at a temperature range of 0°C to 100°C for 1 minute to 3 days, depending on the reactants.

[0044] In another embodiment, the present invention relates to formula I (wherein R1, R2, R3 and R4 are as defined above; Y1, Y2 =H The present invention provides a method for producing a preferred compound having m, n=1, and this method is shown in Scheme II. [ka]

[0045] This method includes the following steps: a) A step of reacting a solution of unsubstituted or substituted 4-nitroaniline in HCl with a solution of sodium nitrite in distilled water at 0°C to form a diazonium salt, and then reacting it with substituted aniline to form a compound having general formula S1; b) A compound having the general formula S2 (wherein Y1, Y2=OH and m, n=1) in dried DCM is reacted with DMT-Cl at room temperature under an inert atmosphere in the presence of a base (DIPEA) to obtain product S3; c) A step of reacting the compound of general formula S3 with succinic anhydride and DMAP in an organic solvent for 12 to 48 hours to obtain compound S4; d) A step of reacting a compound of general formula S1 in HCl with a sodium nitrite solution to form a diazonium salt, and then reacting this with a compound having general formula S4 in the presence of a buffer (NaOAc) to obtain a compound having general formula I; and e) The step of isolating the compound of general formula I from the reaction mixture and purifying it by washing with an organic solvent or by chromatography.

[0046] The reaction is carried out in common organic solvents, particularly CH3CN, dimethyl sulfoxide, water, and tetrahydrofuran, in the presence of a buffer, at a temperature range of 0°C to 100°C, for 1 minute to 3 days depending on the reactants.

[0047] In one embodiment of the present invention, the compound is useful as a fluorescent quencher in chemical and biological sciences.

[0048] In another embodiment of the present invention, the compound exhibits a broad extinction range between 450 nm and 700 nm.

[0049] Furthermore, compounds having general formula I can potentially be used as fluorescent quenchers in chemical and biological sciences, such as for cell imaging applications, fluorescent and non-fluorescent tags, and other useful biological applications (e.g., development of diagnostic kits). [Examples]

[0050] The following examples are for illustrative purposes only and do not represent the scope of the present invention.

[0051] Example 1 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methoxyphenyl)azandiyl)bis(ethane-1-ol) (1) (CDRI-Q2) A solution of compound S2 (R3=OCH3, R4=H) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After the addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. Subsequently, the reaction mixture was filtered and washed with ACN and water (1:1) to obtain the product 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azandiyl)bis(ethane-1-ol) as a purple solid. MP = 205~206 °C, MS (ESI) m / z 525 [M+H] +, 1H NMR (400 MHz, DMSO-d6) δ 8.47 - 8.40 (m, 2H), 8.09 - 8.03 (m, 2H), 7.62 (d, J = 9.3 Hz, 1H), 7.44 (s, 1H), 7.28 (s, 1H), 6.48 (dd, J = 9.4, 2.5 Hz, 1H), 6.40 (d, J = 2.5 Hz, 1H), 5.03 - 4.80 (m, 2H), 3.99 (s, 3H), 3.97 (s, 3H), 3.94 (s, 3H), 3.68 - 3.57 (m, 8H). 13C NMR (101 MHz, DMSO) δ 160.61, 156.28, 154.27, 153.76, 150.62, 148.44, 147.60, 141.34, 134.36, 125.57, 123.88, 118.66, 105.80, 101.37, 100.20, 95.24, 79.64, 58.81, 56.98, 56.80, 56.49, 53.93.

[0052] Example 2 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-ethoxyphenyl)azandiyl)bis(ethane-1-ol) (2) A solution of compound S2 (R4=H, R3=OCH2CH3) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After the addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. The reaction mixture was then filtered and washed with ACN and water (1:1) to obtain the product 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-ethoxyphenyl)azandiyl)bis(ethane-1-ol) as a purple solid. MP= 202~203 ℃, MS (ESI) m / z 539 [M+H] +, 1H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.6 Hz, 2H), 8.05 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 9.3 Hz, 1H), 7.44 (s, 1H), 7.35 (s, 1H), 6.49 (d, J = 9.4 Hz, 1H), 6.44 - 6.35 (m, 1H), 4.88 (t, J = 5.1 Hz, 2H), 4.25 (q, J = 7.0 Hz, 2H), 3.98 (s, 3H), 3.94 (s, 3H), 3.68 - 3.56 (m, 8H), 1.44 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 160.12, 156.28, 154.25, 153.70, 150.63, 148.43, 147.58, 141.26, 134.36, 125.57, 123.88, 118.51, 106.06, 101.24, 100.27, 96.67, 65.10, 58.80, 56.81, 56.67, 53.91, 15.17.

[0053] Example 3 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-propoxyphenyl)azandiyl)bis(ethane-1-ol) (3) A solution of compound S2 (R4=H, R3=OCH2CH2CH3) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After the addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. The reaction mixture was then filtered and washed with ACN and water (1:1) to obtain the product 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-propoxyphenyl)azandiyl)bis(ethane-1-ol) as a purple solid. MP = 202~203 °C, MS (ESI) m / z 553 [M+H] +, 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.6 Hz, 2H), 8.11-7.90 (m, 2H), 7.62 (s, 1H), 7.45-7.19 (m, 2H), 6.74 - 6.31 (m, 2H), 4.41-4.11 (m, 2H), 4.10 - 3.59 (m, 16H), 1.92-1.80 (m, 2H), 1.19-1.02 (m, 3H).

[0054] Example 4 2,2'-((3-butoxy-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azandiyl)bis(ethane-1-ol) (4) A solution of compound S2 (R4=H, R3=OCH2CH2CH2CH3) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After the addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. The reaction mixture was then filtered and washed with ACN and water (1:1) to obtain the product 2,2'-((3-butoxy-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azandiyl)bis(ethane-1-ol) as a purple solid. MS (ESI) m / z 567 [M+H] + , 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.4 Hz, 2H), 8.10-7.90 (m, 2H), 7.61 (s, 1H), 7.79-7.22 (m, 2H), 6.77 - 6.22 (m, 2H), 4.12-3.64 (m, 2H), 4.12 - 3.64 (m, 16H), 2.01-1.75 (m, 2H), 1.67-1.46 (m, 2H), 1.12-0.88 (m, 3H).

[0055] Example 5 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-(hexyloxy)phenyl)azandiyl)bis(ethane-1-ol) (5) A solution of compound S2 (R4=H, R3=OCH2CH2CH2CH2CH2CH3) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After the addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. Subsequently, the reaction mixture was filtered and washed with ACN and water (1:1) to obtain the product 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-(hexyloxy)phenyl)azandiyl)bis(ethane-1-ol) as a purple solid. MP = 198~199 °C, MS (ESI) m / z 595 [M+H] + , 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.5 Hz, 2H), 8.11-7.92 (m, 2H), 7.68-7.53 (m, 1H), 7.45-7.21 (m, 2H), 6.75 - 6.30 (m, 2H), 4.47-4.11 (m, 2H), 4.08-3.60 (m, 16H), 1.99-1.76 (m, 2H), 1.63 - 1.44 (m, 2H), 1.40-1.27 (m, 4H), 0.87 (t, J = 6.5 Hz, 3H).

[0056] Example 6 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methylphenyl)azandiyl)bis(ethane-1-ol) (6) A solution of compound S2 (R4=H, R3=CH3) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. Subsequently, the reaction mixture was filtered and washed with ACN and water (1:1) to obtain the product 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methylphenyl)azandiyl)-bis(ethane-1-ol) as a purple solid. MP = 201~202 °C, MS (ESI) m / z 509 [M+H] + , 1H NMR (400 MHz, DMSO-d6) δ 8.48 - 8.39 (m, 2H), 8.10 - 8.01 (m, 2H), 7.64 (d, J = 9.9 Hz, 1H), 7.44 (s, 1H), 7.37 (s, 1H), 6.75-6.69 (m, 2H), 4.86 (t, J = 5.2 Hz, 2H), 4.00 (s, 3H), 3.95 (s, 3H), 3.65-3.55 (m, 8H), 2.67 (s, 3H). 13C NMR (101 MHz, DMSO) δ 156.22, 153.67, 152.25, 150.76, 148.49, 147.24, 142.54, 142.25, 141.54, 125.57, 123.91, 118.02, 112.68, 110.76, 101.35, 100.35, 58.72, 56.84, 56.80, 53.73, 18.48.

[0057] Example 7 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-2,5-dimethoxyphenyl)azandiyl)bis(ethane-1-ol)(7) A solution of compound S2 (R3=OCH3, R4=OCH3) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. Subsequently, the reaction mixture was filtered and washed with ACN and water (1:1) to obtain the product 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-2,5-dimethoxyphenyl)azandiyl)bis(ethane-1-ol) as a purple solid. MP=230~231 °C, MS (ESI) m / z 555 [M+H] + , 1H NMR (400 MHz, DMSO-d6) δ 8.32 (d, J = 8.8 Hz, 2H), 8.00 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 9.2 Hz, 1H), 7.53(s, 1H), 7.4 (s, 1H), 7.0 (d, J = 2.34 Hz, 1H), 6.66 (dd, J = 2.4, 9.2Hz, 1H), 4.03 (s, 3H), 4.0 (s, 3H), 3.85 (s, 3H), 3.75 (s, 3H), 3.52 (t, J = 4.95 Hz, 4H), 3.23 (t, J = 4.96 Hz, 4H).

[0058] Example 8 2,2'-((3-bromo-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azandiyl)bis(ethane-1-ol) (8) A solution of compound S2 (R4 = H, R3 = Br) in ACN was slowly added to a stirred mixture of the salt of S1 in a solution of NaOAc buffer and ACN (1:1) at 0 °C. After the addition, the reaction mixture was stirred at 0 °C for 30 minutes. The completion of the reaction was monitored by TLC. Then, the reaction mixture was filtered, washed with ACN and water (1:1), and the product 2,2'-((3-bromo-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azanediyl)bis(ethane-1-ol) was obtained as a purple solid. M.P. = 220~221 °C, MS (ESI) m / z 573 [M+H] + , 1H NMR (400 MHz, DMSO-d6) δ 8.32 (d, J = 8.8 Hz, 2H), 8.00 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 9.2 Hz, 1H), 7.53(s, 1H), 7.4 (s, 1H), 7.0 (d, J= 2.34 Hz, 1H), 6.66 (dd, J= 2.4, 9.2Hz, 1H), 4.03 (s, 3H), 4.0 (s, 3H), 3.82 (t, J= 4.95 Hz, 4H), 3.63 (t, J= 4.96 Hz, 4H).

[0059] Example 9 2,2'-((4-((4-((2,6-dichloro-4-nitrophenyl)diazenyl)-2,5-dimethoxyphenyl)diazenyl)-3-methoxyphenyl)azanediyl)bis(ethane-1-ol) (9) A solution of compound S2 (R3=OCH3, R4=H) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. Subsequently, the reaction mixture was filtered and washed with ACN and water (1:1) to obtain 2,2'-((4-((4-((2,6-dichloro-4-nitrophenyl)diazenyl)-2,5-dimethoxyphenyl)diazenyl)-3-methoxyphenyl)azandiyl)bis(ethane-1-ol) as a purple solid. MP = 189~190 °C, MS (ESI) m / z 592 [M+H] + , 1H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 2H), 7.65 (d, J = 9.38 Hz, 1H), 7.37 (s, 1H), 7.31 (s, 1H), 6.53 (d, J = 7.61 Hz, 1H), 6.40 (s, 1H), 4.0-3.94 (m, 6H), 3.92 (s, 3H), 3.69-3.59 (m,8H).

[0060] Example 10 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-hydroxyphenyl)azandiyl)bis(ethane-1-ol) (10) A solution of compound S2 (R3=OH, R4=H) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After the addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. The reaction mixture was then filtered and washed with ACN and water (1:1) to obtain the product 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-hydroxyphenyl)azandiyl)bis(ethane-1-ol) as a purple solid. MP = 203~204 °C, MS (ESI) m / z 551[M+H] +, 1H NMR (400 MHz, DMSO-d6) δ 8.47 - 8.40 (m, 2H), 8.09 - 8.03 (m, 2H), 7.62 (d, J = 9.3 Hz, 1H), 7.44 (s, 1H), 7.28 (s, 1H), 6.48 (dd, J = 9.4, 2.5 Hz, 1H), 6.40 (d, J = 2.5 Hz, 1H), 5.03 - 4.80 (m, 2H), 3.99 (s, 3H), 3.97 (s, 3H), 3.68 - 3.57 (m, 8H).

[0061] Example 11 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methoxyphenyl)amino)ethoxy)-4-oxobutanoic acid (11) A solution of compound S4 (R4=H, R3=OCH3) in ACN was slowly added at 0°C to a stirred mixture of the salt of S1 in NaOAc buffer and ACN (1:1) solution. After addition, the reaction mixture was stirred at 0°C for 30 minutes. The completion of the reaction was monitored by TLC. Subsequently, the reaction mixture was filtered and washed with ACN and water (1:1) to obtain 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methoxyphenyl)amino)ethoxy)-4-oxobutanoic acid as a purple solid. MP = 208~209°C, MS (ESI) m / z 927 [M+H] +, 1H NMR (400 MHz, DMSO-d6) δ 12.1 (brs, 1H), 8.44 (d, J = 8.8 Hz, 2H), 8.06 (d, J = 8.8 Hz, 2H), 7.59-7.57 (m, 1H), 7.44 (s, 1H), 7.36-7.34 (m, 2H), 7.28-7.20 (m, 7H), 7.08 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 9.2 Hz, 4H), 6.47-6.45 (m, 1H), 6.17-6.22-6.20 (m, 1H), 4.29-4.27 (m, 2H), 3.99-3.94 (m, 9H), 3.83-3.68 (m, 10H), 3.28-3.25 (m, 2H), 2.52-2.41 (m, 4H).

[0062] Photophysical testing of compounds of general formula I The photophysical properties of all synthesized compounds 1-6 were tested by UV-vis absorption analysis. Table 1 shows the absorption maxima and quenching range in PCR buffer (pH 7.2). Table 1. Photophysical characteristics of Examples 1-9 [Table 1]

[0063] All of the quenchers synthesized in representative Examples 1-9 showed broad absorption spectra in PCR buffer at room temperature. The absorption spectra of the newly synthesized quencher derivatives (1-9) are shown in Figure 1. Compounds 1 and 2 showed good absorption intensity in the range of 400-750 nm. Similarly, increasing the methylene units of the alkoxy group of general formula I (R4=H, R3=OC1-C6) resulted in a decrease in intensity (Figure 1).

[0064] Comparison of the known quencher BHQ-2 and the novel quencher CDRI-Q2 of the present invention The absorption spectrum of the quencher CDRI-Q2 of the present invention (Example 1) was compared with that of a known commercially available quencher BHQ-2. This data suggests that the quencher CDRI-Q2 of the present invention exhibits broader absorption and better intensity compared to BHQ-2 (Figure 2).

[0065] Extinction study of CDRI-Q2 in the presence of various fluorescent dyes The compound CDRI-Q2 of the present invention effectively quenches the fluorescent dye 5-FAM, which exhibits a maximum emission at 517 nm in PCR buffer (Figure 3).

[0066] The compound CDRI-Q2 of the present invention effectively quenches the fluorescent dye Cy-3, which exhibits a maximum emission at 566 nm in PCR buffer (Figure 4).

[0067] The compound CDRI-Q2 of the present invention effectively quenches the fluorescent dye 5-TAMRA, which exhibits a maximum emission at 583 nm in PCR buffer (Figure 5).

[0068] The compound CDRI-Q2 of the present invention effectively quenches the fluorescent dye CalFluor red, which exhibits an emission maximum at 601 nm in PCR buffer (Figure 6).

[0069] The compound CDRI-Q2 of the present invention effectively quenches the fluorescent dye Texas Red, which exhibits an emission maximum at 603 nm in PCR buffer (Figure 7).

[0070] The compound CDRI-Q2 of the present invention effectively quenches the fluorescent dye Cy-5, which exhibits a maximum emission at 662 nm in PCR buffer (Figure 8).

[0071] Synthesis of dual-labeled probes A mixture of a fluorescent quencher of general formula I containing an acid at the end (3 equivalents), DMAP (0.05 equivalents), triethylamine (13 equivalents), DEC / EDC (10 equivalents), and anhydrous pyridine (2 mL) was mixed with free amine-containing Controlled Pore Glass (CPG beads, 1000 A), and the mixture was shaken at room temperature for 24 hours. The solvent was removed by suction filtration, and the mixture was washed sequentially with pyridine and DCM, and dried under vacuum for several hours. The coupling efficiency was then determined using the detritylation method.

[0072] Fluorescent dye labeling of the 5'-terminus of 3'-quencher-tagged oligonucleotides: We prepared probes for RT-PCR-based diagnostics of COVID-19. CPG-amine (1000 Angs) was tagged with CDRI Q2, a representative novel quencher of general formula I. Oligonucleotides were synthesized using CPG-CDRI-Q2 as described above. Table 2 shows oligonucleotide sequences corresponding to different genes E and RdRp from one host and two viruses (however, not limited to these viral genes). Using solid-phase synthesis with a phosphoramidite system, a hexinyl group was introduced to the 5'-terminus of each 3'-CDRI Q2-tagged oligonucleotide using hexinyl phosphoramidite. Commercially available and in-house synthesized fluorescent dyes (azides) were used.

[0073] Table 2: Oligonucleotide sequences of probes for RNaseP (host) and the E and RdRP genes of the virus [Table 2]

[0074] The azide of the fluorescent dye was bonded to hexynyl oligonucleotide-3'-CDRI Q2 via a copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction (also known as copper-catalyzed alkyne azide cycloaddition reaction (CuAAC)). The copper-catalyzed reaction allows for the specific synthesis of 1,4-disubstituted positional isomers.

[0075] Advantages of using fluorescent dye azide instead of fluorescent dye phosphoramidite Generally, dual-labeled probes are produced using phosphoramidite chemistry by conjugating a fluorescent dye to the 5'-terminus of an oligonucleotide containing a 3'-quencher. These phosphoramidites of fluorescent dyes are stored at -20°C, are unstable at room temperature, and are sensitive to moisture. In this invention, a fluorescent dye azide that is stable at room temperature and essentially non-hygroscopic was used. Triazole-based dual-labeled oligonucleotides with different viral gene sequences (E gene, RdRp, and human gene RNaseP) have not been used for the detection of SARS-CoV-2 or related viral infections using RT-PCR technology. The results of triple RT-PCR experiments are described in Figures 9-11 of the drawings attached to the specification. Details of the different fluorescent dye azides used for labeling are shown in Table 3.

[0076] Table 3: Photophysical properties of different fluorescent dye azides [Table 3]

[0077] Table 4 shows the reagents and stock solution concentrations used in the conjugate chemistry. [Table 4]

[0078] A 500 μM stock solution of 5'-modified oligonucleotide was prepared in nuclease-free water (NFW) (Sigma Cat no.), and a 10 mM stock solution of fluorescent dye was prepared in molecular biological grade DMSO (Sigma).

[0079] For a 100 μL CuAAC reaction, a 50 μM alkyned oligonucleotide solution (10 μL from a 500 μM stock solution in NFW) was sequentially treated with 0.2 M triethylammonium acetate buffer, pH 7.0 (Sigma), followed by the addition of 50 μL of DMSO. The reaction mixture was thoroughly mixed by vortexing. A 150 μM fluorescent dye azide solution (1.5 μL from a 10 mM stock solution in DMSO) and a freshly prepared 0.5 mM ascorbic acid solution in NFW were further added to the reaction mixture, and each reagent was thoroughly mixed after its addition. The reaction mixture was then completely degassed and flushed with argon for approximately 60 seconds. 0.5 mM copper(II)-tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine complex (Cu-TBTA complex, prepared by mixing 5 mg / mL copper(II) sulfate pentahydrate and 10.5 mg / mL TBTA in 55% DMSO) was added to the reaction mixture, thoroughly mixed by vortexing, and then flushed with argon for 60–100 seconds. The reaction mixture was incubated at 22°C for 12–16 hours. After the reaction was complete, 3 volumes of cold acetone were added to precipitate the mixture, and it was stored at -20°C for 30 minutes. Labeled DNA was extracted from the mixture by high-speed centrifugation at 10,000 rpm for 20 minutes at 4°C.

[0080] The pellet obtained in this process was washed twice with cold acetone (1 mL). After the final wash, the pellet was dried by further incubation of the tube at 22°C for approximately 30 minutes. The dried fluorescent dye-labeled oligonucleotide obtained in this process was resuspended in 45 μL of cold NFW for HPLC purification.

[0081] The labeled oligonucleotides were analyzed and purified using HPLC on a Shimadzu dual-pump system equipped with an XTerra MS C18 column (75 x 4.6 mm, packed with 2.5 μm particles, mean pore size 125 Å) along with an Inertsil C4 5 μm guard column (4.0 x 10 mm), a 20 μL sample loop, an RF-20A spectrofluoroscopy detector, and an SPD-10A UV-VIS detector. The fluorescence detector was set to the excitation and emission wavelengths corresponding to the target fluorescent dye, and the UV detector was set to wavelengths of 260 nm and 280 nm. The mobile phase consisted of 0.1 M triethylammonium acetate buffer (pH 7.0, Sigma) and acetonitrile (HPLC grade, Sigma). Oligonucleotides were separated by flowing a 0-60% acetonitrile gradient through the column at a flow rate of 1 mL / min over 30 minutes. Peaks corresponding to both fluorescence and UV detection were manually collected and stored at -20°C. These preserved samples were frozen in liquid nitrogen and lyophilized at 0.08 mbar and -51°C using a CHRiST freeze-drying system. The lyophilized probes were stored at -20°C and used for RT-PCR to detect each gene.

[0082] Demonstration of the application of quencher agents in RT-PCR-based diagnostic methods for SARS-CoV-2 infection. Assay method 1) Extract RNA using a commercially available kit. In the RT-PCR reaction, the first strand of cDNA is synthesized from the RNA molecule using the RT-mix in a single tube, followed by PCR amplification and detection using specific primers and probes. Depending on the amount of target RNA, the template concentration can be used in the range of 0.5 pg to 0.5 μg. Alternatively, this reaction can be performed separately by cDNA synthesis (0.5 μg to 2 μg). The cDNA can be diluted 3 to 5 times and used for PCR amplification and detection using specific primers and probes; 2) The setup and cycling protocol for the real-time PCR reaction shall follow the manufacturer's protocol. Primer (forward and reverse) and probe concentrations can be used from 0.2 μM to 1 μM; and 3) Fluorescent dye-quencher combinations are suitable for detecting target genes in various real-time PCR instruments (ABI, BioRad) that utilize fluorescent dye-specific channels.

[0083] Figure 9 shows RT-PCR data with the cycle threshold (Ct) on the X-axis and RFU on the Y-axis. FAM-RnaseP-BHQ1 (IDT) was used as a positive control. The probe used for detection had CDRI-Q2 at the 3' end and FAM / Texas Red (TR) / Cy5 at the 5' end. This data demonstrates the quenching suitability of CDRI-Q2 in various ranges (520 nm to 670 nm) for accurate RT-PCR detection.

[0084] Multiplex RT-PCR detection of SARS-CoV-2 virus genes E and RdRp, as well as the housekeeping gene RnaseP, was performed using a positive control. The data are shown in Figure 10, with the cycle threshold (Ct) on the X axis and RFU on the Y axis. The data are shown in Figure 10, with the cycle threshold (Ct) on the X axis and RFU on the Y axis. This data demonstrates the quenching compatibility of CDRI-Q2 across various emission ranges (450 nm to 700 nm) of RT-PCR.

[0085] Figure 11 shows the detection of SARS-CoV-2 viral genes E and RdRp, as well as the housekeeping gene RnaseP, by multiplex RT-PCR using positive RNA samples from COVID-19 positive patients; the data shows the cycle threshold (Ct) on the X axis and RFU on the Y axis. FAM-E-CDRI-Q2, TR-RdRp-CDRI-Q2, and Cy5-RnaseP-CDRI-Q2 probes were used. This data demonstrates the quenching compatibility of CDRI-Q2 across various emission ranges (450 nm to 700 nm) of RT-PCR.

[0086] Advantages of the present invention The present invention provides a family of non-fluorescent quenchers with excellent excited state energy, which are well-defined modified quenchers of the known BHQ-2 ("black hole quencher"). According to the literature, BHQ-2 is suitable for dyes that emit in the orange to red region of the visible spectrum (560-670 nm), but is not suitable for FAM. For FAM, it is preferable to use BHQ-1. The present invention provides universal quenchers functionalized to rapidly bind to probe components, and also provides quenchers designed to have a desired broad quenching range covering the entire visible spectrum. The present invention describes the use of azides of fluorescent dyes that are stable at room temperature and are essentially non-hygroscopic.

[0087] Quenching agents may consist of electron-donating and electron-withdrawing groups linked by a π-conjugated network. By altering the conjugation system of the quenching agent and / or incorporating electron-donating and electron-withdrawing groups onto an aromatic skeleton, the spectral properties (e.g., absorbance) can be "tuned" to match the spectral properties (e.g., emission) of one or more fluorescent dyes. The novel quenching agents of the present invention exhibited broad absorption spectra covering the entire visible color gamut. These quenching agents can be used to quench various fluorescent dyes that emit in the 500-750 nm range, such as FAM, cyanine dyes, Texas Red, Calfluor Red, and other fluorescent dyes. Furthermore, they exhibit superior quenching properties (e.g., higher absorbance) compared to other well-known BHQ dyes. Furthermore, this application also encompasses the following aspects. [Aspect 1] General formula I: [C1] JPEG0007880419000014.jpg51131 [In the formula, R is independently selected from the group consisting of hydrogen and halogens; R 1 and R 2 (C) is hydrogen, substituted or unsubstituted 1 -C 4 )alkyl and (C1 -C 6 ) Independently selected from the group consisting of alkoxys; R 3 is hydroxy, halogen, (C 1 -C 6 ) alkoxy, substituted or unsubstituted (C 1 -C 4 ) alkyl, thioalkyl (SC 1 -C 6 ), selected from the group consisting of methylamino and dimethylamino; R 4 (C) 1 -C 6 )alkoxy and substituted or unsubstituted (C 1 -C 4 Selected from the group consisting of alkyl groups; m and n are independently selected from 0 to 3; Y 1 and Y 2 is hydrogen, (C 1 -C 6 [Independently selected from the group consisting of alkyl groups, glycols, substituted or unsubstituted alkylaryls, oxoalkanoates, epoxys, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl esters, phosphoramidites, mononucleotide units, and two or more mononucleotide units having or not having separate phosphate groups or polyphosphate groups linked by nucleoside groups] Compounds thereof, as well as nucleic acid conjugates, complexes, and salts thereof. [Aspect 2] The following compound: i. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methoxyphenyl)azandiyl)bis(ethane-1-ol) (1); ii. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-ethoxyphenyl)azandiyl)bis(ethane-1-ol) (2); iii. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-propoxyphenyl)azandiyl)bis(ethane-1-ol) (3); iv. 2,2'-((3-butoxy-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azandiyl)bis(ethane-1-ol) (4); v. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-(hexyloxy)phenyl)azandiyl)bis(ethane-1-ol) (5); vi. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methylphenyl)azandiyl)bis(ethane-1-ol) (6); vii. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-2,5-dimethoxyphenyl)azandiyl)bis(ethane-1-ol) (7); viii. 2,2'-((3-bromo-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azandiyl)bis(ethane-1-ol) (8); ix. 2,2'-((4-((4-((2,6-dichloro-4-nitrophenyl)diazenyl)-2,5-dimethoxyphenyl)diazenyl)-3-methoxyphenyl)azandiyl)bis(ethane-1-ol) (9); x. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-hydroxyphenyl)azandiyl)bis(ethane-1-ol) (10); xi. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methoxyphenyl)amino)ethoxy)-4-oxobutanoic acid (11); xii. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-ethoxyphenyl)amino)ethoxy)-4-oxobutanoic acid (12); xiii. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-propoxyphenyl)amino)ethoxy)-4-oxobutanoic acid (13); xiv. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(3-butoxy-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)amino)ethoxy)-4-oxobutanoic acid (14); xv. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-(hexyloxy)phenyl)amino)-ethoxy)-4-oxobutanoic acid (15); and xvi. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methylphenyl)amino)-ethoxy)-4-oxobutanoic acid (16), A compound according to embodiment 1, selected from the group consisting of the following. [Aspect 3] The compound according to embodiment 1, wherein the nucleic acid conjugate contains a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3. [Aspect 4] General formula I: [Case 2] JPEG0007880419000015.jpg45114 [In the formula, R is selected from the group consisting of hydrogen and halogens; R 1 and R 2 (C) is hydrogen, substituted or unsubstituted 1 -C 4 )alkyl and (C 1 -C 6 ) Independently selected from the group consisting of alkoxys; R 3 is hydroxy, halogen, (C 1 -C 6 ) alkoxy, substituted or unsubstituted (C 1 -C 4 ) alkyl, thioalkyl (SC 1 -C 6 ), selected from the group consisting of methylamino and dimethylamino; R 4 (C) 1 -C 6 )alkoxy and substituted or unsubstituted (C 1 -C 4 Selected from the group consisting of alkyl groups; m and n are independently selected from 0 to 3; Y 1 and Y 2 is hydrogen, (C 1 -C 6 [Independently selected from the group consisting of alkyl groups, glycols, substituted or unsubstituted alkylaryls, oxoalkanoates, epoxy, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl esters, alkenyl esters, alkynyl esters, aromatic esters, phosphoramidites, mononucleotide units, and two or more mononucleotide units having or not having separate phosphate groups or polyphosphate groups linked by nucleoside groups] A method for producing the compound, as well as its nucleic acid conjugate, complex, and salt, comprising the following steps: [C3] JPEG0007880419000016.jpg92160 a. A step of reacting a solution of unsubstituted or substituted 4-nitroaniline in HCl with a solution of sodium nitrite in distilled water to form a diazonium salt, and then reacting it with substituted aniline to form a compound having general formula S1; b. A step of reacting a substituted aniline mixture with a substituted alkyl halide in the presence of a base to form a compound having the general formula S2; c. A step of reacting a compound having general formula S1 in HCl with a sodium nitrite solution to form a diazonium salt, and then reacting this with a compound having general formula S2 in the presence of NaOAc buffer to obtain a compound having general formula I; and d. The step of isolating the compound of general formula I from the reaction mixture and purifying it by washing with an organic solvent or by chromatography. A manufacturing method that includes this. [Aspect 5] Processes a to c are CH 3 The method for producing the product according to embodiment 4, which is carried out for 1 minute to 3 days at a temperature range of 0 to 100°C in the presence of an organic solvent selected from CN, dimethyl sulfoxide, water, and tetrahydrofuran. [Aspect 6] The method involves the following steps: [C4] JPEG0007880419000017.jpg83161 a) Compound S2(Y) in dried DCM 1 、Y 2 The process involves reacting a compound (where =H and m, n=1) with DMT-Cl at room temperature in an inert atmosphere in the presence of a base (DIPEA) to obtain compound S3; b) A step of reacting S3 with succinic anhydride and DMAP in an organic solvent for 12 to 48 hours to obtain compound S4; and c) A step of reacting the diazonium salt of S1 with S4 to obtain a compound having general formula I, The method according to embodiment 3, including the method described in embodiment 3. [Aspect 7] Q is a method for producing a conjugate compound of general formula I, which is a quenching compound of formula 1, and the steps are as follows: [5] JPEG0007880419000018.jpg149164 a. A step in which the compound of formula S4 is coupled with the amine functional group of the solid support CPG beads of formula S5 to produce S6, and then the DMT group is deprotected to obtain S7; b. A step in which S7 is reacted with a nucleotide phosphoramidite to form oligonucleotide S8, and then 5'-modified with a hexynyl phosphoramidite to obtain product S9; c. A step of treating S9 with a base to cleave oligonucleotides from a solid support to obtain S10; and d. A step of reacting S10 with a fluorescent dye azide to obtain an oligonucleotide probe of general formula S12. A manufacturing method that includes this. [Aspect 8] The compound according to embodiment 1, wherein the compound is used in combination with acetyl, azide, oxoalkanoic acid, epoxy, N-hydroxysuccinimide ester, N-hydroxybenzotriazole ester, acid halide, acylimidazole, thioester, p-nitrophenyl ester, alkyl ester, phosphoramidite, mononucleotide unit, or two or more mononucleotide units having or not having separate phosphate groups or polyphosphate groups linked by nucleoside groups. [Aspect 9] A method for detecting nucleic acids (DNA, RNA), peptides, chemical substances, pharmaceuticals, microorganisms, and other diagnostically important biological substances using the compound described in Embodiment 1. [Aspect 10] A method for detecting substances, hormones, pathogenic microorganisms, viruses, antibodies, enzymes, and nucleic acids using the compound described in Embodiment 1. [Aspect 11] The compound according to Embodiment 1, wherein the compound is useful for preparing single-labeled or double-labeled probes and analyzing them in single, double, and multiple RT-PCR or other relevant detection systems.

Claims

1. General formula I: 【Chemistry 1】 [In the formula, R is independently selected from the group consisting of hydrogen and halogens; R 1 and R 2 (C) is hydrogen, substituted or unsubstituted 1 -C 4 )alkyl and (C 1 or C 3-6 ) Independently selected from the group consisting of alkoxys; R 3 is selected from the group consisting of hydroxy, halogen, (C 1 -C 6 ) alkoxy, substituted or unsubstituted (C 1 -C 4 ) alkyl, thioalkyl (SC 1 -C 6 ), methylamino and dimethylamino; R 4 (C) 1 -C 6 )alkoxy and substituted or unsubstituted (C 1 -C 4 Selected from the group consisting of alkyl groups; m and n are independently selected from 0 to 3; Y 1 and Y 2 is hydrogen, (C 1 -C 6 [Independently selected from the group consisting of alkyl groups, glycols, substituted or unsubstituted alkylaryls, oxoalkanoates, epoxys, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl esters, phosphoramidites, mononucleotide units, and two or more mononucleotide units having or not having separate phosphate groups or polyphosphate groups linked by nucleoside groups] Compounds thereof, or nucleic acid conjugates, complexes, or salts thereof.

2. i. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methoxyphenyl)azandiyl)bis(ethane-1-ol) (1); ii. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-ethoxyphenyl)azandiyl)bis(ethane-1-ol) (2); iii. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-propoxyphenyl)azandiyl)bis(ethane-1-ol) (3); iv. 2,2'-((3-butoxy-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azandiyl)bis(ethane-1-ol) (4); v. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-(hexyloxy)phenyl)azandiyl)bis(ethane-1-ol) (5); vi. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methylphenyl)azandiyl)bis(ethane-1-ol) (6); vii. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-2,5-dimethoxyphenyl)azandiyl)bis(ethane-1-ol) (7); viii. 2,2'-((3-bromo-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)azandiyl)bis(ethane-1-ol) (8); ix. 2,2'-((4-((4-((2,6-dichloro-4-nitrophenyl)diazenyl)-2,5-dimethoxyphenyl)diazenyl)-3-methoxyphenyl)azandiyl)bis(ethane-1-ol) (9); x. 2,2'-((4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-hydroxyphenyl)azandiyl)bis(ethane-1-ol) (10); xi. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methoxyphenyl)amino)ethoxy)-4-oxobutanoic acid (11); xii. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-ethoxyphenyl)amino)ethoxy)-4-oxobutanoic acid (12); xiii. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-propoxyphenyl)amino)ethoxy)-4-oxobutanoic acid (13); xiv. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(3-butoxy-4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)amino)ethoxy)-4-oxobutanoic acid (14); xv. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-(hexyloxy)phenyl)amino)-ethoxy)-4-oxobutanoic acid (15); and xvi. 4-(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(4-((2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)phenyl)diazenyl)-3-methylphenyl)amino)-ethoxy)-4-oxobutanoic acid (16), The compound according to claim 1, or a nucleic acid conjugate, complex, or salt thereof, selected from the group consisting of such nucleic acid conjugates, complexes, and salts.

3. A nucleic acid conjugate of the compound according to claim 1, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

4. General formula I: 【Chemistry 2】 [In the formula, R is selected from the group consisting of hydrogen and halogens; R 1 and R 2 (C) is hydrogen, substituted or unsubstituted 1 -C 4 )alkyl and (C 1 or C 3-6 ) Independently selected from the group consisting of alkoxys; R 3 is hydroxy, halogen, (C 1 -C 6 ) alkoxy, substituted or unsubstituted (C 1 -C 4 ) alkyl, thioalkyl (SC 1 -C 6 ), selected from the group consisting of methylamino and dimethylamino; R 4 (C) 1 -C 6 )alkoxy and substituted or unsubstituted (C 1 -C 4 Selected from the group consisting of alkyl groups; m and n are independently selected from 0 to 3; Y 1 and Y 2 is hydrogen, (C 1 -C 6 [Independently selected from the group consisting of alkyl groups, glycols, substituted or unsubstituted alkylaryls, oxoalkanoates, epoxys, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl esters, phosphoramidites, mononucleotide units, and two or more mononucleotide units having or not having separate phosphate groups or polyphosphate groups linked by nucleoside groups] A method for producing the compound, or its nucleic acid conjugate, complex, or salt, comprising the following steps: 【Transformation 3】 a. A step of reacting a solution of unsubstituted or substituted 4-nitroaniline in HCl with a solution of sodium nitrite in distilled water to form a diazonium salt, and then reacting it with substituted aniline to form a compound having general formula S1; b. A step of reacting a substituted aniline mixture with a substituted alkyl halide in the presence of a base to form a compound having the general formula S2; c. A step of reacting a compound having general formula S1 in HCl with a sodium nitrite solution to form a diazonium salt, and then reacting this with a compound having general formula S2 in the presence of NaOAc buffer to obtain a compound having general formula I; and d. The step of isolating the compound of general formula I from the reaction mixture and purifying it by washing with an organic solvent or by chromatography. A manufacturing method that includes this.

5. Processes a to c are CH 3 The method for producing the product according to claim 4, which is carried out for 1 minute to 3 days at a temperature range of 0 to 100°C in the presence of an organic solvent selected from CN, dimethyl sulfoxide, water, and tetrahydrofuran.

6. General formula I: 【Chemistry 4】 [In the formula, R is selected from the group consisting of hydrogen and halogens; R 1 and R 2 (C) is hydrogen, substituted or unsubstituted 1 -C 4 )alkyl and (C 1 or C 3-6 ) Independently selected from the group consisting of alkoxys; R 3 is hydroxy, halogen, (C 1 -C 6 ) alkoxy, substituted or unsubstituted (C 1 -C 4 ) alkyl, thioalkyl (SC 1 -C 6 ), selected from the group consisting of methylamino and dimethylamino; R 4 (C) 1 -C 6 )alkoxy and substituted or unsubstituted (C 1 -C 4 [Selected from the group consisting of alkyl groups] A method for producing the compound, or its nucleic acid conjugate, complex, or salt, comprising the following steps: 【Transformation 5】 a) Compound S2(Y) in dried DCM 1 , Y 2 The process involves reacting a compound (where =H and m, n = 1) with DMT-Cl in the presence of a base (DIPEA) under an inert atmosphere at room temperature to obtain compound S3; b) A step of reacting S3 with succinic anhydride and DMAP in an organic solvent for 12 to 48 hours to obtain compound S4; and c) A step of reacting the diazonium salt of S1 with S4 to obtain a compound having general formula I, A manufacturing method that includes this.

7. Oligonucleotides and general formula I: 【Transformation 6】 [In the formula, R is selected from the group consisting of hydrogen and halogens; R 1 and R 2 (C) is hydrogen, substituted or unsubstituted 1 -C 4 )alkyl and (C 1 or C 3-6 ) Independently selected from the group consisting of alkoxys; R 3 is hydroxy, halogen, (C 1 -C 6 ) alkoxy, substituted or unsubstituted (C 1 -C 4 ) alkyl, thioalkyl (SC 1 -C 6 ), selected from the group consisting of methylamino and dimethylamino; R 4 (C) 1 -C 6 )alkoxy and substituted or unsubstituted (C 1 -C 4 [Selected from the group consisting of alkyl groups] A method for producing a conjugate with a compound, comprising the following steps: 【Transformation 7】 [In the scheme, Q represents the quenched portion of the compound of general formula I.] a. A step in which the compound of formula S4 is coupled with the amine functional group of the solid support CPG beads of formula S5 to produce S6, and then the DMT group is deprotected to obtain S7; b. A step in which S7 is reacted with a nucleotide phosphoramidite to form oligonucleotide S8, and then 5'-modified with a hexynyl phosphoramidite to obtain product S9; c. A step of treating S9 with a base to cleave oligonucleotides from a solid support to obtain S10; and d. A step of reacting S10 with a fluorescent dye azide to obtain an oligonucleotide probe of general formula S12. A manufacturing method that includes this.

8. The compound according to claim 1, or its nucleic acid conjugate, complex, or salt, functionalized with acetyl, azide, oxoalkanoic acid, epoxy, N-hydroxysuccinimide ester, N-hydroxybenzotriazole ester, acid halide, acylimidazole, thioester, p-nitrophenyl ester, alkyl ester, phosphoramidite, mononucleotide unit, or two or more mononucleotide units having or not having separate phosphate groups or polyphosphate groups linked by nucleoside groups.

9. A composition for detecting nucleic acids (DNA, RNA), peptides, chemicals, pharmaceuticals, microorganisms, and other diagnostically important biological substances, comprising a compound according to any one of claims 1 to 3 and 8 or its nucleic acid conjugate, complex, or salt thereof.

10. A composition for detecting substances, hormones, pathogenic microorganisms, viruses, antibodies, enzymes, and nucleic acids, comprising a compound according to any one of claims 1 to 3 and 8 or its nucleic acid conjugate, complex, or salt.

11. A composition for preparing single-labeled or double-labeled probes comprising a compound or its nucleic acid conjugate, complex, or salt according to any one of claims 1 to 3 and 8, and for analyzing them in single, double, and multiple RT-PCR or other relevant detection systems.

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