Non-fluorescent compounds and their uses

Abstract

This invention relates to non-fluorescent compounds useful as quenchers for fluorescent compounds that exhibit luminescence properties at excitation energy levels, and to various applications thereof.

Description

[Technical Field]

[0001] This invention relates to non-fluorescent compounds useful as quenchers for fluorescent compounds that exhibit luminescence properties at excitation energy levels, and to various applications thereof. [Background technology]

[0002] A quencher is a non-fluorescent compound that can quench the emission properties of a fluorescent compound in a specific wavelength range.

[0003] The fluorescence quenching caused by the nonfluorescent compound and / or the chemical moiety contained in the nonfluorescent compound can be manifested through various mechanisms such as static quenching or dynamic quenching.

[0004] Typical examples of static quenching include ground state quenching, while typical examples of dynamic quenching include fluorescence resonance energy transfer (FRET) and photo-induced electron transfer.

[0005] Of the aforementioned mechanisms of quenching, the most common is FRET, which can occur when the emission spectrum of a fluorescent compound and the absorption spectrum of a quencher overlap, and the fluorescent compound and the quencher are within a sufficient distance (the so-called Forster distance) to exhibit quenching. In other words, since FRET quenching occurs in the region where the emission spectrum of the fluorescent compound and the absorption spectrum of the quencher overlap in wavelength, it is difficult for a single quencher designed to induce FRET quenching to exhibit uniform quenching characteristics across a wide wavelength band. Therefore, a single quencher designed to induce FRET quenching generally exhibits quenching characteristics limited to a narrow wavelength band, or in the emission spectrum region of the fluorescent compound where it is relatively easier to exhibit quenching characteristics.

[0006] For example, the commercially available quencher BHQ-1 can absorb light in the 500-550 nm wavelength range and can quench the emission properties of fluorescein, which emits light at a wavelength of approximately 520 nm. BHQ-3 can absorb light in the 650-700 nm wavelength range and, while it is not able to quench the emission properties of fluorescein, it can quench the emission properties of cy5, which emits light at a wavelength of approximately 670 nm.

[0007] Thus, because a single quencher exhibits effective quenching properties within a narrow wavelength range, the types of fluorescent compounds that can be used in combination with a single quencher are limited.

[0008] Furthermore, ground state quenching, one of the mechanisms of quenching, differs from FRET in that the emission spectrum of the fluorescent compound and the absorption spectrum of the quencher can be expressed without wavelength overlap. Generally, ground state quenching is expressed when the fluorescent compound and the quencher are at a sufficient distance from each other and form a ground state complex, and is therefore also called contact quenching. The ground state complex can be formed when the fluorescent compound and the quencher are at a sufficiently close distance from each other within a double-labeled oligonucleotide, where both ends are labeled with the fluorescent compound and the quencher, respectively, before hybridization with the target nucleic acid sequence.

[0009] Such ground-state quenching does not require wavelength overlap between the emission spectrum of the fluorescent compound and the absorption spectrum of the quencher. Therefore, if a single quencher can form a complex with multiple fluorescent compounds having various emission wavelength bands and form a ground state, the single quencher can exhibit quenching properties across a broad wavelength band, even outside the emission spectrum region of the fluorescent compound.

[0010] Therefore, if both static and dynamic quenching are possible using a single non-fluorescent compound, it is expected that quenching phenomena can be induced with various fluorescent compounds or across a wide wavelength range. [Overview of the project] [Problems that the invention aims to solve]

[0011] As described above, the present invention aims to provide a non-fluorescent compound that can be used as a quencher across a wide range of fluorescent compounds or wavelengths by enabling both static and dynamic quenching by a single non-fluorescent compound.

[0012] The present invention also aims to provide a conjugate using the non-fluorescent compound as a quencher, particularly a conjugate for nucleic acid labeling.

[0013] The present invention also aims to provide a composition for nucleic acid detection, a support for nucleic acid detection, and a nucleic acid detection method using the conjugate using the non-fluorescent compound as a quencher.

Means for Solving the Problems

[0014] According to one aspect of the present invention, a non-fluorescent compound represented by the following Chemical Formula 1 is provided, and the non-fluorescent compound can reduce fluorescence of various fluorescent compounds or in a wide wavelength band by at least one mechanism selected from static quenching and dynamic quenching.

Chem.

[0015] Here, Ar is C5-C 50 aryl, C2-C 50 heteroaryl or C5-C 50 aliphatic-aromatic mixed ring, 50 and the Ar may be substituted with at least one R″, R1 to R6, R′ and R″ are each independently hydrogen, optionally substituted C1-C alkyl, optionally substituted C1-C 40 alkyl, optionally substituted C1-C 40 heteroalkyl, optionally substituted C2-C 40 alkenyl, optionally substituted C2-C 40 alkynyl, optionally substituted C3-C 20 cycloalkyl, optionally substituted C3-C 20 cycloalkenyl, optionally substituted C2-C 20 heterocycloalkyl, hydroxy, oxide (-O - )、optionally substituted C1-C40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso(-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s Selected from, R x This is a reactive group, or a group in which at least one reactive group is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms. The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO2 -), functional groups selected from carboxylates, hydroxyls, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamide, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides, R s This is either a carrier molecule or a group in which at least one carrier molecule is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms. R7 and R8 are, independently, hydrogen, or C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, R x and R s Selected from, X is CR 20 R 21 , NR 22 O, S, R 20~R 22 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C3-C 30 Cycloalkyl, possibly substituted C3-C 30 Heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, R x and R s They may be selected from or bonded to each other to form a 5-7 atom ring, n is an integer between 0 and 5. Two adjacent groups from R1 to R6 can bond to each other and form a 5-7 atom ring, which may also be substituted. The non-fluorescent compound represented by the chemical formula 1 contains at least one R x or R s It exists.

[0016] According to another aspect of the present invention, a conjugate comprising a non-fluorescent compound as defined herein as a quencher is provided.

[0017] According to yet another aspect of the present invention, a nucleic acid detection composition comprising a conjugate as defined herein is provided.

[0018] According to yet another aspect of the present invention, a nucleic acid detection support is provided which comprises a non-fluorescent compound as defined herein as a quencher.

[0019] According to yet another aspect of the present invention, a nucleic acid detection method is provided using a conjugate containing a non-fluorescent compound as defined herein as a quencher. [Effects of the Invention]

[0020] The non-fluorescent compounds defined in this application can induce quenching phenomena for various fluorescent compounds by enabling both static and dynamic quenching. Furthermore, unlike conventional quenchers that operate by dynamic quenching, the non-fluorescent compounds defined in this application can induce quenching phenomena by static quenching as well, thereby enabling quenching phenomena to be induced over an even wider wavelength range.

[0021] As a result, the non-fluorescent compounds defined in this application have a broad wavelength range over which they can exhibit effective quenching properties even as a single substance, which is an advantage because they can be used in combination with a variety of fluorescent compounds along with a single quencher. [Brief explanation of the drawing]

[0022] [Figure 1] This is a schematic diagram of the hybrid of the forward and reverse probes manufactured in Experimental Example 2. [Figure 2] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 4. [Figure 3] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 4. [Figure 4] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 5. [Figure 5] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 5. [Figure 6] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 5. [Figure 7] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 6. [Figure 8] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 6. [Figure 9] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 7. [Figure 10] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 7. [Figure 11] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 7. [Figure 12] This figure shows the results of a PCR experiment using a double-labeled oligonucleotide, as shown in Experimental Example 7. [Figure 13] This figure shows the results of a PCR experiment using triple-labeled oligonucleotides, as shown in Experimental Example 8. [Modes for carrying out the invention]

[0023] For the convenience of understanding the present invention, certain terms are defined herein. Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by a person of ordinary skill in the art.

[0024] Furthermore, unless otherwise specified, singular terms shall include their plural forms, and plural terms shall include their singular forms.

[0025] Non-fluorescent compounds According to one aspect of the present invention, a non-fluorescent compound is provided that can reduce fluorescence by at least one mechanism selected from static quenching and dynamic quenching. The wavelength range in which the non-fluorescent compound can be quenched may vary depending on the emission spectrum of its counterpart, the fluorescent compound, but it can exhibit quenching properties in the 200-1000 nm wavelength range.

[0026] Furthermore, the non-fluorescent compounds defined in this application can quench the luminescence properties of fluorescent compounds through both static quenching and dynamic quenching.

[0027] For example, the non-fluorescent compound can reduce the fluorescence of a fluorescent compound having an absorption or emission spectrum of 300 nm to 900 nm, preferably 550 nm to 900 nm, more preferably 550 nm to 800 nm, by dynamic quenching. Specifically, the dynamic quenching may be by a FRET mechanism. Alternatively, the non-fluorescent compound can reduce the fluorescence of a fluorescent compound having an absorption or emission spectrum of 200 nm to 1000 nm, preferably 300 nm to 900 nm, by static quenching. Specifically, the static quenching may be induced by the non-fluorescent compound forming a ground state complex with the fluorescent compound.

[0028] Therefore, the non-fluorescent compounds defined in this application have the advantage of being able to reduce fluorescence over a wide wavelength range (e.g., 200-1000 nm) through two quenching mechanisms (dynamic quenching and static quenching), and can be used as counterparts to a wider variety of fluorescent compounds than quenchers that reduce fluorescence through only a single quenching mechanism.

[0029] The non-fluorescent compound is represented by the following chemical formula 1. Furthermore, the chemical formula 1 may exist as a resonance structure represented by the following chemical formula 1-1. [ka]

[0030] Here, Ar is C5-C 50 Aryl, C2-C 50 Heteroaryl or C5-C 50 It is an aliphatic-aromatic mixed ring, where n is an integer from 0 to 5.

[0031] In this application, aryl means an unsaturated aromatic ring containing a monocyclic or polycyclic ring (preferably 1 to 6 rings) linked to each other by condensation or covalent bonds (e.g., single bonds), and heteroaryl means an unsaturated aromatic ring in which at least one carbon atom in the aryl is substituted with a heteroatom (e.g., nitrogen, oxygen, or sulfur).

[0032] Non-restrictive examples of aryls include phenyl, biphenyl, terphenyl, naphthyl, anthryl, phenanthrenyl, pyrenyl, fluoranthenyl, and their condensed analogues.

[0033] Non-restrictive examples of heteroaryls include furyl, tetrahydrofuryl, pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl, pyridyl, pyridazinyl, triazinyl, piperidinyl, and morpholinyl. linyl), thiomorpholinyl, pyrazinyl, piperazinyl, pyrimidinyl, naphthyridinyl, benzofuranyl, benzothienyl, indolyl, indolinyl, indazolyl, quinolidinyl, isoquinolinyl, cinolinyl There are compounds such as cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazoyl, acridinyl, phenadinyl, phenothizinyl, phenoxadinyl, prinyl, benzimidazolyl, and benzothiazolyl, as well as analogs formed by their condensation.

[0034] In this application, an aliphatic-aromatic mixed ring means a polycyclic ring (preferably 2 to 6 rings) in which an aliphatic ring and an aromatic ring are linked to each other by condensation or covalent bonds (e.g., single bonds). In this application, an aliphatic ring means a saturated hydrocarbon ring (cycloalkyl) or a saturated hydrocarbon ring (heterocycloalkyl) in which at least one carbon atom within the hydrocarbon ring is substituted with a heteroatom, and corresponds to an alkyl or heteroalkyl cyclic structure, respectively.

[0035] Non-restrictive examples of aliphatic rings include cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl.

[0036] For example, the aforementioned Ar can be selected from the following [Ar-1] to [Ar-6]. [ka]

[0037] In [Ar-1] to [Ar-6], * indicates the position of carbon atoms that can condense into a pentatomic ring containing nitrogen in chemical formula 1, and Y is independently CR. 23 R 24 , NR 25 It is either O or S.

[0038] The aforementioned R 23 ~R 25 C1-C may be substituted independently of each other. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C3-C 30 Cycloalkyl, possibly substituted C3-C 30 Heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, Rx and R s They can be selected from or bonded to each other to form a 5-7 atom ring, which may be substituted.

[0039] The Ar may be substituted with at least one R''. That is, the Ar is a substituted or unsubstituted ring. If the Ar is a substituted ring, at least one carbon atom in the Ar may be bonded to R''. Also, if the Ar is a substituted ring, the R'' bonded to at least one carbon atom in the Ar does not substantially reduce the quenching properties of the nonfluorescent compound represented by chemical formula 1. Similarly, [Ar-1] to [Ar-6] may be substituted with at least one R''.

[0040] Various variations of Ar in chemical formula 1 can be seen from the structures of Dye 1 to Dye 97 attached to this application.

[0041] R1~R6, R′ and R″ are each independently hydrogen, or C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C50 Alkyl, optionally substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, optionally substituted C3-C 40 Cycloalkylthio, optionally substituted C2-C 40 Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonate ester, optionally substituted sulfonamide, substituted ester, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s selected from. The functional groups according to the above definition do not substantially reduce the quenching characteristics of the non-fluorescent compound represented by the chemical formula 1.

[0042] In the non-fluorescent compound represented by the chemical formula 1, when the Ar has the structure of [Ar-1] or [Ar-2], NH4 + , PO4 - and SO3 - (including SO3H, alkyl sulfonate, arylsulfonate, etc.) and the like are preferably not included. Thereby, the non-fluorescent compound in which the Ar has the structure of [Ar-1] or [Ar-2] substantially exhibits lipophilicity. The fact that the non-fluorescent compound substantially exhibits lipophilicity means that the non-fluorescent compound exhibits a solubility of 30% or less, 20% or less, or 10% or less in an aqueous solvent, or does not dissolve at all.

[0043] The hydrogen used in the present application may be light hydrogen (1H), deuterium (2H) or tritium (3H) having a mass number of 1.

[0044] In this application, heteroatoms refer to atoms other than carbon and hydrogen, and more specifically, the heteroatoms are atoms that can be used in place of carbon in a backbone structure made of carbon (more specifically, hydrocarbons), such as nitrogen, oxygen, sulfur, phosphorus, silicon, or selenium.

[0045] In this application, C a -C b The functional group represented by means a functional group having a to b carbon atoms.

[0046] For example, C a -C b Alkyl refers to a saturated aliphatic functional group having a to b carbon atoms, including linear alkyl and branched alkyl groups. Branched alkyl refers to a saturated aliphatic functional group in which another linear alkyl group is bonded to any carbon atom of an unbranched linear alkyl group.

[0047] Specifically, alkyl groups may be methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pento-1-yl, pento-2-yl, pento-3-yl, 3-methylbuto-1-yl, 3-methylbuto-2-yl, 2-methylbuto-2-yl, 2,2,2-trimethyletho-1-yl, n-hexyl, n-heptyl, and n-octyl.

[0048] In this application, heteroalkyl means a stable linear or branched alkyl group having a total number of atoms as specified and at least one heteroatom (e.g., 1 to 15) selected from the group consisting of O, N, Si, and S. The carbon and heteroatoms of the heteroalkyl group can be oxidized (e.g., to form ketones, N-oxides, sulfones, etc.), and the nitrogen atom can be quaternized. Non-restrictive examples of heteroalkyl groups include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)2, -C(=O)-NH-CH2-CH2-NH-CH3, -C(=O)-N(CH3)-CH2-CH2-N(CH3)2, -C(=O)-NH-CH2-CH2-NH-C(=O)-CH2-CH3, -C(=O)-N(CH3)-CH2-CH2-N(CH3)-C(=O)-CH2-CH3, -O-CH2-CH2-CH2-NH(CH3), -O-CH2-CH2-CH2-N(CH3)2, -O-CH2-CH2-CH2-NH-C(= O)-CH2-CH3, -O-CH2-CH2-CH2-N(CH3)-C(=O)-CH2-CH3, -CH2-CH2-CH2-NH(CH3), -O-CH2-CH2-CH2-N(CH3)2, -CH2-CH2-CH2-NH-C(=O)-CH2-CH3, -CH2-CH2-CH2- Includes N(CH3)-C(=O)-CH2-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(=O)-CH2-CH3, -CH2-CH2-S(O)2-CH3, -CH2-CH2-O-CF3, and -Si(CH3)3.

[0049] In this application, alkoxy includes all -O-(alkyl) groups and -O-(unsubstituted cycloalkyl) groups, and specifically includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, etc.

[0050] In this application, acyl refers to -C(=O)-alkyl, -C(=O)-heteroalkyl, -C(=O)-cycloalkyl (substituted or unsubstituted), and -C(=O)-heterocycloalkyl (substituted or unsubstituted), and the alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl bonded to the carbonyl carbon of the acyl is as defined in this application. Non-restrictive examples of acyls include -C(=O)CH3, -C(=O)CH2CH3, -C(=O)CH(CH3)2, -C(=O)C(CH3)3, -C(=O)-phenyl (substituted or unsubstituted), -C(=O)-cyclopropyl (substituted or unsubstituted), -C(=O)-cyclobutyl (substituted or unsubstituted), -C(=O)-cyclopentyl (substituted or unsubstituted), -C(=O)-cyclohexyl (substituted or unsubstituted), and -C(=O)-pyridyl (substituted or unsubstituted).

[0051] Furthermore, in this application, halogen means fluoro(-F), chloro(-Cl), bromo(-Br), or iodo(-I), and alkyl halide or haloalkyl means alkyl substituted with the aforementioned halogens. For example, halomethyl means methyl (-CH2X, -CHX2, or -CX3) in which at least one of the hydrogen atoms of methyl is substituted with a halogen. Therefore, alkyl halide means a functional group in which at least one halogen is bonded to at least one of the carbon atoms constituting the alkyl.

[0052] In this application, alphaalkyl is a functional group in which an aryl is substituted on the alkyl carbon, -(CH2) n Ar is a general term for alkyl groups. Examples of alkyl groups include benzyl (-CH2C6H5) and phenethyl (-CH2CH2C6H5).

[0053] In the present application, "may be substituted" has the same meaning as the term "substituted or unsubstituted", and means that one or more hydrogen atoms present in any functional group may each be independently substituted with a non-hydrogen substituent. However, when any functional group is a substituted functional group, the functional group does not include any substituent or substitution pattern that is sterically impossible to achieve.

[0054] When any functional group among R1 to R6, R' and R" is a substituted functional group, at least one substituent is bonded to any carbon atom (in the case of a functional group having no carbon atom, any atom to which the substituent can be bonded (e.g., nitrogen, oxygen or sulfur, etc.)) in the functional group.

[0055] The substituent is C1-C 40 alkyl, C1-C 40 heteroalkyl, C2-C 40 alkenyl, C2-C 40 alkynyl, C3-C 20 cycloalkyl, C3-C 20 cycloalkenyl, C2-C 20 heterocycloalkyl, optionally substituted C5-C 50 aryl, C2-C 50 heteroaryl, C5-C 50 aralkyl, R x and R s selected from, preferably C1-C 40 alkyl, C1-C 40 heteroalkyl, C2-C 40 alkenyl, C2-C 40 alkynyl, R x and R s selected from, more preferably C1-C 40 alkyl, C1-C 40 heteroalkyl, R x and R s selected from.

[0056] R xThis is a reactive group, or a group in which at least one reactive group is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms.

[0057] The aforementioned backbone refers to a series of bonds used to link a reactive group or carrier molecule to the substrate represented by chemical formula 1.

[0058] For example, the backbone may have an alkyl or heteroalkyl group containing 1 to 40 carbon atoms as its main chain. Furthermore, the main chain may also contain multiple alkyl or heteroalkyl groups linked together via intermediate functional groups such as amides or esters.

[0059] The backbone may contain at least one heteroatom selected from O, S, N, P, and Si. The backbone may also be linear or nonlinear and may consist of any combination of single, double, or triple bonds.

[0060] The aforementioned backbone may be, for example, alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C(O)-, -C(O)O-, or -NR. L -, -O-, -S-, -C(O)NR L -, -S(O) O -, -S(O)NR L -, S(O2)NR L -,-P(O) P -and may be selected from the following structural formulas (a) to (o), or composed of a combination thereof. [ka]

[0061] Here, R L is hydrogen, C1-C 40 Alkyl and C1-C 40Selected from heteroalkyl groups, where each q is an integer from 0 to 10, which may be the same or different, each o is an integer from 1 to 2, and each p is an integer from 1 to 4.

[0062] The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO2 - The functional group is selected from carboxylates, hydroxyls, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamide, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides.

[0063] Furthermore, the reactive group may not have to participate in any reaction if it is protected by a protecting group.

[0064] The protecting group is introduced by chemically converting a reactant to confer reaction selectivity to at least some of the reactants during a continuous chemical or biological reaction process.

[0065] Examples of the protecting groups include alcohol-derived protecting groups, amine-derived protecting groups, carbonyl-derived protecting groups, carboxylic acid-derived protecting groups, phosphate-derived protecting groups, or alkyne-derived protecting groups.

[0066] For example, if the reactive group is hydroxyl, acetyl, benzoyl, benzyl, β-methoxyethoxymethyl ether, dimethoxytrityl, methoxymethyl ether, methoxytrityl, p-methoxybenzyl ether, p-methoxyphenyl ether, methylthiomethyl ether, silyl ether, trityl, or analogs thereof can be used as protecting groups. If the reactive group is amino, tert-butylcarbamate, benzylcarbamate, acetamide, phthalimide, p-toluenesulfonamide, or analogs thereof can be used as protecting groups. x If the protecting group is carbonyl or carboxyl, acetals, ketals, dithianes, methyl esters, benzyl esters, tert-butyl esters, silyl esters, or analogues thereof can be used. For other examples of preferred protecting groups, please refer to the following reference (Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991; https: / / en.wikipedia.org / wiki / Protecting_group ).

[0067] R s This is a carrier molecule, or a group to which at least one carrier molecule is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms, and the definition of the backbone is R x It is identical to [the other one].

[0068] A carrier molecule refers to a substance that transports the non-fluorescent compound represented by the chemical formula 1.

[0069] The carrier molecule includes amino acids, amino acid polymers, peptides, proteins, neurotoxins, phallotoxins, cytokines, toxins, protease substrates, protein kinase substrates, enzymes, antibodies, antibody fragments, lectins, glycoproteins, histones, albumins, lipoproteins, avidin, streptavidin, protein A, protein G, protein L, phycobiliproteins, fluorescent proteins, hormones, growth factors, and nucleic acid bases. The base can be selected from nucleosides, nucleotides, nucleic acid polymers, nucleotide analogs, nucleoside analogs, nucleoside triphosphates, deoxynucleoside triphosphates (dNTPs), dideoxynucleoside triphosphates (ddNTPs), organic or inorganic nanoparticles, organic or inorganic microparticles, heptenes, carbohydrates, polysaccharides, lipids, ion complexing moieties such as crown ethers, PEG groups, organic polymers, and inorganic polymers.

[0070] Furthermore, the carrier molecule does not need to participate in any reaction if it is protected by a protecting group. In this case, protection of the carrier molecule by a protecting group means that the functional groups present in the carrier molecule are protected by the protecting group. The definition of the protecting group for the carrier molecule is the same as that of the protecting group for the reactant group described above.

[0071] In this application, a derivative refers to a similar compound obtained by chemically altering a part of any compound.

[0072] A carboxyl derivative is a functional group that can be converted into a carboxyl group, and it may be an N-hydroxysuccinimide ester, an N-hydroxybenzotriazole ester, an acyl halide, an acyl imidazole, a thioester, an alkyl ester, an alkenyl ester, an alkynyl ester or an aromatic ester.

[0073] The carboxyl derivative may be substituted, for example, it may be a trifluoromethyl ester, a pentafluorophenyl ester, a p-nitrophenyl ester, a tetrafluorophenyl ester, a sulfo-succinimidyl ester, a sulfodichlorophenyl ester, or a sulfotetrafluorophenyl ester.

[0074] The 1,2,4,5-tetrazine derivative may be 3,6-dimethyl-1,2,4,5-tetrazine, 3,6-diphenyl-1,2,4,5-tetrazine or 3-methyl-6-phenyl-1,2,4,5-tetrazine.

[0075] The cycloalkyne derivative may be a compound that participates in a bipolar cycloaddition, an inverse-electron demand Diels-Alder or a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. For example, as the cycloalkyne derivative participating in the SPAAC reaction, cyclooctynes such as OCT, COMBO(ALO), MOFO, DIFO, DIBO, BARAC, DIBAC(ADIBO), DBCO, DIMAC, BCN or TMTH can be used.

[0076] In addition, the exemplification of any derivative specified in the present application can be confirmed through the literature known in the technical field.

[0077] The parent diene is an alkene or an alkyne capable of undergoing a Diels-Alder reaction with a diene. Specifically, sp 2-These may be alkenes or alkynes in which an electron-withdrawing group is directly linked to a hybrid carbon or sp-hybrid carbon.

[0078] An electron-withdrawing group is a functional group that tends to attract electrons through inductive or resonance effects, and can also be called a deactivating group. Examples of electron-withdrawing groups include trifluoromethylsulfonyl (-SO2CF3) and substituted or unsubstituted ammonium (-NR3). + Examples include nitro, sulfonic acid (-SO3H), sulfonyl (-SO2R), nitrile, trihalomethyl (-CF3, -CCl3, -CBr3, -CI3), haloformyl (-COCl, -COBr, -COI), formyl (-CHO), acyl (-COR), carboxyl (-CO2H), substituted ester (-CO2R), substituted or unsubstituted aminocarbonyl (-CONR2), and nitroso (-N=O). Furthermore, unless otherwise defined in this application, the electron-withdrawing groups may include other functional groups that tend to attract electrons, in addition to the functional groups exemplified above.

[0079] R7 and R8 are, independently, hydrogen, or C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, R x and R s Selected from.

[0080] X is CR 20 R 21 , NR 22 , O or S. X is CR 20 R 21 If R 20 and R 21 C1-C may be substituted independently of each other. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C3-C 30 Cycloalkyl, possibly substituted C3-C 30 Heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, R x and R s Selected from.

[0081] X is NR 22 If R 22 C1-C may be substituted independently of each other. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C3-C 30 Cycloalkyl, possibly substituted C3-C 30 Heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, R x or R s That is the case.

[0082] n is an integer between 0 and 5. If n is 0, there is one R'; if n is 1 or greater, there are multiple R's. If multiple R's exist in the polymer when n is 1 or greater, these may each be independently hydrogen-substituted C1-C. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso(-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s Selected from.

[0083] Two adjacent groups from R1 to R6 can bond to each other and form a 5- to 7-atom ring, which may also be substituted.

[0084] The ring formed by the bonding of two adjacent groups from R1 to R6 may be an aliphatic ring (cycloalkyl, heterocycloalkyl) or an aromatic ring (aryl, heteroaryl). The ring may also be monocyclic or polycyclic, and if the ring is polycyclic, it may be an aliphatic-aliphatic mixed ring, an aliphatic-aromatic mixed ring, or an aromatic-aromatic mixed ring. As mentioned above, a mixed ring means a polycyclic ring (preferably 2 to 6 rings) in which identical or different rings are linked by condensation or covalent bonds (e.g., single bonds).

[0085] The ring formed when two adjacent groups from R1 to R6 are bonded to each other is C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Alkinyl, C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl, C2-C 20 Heterocycloalkyl, possibly substituted C5-C 50 Aryl, C2-C 50 Heteroaryl, C5-C 50 Alalkyl, R x and R s It may be substituted with a functional group selected from the following.

[0086] A modified example in which two adjacent groups from R1 to R6 bond to each other to form a 5- to 7-atom ring can be seen from the structures of Dye 1 to Dye 97 attached to this application.

[0087] When the Ar is substituted with multiple R'' groups, two adjacent R'' groups can bond to each other to form a 5-7 atom ring, which may also be substituted.

[0088] The ring formed by the bonding of two adjacent groups of R″ may be an aliphatic ring (cycloalkyl, heterocycloalkyl) or an aromatic ring (aryl, heteroaryl). The ring may also be monocyclic or polycyclic, and if the ring is polycyclic, it may be an aliphatic-aliphatic mixed ring, an aliphatic-aromatic mixed ring, or an aromatic-aromatic mixed ring. As mentioned above, a mixed ring means a polycyclic ring (preferably 2 to 6 rings) in which identical or different rings are linked by condensation or covalent bonds (e.g., single bonds).

[0089] The ring formed by two adjacent groups of R″ bonding to each other is C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Alkinyl, C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl, C2-C 20 Heterocycloalkyl, possibly substituted C5-C 50 Aryl, C2-C 50 Heteroaryl, C5-C 50 Alalkyl, R x and R s It may be substituted with a functional group selected from the following.

[0090] A modified form in which two adjacent R″ groups bond to each other to form a 5- to 7-atom ring can be seen from the structures of Dye 1 to Dye 97 attached to this application.

[0091] R 20 ~R 22 Two of these adjacent groups can bond to each other to form a 5- to 7-atom ring, which may also be substituted.

[0092] R 20 ~R 22The ring formed by the bonding of two adjacent groups may be an aliphatic ring (cycloalkyl, heterocycloalkyl) or an aromatic ring (aryl, heteroaryl). The ring may also be monocyclic or polycyclic, and if polycyclic, it may be an aliphatic-aliphatic mixed ring, an aliphatic-aromatic mixed ring, or an aromatic-aromatic mixed ring. As mentioned above, a mixed ring means a polycyclic ring (preferably 2 to 6 rings) in which identical or different rings are linked by condensation or covalent bonds (e.g., single bonds).

[0093] R 20 ~R 22 The ring formed when two adjacent groups are bonded to each other is C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Alkinyl, C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl, C2-C 20 Heterocycloalkyl, possibly substituted C5-C 50 Aryl, C2-C 50 Heteroaryl, C5-C 50 Alalkyl, R x and R s It may be substituted with a functional group selected from the following.

[0094] R 20 ~R 22 A modified form in which two adjacent groups bond to each other to form a 5- to 7-atom ring can be seen from the structures of Dye 1 to Dye 97 attached to this application.

[0095] Thus, when two adjacent groups of the nonfluorescent compound represented by chemical formula 1 bond to each other to form a ring, the ring may be phenyl, naphthyl, carbazole, fluorene, dibenzofuran, dibenzothiophene, fluorantene, etc., and is not necessarily limited to these.

[0096] In addition, the non-fluorescent compound represented by the chemical formula 1 has at least one R x or R s characterized by its presence.

[0097] The non-fluorescent compound represented by chemical formula 1 may further contain a counter ion. The counter ion is an organic or inorganic anion and can be appropriately selected considering factors such as the solubility and stability of the quencher.

[0098] Specifically, as the counter ion, inorganic acid anions such as hexafluorophosphate ion, halogen ion, phosphate ion, perchlorate ion, periodate ion, hexafluoroantimonate ion, hexafluorostannate ion, fluoroborate ion and tetrafluoroborate ion, and organic acid ions such as thiocyanate ion, benzenesulfonate ion, naphthalenesulfonate ion, p-toluenesulfonate ion, alkylsulfonate ion, benzenecarboxylate ion, alkylcarboxylate ion, trihaloalkylcarboxylate ion, alkylsulfonate ion, trihaloalkylsulfonate ion and nicotinate ion can be used. Furthermore, metal compound ions such as bisphenyldithiol, thiobisphenol chelate and bisdiol-α-diketone, metal ions such as sodium and potassium, and quaternary ammonium salts can also be used as the counter ion.

[0099] In other embodiments, the non-fluorescent compound may be represented by the following chemical formula 2. The non-fluorescent compound represented by the following chemical formula 2 has a structure in which Ar is [Ar-1] among the non-fluorescent compounds represented by the chemical formula 1.

Chemical formula

[0100] In Chemical formula 2, R1 to R8, R′, R x , R s , n, X, R 20 ~R 22The definition of is the same as that of chemical formula 1, and all other definitions are also the same as those of chemical formula 1 unless otherwise defined.

[0101] R9~R 12 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso(-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s Selected from.

[0102] R9~R 12 Two of these adjacent groups can bond to each other to form a 5- to 7-atom ring, which may also be substituted.

[0103] The non-fluorescent compound represented by the chemical formula 2 contains at least one R x or R s It exists.

[0104] In yet another embodiment, the non-fluorescent compound may be represented by the following chemical formula 3. The non-fluorescent compound represented by the following chemical formula 2 is R among the non-fluorescent compounds represented by the chemical formula 3. 10 and R 11 It has a structure in which these are bonded together to form ring A. [ka]

[0105] In chemical formula 3, R1 to R8, R′, R'', R x , R s n, X, R 20 ~R 22 The definition of is the same as that of chemical formula 1, and all other definitions are also the same as those of chemical formula 1 unless otherwise defined.

[0106] Ring A is C5-C 50 Aryl, C2-C 50Heteroaryl or C5-C 50 It is an aliphatic-aromatic mixed ring, and ring A may be substituted with at least one R''.

[0107] R9 and R 12 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso(-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s Selected from.

[0108] R″, R9 and R 12 Two of these adjacent groups can bond to each other to form a 5- to 7-atom ring, which may also be substituted.

[0109] The non-fluorescent compound represented by the chemical formula 3 contains at least one R x or R s It exists.

[0110] In yet another embodiment, the non-fluorescent compound may be represented by the following chemical formula 4. The non-fluorescent compound represented by the following chemical formula 4 is one of the non-fluorescent compounds represented by the chemical formula 2, with R 11 and R 12 It has a structure in which these elements are bonded together to form ring B. [ka]

[0111] In chemical formula 4, R1 to R8, R′, R″, R x , R s n, X, R 20 ~R 22 The definition of is the same as that of chemical formula 1, and all other definitions are also the same as those of chemical formula 1 unless otherwise defined.

[0112] Ring B is C5-C 50 Aryl, C2-C 50Heteroaryl or C5-C 50 It is an aliphatic-aromatic mixed ring, and ring B may be substituted with at least one R''.

[0113] R9 and R 10 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso(-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s Selected from.

[0114] R″ and R 10 These atoms can form a 5-7 atom ring, which may be bonded to each other and substituted.

[0115] The non-fluorescent compound represented by the chemical formula 4 contains at least one R x or R s It exists.

[0116] In yet another embodiment, the non-fluorescent compound may be represented by the following chemical formula 5. The non-fluorescent compound represented by the following chemical formula 5 is one of the non-fluorescent compounds represented by chemical formula 2, with R9 and R 10 It has a structure in which two atoms are bonded together to form a ring C. [ka]

[0117] In chemical formula 5, R1 to R8, R′, R'', R x , R s n, X, R 20 ~R 22 The definition of is the same as that of chemical formula 1, and all other definitions are also the same as those of chemical formula 1 unless otherwise defined.

[0118] Ring C is C5-C 50 Aryl, C2-C 50 Heteroaryl or C5-C 50It is an aliphatic-aromatic mixed ring, wherein the ring C may be substituted with at least one R''.

[0119] R 11 and R 12 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso(-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s Selected from.

[0120] R″ and R 11 These atoms can form a 5-7 atom ring, which may be bonded to each other and substituted.

[0121] The non-fluorescent compound represented by the chemical formula 5 contains at least one R x or R s It exists.

[0122] In yet another embodiment, the nonfluorescent compound may be represented by the following chemical formula 6. The nonfluorescent compound represented by the following chemical formula 6 has a structure in which Ar is fused with X in the nonfluorescent compound represented by chemical formula 1. [ka]

[0123] In chemical formula 6, R1 to R8, R', R x , R s The definitions of and n are the same as those in Chemical Formula 1, and all other definitions are also the same as those in Chemical Formula 1 unless otherwise defined.

[0124] R D Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso(-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s It is selected from the range, and o is an integer from 0 to 6.

[0125] The non-fluorescent compound represented by the chemical formula 6 contains at least one R x or R s It exists.

[0126] The non-fluorescent compound represented by chemical formula 6 is NH4 + , PO4 - and SO3 - It is preferable that the compound does not contain water-soluble functional groups such as SO3H, alkyl sulfonate, or arylsulfonate. This makes the non-fluorescent compound represented by chemical formula 6 substantially lipophilic. Substantially lipophilic means that the non-fluorescent compound has a solubility of 30% or less, 20% or less, or 10% or less in an aqueous solvent, or does not dissolve at all.

[0127] In yet another embodiment, the nonfluorescent compound may be represented by the following chemical formula 7. The nonfluorescent compound represented by the following chemical formula 7 has a structure in which Ar is represented by the following chemical formula 8 among the nonfluorescent compound represented by chemical formula 1. [ka]

[0128] In chemical formula 7, R1 to R7, R′, R x , R s n, X, R 20 ~R 22 The definition of is the same as that of chemical formula 1, and all other definitions are also the same as those of chemical formula 1 unless otherwise defined.

[0129] a and b in the above chemical formula 7 are R in the following chemical formula 8. 31 ~R 35 It condenses with two adjacent elements. [ka]

[0130] R 31 ~R 35 Of these, those that do not condense with a and b of chemical formula 7 are, independently, hydrogen and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso(-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R x and R s Selected from.

[0131] R 31 and R 35Of these, two adjacent groups that do not condense with a and b of chemical formula 7 can bond to each other to form a 5- to 7-atom ring, which may be substituted.

[0132] Q is hydrogen, and C1-C may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, possibly substituted C5-C 50 Alalkyl, R x and R s Selected from.

[0133] The non-fluorescent compound represented by the chemical formula 7 contains at least one R x or R s It exists.

[0134] Specific examples of compounds as defined in this application are as follows. However, the following example compounds are intended to aid in understanding the compounds as defined in this application and are not intended to limit the scope of the compounds as defined in this application. Compounds that are considered equivalent to the following example compounds, and compounds that are considered equivalent to the compounds as defined in this application, can all be expected to exhibit the effects of the compounds as defined in this application.

[0135] Furthermore, the remaining example compounds, excluding those whose synthesis steps are described in detail through the manufacturing examples disclosed herein, can also be synthesized by referring to the manufacturing examples disclosed herein, or by synthetic methods known to the art, referring to the content defined herein. [ka] JPEG2026512495000014.jpg241170JPEG2026512495000015.jpg208170JPEG2026512495000016.jpg201170JPEG20265124950 00017.jpg240170JPEG2026512495000018.jpg213170JPEG2026512495000019.jpg222170JPEG2026512495000020.jpg103170

[0136] Applications of non-fluorescent compounds The present invention relates to non-fluorescent compounds useful as quenchers for fluorescent compounds that exhibit luminescence properties at excitation energy levels, and to various applications thereof. According to other aspects of the present invention, various applications of non-fluorescent compounds are provided that can quench the luminescence properties of fluorescent compounds by static quenching and dynamic quenching.

[0137] In one embodiment, a conjugate can be provided that includes a non-fluorescent compound as defined in this application as a quencher.

[0138] The conjugate can be used for biomolecule labeling. The conjugate can target biomolecules such as antibodies, lipids, proteins, peptides, carbohydrates, and nucleic acids (including nucleotides and nucleosides). Specific examples of lipids include fatty acids, phospholipids, and lipopolysaccharides, while specific examples of carbohydrates include monosaccharides, disaccharides, and polysaccharides (e.g., dextran).

[0139] The conjugate may be a nucleotide conjugate, a probe, a primer, and / or a peptide. The nonfluorescent compound may be bound to any end (5'-terminus or 3'-terminus) or any position inside the conjugate.

[0140] If the conjugate is a nucleotide conjugate, the conjugate may include an oligonucleotide.

[0141] The oligonucleotide refers to a polymer of 1 to several hundred nucleotides. The phosphodiester bond in the oligonucleotide may be modified or replaced with methylphosphonate, phosphorothioate (PTO), boranophosphate, phosphorylguanidine, guanidinopropylphosphoramidate, thiazole, guanidinium, etc. The oligonucleotide may also contain one or more modified nucleotides. For example, the modified nucleotide may include sugar-modified PNA, BNA (e.g., LNA, ENA, etc.), HNA, ANA, CeNA, and GNA, or cases where any substituent (e.g., MOE, alkyl, halogen, etc.) is introduced to the sugar.

[0142] Furthermore, modified nucleotides and nucleosides may also contain nuclear bases modified with adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U), for example, inosine, xanthine, hypoxanthine, nebularin, isoguanisine, tubercidine, 2-aminoadenine, 2-haloadenine, 2-alkyladenine, 2-methylaminoadenine, 6-methyladenine, 8-haloadenine, 8-aminoadenine, 8-thioadenine, diazaadenine, 8-hydroxyadenine and other substituted adenines, 5-halouracil, 4-thiouracil, 5-trifluoromethyluracil, shudouracil, 2- Thiouracil, 5-halouracil and other substituted uracils, 5-halocytosine, 5-trifluorocytosine, 6-azacytosine and other substituted cytosines, 2-thiothymine, 6-azacymine and other substituted thymines, 8-haloguanine, 8-aminoguanine, 8-thioguanine, 8-thioalkylguanine, 8-hydroxyguanine, 8-azacuanine, 7-deazaguanine, 7-methylguanine and other substituted guanines, pyrazolo[3,4-d]pyrimidine, 2,6-diaminopurine, phenoxazine, 2-aminopurine, 3-nitropyrrole, 5-hydroxybutynyluridine, Super A (registered trademark), Super G (registered trademark), Super T (registered trademark), Super D TM It may also include things like the following. Furthermore, for examples of modified nuclear bases, you can refer to the following reference (US Pat. Nos. 11, 155, 713; Current Topics in Medicinal Chemistry, Volume 7, Number 7, Wojciechowski, Filip, E. Hudson, Robert H, 2007, pp. 667-679).

[0143] In this application, the nuclear bases include modified nuclear bases.

[0144] An oligonucleotide labeled with one non-fluorescent compound or one fluorescent compound can be called a single-labeled oligonucleotide, and may, for example, be an oligonucleotide in which the non-fluorescent compound is labeled at the 3'-terminus.

[0145] Furthermore, the conjugate may also include a quencher containing a non-fluorescent compound as defined in this application and a phosphor. Here, the phosphor can be a known fluorescent compound suitable for biomolecule labeling. The fluorescent compound may be bound to any end (5'-terminus or 3'-terminus) or any position inside the conjugate.

[0146] Oligonucleotides labeled with two non-fluorescent or fluorescent compounds can be called double-labeled oligonucleotides, and may, for example, be oligonucleotides in which the fluorescent compound is labeled at the 5'-terminus and the non-fluorescent compound is labeled at the 3'-terminus.

[0147] As the fluorescent compound, at least one selected from coumarin, cyanine, bolus, furosein, rhodamine, pyrene, carbopyronine, oxazine, xanthene, thioxanthene, acridine, and / or derivatives thereof can be used.

[0148] A more specific example of the aforementioned fluorescent compound is 6-FAM TM , TET TM , JOE TM , VIC(registered trademark), HEX TM NED TM PET (registered trademark), ROX TM TAMRA TM , TET TMTexas Red (registered trademark), SUN, MAX, ABY, JUN, LIZ, TAZ, CAL Fluor (registered trademark) Gold 540, CAL Fluor (registered trademark) Orange 560, CAL Fluor (registered trademark) Red 590, CAL Fluor (registered trademark) Red 610, CAL Fluor (registered trademark) Red 635, Cy (registered trademark) (cyanine) 3, Cy (registered trademark) 3.5, Cy (registered trademark) 5, Cy (registered trademark) 5.5, Cy (registered trademark) 7, Cy (registered trademark) 7.5, Quasar (registered trademark) 570, Quasar (registered trademark) 670, Quasar (registered trademark) 705, Rhodamine Green TM Rhodamine Red TM LightCycler® Cyan 500, LightCycler® Red 610, LightCycler® Red 640, LightCycler® Red 670, LightCycler® Red 705, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Alexa Fluor® dyes 350, 405, 488, 532, 546, 555, 568, 594, 610, 647, 680 and Alexa Fluor® 750, BODIPY® dyes, Epoch Blue, AMCA, Marina Blue®, Pacific Blue TM Pacific Green TM Pacific Orange TM Yakima Yellow TM , ATTO dyes 390, 425, 465, 488, 495, 514, 520, 532, Rho6G, 542, 550, 565, Rho3B, Rho11, Rho12, Thio12, Rho10 1, 590, 594, Rho13, 610, 620, Rho14, 633, 643, 647, 647N, 655, Oxa12, 665, 680, 700, 725, and ATTO 740, , Oyster 645, SFC TM -V, SFC TM-N, SFC TM 574, SFC TM 647, SFC TM -C610, SFC TM 620, SFC TM 670, SFC TM 705, Chamel TM 560, Chamel TM 610, Chamel TM 670 and Chamel TM Examples include the 705.

[0149] The conjugate may include a first quencher comprising a non-fluorescent compound as defined herein; a second quencher selected from azo, coumarin, cyanine, bolus, furosein, rhodamine, pyrene, carbopyronine, benzo[c,d]indole, oxazine, xanthene, thioxanthene, acridine and derivatives thereof; and a phosphor.

[0150] The first quencher, the second quencher, and the phosphor may be bonded to any end (5'-end or 3'-end) or any position within the conjugate.

[0151] An oligonucleotide labeled with three non-fluorescent or fluorescent compounds can be called a triple-labeled oligonucleotide, for example, in which the fluorescent compound is labeled at the 5'-terminus, the first quencher is labeled at the 3'-terminus, and the second quencher is inserted between the oligonucleotides.

[0152] As the second quencher, at least one selected from azo, coumarin, cyanine, bolus, furosein, rhodamine, pyrene, carbopyronine, benzo[c,d]indole, oxazine, xanthene, thioxanthene, acridine, and derivatives thereof can be used.

[0153] More specific examples of the second quencher mentioned above include dabcyl and Eclipse. TM Black Hole Quencher TM BHQ0(BHQnova), Black Hole Quencher TM BHQ1, Black Hole Quencher TM BHQ2, Black Hole Quencher TM BHQ3, BlackBerry TM Quencher 650 (BBQ650) TM ), Iowa Black TM FQ (IABkFQ), SFC TM Q1, SFC TM Q2, Iowa Black TM RQ-n1, Iowa Black TM RQ-n2 and Iowa Black TM RQSp, TAMRA, Deep Dark Quencher I (DDQ I), Deep Dark Quencher II (DDQ II), QXL TM 520, QXL TM 570, QXL TM 610, QXL TM 670, IRQXL TM IRDye TM QC-1, QSY TM QSY TM 2. Examples include BMN-Q460, BMN-Q535, BMN-Q1, BMN-Q2, BMN-Q590, BMN-Q620, and BMN-Q651.

[0154] Furthermore, the conjugate may further contain a minor groove binder (MGB) to improve its binding affinity to nucleic acids. When a conjugate further containing MGB is used, the PCR amplification effect can be improved.

[0155] The conjugates defined in this application can be used in a variety of applications in the chemical and biological fields. They are particularly useful in real-time polymerase chain reactions or microassays, but are not limited to these applications.

[0156] In other embodiments, nucleic acid detection compositions comprising the conjugate defined in this application can be provided.

[0157] A nucleic acid detection composition according to one embodiment of the present invention may further include, together with the conjugate defined herein, an enzyme for reaction with a target nucleic acid, a solvent (such as a buffer solution), and other reagents.

[0158] Here, the solvent can be a buffer selected from the group consisting of phosphate buffers, carbonate buffers, and Tris buffers, an organic solvent selected from dimethyl sulfoxide, dimethylformamide, dichloromethane, methanol, ethanol, and acetonitrile, or water, and the solubility can be adjusted by introducing various functional groups to the quencher depending on the type of solvent.

[0159] In yet another embodiment, a nucleic acid detection support can be provided, comprising a quencher containing a non-fluorescent compound as defined in this application, a support, and a connecting portion connecting the quencher and the support. Biomolecules (nucleic acids) in a sample can be immobilized on the support by interaction with the quencher immobilized on the support.

[0160] The support can be manufactured from at least one selected from glass (e.g., CPG), cellulose, nylon, acrylamide gel, dextran, polystyrene, alginate, collagen, peptide, fibrin, hyaluronic acid, agarose, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polyethylene glycol diacrylate, gelatin, Matrigel, polylactic acid, carboxymethylcellulose, dextran, chitosan, latex, and Sepharose, and may be in the form of beads or membranes.

[0161] The aforementioned connecting portion is the part that connects the quencher and the support, and any material that can connect the quencher and the support can be used as the connecting portion intended in this application.

[0162] For example, the connecting portion is a substituted or unsubstituted C1-C 30 Alkyl, substituted or unsubstituted C3-C 30 Cycloalkyl, substituted or unsubstituted C2-C containing at least one heteroatom 30 Heteroalkyl, substituted or unsubstituted C2-C containing at least one heteroatom 30 Heterocycloalkyl, substituted or unsubstituted C2-C 30 Alkenyl, substituted or unsubstituted C6-C 30 Aryl, substituted or unsubstituted C3-C 30 The materials can be selected from heteroaryls, amides (-CONH-), esters (-COO-), ketones (-CO-), nucleosides, and any combination thereof. The coupling serves only to connect the quencher and the support and does not substantially affect other reactions or fluorescence and quenching effects of the quencher or phosphor.

[0163] An example of a connection structure between the support and the quencher via the aforementioned connecting portion is shown below. In the above example, "Quencher" means "quencher (extinguishing photon)" and "dye" means phosphor. [ka] JPEG2026512495000022.jpg110170

[0164] Nucleic acid detection method According to yet another aspect of the present invention, a method is provided for labeling a target biomolecule, which is a nucleic acid, using a conjugate comprising a non-fluorescent compound and a fluorescent compound as defined herein.

[0165] Furthermore, by introducing appropriate reactive groups into non-fluorescent and / or fluorescent compounds depending on the type of target biomolecule, a method for labeling biomolecules using target-specific interactions can be realized. Moreover, a method for identifying biomolecules labeled with the conjugate defined in this application by electrophoresis can also be realized.

[0166] In one embodiment, the nucleic acid detection method may include the steps of (a) preparing a reaction mixture comprising a target nucleic acid, reagents necessary for amplifying the target nucleic acid, and a conjugate as defined herein; (b) amplifying the target nucleic acid in the reaction mixture; and (c) measuring the fluorescence intensity of the reaction mixture.

[0167] The conjugate used in the above embodiment includes a quencher containing at least a non-fluorescent compound as defined herein and a phosphor.

[0168] Step (b) may include (b-1) a step in which the nucleotide conjugate hybridized to the target nucleic acid is extended by polymerase; (b-2) a step in which the quencher and phosphor of the nucleotide conjugate are separated from the target nucleic acid by the exonuclease activity of the polymerase; and (b-3) a step in which the phosphor released from the quencher emits fluorescence. In addition, in step (b) above, the target nucleic acid is subjected to: Strand Displacement Amplification (SDA), Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real-time Polymerase Chain Reaction, Allele-specific Polymerase Chain Reaction, Ligase Chain Reaction (LCR), Rolling Circle Amplification (RCA), Isothermal Multiple Displacement Amplification (IMDA), Recombinase Polymerase Amplification (RPA), Self-Sustained Sequence Replication (3SR), Single Primer Isothermal Amplification (SPIA), and Multiple Displacement Amplification (MDA). Amplification, whole-genome amplification (WGA), cross-priming amplification (CPA), signal-mediated amplification of RNA technology (SMART), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HDA).The amplification may be performed by a method selected from Dependent Amplification.

[0169] The method may further include a step (d) in which the amount of amplification of the target nucleic acid is measured from the fluorescence intensity measured in step (c).

[0170] The following describes specific embodiments of the present invention. However, the embodiments described below are provided solely to illustrate or explain the present invention and should not be considered limiting.

[0171] Synthesis example: Synthesis of non-fluorescent compounds Synthesis of Dye 1 [ka]

[0172] Synthesis of intermediate 1-1 Intermediate 1-1 was synthesized by referring to Dyes and Pigments, 2012, vol.93, #1-3, pp.1506-1511.

[0173] Synthesis of intermediate 1-1 Intermediates 1 and 2 were synthesized by referring to Journal of Photochemistry and Photobiology A:Chemistry 2008, vol.200, #2-3, pp.438-444.

[0174] Synthesis of Dye 1 In a 100 mL single-port reactor, combine intermediate 1-1 (2.0 g, 4.8 mmol), intermediate 1-2 (1.95 g, 4.8 mmol), and pyridine (20 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 1 (1.2 g). The obtained Dye 1 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.72(t, 1H, J=12.8Hz), 8.52(d, 1H, J=8.0Hz), 8.40(d, 1 H, J=8.22(d, 1H, J=8.4Hz), 8.03(t, 1H, J=7.2Hz), 7.79(d, 1H, J=8.4Hz), 7.68(t, 1 H, J=8.4Hz), 7.57-7.55(m, 2H), 7.45-7.29(m, 2H), 7.00(d, 1H, J=13.6Hz), 6.83(d , 1H, J=12.8Hz), 4.50(t, 2H, J=7.6Hz), 3.82(s, 3H), 2.54(m, 2H), 2.03-1.77(m, 6H)

[0175] Dye 2 synthesis [ka]

[0176] Synthesis of intermediate 2-1 Intermediate 2-1 was synthesized by referring to Organic and Biomolecular Chemistry, 2011, vol.9, #11, pp.4199-4204.

[0177] Dye 2 synthesis In a 100 mL single-port reactor, combine intermediate 2-1 (3.0 g, 6.9 mmol), intermediate 1-2 (2.81 g, 6.9 mmol), and pyridine (30 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 2 (2.2 g). The obtained Dye 2 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.75(t, 1H, J=12.8Hz), 8.50(d, 1H, J=8.0Hz), 8.39(d, 1 H, J=8.21(d, 1H, J=8.4Hz), 8.00(t, 1H, J=7.2Hz), 7.76(d, 1H, J=8.4Hz), 7.68(t, 1 H, J=8.4Hz), 7.59-7.51(m, 2H), 7.49-7.25(m, 2H), 7.01(d, 1H, J=13.6Hz), 6.88(d , 1H, J=12.8Hz), 4.51(t, 2H, J=7.6Hz), 3.80(s, 3H), 2.53(m, 2H), 2.10-1.76(m, 6H)

[0178] Dye 3 synthesis [ka]

[0179] Synthesis of intermediate 3-1 Intermediate 3-1 was synthesized by referring to Angewandte Chemie-International Edition, 2020, vol.59, #10, pp.3948-3951.

[0180] Synthesis of intermediate 3-2 In a 250 mL single-port reactor, intermediate 3-1 (5.0 g, 17 mmol), N,N-diphenylformamidine (4.1 g, 21 mmol), and acetic anhydride (40 mL) were added and stirred at 110 °C for 1 hour. After cooling, ethyl acetate (100 mL) was added and the mixture was vigorously stirred. The resulting solid was filtered to synthesize intermediate 3-2.

[0181] Dye 3 synthesis In a 100 mL single-port reactor, combine intermediate 3-2 (2.0 g, 4.6 mmol), intermediate 1-2 (1.89 g, 4.6 mmol), and pyridine (20 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 3 (0.8 g). The obtained Dye 3 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.70(t, 1H, J=12.8Hz), 8.48(d, 1H, J=8.0Hz), 8.37(d, 1 H, J=8.21(d, 1H, J=8.4Hz), 8.00(t, 1H, J=7.2Hz), 7.74(d, 1H, J=8.4Hz), 7.66(t, 1 H, J=8.4Hz), 7.55-7.50(m, 2H), 7.49-7.25(m, 2H), 7.00(d, 1H, J=13.6Hz), 6.86(d , 1H, J=12.8Hz), 4.50(t, 2H, J=7.6Hz), 3.88(s, 6H), 2.51(m, 2H), 2.10-1.75(m, 6H)

[0182] Dye 4 synthesis [ka]

[0183] Synthesis of intermediate 4-1 Intermediate 4-1 was synthesized by referring to the Journal of the American Chemical Society, 2001, vol. 123, #2, pp. 361-362.

[0184] Synthesis of intermediate 4-2 Intermediate 4-1 (5.0 g, 17 mmol), intermediate 1-2 (4.1 g, 21 mmol), and pyridine (50 mL) were placed in a 100 mL single-port reactor and stirred at 50°C for 1 hour. After concentration, intermediate 4-2 was synthesized by column purification.

[0185] Dye 4 synthesis In a 100 mL single-port reactor, combine intermediate 4-2 (2.7 g, 4.2 mmol), HSTU (O-(N-Succinimidyl)-N,N,N',N'-tetramethyl uronium hexafluorophosphate) (1.81 g, 5.0 mmol), triethylamine (1.76 mL, 12.6 mmol), and dimethylformamide (27 mL). Stir at room temperature for 30 minutes. After concentration, column chromatography was performed to synthesize Dye 4 (2.1 g). The obtained Dye 4 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.77(t, 1H, J=12.8Hz), 8.54(d, 1H, J=8.0Hz), 8.38(d, 1H, J=8.2 9Hz), 8.22(d, 1H, J=8.4Hz), 8.03(t, 1H, J=7.2Hz), 7.71(d, 1H, J=8.4Hz), 7.65(t, 1H, J=8.4 Hz), 7.61-7.52(m, 2H), 7.47-7.15(m, 2H), 7.00(d, 1H, J=13.6Hz), 6.87(d, 1H, J=12.8Hz), 4 .50(t, 2H, J=7.6Hz), 3.90(s, 4H), 3.82(s, 3H), 2.53(m, 2H), 2.12(s, 6H), 2.10-1.76(m, 6H)

[0186] Dye 5 synthesis [ka]

[0187] Synthesis of intermediate 5-1 Intermediate 5-1 was synthesized by referring to Journal of Medicinal Chemistry, 2022, vol.65, #1, pp.811-823.

[0188] Dye 5 synthesis In a 100 mL single-port reactor, combine intermediate 5-1 (3.0 g, 6.4 mmol), intermediate 1-2 (2.6 g, 6.4 mmol), and pyridine (30 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 5 (1.7 g). The obtained Dye 5 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.56 (d, 1H, J=8.0Hz), 8.49 (t, 2H, J=15.6Hz), 8.33 (d, 1H, J=8 .30Hz), 8.22(d, 1H, J=8.4Hz), 8.00(t, 1H, J=7.2Hz), 7.75(d, 1H, J=8.4Hz), 7.63(t, 1H, J=8.4Hz), 7.60-7.50(m, 2H), 7.45-7.17(m, 2H), 6.90(t, 1H, J=13.6Hz), 6.36(d, 2H, J=1 3.6Hz), 4.52(t, 2H, J=7.6Hz), 3.80(s, 3H), 2.53(m, 2H), 2.12(s, 6H), 2.10-1.76(m, 6H)

[0189] Dye 6 synthesis [ka]

[0190] Synthesis of intermediate 6-1 Intermediate 6-1 was synthesized by referring to the Journal of the American Chemical Society, 2011, vol. 133, #1, pp. 51-55.

[0191] Synthesis of intermediate 6-2 Intermediate 6-2 was synthesized by referring to Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0192] Dye 6 synthesis In a 100 mL single-port reactor, combine intermediate 6-1 (2.0 g, 3.7 mmol), intermediate 6-2 (1.1 g, 3.7 mmol), and pyridine (30 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 6 (1.1 g). The obtained Dye 6 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.76(t, 1H, J=12.8Hz), 8.51(d, 1H, J=8.0Hz), 8.37(d, 1H, J= 8.0Hz), 8.22(d, 1H, J=8.4Hz), 8.04(t, 1H, J=7.2Hz), 7.70(d, 1H, J=8.4Hz), 7.62(t, 1H, J=8.4Hz), 7.59-7.50(m, 2H), 7.45-7.13(m, 2H), 6.98(d, 1H, J=13.6Hz), 6.86(d, 1H, J=1 2.8Hz), 4.26(t, 2H, J=7.6Hz), 3.85(s, 3H), 2.51(m, 2H), 2.13(s, 6H), 2.10-1.75(m, 6H)

[0193] Dye 7 synthesis [ka]

[0194] Synthesis of intermediate 7-1 In a 250 mL single-port reactor, combine intermediate 6-2 (5.0 g, 16 mmol), N,N-diphenylformamidine (3.8 g, 21 mmol), and acetic anhydride (40 mL), and stir at 110 °C for 1 hour. After cooling, add ethyl acetate (100 mL) and stir vigorously. Filter the resulting solid to synthesize intermediate 7-1.

[0195] Synthesis of intermediate 7-2 Intermediate 7-2 was synthesized by referring to WO2022-191485 A1.

[0196] Dye 7 synthesis In a 100 mL single-port reactor, combine intermediate 7-1 (3.0 g, 6.6 mmol), intermediate 7-2 (2.8 g, 6.6 mmol), and pyridine (30 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 7 (1.5 g). The obtained Dye 7 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.73(t, 1H, J=12.8Hz), 8.36-8.28(m, 2H), 8.00(t , 1H, J=7.6Hz), 7.80Hz), 8.21(d, 1H, J=8.0Hz), 7.74-7.37(m, 6H), 6.86(d, 2) H, J=13.6Hz), 4.03(q, 2H, J=7.6Hz), 3.85(s, 3H), 3.80(s, 3H), 2.53(m, 2), 2 .10-1.75(m, 9H)1.19(t, 3H, J=8.0Hz), 1.02-0.95(m, 1H), 0.69-0.64(m, 1H)

[0197] Dye 8 synthesis [ka]

[0198] Synthesis of intermediate 8-1 Intermediate 8-1 was synthesized by referring to US10473666, 2019, B2.

[0199] Synthesis of intermediate 8-2 In a 250 mL single-port reactor, intermediate 8-1 (10 g, 40.8 mmol), iodomethane (8.68 g, 61.2 mmol), and acetonitrile (100 mL) were added and stirred under reflux for 24 hours. After cooling, ethyl acetate (100 mL) was added and stirred for 10 minutes. The resulting solid was filtered to synthesize intermediate 8-2.

[0200] Synthesis of intermediate 8-3 Intermediate 8-2 (3.0g, 7.7 mmol), intermediate 7-1 (3.5g, 7.7 mmol), and pyridine (30 mL) were placed in a 100 mL single-port reactor and stirred at 50°C for 1 hour. After concentration, intermediate 8-3 was synthesized by column purification.

[0201] Dye 8 synthesis In a 100 mL single-port reactor, combine intermediate 8-3 (2.0 g, 3.5 mmol), 2 N sodium hydroxide aqueous solution (5 mL), and methanol (2 mL), and stir at room temperature for 24 hours. After concentration, add 2 M hydrochloric acid aqueous solution (10 mL) and dichloromethane (50 mL) and stir. After separating the organic layer, filtration was performed by column chromatography to synthesize Dye 8 (0.8 g). The obtained Dye 8 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.73(t, 1H, J=12.8Hz), 8.36-8.28(m, 2H), 8.00(t, 1H, J=7.6Hz), 7.80(d, 1H, J=8.0Hz), 7.74-7.37(m, 5H), 6.86(d, 2H, J=13.6Hz), 3.96(s, 2H), 3.85(s, 3H), 3.80(s, 3H), 2.12(s, 6H)

[0202] Dye 9 synthesis [ka]

[0203] Synthesis of intermediate 9-1 Intermediate 9-1 was synthesized by referring to US2011 / 152538, 2011, A1.

[0204] Synthesis of intermediate 9-2 In a 250 mL three-neck reactor, intermediate 9-1 (10 g, 38 mmol) and tetrahydrofuran (100 mL) were added and stirred at -78°C for 5 minutes under a nitrogen stream. 1.6 M butyllithium (24 mL, 38 mmol) was added and stirred for 1 hour. Dry ice (5 g) was added and stirred at room temperature for 1 hour. After concentration, intermediate 9-2 was synthesized by column purification.

[0205] Synthesis of intermediate 9-3 In a 250 mL single-port reactor, intermediate 9-2 (6 g, 23 mmol), iodoethyl (3.9 g, 25 mmol), potassium carbonate (9.0 g, 34 mmol), and N,N-dimethylformamide (60 mL) were added and stirred at room temperature for 24 hours. After filtering the solid, the mixture was concentrated. Intermediate 9-3 was synthesized by column purification.

[0206] Synthesis of intermediate 9-4 Intermediate 9-4 was synthesized by referring to Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0207] Dye 9 synthesis In a 50 mL single-port reactor, combine intermediate 9-4 (1.0 g, 2.6 mmol), intermediate 4-1 (1.2 g, 2.6 mmol), and pyridine (10 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 9 (0.7 g). The obtained Dye 9 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.72(t, 1H, J=12.8Hz), 8.50(d, 1H, J=8.0Hz), 8.34(d, 1H, J=8.0Hz), 8.18(d, 1H, J=8.4Hz), 7.69(d, 1H, J=8.4Hz), 7. 60(t, 1H, J=8.4Hz), 7.57-7.50(m, 2H), 7.45-7.10(m, 2H), 6.98(d, 1H, J =13.6Hz), 6.86(d, 1H, J=12.8Hz), 3.97(s, 3H), 3.85(s, 3H), 2.13(s, 6H)

[0208] Dye 10 synthesis [ka]

[0209] Synthesis of intermediate 10-1 Intermediate 10-1 was synthesized by referring to Chemical Communications, 2016, vol.52, #90, pp.13307-13310.

[0210] Synthesis of intermediate 10-2 In a 250 mL three-neck reactor, intermediate 10-1 (4.75 g, 18.2 mmol), 4-(ethoxycarbonyl)phenylboronic acid (5.0 g, 27.8 mmol), potassium carbonate (5.0 g, 36.4 mmol), Pd(PPh3)4 (0.1 g, 0.09 mmol), 1,2-dimethoxyethane (30 mL), water (9 mL), and N,N-dimethylformamide (65 mL) were added and stirred at 85 °C for 12 hours. After cooling, the solid was filtered through cellulite. After concentrating the filtrate, intermediate 10-2 was synthesized by column purification.

[0211] Synthesis of intermediate 10-3 Intermediate 10-3 was synthesized by referring to Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0212] Dye 10 synthesis In a 50 mL single-port reactor, combine intermediate 10-3 (2.0 g, 4.5 mmol), intermediate 4-1 (2.0 g, 4.5 mmol), and pyridine (20 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 10 (1.1 g). The obtained Dye 10 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.75 (t, 1H, J=12.8Hz), 8.49 (d, 1H, J=8.0Hz), 8.33 (d, 1H, J=8.0Hz), 8.16(d, 1H, J=8.4Hz), 8.14(d, 2H, J=8.7Hz), 7.67(d, 1H, J= 8.4Hz), 7.60(t, 1H, J=8.4Hz), 7.60-7.49(m, 2H), 7.47-7.12(m, 4H), 7.00(d, 1H, J=13.6Hz), 6.88(d, 1H, J=12.8Hz), 3.96(s, 3H), 3.84(s, 3H), 2.11(s, 6H)

[0213] Dye 11 synthesis [ka]

[0214] Synthesis of intermediate 11-1 Intermediate 11-1 was synthesized by referring to US9150922, 2015, B2.

[0215] Dye 11 synthesis Dye 11 was synthesized from intermediates 11-1 and 6-2 using the method described in US9150922, 2015, B2. The obtained Dye 11 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.49-8.37(m, 3H), 8.33(d, 1H, J=7.4Hz), 8.21-8.00(m, 2H), 7.67(d, 1H, J=8.4Hz), 7.6 0(t, 1H, J=8.4Hz), 7.53-7.26(m, 6H), 7.00(d, 1H, J=13.6Hz), 6.85(m, 2H), 4.22(s, 3H), 3.85(s, 3H), 2.11(s, 6H)

[0216] Synthesis of Dye 12 [ka]

[0217] Synthesis of intermediate 12-1 Intermediate 12-1 was synthesized by referring to US2002 / 77487, 2002, A1.

[0218] Synthesis of Dye 12 In a 50 mL single-port reactor, combine intermediate 12-1 (2.0 g, 5.0 mmol), intermediate 7-1 (2.26 g, 5.0 mmol), and pyridine (20 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 12 (0.9 g). The obtained Dye 12 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.87(m, 2H), 8.53-8.33(m, 3H), 8.20(d, 1H, J=7.2Hz), 7.67(d, 1H, J=8.4Hz), 7.60(t, 1H, J=8 .4Hz), 7.42-7.26(m, 2H), 6.85(m, 2H), 4.52(q, 2H, J=7.2Hz), 3.85(s, 3H), 2.52(m, 2H), 2.11(s, 6H), 2.07-1.71(m, 6H)

[0219] Dye 13 synthesis [ka]

[0220] Synthesis of intermediate 13-1 Intermediate 13-1 was synthesized by referring to Journal of Organic Chemistry, 2018, vol.83, #8, pp.4389-4401.

[0221] Synthesis of intermediate 13-2 In a 1L four-port reactor, intermediate 13-1 (50g, 0.202mol), ethyl 4-piperidine carboxylate (38g, 0.242mol), Pd2(dba)3 (7.4g, 0.008mol), BINAP (5.0g, 0.008mol), sodium tert-butoxide (27g, 0.282mol), and toluene (500mL) were added and stirred under reflux for 12 hours. After cooling, the solid was filtered through cellulite. After concentrating the filtrate, intermediate 13-2 was synthesized by column purification.

[0222] Synthesis of intermediate 13-3 Intermediate 13-3 was synthesized from intermediate 13-2 by referring to the method in Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0223] Dye 13 synthesis In a 50 mL single-port reactor, combine intermediate 13-3 (0.7 g, 1.6 mmol), intermediate 4-1 (0.72 g, 1.6 mmol), and pyridine (15 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 13 (0.3 g). The obtained Dye 13 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.75 (t, 1H, J=12.6Hz), 8.49 (d, 1H, J=8.0Hz), 8. 31(d, 1H, J=8.28(d, 1H, J=8.4Hz), 7.73-7.63(m, 2H), 7.57-7.52(m, 2H), 7.4 5-7.15(m, 2H), 6.99(d, 1H, J=13.6Hz), 6.88(d, 1H, J=12.8Hz), 3.95(s, 3H), 3.81(s, 3H), 3.44(m, 4H), 2.49-2.44(m, 1H), 2.10(s, 6H), 1.88-1.61(m, 4H)

[0224] Dye 14 synthesis [ka]

[0225] Synthesis of intermediate 14-1 3-nitro-9H-carbazole (20g, 94 mmol), ethyl 4-bromobutyrate (22g, 113 mmol), potassium carbonate (26g, 190 mmol), and N,N-dimethylformamide (200 mL) were placed in a 500 mL single-port reactor and stirred at 50°C for 12 hours. After concentration, dichloromethane (200 mL) and water (100 mL) were added and stirred for 5 minutes, after which the organic layer was separated. Anhydrous sodium sulfate was added and stirred for 5 minutes, then filtered. After concentrating the filtrate, intermediate 14-1 was synthesized by column purification.

[0226] Synthesis of intermediate 14-2 Intermediate 14-1 (12g, 36 mmol), 5% Pd / C (0.6g), and methane (120mL) were placed in a 500mL single-port reactor and stirred under a hydrogen stream for 24 hours. After filtering through cellulite, the filtrate was concentrated to synthesize intermediate 14-2.

[0227] Synthesis of intermediate 14-3 Intermediate 14-3 was synthesized from intermediate 14-2 by referring to the method of KR2020 / 67733,2020,A.

[0228] Dye 14 synthesis In a 50 mL single-port reactor, combine intermediate 14-3 (1.6 g, 3.2 mmol), intermediate 7-1 (1.4 g, 3.2 mmol), and pyridine (15 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 14 (0.7 g). The obtained Dye 14 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.72(t, 1H, J=12.8Hz), 8.52(d, 1H, J=8.0Hz), 8.40(d, 1H, J=7.8Hz) , 8.22(d, 1H, J=8.4Hz), 8.03(t, 1H, J=7.2Hz), 7.86-7.62(m, 7H), 7.46-7.40(m, 2H), 7.00(d, 1 H, J=13.6Hz), 6.83(d, 1H, J=12.8Hz), 4.43(t, 2H, J=7.6Hz), 4.12(q, 2H, J=7.6Hz), 3.95(s, 3H) ), 3.82(s, 3H), 2.25(t, 2H, J=7.2Hz), ), 2.12(s, 6H), 1.91-1.87(m, 2H), 1.33(t, 3H, J=7.6Hz)

[0229] Dye 15 synthesis [ka]

[0230] Synthesis of intermediate 15-1 Intermediate 15-1 was synthesized by referring to the Journal of the American Chemical Society, 2011, vol. 133, #1, pp. 51-55.

[0231] Dye 15 synthesis In a 100 mL single-port reactor, intermediate 15-1 (2.0 g, 3.5 mmol), intermediate 6-2 (1.1 g, 3.5 mmol), and pyridine (20 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 15 was synthesized by column purification (0.9 g). The obtained Dye 15 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.54(d, 1H, J=8.0Hz), 8.49(t, 2H, J=15.6Hz), 8.32(d, 1H, J= 7.6Hz), 8.31(d, 1H, J=8.2Hz), 8.00(t, 1H, J=7.0Hz), 7.72(d, 1H, J=8.4Hz), 7.61(t, 1H, J=8.4Hz), 7.60-7.50(m, 2H), 7.44-7.14(m, 2H), 6.87(t, 1H, J=13.6Hz), 6.36(d, 2H, J=1 3.6Hz), 4.23(t, 2H, J=7.2Hz), 3.83(s, 3H), 2.23(m, 2H), 2.12(s, 6H), 2.10-1.76(m, 6H)

[0232] Dye 16 synthesis [ka]

[0233] Synthesis of intermediate 16-1 Intermediate 16-1 was synthesized by referring to CN113336743,2021,A.

[0234] Dye 16 synthesis In a 100 mL single-port reactor, intermediate 16-1 (3.0 g, 6.6 mmol), intermediate 7-1 (3.0 g, 6.6 mmol), and pyridine (30 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 16 was synthesized by column purification (2.1 g). The obtained Dye 16 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.89(t, 1H, J=12.6Hz), 8.23-8.00(m, 5H), 7.89(t, 1H, J=7.2Hz), 7.77-7.47(m, 7H), 7.19(d, 1H, J=7.2Hz), 4.51(t, 2H, J=8.4Hz), 3.97(s, 3H), 2.54(t, 2H, J=6.8Hz), 2.12(s, 6H), 2.03-1.77(m, 6H)

[0235] Dye 17 synthesis [ka]

[0236] Synthesis of intermediate 17-1 Intermediate 17-1 was synthesized by referring to US2020 / 0224257A1.

[0237] Dye 17 synthesis In a 100 mL single-port reactor, intermediate 17-1 (3.0 g, 5.8 mmol), intermediate 7-1 (2.6 g, 5.8 mmol), and pyridine (30 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 17 was synthesized by column purification (1.7 g). The obtained Dye 17 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.75(t, 1H, J=12.8Hz), 8.49(d, 1H, J=7.8Hz), 8.44(d, 1H, J=7.6Hz), 8182(d, 1H, J=8.2Hz), 8.01(t, 1H, J=7.6Hz), 7.86-7.40(m, 8H), 6. 68(m, 2H), 4.58(q, 2H, J=6.8Hz), 4.44(t, 2H, J=7.2Hz), 4.21(q, 2H, J=7.8Hz), 3. 52(s, 3H), 2.25(t, 2H, J=7.2Hz), 2.11(s, 6H), 1.93(m, 2H), 1.32(t, 3H, J=7.8Hz)

[0238] Dye 18 synthesis [ka]

[0239] Synthesis of intermediate 18-1 Intermediate 18-1 was synthesized by referring to Dyes and Pigments, 2022, vol.197, art.no.109874.

[0240] Synthesis of intermediate 18-2 In a 250 mL single-port reactor, intermediate 18-1 (10 g, 3.1 mmol), malonaldehyde dianilide hydrochloride (8.8 g, 34 mmol), and acetic anhydride (100 mL) were added, and the mixture was stirred under reflux for 1 hour. After cooling, ethyl acetate (100 mL) was added to the reactor and the mixture was stirred for 5 minutes. The resulting solid was filtered to synthesize intermediate 18-2.

[0241] Synthesis of intermediate 18-3 Intermediate 18-3 was synthesized by referring to US2022 / 274960,2022,A1.

[0242] Dye 18 synthesis Add intermediate 18-2 (1.9g, 3.9 mmol), intermediate 18-3 (2.0g, 3.9 mmol), and pyridine (20 mL) to a 100 mL single-port reactor and stir at 50°C for 1 hour. After concentration, Dye 18 was synthesized by column purification (0.7g). The obtained Dye 18 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.54 (d, 1H, J=8.0Hz), 8.49 (m, 2H), 8.32 (d, 1H, J=8. 0Hz), 8.00(t, 1H, J=7.0Hz), 7.81-7-40(m, 9H), 6.87(t, 1H, J=13.6Hz), 6.52(d, 1H, J=12.8Hz) 6.36(d, 1H, J=13.6Hz), 4.58(q, 2H, J=6.8Hz), 4.23(t, 2H, J=7.2H z), 2.25(t, 2H, J=7.2Hz), 1.93-1.75(s, 9H), 1.56(s, 6H), 1.35(t, 3H, J=7.2Hz)

[0243] Dye 19 synthesis [ka]

[0244] Synthesis of intermediate 19-1 Intermediate 19-1 was synthesized by referring to KR2020 / 67732,2020,A.

[0245] Synthesis of intermediate 19-2 In a 250 mL single-port reactor, intermediate 19-1 (10 g, 30 mmol), 6-bromohexanoic acid (8.9 g, 46 mmol), and 1,2-dichlorobenzene (100 mL) were added and stirred at 150 °C for 2 hours. After cooling, acetone (100 mL) was added to the reactor and stirred for 5 minutes. The resulting solid was filtered to synthesize intermediate 19-2.

[0246] Dye 19 synthesis Add intermediate 19-2 (2.0g, 3.8 mmol), intermediate 18-2 (1.9g, 3.8 mmol), and pyridine (20 mL) to a 100 mL single-port reactor and stir at 50°C for 1 hour. After concentration, Dye 19 was synthesized by column purification (0.3g). The obtained Dye 19 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.48-8.51(m, 3H), 8.40(d, 1H, J=7.8Hz), 8.22(d, 1H, J= 8.4Hz), 8.03(t, 1H, J=7.6Hz), 7.86-7.40(m, 6H), 7.19(t, 1H, J=8.0Hz), 7.09(t, 1 H, 8.4Hz), 6.75-6.53(m, 3H), 4.41(t, 2H, J=7.2Hz), 4.23(q, 2H, J=6.8Hz), 3.91(s , 3H), 2.53(t, 2H, J=7.2Hz), 2.13(s, 6H), 1.90-1.50(m, 9H), 1.33(t, 3H, J=7.8Hz)

[0247] Dye 20 synthesis [ka]

[0248] Synthesis of intermediate 20-1 Intermediate 20-1 was synthesized by referring to WO2017146187A1.

[0249] Synthesis of intermediate 20-2 In a 250 mL single-port reactor, intermediate 20-1 (10 g, 35 mmol), 6-bromohexanoic acid (10.3 g, 53 mmol), and 1,2-dichlorobenzene (100 mL) were added and stirred at 150 °C for 2 hours. After cooling, acetone (100 mL) was added to the reactor and stirred for 5 minutes. The resulting solid was filtered to synthesize intermediate 20-2.

[0250] Dye 20 synthesis In a 100 mL single-port reactor, combine intermediate 20-2 (3.0 g, 6.3 mmol), intermediate 7-1 (2.9 g, 6.3 mmol), and pyridine (30 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 20 (2.3 g). The obtained Dye 20 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.59-8.52(m, 4H), 8.38(d, 1H, J=8.0Hz), 8.30(d, 1H), 8.22(d, 1H, J=8.2Hz), 8.14-8.10(m, 2H), 8.03(t, 1H, J=7.2Hz), 7.90-7. 65(m, 3H), 7.53-7.42(m, 2H), 6.83(m, 2H), 4.28(t, 2H, J=6.8Hz), 3.83(s, 3H) , 2.52(t, 2H, J=7.6Hz), 2.10(s, 6H), 1.98-1.53(m, 6H), 1.30(t, 3H, J=7.6Hz)

[0251] Dye 21 synthesis [ka]

[0252] Dye 21 synthesis In a 100 mL single-port reactor, combine intermediates 1-2 (2.0 g, 4.9 mmol), 7-1 (2.2 g, 4.9 mmol), and pyridine (10 mL), and stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 21 (1.7 g). The obtained Dye 21 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ9.14(t, 1H, J=12.8Hz), 8.64(d, 2H, J=7.2Hz), 8.33(d, 2H, J=7.6Hz), 8.06-8.01(m, 2H), 7.85(d, 2H, J =7.0Hz), 7.70-7.67(m, 4H), 7.17(d, 2H, J=13.2Hz), 4.33(t, 2H, J=7.2Hz), 3.92(s, 3H), 2.51(t, 2H, J=7.6Hz), 1.92-1.43(m, 6H)

[0253] Synthesis of Dye 22 [ka]

[0254] Synthesis of intermediate 22-1 Intermediate 22-1 was synthesized by referring to Organic Letters, 2019, vol.21, #14, pp. 5694-5698.

[0255] Synthesis of intermediate 22-2 Intermediate 22-2 was synthesized from intermediate 22-1 by referring to the method in Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0256] Synthesis of Dye 22 In a 100 mL single-port reactor, intermediate 22-2 (1.3 g, 3.7 mmol), intermediate 6-1 (2.0 g, 3.7 mmol), and pyridine (13 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 22 was synthesized by column purification (0.6 g). The obtained Dye 221 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.54(t, 1H, J=12.8Hz), 8.37(d, 1H, J=7.8Hz) 8.30(d, 1H, J=8.2Hz), 8.04(t, 1H, J=7.2Hz), 7.70-7.50(m, 5H), 7.45 -7.10(m, 4H), 7.00(d, 1H, J=13.6Hz), 6.88(d, 1H, J=12.8Hz), 4.28(t, 2H, J=7.2Hz), 3.84(s, 3H), 2.51(m, 2H), 2.13(s, 6H), 2.09-1.75(m, 6H)

[0257] Synthesis of Dye 23 [ka]

[0258] Synthesis of intermediate 23-1 Intermediate 23-1 was synthesized by referring to EP3882242,2021,A1.

[0259] Synthesis of intermediate 23-2 Intermediate 23-2 was synthesized from intermediate 23-1 by referring to the method in Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0260] Synthesis of Dye 23 In a 100 mL single-port reactor, intermediate 23-2 (3.2 g, 9.0 mmol), intermediate 6-1 (4.9 g, 9.0 mmol), and pyridine (30 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 23 was synthesized by column purification (1.9 g). The obtained Dye 23 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ9.07(d, 1H, J=8.4Hz), 8.72(t, 1H, J=12.8Hz), 8.62(d, 1 H, J=8.0Hz), 8.41(d, 1H, J=7.8Hz), 8.18(d, 1H, J=8.4Hz), 7.62(t, 1H, J=8.4Hz), 7 .64-7.50(m, 2H), 7.48-7.28(m, 2H), 6.98(d, 1H, J=12.8Hz), 6.84(d, 1H, J=12.8Hz) ), 4.24(t, 2H, J=7.6Hz), 3.97(s, 3H), 2.52(m, 2H), 2.10(s, 6H), 2.11-1.73(m, 6H)

[0261] Synthesis of Dye 24 [ka]

[0262] Synthesis of intermediate 24-1

[0263] Intermediate 24-1 was synthesized from intermediate 9-1 by referring to the method in Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0264] Synthesis of Dye 24 In a 100 mL single-port reactor, intermediate 24-1 (2.6 g, 6.7 mmol), intermediate 6-1 (3.7 g, 6.7 mmol), and pyridine (30 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 24 was synthesized by column purification (1.9 g). The obtained Dye 24 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.70 (t, 1H, J=12.8Hz), 8.58 (d, 1H, J=8.0Hz), 8.39 (d, 1H, J=7.2Hz), 8.24(d, 1H, J=8.0Hz), 7.89(d, 1H, J=8.4Hz), 7.62(t, 1H, J= 8.4Hz), 7.59-7.28(m, 4H), 6.82(d, 1H, J=12.8Hz), 6.51(d, 1H, J=12.6Hz), 4. 22(t, 2H, J=7.8Hz), 3.83(s, 3H), 2.51(m, 2H), 2.13(s, 6H), 2.10-1.59(m, 6H)

[0265] Synthesis of Dye 25 [ka]

[0266] Synthesis of intermediate 25-1 Intermediate 25-1 was synthesized by referring to the method described in Journal of Organic Chemistry USSR 1982, vol.18, #2, pp.380-386.

[0267] Synthesis of Dye 25 In a 100 mL single-port reactor, intermediate 25-1 (2.0 g, 5.7 mmol), intermediate 6-1 (3.1 g, 5.7 mmol), and pyridine (20 mL) were added, and the mixture was stirred at 50°C for 1 hour. After concentration, Dye 25 was synthesized by column purification (0.8 g). The obtained Dye 25 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.64(t, 1H, J=13.6Hz), 8.55(d, 1H, J=8.0Hz), 8.34(d, 1H, J=7 .6Hz), 8.22(d, 1H, J=7.8Hz), 7.86(d, 1H, J=8.4Hz), 7.60(t, 1H, J=8.4Hz), 7.53-7.22(m, 4H), 6.80(d, 1H, J=12.8Hz), 6.50(d, 1H, J=12.6Hz), 4.22(t, 2H, J=7.6Hz), 4.15(q, 2H, J =7.8Hz), 3.90(s, 3H), 2.51(m, 2H), 2.11(s, 6H), 2.11-1.50(m, 6H), 1.32(t, 3H, J=7.2Hz)

[0268] Synthesis of Dye 26 [ka]

[0269] Synthesis of intermediate 26-1 In a 500 mL four-neck reactor, intermediate 9-1 (20 g, 0.077 mol), N-methylaniline (9.8 g, 0.092 mol), Pd2(dba)3 (2.8 g, 0.003 mol), BINAP (1.9 g, 0.003 mol), sodium tert-butoxide (10.3 g, 0.107 mol), and toluene (200 mL) were added and stirred under reflux for 12 hours. After cooling, the solid was filtered through cellulite. After concentrating the filtrate, intermediate 26-1 was synthesized by column purification.

[0270] Synthesis of intermediate 26-2 Intermediate 26-2 was synthesized from intermediate 26-1 by referring to the method in Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0271] Synthesis of Dye 26 In a 100 mL single-port reactor, intermediate 26-2 (3.0 g, 7.2 mmol), intermediate 6-1 (4.0 g, 7.2 mmol), and pyridine (30 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 26 was synthesized by column purification (1.9 g). The obtained Dye 26 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.63(t, 1H, J=13.6Hz), 8.52(d, 1H, J=7.8Hz), 8.32(d , 1H, J=7.8Hz), 8.21(d, 1H, J=7.8Hz), 7.84(d, 1H, J=8.2Hz), 7.67(t, 1H, J=8.2Hz ), 7.58-7.22(m, 9H), 6.96(d, 1H, J=12.6Hz), 6.69(d, 1H, J=13.2Hz), 4.22(t, 2H, J=7.6Hz), 3.84(s, 3H), 3.42(s, 3H), 2.53(m, 2H), 2.12(s, 6H), 2.10-1.47(m, 6H)

[0272] Synthesis of Dye 27 [ka]

[0273] Synthesis of intermediate 27-1 In a 500 mL four-neck reactor, intermediate 9-1 (20 g, 0.077 mol), piperidine (7.8 g, 0.092 mol), Pd2(dba)3 (2.8 g, 0.003 mol), BINAP (1.9 g, 0.003 mol), sodium tert-butoxide (10.3 g, 0.107 mol), and toluene (200 mL) were added and stirred under reflux for 12 hours. After cooling, the solid was filtered through cellulite. After concentrating the filtrate, intermediate 27-1 was synthesized by column purification.

[0274] Synthesis of intermediate 27-2 Intermediate 27-2 was synthesized from intermediate 27-1 by referring to the method in Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0275] Synthesis of Dye 27 In a 100 mL single-port reactor, intermediate 27-2 (3.0 g, 7.6 mmol), intermediate 6-1 (4.2 g, 7.2 mmol), and pyridine (30 mL) were added, and the mixture was stirred at 50°C for 1 hour. After concentration, Dye 27 was synthesized by column purification (1.6 g). The obtained Dye 27 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.67(t, 1H, J=13.6Hz), 8.56(d, 1H, J=7.6Hz), 8.37(d , 1H, J=8.0Hz), 8.24(d, 1H, J=7.6Hz), 7.84(d, 1H, J=8.0Hz), 7.67(m, 1H), 7.55- 7.20(m, 4H), 6.87(d, 1H, J=12.6Hz), 6.70(d, 1H, J=12.6Hz), 4.24(t, 2H, J=7.6H z), 3.84(s, 3H), 3.40-3.34(m, 4H), 2.53(m, 2H), 2.12(s, 6H), 2.10-1.47(m, 12H)

[0276] Dye 28 synthesis [ka]

[0277] Synthesis of intermediate 28-1 In a 100 mL single-port reactor, combine Dye 6 (2 g, 3.4 mmol), 2-(methylamino)ethanol (0.3 g, 4.1 mmol), HATU (1.9 g, 5.1 mmol), triethylamine (1.4 mL, 10.1 mmol), and dimethylformamide (20 mL), and stir at room temperature for 1 hour. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 28-1.

[0278] Dye 28 synthesis In a 25 mL single-port reactor, combine intermediate 28-1 (1.8 g, 2.8 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.85 g, 3.6 mmol), triethylamine (0.77 mL, 5.5 mmol), and dichloromethane (20 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize Dye 28 (1.5 g). The obtained Dye 28 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.75(t, 1H, J=12.6Hz), 8.50(d, 1H, J=8.0Hz), 8.35(d, 1H, J=8.0Hz), 8.21(d, 1H, J=8.2Hz), 8.03(t, 1H, J=7.2Hz), 7.70(d, 1H, J=8.4Hz), 7.60(t, 1H, J=8.2Hz), 7.60-7.50(m, 2H), 7. 44-7.12(m, 2H), 6.97(d, 1H, J=13.6Hz), 6.84(d, 1H, J=12.8Hz), 4.25(t, 2H, J=7.4Hz), 3.83(s, 3H), 3.5 0-3.42(m, 11H), 2.69(t, 2H, J=5.6Hz), 2.48(m, 2H), 2.13(s, 6H), 2.10-1.75(m, 6H), 1.20-1.18(m, 12H)

[0279] Dye 29 synthesis [ka]

[0280] Synthesis of intermediate 29-1 In a 100 mL single-port reactor, combine Dye 6 (3.0 g, 5.1 mmol), 4-piperidinemethanol (0.70 g, 6.1 mmol), HATU (2.9 g, 7.6 mmol), triethylamine (2.1 mL, 15.2 mmol), and dimethylformamide (30 mL), and stir at room temperature for 1 hour. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 29-1.

[0281] Dye 29 synthesis In a 25 mL single-port reactor, combine intermediate 29-1 (2.6 g, 3.8 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (1.2 g, 4.9 mmol), triethylamine (1 mL, 7.5 mmol), and dichloromethane (25 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize Dye 29 (2.2 g). The obtained Dye 29 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.77(t, 1H, J=12.8Hz), 8.50(d, 1H, J=7.8Hz), 8.33(d, 1H, J=7.8Hz), 8.27(d, 1H, J=7.3Hz), 8.03(t, 1H, J=7.8Hz), 7.70(d, 1H, J=7.8Hz), 7.63(t, 1H, J=8.2Hz), 7.61-7.45(m, 2H), 7. 45-7.10(m, 2H), 6.94(d, 1H, J=12.8Hz), 6.84(d, 1H, J=12.8Hz), 4.25(t, 2H, J=7.4Hz), 3.83(s, 3H), 3.7 0-3.48(m, 10H), 2.78(t, 2H, J=5.8Hz), 2.50(m, 2H), 2.11(s, 6H), 1.89-1.44(m, 11H), 1.21-1.11(m, 12H)

[0282] Dye 30 synthesis [ka]

[0283] Synthesis of intermediate 30-1 Intermediate 30-1 was synthesized by referring to Journal of the American Chemical Society 2004, vol.126, #27, pp.8364-8365.

[0284] Synthesis of intermediate 30-2 In a 100 mL single-port reactor, combine Dye 6 (3.0 g, 5.1 mmol), intermediate 30-1 (2.47 g, 6.1 mmol), HATU (2.9 g, 7.6 mmol), triethylamine (2.1 mL, 15.2 mmol), and dimethylformamide (30 mL), and stir at room temperature for 1 hour. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 30-2.

[0285] Dye 30 synthesis In a 25 mL single-port reactor, combine intermediate 30-2 (2.4 g, 2.0 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.62 g, 4.9 mmol), triethylamine (0.6 mL, 4.1 mmol), and dichloromethane (25 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize Dye 30 (1.1 g). The obtained Dye 30 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.76 (t, 1H, J=12.8Hz), 8.52 (d, 1H, J=7.8Hz), 8.34 (d, 1H, J=7.8Hz), 8.2 8(d, 1H, J=7.4Hz), 8.01(t, 1H, J=7.2Hz), 7.71-7.63(m, 2H), 7.61-7.45(m, 2H), 7.45-7.10(m, 11H), 6.94(d, 1H, J=12.8Hz), 6.85-6.82(m, 5H), 4.24(t, 2H, J=7.4Hz), 3.84(s, 3H), 3.77(s, 6H), 3.59-3 .41(m, 12H), 2.76(t, 2H, J=6.2Hz), 2.51(m, 2H), 2.11(s, 6H), 1.86-1.44(m, 6H), 1.23-1.14(m, 12H)

[0286] Synthesis of Dye 31 [ka]

[0287] Synthesis of intermediate 31-1 Intermediate 31-1 was synthesized by referring to US2020 / 369703,2020,A1.

[0288] Synthesis of intermediate 31-2 In a 100 mL single-port reactor, combine Dye 6 (4.0 g, 6.8 mmol), intermediate 31-1 (3.2 g, 8.1 mmol), HATU (3.9 g, 10.1 mmol), triethylamine (2.8 mL, 20.3 mmol), and dimethylformamide (40 mL), and stir at room temperature for 1 hour. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 31-2.

[0289] Synthesis of Dye 31 In a 25 mL single-port reactor, combine intermediate 31-2 (2.5 g, 2.6 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.8 g, 3.4 mmol), triethylamine (0.7 mL, 5.2 mmol), and dichloromethane (25 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize Dye 31 (1.3 g). The obtained Dye 31 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.77(t, 1H, J=12.8Hz), 8.50(d, 1H, J=7.6Hz), 8.36(d, 1H, J=8.0Hz), 8.2 5(d, 1H, J=7.2Hz), 8.00(t, 1H, J=7.2Hz), 7.72-7.61(m, 2H), 7.59-7.45(m, 2H), 7.44-7.10(m, 11H) , 6.97(d, 1H, J=13.2Hz), 6.87-6.80(m, 5H), 4.22(t, 2H, J=7.4Hz), 3.83(s, 3H), 3.75(s, 6H), 3.55- 3.40(m, 8H), 2.76(t, 2H, J=6.0Hz), 2.50(m, 2H), 2.13(s, 6H), 1.90-1.40(m, 6H), 1.21-1.11(m, 12H)

[0290] Synthesis of Dye 32 [ka]

[0291] Synthesis of intermediate 32-1 Intermediate 32-1 was synthesized by referring to Bioorganic and Medicinal Chemistry, 2016, vol.24, #1, pp.26-32.

[0292] Synthesis of intermediate 32-2 In a 100 mL single-port reactor, combine Dye 6 (4.0 g, 6.8 mmol), intermediate 32-1 (3.4 g, 8.1 mmol), HATU (3.9 g, 10.1 mmol), triethylamine (2.8 mL, 20.3 mmol), and dimethylformamide (40 mL), and stir at room temperature for 1 hour. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 32-2.

[0293] Synthesis of Dye 32 In a 25 mL single-port reactor, combine intermediate 32-2 (2.7 g, 2.7 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.84 g, 3.5 mmol), triethylamine (0.8 mL, 5.4 mmol), and dichloromethane (30 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize Dye 32 (1.7 g). The obtained Dye 32 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.77(m, 1H), 8.49(d, 1H, J=7.6Hz), 8.35(d, 1H, J=7.8Hz), 8.27(d, 1H) , J=7.6Hz), 7.98(t, 1H, J=7.6Hz), 7.70-7.45(m, 4H), 7.44-7.09(m, 11H), 6.95(d, 1H, J=13.2Hz) ), 6.89-6.80(m, 5H), 4.56(m, 1H), 4.22(t, 2H, J=7.4Hz), 3.85(s, 3H), 3.74(s, 6H), 3.55-3.32( m, 10H), 2.76(t, 2H, J=6.0Hz), 2.50(m, 2H), 2.13(s, 6H), 1.90-1.40(m, 8H), 1.21-1.11(m, 12H)

[0294] Dye 33 synthesis [ka]

[0295] Synthesis of intermediate 33-1 Intermediate 33-1 was synthesized by referring to KR2016 / 90242, 2016, A.

[0296] Synthesis of intermediate 33-2 Intermediate 33-2 was synthesized from intermediate 33-1 by referring to the method in Angew. Chem. Int. Ed. 2009, 48, 4222-4225.

[0297] Synthesis of intermediate 33-3 Intermediate 33-3 was synthesized by referring to Organic Letters, 2001, vol.3, #16, pp.2591-2594.

[0298] Synthesis of intermediates 33-4 Intermediate 33-2 (12g, 50.2 mmol), intermediate 33-3 (17.2g, 75.3 mmol), and acetonitrile (120 mL) were placed in a 250 mL single-port reactor and stirred under reflux at room temperature for 12 hours. After concentration, intermediate 33-4 was synthesized by column purification.

[0299] Synthesis of intermediates 33-5 In a 500 mL single-port reactor, combine intermediate 33-4 (18.0 g, 39 mmol), N,N-diphenylformamidine (8.3 g, 42 mmol), and acetic anhydride (200 mL), and stir at 110 °C for 1 hour. After cooling, pour the reaction mixture into ethyl acetate (500 mL) and stir vigorously. Filter the resulting solid to synthesize intermediate 33-5.

[0300] Dye 33 synthesis In a 100 mL single-port reactor, combine intermediate 33-5 (3.0 g, 4.9 mmol), intermediate 23-2 (1.7 g, 4.9 mmol), and pyridine (30 mL), and stir at 50°C for 1 hour. After concentration, add 6N hydrochloric acid aqueous solution (20 mL) and stir at room temperature for 12 hours. After concentration, column purification was performed to synthesize Dye 33 (1.6 g). The obtained Dye 33 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ9.08(d, 1H, J=8.2Hz), 8.71(t, 1H, J=12.6Hz), 8.61(d, 1H, J=8.0Hz), 8.40(d, 1H, J=7.8Hz), 8.18(d, 1H, J=8 .4Hz), 7.88-7.40(m, 7H), 6.97(d, 1H, J=12.6Hz), 6.83(d, 1H, J=12.6Hz), 4.25(t, 2H, J=7.6Hz), 3.86(m, 2H), 3.97(s, 3H), 2.09(m, 2H)

[0301] Dye 34 synthesis [ka]

[0302] Synthesis of intermediate 34-1 Intermediate 34-1 was synthesized by referring to Chemistry of Heterocyclic Compounds, 1988, vol.24, pp.87-92.

[0303] Synthesis of intermediate 34-2 Intermediate 34-1 (10g, 37.7 mmol), intermediate 33-3 (12.9g, 56.6 mmol), and acetonitrile (100 mL) were placed in a 250 mL single-port reactor and stirred under reflux at room temperature for 12 hours. After concentration, intermediate 34-2 was synthesized by column purification.

[0304] Synthesis of intermediate 34-3 In a 250 mL single-port reactor, combine intermediate 34-3 (15.0 g, 30 mmol), N,N-diphenylformamidine (6.6 g, 33 mmol), and acetic anhydride (150 mL), and stir at 110 °C for 1 hour. After cooling, pour the reaction mixture into ethyl acetate (500 mL) and stir vigorously. Filter the resulting solid to synthesize intermediate 34-3.

[0305] Dye 34 synthesis In a 100 mL single-port reactor, combine intermediate 33-5 (3.0 g, 4.7 mmol), intermediate 25-1 (1.7 g, 4.7 mmol), and pyridine (30 mL), and stir at 50°C for 1 hour. After concentration, add 6N hydrochloric acid aqueous solution (20 mL) and stir at room temperature for 12 hours. After concentration, column purification was performed to synthesize Dye 34 (1.8 g).

[0306] The obtained Dye 34 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.62 (t, 1H, J=12.8Hz), 8.60 (d, 1H, J=8.0Hz), 8.40 (d, 1H, J=7.8Hz), 8.23-8.01 (m, 2H) 7.93- 7.40(m, 8H), 6.67(m, 2H), 4.25(t, 2H, J=7.6Hz), 4.22(q, 2H, J=7.2Hz), 3.86(m, 2H), 2.09(m, 2H), 1.18(t, 2H, J=7.2Hz)

[0307] Dye 36 synthesis [ka]

[0308] Synthesis of intermediate 36-1 Intermediate 36-1 was synthesized by referring to CN115304539,2022,A.

[0309] Synthesis of intermediate 36-2 In a 250 mL single-port reactor, intermediate 36-1 (10 g, 25 mmol), malonaldehyde dianilide hydrochloride (7.1 g, 27 mmol), and acetic anhydride (100 mL) were added, and the mixture was stirred under reflux for 1 hour. After cooling, ethyl acetate (500 mL) was added to the reactor and the mixture was stirred for 5 minutes. The resulting solid was filtered to synthesize intermediate 36-2.

[0310] Synthesis of intermediate 36-3 Intermediate 36-3 was synthesized from 4-bromobutanoic acid by following the method of US2022 / 274960,2022,A1.

[0311] Synthesis of intermediate 36-4 Intermediate 36-2 (4.0 g, 7.0 mmol), intermediate 36-3 (3.1 g, 7.0 mmol), and pyridine (40 mL) were placed in a 100 mL single-port reactor and stirred at 50°C for 1 hour. After concentration, intermediate 36-4 was synthesized by column purification.

[0312] Dye 36 synthesis In a 100 mL single-port reactor, combine intermediate 36-4 (2.0 g, 2.8 mmol), HSTU (O-(N-Succinimidyl)-N,N,N',N'-tetramethyl uronium hexafluorophosphate) (1.6 g, 3.6 mmol), triethylamine (1.2 mL, 8.3 mmol), and dimethylformamide (20 mL). Stir at room temperature for 30 minutes. After concentration, column chromatography was performed to synthesize Dye 36 (1.7 g). The obtained Dye 36 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.70(t, 1H, J=12.8Hz), 8.54(d, 1H, J=8.0Hz), 8.38(d, 1H, J=7.8Hz), 8.23-7.40(m, 10H), 6.76(m, 2H), 4.25( t, 2H, J=7.2Hz), 4.22(q, 2H, J=7.2Hz), 2.85(s, 4H), 2.50(t, 2H, J=7.6Hz), 2.09(m, 2H), 1.76(s, 6H), 1.73(s, 6H)1.21(t, 2H, J=7.2Hz)

[0313] Dye 38 synthesis [ka]

[0314] Synthesis of intermediate 38-1 Intermediate 38-1 was synthesized by referring to KR2015 / 130206,2015 A1.

[0315] Synthesis of intermediate 38-2 In a 2L four-neck reactor, intermediate 38-1 (70g, 0.256mol), benzophenolhydrazine (60g, 0.229mol), Pd2(dba)3 (9.4g, 0.010mol), BINAP (6.4g, 0.010mol), sodium-tert-butoxide (35g, 0.359mol), and toluene (700mL) were added and stirred at 100°C for 12 hours. The mixture was placed on cellulite and the solid was filtered while still hot. After concentration, intermediate 38-2 was synthesized by column purification.

[0316] Synthesis of intermediate 38-3 Intermediate 38-2 (80g, 0.206mol), 7-methyl-8-oxononanoic acid (57.6g, 0.309mol), concentrated hydrochloric acid (160mL), and ethanol (640mL) were placed in a 1L single-port reactor and stirred under reflux for 12 hours. After concentration, water (500mL) was added to the reactor and extracted with ethyl acetate (500mL x 2). Anhydrous magnesium sulfate was added to the organic layer and stirred for 5 minutes, after which the solid was filtered. After concentration, intermediate 38-3 was synthesized by column purification.

[0317] Synthesis of intermediate 38-4 In a 250 mL single-port reactor, intermediate 38-3 (10 g, 24.8 mmol), iodomethane (5.3 g, 37.2 mmol), and acetonitrile (100 mL) were added and stirred under reflux for 24 hours. After cooling, ethyl acetate (200 mL) was added and stirred for 10 minutes. The resulting solid was filtered to synthesize intermediate 38-4.

[0318] Synthesis of intermediate 38-5 In a 250 mL single-port reactor, intermediate 27-2 (8 g, 20 mmol), malonaldehyde dianilide hydrochloride (5.8 g, 22 mmol), and acetic anhydride (80 mL) were added and stirred under reflux for 1 hour. After cooling, ethyl acetate (300 mL) was added to the reactor and stirred for 5 minutes. The resulting solid was filtered to synthesize intermediate 38-5.

[0319] Dye 38 synthesis In a 100 mL single-port reactor, combine intermediate 38-4 (2.0 g, 3.7 mmol), intermediate 38-5 (2.1 g, 3.7 mmol), and pyridine (20 mL). Stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 38 (0.7 g). The obtained Dye 38 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.52-8.50(m, 3H), 8.38(d, 1H, J=7.8Hz), 8.20-7.22(m, 9H), 6.87(t, 1H, J=12.6Hz), 6.35(d, 2H, J=13.6 Hz), 3.98(s, 3H), 3.80(s, 3H), 3.44-3.40(m, 4H), 2.54(t, 2H, J=7.6Hz), 2.10-1.75(m, 21H), 1.02-0.95(m, 1H), 0.69-0.64(m, 1H)

[0320] Synthesis of Dye 41 [ka]

[0321] Synthesis of intermediate 41-1 2-methylnaphthyl[1,2-d]oxazole (10 g, 54.6 mmol), iodomethane (11.6 g, 81.9 mmol), and acetonitrile (100 mL) were placed in a 250 mL single-port reactor and stirred under reflux at room temperature for 12 hours. After concentration, intermediate 41-1 was synthesized by column purification.

[0322] Synthesis of intermediate 41-2 In a 500 mL single-port reactor, combine intermediate 41-1 (12.0 g, 37 mmol), N,N-diphenylformamidine (8.0 g, 41 mmol), and acetic anhydride (120 mL), and stir at 110 °C for 1 hour. After cooling, pour the reaction mixture into ethyl acetate (600 mL) and stir vigorously. Filter the resulting solid to synthesize intermediate 41-2.

[0323] Synthesis of Dye 41 In a 100 mL single-port reactor, combine intermediate 41-2 (4.0 g, 8.5 mmol), intermediate 1-2 (3.5 g, 8.5 mmol), and pyridine (40 mL). Stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 41 (1.4 g). The obtained Dye 41 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.60(t, 1H, J=12.6Hz), 8.51(d, 1H, J=8.0Hz), 8.40(d, 1H, J=7.8Hz), 8.20-8.18(m, 3H), 7.93-7.90(m, 3H), 7.62-7.34(m, 4H), 6.97(d, 1H, J=12.6Hz), 6.83(d, 1H, J=12.6Hz), 4.25(t, 2H, J=7.6Hz), 3.87(s, 3H), 2.54(m, 2H), 2.08-1.73(m, 6H)

[0324] Synthesis of Dye 42 [ka]

[0325] Synthesis of intermediate 42-1 Intermediate 42-1 was synthesized by referring to US2020 / 0224257A1.

[0326] Synthesis of Dye 42 Add intermediate 42-1 (3.0g, 5.8 mmol), intermediate 7-1 (2.6g, 5.8 mmol), and pyridine (30 mL) to a 100 mL single-port reactor and stir at 50°C for 1 hour. After concentration, Dye 42 was synthesized by column purification (1.7g). The obtained Dye 42 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.70(t, 1H, J=13.2Hz), 8.52(d, 1H, J=8.0Hz), 8.40(d, 1H, J =7.8Hz), 8.22(d, 1H, J=8.4Hz), 8.03(t, 1H, J=7.6Hz), 7.86-7.40(m, 8H), 7.00(d, 1H, J=14.0Hz), 6.81(d, 1H, J=12.8Hz), 4.58(q, 2H, J=6.8Hz), 4.44(t, 2H, J=7.2Hz), 3.76 (s, 3H), 2.25(t, 2H, J=7..2Hz), 2.11(s, 6H), 1.93-1.50(m, 6H), 1.35(t, 3H, J=7.2Hz)

[0327] Synthesis of Dye 43 [ka]

[0328] Synthesis of intermediate 43-1 Intermediate 43-1 was synthesized by referring to KR2015 / 130206,2015,A1.

[0329] Synthesis of intermediate 43-2 Intermediate 43-2 was synthesized by referring to the synthesis methods of intermediates 38-2 and 38-3.

[0330] Synthesis of intermediate 43-3 In a 250 mL single-port reactor, combine intermediate 43-2 (10 g, 38.1 mmol), iodoethane (8.9 g, 57.2 mmol), and acetonitrile (100 mL), and stir under reflux for 24 hours. After cooling, add ethyl acetate (200 mL) and stir for 10 minutes. Filter the resulting solid to synthesize intermediate 43-3.

[0331] Synthesis of intermediate 43-4 In a 250 mL single-port reactor, intermediate 43-3 (6.0 g, 14 mmol), N,N-diphenylformamidine (3.1 g, 16 mmol), and acetic anhydride (60 mL) were added and stirred at 110 °C for 1 hour. After cooling, ethyl acetate (200 mL) was added and the mixture was vigorously stirred. The resulting solid was filtered to synthesize intermediate 43-4.

[0332] Synthesis of Dye 43 In a 100 mL single-port reactor, intermediate 43-4 (3.0 g, 5.3 mmol), intermediate 9-4 (1.9 g, 5.3 mmol), and pyridine (30 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 43 was synthesized by column purification (0.4 g). The obtained Dye 43 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ9.03(d, 1H, J=8.0Hz), 8.70(t, 1H, J=12.6Hz), 8.51(d, 1H, J=8.0Hz), 8.22(d, 1H, J=8.4Hz), 7.98(m, 1H), 7.86-7.4 0(m, 7H), 7.00(d, 1H, J=14.0Hz), 6.81(d, 1H, J=12.6Hz), 4.55(q, 2H, J=7.2Hz), 3.87(s, 3H), 3.76(s, 3H), 2.10(s, 6H), 1.35(t, 3H, J=7.2Hz)

[0333] Dye 47 synthesis [ka]

[0334] Synthesis of intermediate 47-1 Intermediate 47-1 was synthesized by referring to KR2015 / 130206,2015,A1.

[0335] Synthesis of intermediate 47-2 Intermediate 47-2 was synthesized by referring to the synthesis methods of intermediates 38-2 and 38-3.

[0336] Synthesis of intermediate 47-3 In a 250 mL single-port reactor, combine intermediate 47-2 (10 g, 30 mmol), iodomethane (6.4 g, 45 mmol), and acetonitrile (100 mL), and stir under reflux for 24 hours. After cooling, add ethyl acetate (100 mL) and stir for 10 minutes. Filter the resulting solid to synthesize intermediate 47-3.

[0337] Synthesis of intermediate 47-4 Intermediate 47-4 was synthesized by referring to US2022 / 59772,2022,A1.

[0338] Synthesis of intermediates 47-5 Intermediate 47-5 was synthesized from intermediate 47-4 by referring to the method in Bioconjugate Chemistry 2019, vol.30, #10, pp.2647-2663.

[0339] Synthesis of intermediates 47-6 In a 250 mL single-port reactor, intermediate 47-5 (5.0 g, 13 mmol), N,N-diphenylformamidine (2.8 g, 14 mmol), and acetic anhydride (50 mL) were added and stirred at 110 °C for 1 hour. After cooling, ethyl acetate (100 mL) was added and the mixture was vigorously stirred. The resulting solid was filtered to synthesize intermediate 47-6.

[0340] Dye 47 synthesis In a 100 mL single-port reactor, intermediate 47-3 (2.7 g, 5.7 mmol), intermediate 47-6 (3.0 g, 5.7 mmol), and pyridine (30 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 47 was synthesized by column purification (1.1 g). The obtained Dye 47 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.54(t, 1H, J=12.6Hz), 8.49(d, 1H, J=8.0Hz), 8.38(d, 1H, J=7.8Hz), 8.22(d, 1H, J=8.0Hz), 8.02(d, 1H, J=7.8Hz), 7 .84-7.38(m, 13H), 6.81(m, 2H), 5.87(s, 2H), 3.97(s, 3H), 3.86(m, 2H) , 3.80(s, 3H), 2.10-1.75(m, 11H), 1.02-0.95(m, 1H), 0.69-0.64(m, 1H)

[0341] Dye 48 synthesis [ka]

[0342] Synthesis of intermediate 48-1 Intermediate 48-1 was synthesized from intermediate 34-1 by referring to the method in US2020 / 0224257A1.

[0343] Dye 48 synthesis In a 100 mL single-port reactor, intermediate 48-1 (2.0 g, 3.9 mmol), intermediate 7-1 (1.8 g, 3.9 mmol), and pyridine (20 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 48 was synthesized by column purification (0.3 g).

[0344] The obtained Dye 48 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.62 (t, 1H, J=12.8Hz), 8.60 (d, 1H, J=8.0Hz), 8.40 (d, 1H, J=7.8Hz), 8.23-8.01 (m, 2H) 7.9 3-7.40(m, 9H), 6.67(m, 2H), 4.20(t, 2H, J=7.6Hz), 3.77(s, 3H), 2.66(t, 2H, J=7.6Hz), 2.11(s, 6H), 1.92-1.67(m, 6H)

[0345] Dye 55 synthesis [ka]

[0346] Synthesis of intermediate 55-1 Intermediate 55-1 was synthesized by referring to IN202241025324.

[0347] Synthesis of intermediate 55-2 Intermediate 55-2 was synthesized from intermediate 55-1 by referring to the method in Angew. Chem. Int. Ed. 2009, 48, 4222-4225.

[0348] Synthesis of intermediate 55-3 In a 250 mL single-port reactor, combine intermediate 55-2 (10 g, 32 mmol), iodoethane (7.4 g, 48 mmol), and acetonitrile (100 mL), and stir under reflux for 24 hours. After cooling, add ethyl acetate (100 mL) and stir for 10 minutes. Filter the resulting solid to synthesize intermediate 55-3.

[0349] Synthesis of intermediate 55-4 In a 250 mL single-port reactor, intermediate 55-3 (5.0 g, 11 mmol), N,N-diphenylformamidine (2.3 g, 12 mmol), and acetic anhydride (50 mL) were added and stirred at 110 °C for 1 hour. After cooling, ethyl acetate (100 mL) was added and the mixture was vigorously stirred. The resulting solid was filtered to synthesize intermediate 55-4.

[0350] Dye 55 synthesis Add intermediate 55-4 (2.0g, 3.2 mmol), intermediate 1-2 (1.3g, 3.2 mmol), and pyridine (20 mL) to a 100 mL single-port reactor and stir at 50°C for 1 hour. After concentration, Dye 55 was synthesized by column purification (0.2g). The obtained Dye 55 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.64(t, 1H, J=12.8Hz), 8.54(d, 1H, J=8.0Hz), 8.45-8.40(m, 2H), 8.23-8.01(m, 2H), 7.85-7.68(m, 4H), 7.28- 6.96(m, 10H), 6.67(m, 2H), 4.28(q, 2H, J=7.2Hz), 4.20(t, 2H, J=7.6Hz), 2.57(t, 2H, J=7.2Hz), 1.98-1.69(m, 6H), 1.49(t, 3H, J=7.2Hz)

[0351] Dye 57 synthesis [ka]

[0352] Synthesis of intermediate 57-1 In a 250 mL single-port reactor, intermediate 20-1 (8.0 g, 28 mmol), iodopropane (7.2 g, 42 mmol), and acetonitrile (80 mL) were added and stirred under reflux for 24 hours. After cooling, ethyl acetate (100 mL) was added and stirred for 10 minutes. The resulting solid was filtered to synthesize intermediate 57-1.

[0353] Synthesis of intermediate 57-2 In a 250 mL single-port reactor, intermediate 57-1 (6.0 g, 13 mmol), N,N-diphenylformamidine (2.9 g, 15 mmol), and acetic anhydride (50 mL) were added and stirred at 110 °C for 1 hour. After cooling, ethyl acetate (100 mL) was added and the mixture was vigorously stirred. The resulting solid was filtered to synthesize intermediate 57-2.

[0354] Dye 57 synthesis Add intermediate 57-2 (3.0g, 5.0 mmol), intermediate 9-4 (1.8g, 5.0 mmol), and pyridine (30 mL) to a 100 mL single-port reactor and stir at 50°C for 1 hour. After concentration, Dye 57 was synthesized by column purification (1.4g). The obtained Dye 57 1The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ9.03(d, 1H, J=8.0Hz), 8.60-8.52(m, 3H), 8.38(d , 1H, J=8.0Hz), 8.31(d, 1H), 8.22(d, 1H, J=8.0Hz), 8.15-8.10(m, 2H), 8.03( t, 1H, J=7.2Hz), 7.90-7.62(m, 2H), 7.54-7.40(m, 2H), 6.83(m, 2H), 4.28(t , 2H, J=7.2Hz), 3.85(s, 3H), 1.96(m, 2H), 2.10(s, 6H), 1.35(t, 3H, J=7.6Hz)

[0355] Dye 59 synthesis [ka]

[0356] Synthesis of intermediate 59-1 Intermediate 59-1 was synthesized by referring to KR2020 / 67733,2020,A.

[0357] Synthesis of intermediate 59-2 Intermediate 59-2 was synthesized by referring to EP1221465, 2002, A1.

[0358] Dye 59 synthesis In a 100 mL single-port reactor, combine intermediate 59-1 (4.3 g, 10.0 mmol), intermediate 6-2 (3.0 g, 10.0 mmol), intermediate 59-2 (1.9 g, 5.0 mmol), and pyridine (30 mL). Stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 59 (0.4 g). The obtained Dye 59 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.44(d, 2H, J=12.8Hz), 8.49(d, 1H, J=8.0Hz), 8.38(d, 1H, J=7.8Hz), 8.22(d, 1H, J=8.0Hz), 8.02(d, 1H, J=7.8Hz), 7.95-7.3 8(m, 12H), 6.34(d, 2H, J=13.2Hz), 4.26(q, 2H, J=7.8Hz), 4.02(q, 2H, J=7.6 Hz), 3.80(s, 3H), 2.10(s, 6H), 1.37(t, 3H, J=7.6Hz), 1.09(t, 3H, J=7.8Hz)

[0359] Dye 60 synthesis [ka]

[0360] Synthesis of intermediate 60-1 Intermediate 60-1 was synthesized by referring to Journal of the Indian Chemical Society, 1968, vol. 45, pp. 799-809.

[0361] Dye 60 synthesis In a 100 mL single-port reactor, combine intermediate 60-1 (2.0 g, 4.3 mmol), intermediate 1-2 (1.8 g, 4.3 mmol), and pyridine (20 mL). Stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 60 (0.1 g).

[0362] The obtained Dye 60 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.69(t, 1H, J=12.8Hz), 8.50(d, 1H, J=7.2Hz), 8.40( d, 1H, J=8.0Hz), 8.20(d, 1H, J=8.2Hz), 7.88(t, 1H, J=7.2Hz), 7.75-7.53(m, 4H ), 7.47-7.25(m, 3H), 7.00(d, 1H, J=7.0Hz), 6.88(d, 1H, J=13.4Hz), 6.52(d, 1H , J=13.2Hz), 4.46(q, 2H, J=7.6Hz), 3.97(s, 3), 2.55(m, 2H), 2.08-1.72(m, 6H)

[0363] Dye 61 synthesis [ka]

[0364] Synthesis of intermediate 61-1 Intermediate 61-1 was synthesized by referring to US2166736,1937,A.

[0365] Dye 61 synthesis In a 100 mL single-port reactor, intermediate 61-1 (4.0 g, 8.0 mmol), intermediate 1-2 (3.3 g, 8.0 mmol), and pyridine (40 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 61 was synthesized by column purification (2.3 g). The obtained Dye 61 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.73(t, 1H, J=13.4Hz), 8.52(d, 1H, J=7.8Hz), 8.42(d, 1H, J= 8.2Hz), 8.22(d, 1H, J=8.4Hz), 8.00(t, 1H, J=7.2Hz), 7.76-7.51(m, 4H), 7.49-7.25(m, 3H), 7.01(d, 1H, J=7.2Hz), 6.88(d, 1H, J=12.8Hz), 6.52(d, 1H, J=13.2Hz), 4.51(q, 2H, J=7.6Hz), 4.22(t, 2H, J=7.8Hz), 2.57(m, 2H), 2.10-1.74(m, 6H), 1.33(t, 3H, J=7.8Hz)

[0366] Synthesis of Dye 62 [ka]

[0367] Synthesis of intermediate 62-1 Intermediate 62-1 was synthesized by referring to the synthesis methods of intermediates 38-2 and 38-3.

[0368] Synthesis of intermediate 62-2 In a 250 mL single-port reactor, intermediate 62-1 (6 g, 17 mmol), iodomethane (3.7 g, 26 mmol), and acetonitrile (60 mL) were added and stirred under reflux for 24 hours. After cooling, ethyl acetate (100 mL) was added and stirred for 10 minutes. The resulting solid was filtered to synthesize intermediate 62-2.

[0369] Synthesis of Dye 62 Add intermediate 62-2 (2.0g, 4.0 mmol), intermediate 7-1 (1.9g, 48.0 mmol), and pyridine (20 mL) to a 100 mL single-port reactor and stir at 50°C for 1 hour. After concentration, Dye 62 was synthesized by column purification (1.1g). The obtained Dye 62 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.55(t, 1H, J=13.4Hz), 8.43(d, 1H, J=7.80Hz), 8.32(d, 1H, J=7.8Hz), 8.20(d, 1H, J =8.0Hz), 8.00(d, 1H, J=7.2Hz), 7.86-7.39(m, 8H), 6.82(m, 2H), 5.87(s, 2H), 4.16(s, 3H), 3.86(s, 3H), 3.82(m 2H), 2.10-1.75(m, 15H), 1.02-0.95(m, 1H), 0.69-0.64(m, 1H)

[0370] Dye 63 synthesis [ka]

[0371] Synthesis of intermediate 63-1 Intermediate 63-1 was synthesized by referring to Chemical Communications, 2016, vol.52, #90, pp.13307-13310.

[0372] Synthesis of intermediate 63-2 In a 250 mL three-neck reactor, intermediate 63-1 (4.75 g, 18.2 mmol), 4-(ethoxycarbonyl)phenylboronic acid (5.0 g, 27.8 mmol), potassium carbonate (5.0 g, 36.4 mmol), Pd(PPh3)4 (0.1 g, 0.09 mmol), 1,2-dimethoxyethane (30 mL), water (9 mL), and N,N-dimethylformamide (65 mL) were added and stirred at 85 °C for 12 hours. After cooling, the solid was filtered through cellulite. After concentrating the filtrate, intermediate 63-2 was synthesized by column purification.

[0373] Synthesis of intermediate 63-3 Intermediate 63-3 was synthesized by referring to Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0374] Dye 63 synthesis In a 50 mL single-port reactor, intermediate 63-3 (3.0 g, 7.1 mmol), intermediate 43-4 (4.0 g, 7.1 mmol), and pyridine (20 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 63 was synthesized by column purification (1.3 g). The obtained Dye 63 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.63 (t, 1H, J=12.8Hz), 8.43 (d, 1H, J=8.0Hz), 8.37 (d, 1H, J=7.8Hz), 8.16(d, 1H, J=8.0Hz), 8.14(d, 2H, J=8.0Hz), 7.78-7.49(m, 6H), 7.45-7.12(m, 4H), 6.88(d, 1H, J=12.8Hz), 6.54(d, 1H, J=13.4Hz), 4.52( q, 2H, J=7.8Hz), 3.83(s, 3H), 3.76(s, 3H), 2.11(s, 6H), 1.35(t, 3H, J=7.8Hz)

[0375] Dye 66 synthesis [ka]

[0376] Synthesis of intermediate 66-1 Intermediate 66-1 was synthesized by referring to Journal of Organic Chemistry, 2018, vol.83, #8, pp.4389-4401.

[0377] Synthesis of intermediate 66-2 In a 1L four-port reactor, combine intermediate 66-1 (50g, 0.202mol), ethyl 4-piperidine carboxylate (38g, 0.242mol), Pd2(dba)3 (7.4g, 0.008mol), BINAP (5.0g, 0.008mol), sodium tert-butoxide (27g, 0.282mol), and toluene (500mL), and stir under reflux for 12 hours. After cooling, the solid was filtered through a cellulite layer. The filtrate was concentrated and then purified by column chromatography to synthesize intermediate 66-2.

[0378] Synthesis of intermediate 66-3 Intermediate 66-3 was synthesized from intermediate 66-2 by referring to the method in Bioconjugate Chemistry, 2019, vol.30, #10, pp.2647-2663.

[0379] Synthesis of intermediate 66-4 Intermediate 66-4 was synthesized by referring to Chemistry of Heterocyclic Compounds, 1988, vol.24, pp.87-92.

[0380] Synthesis of intermediate 66-5 In a 250 mL single-port reactor, intermediate 66-4 (10.0 g, 38 mmol), iodomethane (8.0 g, 57 mmol), and acetonitrile (100 mL) were added and stirred under reflux for 24 hours. After cooling, ethyl acetate (100 mL) was added and stirred for 10 minutes. The resulting solid was filtered to synthesize intermediate 66-5.

[0381] Synthesis of intermediate 66-6 In a 250 mL single-port reactor, intermediate 66-5 (5.0 g, 12 mmol), N,N-diphenylformamidine (2.6 g, 14 mmol), and acetic anhydride (50 mL) were added and stirred at 110 °C for 1 hour. After cooling, ethyl acetate (100 mL) was added and vigorously stirred. The resulting solid was filtered to synthesize intermediate 66-6.

[0382] Dye 66 synthesis In a 50 mL single-port reactor, intermediate 66-3 (3.0 g, 6.9 mmol), intermediate 66-6 (3.8 g, 6.9 mmol), and pyridine (30 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 66 was synthesized by column purification (0.9 g). The obtained Dye 66 1 The H-NMR results are as follows: 16 .67(d, 1H, J=13.6Hz), 6.51(d, 1H, J=12.8Hz), 3.95(s, 3H), 3.81(s, 3H), 3.44(m, 4H), 2.49-2.44(m, 1H), 2.10(s, 6H), 1.88-1.61(m, 4H)

[0383] Dye 77 synthesis [ka]

[0384] Synthesis of intermediate 77-1 Intermediate 77-1 was synthesized by referring to KR2020 / 67732,2020,A.

[0385] Synthesis of intermediate 77-2 In a 250 mL single-port reactor, intermediate 77-1 (10 g, 30 mmol), 6-bromohexanoic acid (8.9 g, 46 mmol), and 1,2-dichlorobenzene (100 mL) were added and stirred at 150 °C for 2 hours. After cooling, acetone (100 mL) was added to the reactor and stirred for 5 minutes. The resulting solid was filtered to synthesize intermediate 77-2.

[0386] Dye 77 synthesis In a 100 mL single-port reactor, intermediate 77-2 (2.0 g, 3.8 mmol), intermediate 18-2 (1.9 g, 3.8 mmol), and pyridine (20 mL) were added and stirred at 50°C for 1 hour. After concentration, Dye 77 was synthesized by column purification (0.7 g). The obtained Dye 77 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.48-8.51(m, 3H), 8.40(d, 1H, J=7.8Hz), 8.22(d, 1H, J= 8.4Hz), 8.03(t, 1H, J=7.6Hz), 7.86-7.40(m, 4H), 7.19(t, 1H, J=8.0Hz), 7.09(t, 1 H, 8.4Hz), 6.75-6.53(m, 3H), 4.41(t, 2H, J=7.2Hz), 4.23(q, 2H, J=6.8Hz), 3.61(s , 3H), 2.53(t, 2H, J=7.2Hz), 2.13(s, 6H), 1.90-1.50(m, 12H), 1.33(t, 3H, J=7.8Hz)

[0387] Dye 82 synthesis [ka]

[0388] Synthesis of intermediate 82-1 Intermediate 82-1 was synthesized by referring to Dyes and Pigments, 2014, vol.101, pp.1-8.

[0389] Synthesis of intermediate 82-2 Intermediate 82-2 was synthesized by referring to Journal of the American Chemical Society, 2011, vol.133, #40, pp.15870-15873.

[0390] Dye 82 synthesis In a 100 mL single-port reactor, combine intermediate 82-1 (4.7 g, 10.0 mmol), intermediate 6-2 (3.0 g, 10.0 mmol), intermediate 82-2 (1.6 g, 5.0 mmol), and pyridine (30 mL). Stir at 50°C for 1 hour. After concentration, column purification was performed to synthesize Dye 82 (0.3 g). The obtained Dye 82 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.54(d, 2H, J=13.4Hz), 8.49(m, 2H), 8.32(d, 1H, J=8.0Hz), 8.02(t, 1H, J=7.2Hz), 7.79-7.38(m, 8H), 6.58(d, 2) H, J=12.8Hz), 4.39(s, 3H), 3.76(s, 3H), 2.82(t, 2H, J=7.8Hz), 2.17(t, 2H, J=7.8Hz), 2.07-1.99(m, 4H), 1.73(s, 6H), 0.89-0.67(m, 10H)

[0391] The photophysical properties of the non-fluorescent compounds synthesized using the aforementioned synthesis examples, measured under DMSO solvent, are shown in Table 1 below. [Table 1]

[0392] Referring to Table 1 above, it can be expected that the non-fluorescent compounds defined in this application will exhibit a quenching effect on the luminescence properties of fluorescent compounds via the FRET mechanism in the wavelength range of approximately 550 to 900 nm.

[0393] Experimental Example 1. Evaluation of the spectral change with pH of a conjugate (oligonucleotide) with a non-fluorescent compound single-labeled at the 3'-terminus. Conjugates were prepared by labeling the 3'-terminus of oligonucleotides with Dye 2, Dye 16, and Dye 17, non-fluorescent compounds synthesized using the aforementioned synthesis examples, and the commercially available quenchers BHQ1, BHQ2, and BHQ3, respectively. The spectral changes of these conjugates with respect to pH were then confirmed. [Table 2]

[0394] Specifically, the conjugate uses Controlled Pore Glass (CPG) substituted with a non-fluorescent compound as a support, and is manufactured using MerMade. TMThe DNA was synthesized using a 48X DNA synthesizer so that 10 thymine molecules were attached sequentially starting from the 3' position (see Table 2).

[0395] Next, each non-fluorescent compound labeled T10 oligonucleotide was separated from the stationary phase support using a standard method (NH4OH, 30 v / v% in H2O), and then RP-HPLC was applied to separate only the FLP (Full-Length Product), which was then dried.

[0396] The dried conjugates were titrated at pH 2, 5, 6, 8, and 10 using a pH meter, and all were diluted to 3 μM. The spectral changes of all solutions were measured at room temperature using a UV spectrophotometer. [Table 3]

[0397] Referring to Table 3 above, for Ex 1-4 to Ex 1-6, the change in lambda max becomes larger as the pH decreases, and the standard deviation changes over a wide range of approximately 6 to 17 nm in the pH range of 2 to 10. In contrast, for Ex 1-1 to Ex 1-3, the spectrum is maintained constant within the pH range of 2 to 10, and the effect of pH is extremely small. It was confirmed that the properties of the non-fluorescent compound are maintained over a wide pH range of approximately 0 to 0.4 in standard deviation.

[0398] Experimental Example 2. Evaluation of quenching efficiency based on the distance between the quencher and the phosphor. As shown in Table 4 below, a forward probe (hereinafter referred to as "fp") and a reverse probe (hereinafter referred to as "rp") were synthesized. The reverse probe shown in Table 4 has a complementary sequence to all or part of the forward probe.

[0399] For reference, rp 1-1, rp 2-1, and rp 3-1 are hybridized with fp1 and fp2, respectively, to form a blunt-ended hybrid; rp 1-2, rp 2-2, and rp 3-2 are hybridized with fp1 and fp2, respectively, to form a 5-mer staggered hybrid; and rp 1-3, rp 2-3, and rp 3-3 are hybridized with fp1 and fp2, respectively, to form a 10-mer staggered hybrid. Schematic diagrams of the blunt-ended hybrid, 5-mer staggered hybrid, and 10-mer staggered hybrid are shown in Figure 1. Here, the blunt-ended hybrid is a hybrid designed to confirm static quenching, i.e., quenching caused by the formation of a ground state complex between the quencher and the phosphor, while the staggered hybrid is a hybrid designed to confirm dynamic quenching, particularly quenching caused by FRET.

[0400] The forward-direction probe and the reverse-direction probe were synthesized using the same method as described in Experimental Example 1. [Table 4]

[0401] To evaluate the quenching efficiency with respect to the distance between the quencher and the phosphor, first, the fluorescence intensity of fp1 and fp2, which are conjugates labeled only with phosphor at the 5' end, was measured. Next, the forward and reverse probes were hybridized in the combinations shown in Table 5 below, and the fluorescence intensity was measured again. The two measurement results were then substituted into Equation 1 below to evaluate the quenching efficiency.

number

[0402] Phosphor-labeled oligonucleotides were diluted to a concentration of 0.3 μM using hybridization buffer (final concentration: NaCl 50 mM, MgCl 25 mM, Tris-HCl 10 mM), and the fluorescence intensity was measured using a Varioskan LUX Multimode microplate reader (Thermo Fisher Scientific) (6-FAM:λ). ex 495nm / λ em 520nm, Cy5:λ ex 650nm / λ em (665 nm). In addition, the phosphor-labeled oligonucleotide and quencher-labeled oligonucleotide were diluted with hybridization buffer to concentrations of 0.3 μM and 0.6 μM, respectively, and then hybridized. The amount of fluorescence was measured using a Varioskan LUX Multimode microplate reader, depending on the distance between the phosphor and the quencher. [Table 5]

[0403] Cy5, a commercially available fluorescent compound, is a representative RED region phosphor with maximum emission at 650 nm, and Dye 2, Dye 16, and Dye 17 have maximum absorption above 650 nm. Therefore, the overlap of the emission-absorption spectra of the phosphor and the quencher can induce quenching via the FRET mechanism. Referring to the results in Table 5, while the difference in quenching efficiency due to the distance between the phosphor and the quencher is only 1-3%, it can be confirmed that the non-fluorescent compounds defined in this application can induce quenching not only through dynamic quenching, which is represented by the FRET mechanism, but also through static quenching.

[0404] While the commercially available fluorescent compound 6-FAM is a typical short-wavelength phosphor with maximum emission at 520 nm, Dye 2, Dye 16, and Dye 17 have maximum absorption above 650 nm, resulting in minimal overlap between the emission-absorption spectra of the phosphor and the quencher.

[0405] Referring to the results in Table 5, it can be predicted that as the distance between the phosphor and the quencher increases, the quenching efficiency decreases by 15-20%. This suggests that the interaction between the phosphor and the quencher as they approach each other causes them to form a dimer, which in turn leads to quenching due to static quenching.

[0406] Experimental Example 3. Evaluation of binding stability using combinations of phosphors and quenchers. As shown in Table 6 below, a forward probe (hereinafter referred to as "fp") and a reverse probe (hereinafter referred to as "rp") were synthesized. The forward probe and the reverse probe were synthesized using the same method as described in Experimental Example 1. [Table 6]

[0407] To evaluate the binding stability of the phosphor and quencher combinations, the forward and reverse probes were hybridized using the combinations shown in Table 7 below. Then, the melting temperature (Tm) was measured from the Melt Curve while increasing the temperature from 37°C to 95°C in 0.5°C increments.

[0408] The hybridization of the forward and reverse probes was performed by diluting them to a concentration of 0.3 μM with hybridization buffer (final concentration: NaCl 50 mM, MgCl 25 mM, Tris-HCl 10 mM). The hybrid was prepared using SYBR Green at 1X, and then the Tm was measured using a CFX96 PCR machine (Biorad). [Table 7]

[0409] Referring to the results in Table 7, it can be confirmed that the Tm of the quencher-labeled oligonucleotide hybrid increased by approximately 3°C compared to the quencher-labeled oligonucleotide hybrid (Ex 3-1, 3-2). This result is presumed to be because the binding affinity of the double-stranded oligonucleotide created by the affinity between the phosphor and the quencher became more stable when a non-fluorescent compound as defined in this application was used as the quencher.

[0410] Experimental Example 4. Evaluation of signal-to-noise ratio (S / N ratio) of PCR using double-labeled oligonucleotides. Based on the experimental examples described above, double-labeled oligonucleotides were prepared using the combinations shown in Table 8 below. The 5' end was labeled with a commercially available fluorescent compound, 6-FAM, HEX, TAMRA, Cy5, or Cy5.5, and the 3' end was labeled with a commercially available quencher, BHQ-1, BHQ-2, or BHQ-3, or a non-fluorescent compound as defined in this application, Dye 2, Dye 16, or Dye 17. The PCR characteristics and signal-to-noise ratio (S / N ratio) were evaluated by PCR analysis using these double-labeled oligonucleotides (see Table 9). Real-time PCR against BQCV (black queen cell virus) plasmid DNA was performed twice on a 10 fg / ul template (using Bio-Rad, CFX-96™ Touch). The evaluation results are shown in Figures 2 and 3. [Table 8] [Table 9]

[0411] Referring to Figures 2 and 3, when comparing the use of commercially available phosphors such as 6-FAM, HEX, TAMRA, Cy5, and Cy5.5 with commercially available quenchers such as BHQ-1, BHQ-2, or BHQ-3, it was confirmed that using the non-fluorescent compounds defined in this application as quenchers resulted in lower Ct values ​​and higher S / N ratios.

[0412] Based on the results described above, it was confirmed that the non-fluorescent compounds defined in this application, even as a single compound, can be used as quenchers for various phosphors having different emission spectra. Furthermore, from the perspective of molecular diagnostics, it can be expected that detection ease and accuracy will be higher than commercially available quenchers, even when the target gene in the sample is present at a relatively low concentration. In particular, it was confirmed that the non-fluorescent compounds defined in this application can act as molecular diagnostic probes by static quenching, even when there is little wavelength overlap with 6-FAM.

[0413] Experimental Example 5. Evaluation of the sequence dependence of double-labeled oligonucleotides. Based on the experimental examples described above, double-labeled oligonucleotides were prepared using the combinations shown in Table 10 below. These oligonucleotides were labeled with Cy5, a commercially available fluorescent compound, at the 5' end, and with BHQ-2, a commercially available quencher, and Dye 6, Dye 18, or Dye 60, a non-fluorescent compound as defined in this application, at the 3' end. The PCR characteristics and signal-to-noise ratio (S / N ratio) were evaluated by PCR analysis using these double-labeled oligonucleotides (see Table 9). The evaluation results are shown in Table 11 and Figures 4 to 6. [Table 10] [Table 11]

[0414] Referring to Table 11 and Figures 4-9, the range of Ct values ​​for the non-fluorescent compounds defined in this application is -0.41 < ΔCt < 0.03, confirming that they have sensitivity equivalent to or better than the commercially available quencher BHQ-2, and exhibit lower background and a higher S / N ratio than the commercially available quencher.

[0415] Experimental Example 6. Evaluation of reducing agent resistance of double-labeled oligonucleotides. Based on the experimental examples described above, double-labeled oligonucleotides were prepared using the combinations shown in Table 12 below. These oligonucleotides were labeled with a commercially available fluorescent compound, 6-FAM, Cy5, or Cy5.5, at the 5' end, and with a commercially available quencher, BHQ-1, BHQ-2, or BHQ-3, or a non-fluorescent compound as defined in this application, Dye 6, Dye 48, or Dye 60, at the 3' end. When 100 mM DTT (1,4-dithiothreitol) was added as a reducing agent and the reaction was carried out overnight for 24 hours, the change in purity by HPLC was measured. Specifically, the purity of each oligonucleotide labeled with dried Ex 6-1 to Ex 6-6 was measured immediately after quantification in a 100 mM DTT solution to 100 μM, and the results were obtained under room temperature conditions (21°C). 24-hour storage Afterward, the purity was measured using the same method. The evaluation results are shown in Figures 7 and 8. [Table 12]

[0416] Referring to Figures 7 and 8, it was confirmed that oligonucleotides using the non-fluorescent compound defined in this application as a quencher showed only a slight change in purity, ranging from a minimum of 0.4% to a maximum of approximately 1.6% under the influence of a reducing agent, while oligonucleotides using commercially available quenchers BHQ-1, BHQ-2, or BHQ-3 showed a rapid change in purity, ranging from a minimum of 69.7% to a maximum of approximately 86.7% under the influence of a reducing agent. In other words, the non-fluorescent compound defined in this application maintains a stable state even in the presence of a reducing agent due to its high durability, and is expected to be usable in a wider range of molecular diagnostic fields compared to commercially available quenchers.

[0417] Experimental Example 7. Comparison of the efficiency of double-labeled oligonucleotides in the presence of MGB. Based on the aforementioned experimental examples, double-labeled oligonucleotides were prepared using the combinations shown in Table 13 below. These oligonucleotides were labeled with a commercially available fluorescent compound (6-FAM, HEX, TAMRA, or Cy5) at the 5' end and with a non-fluorescent compound (Dye 12, Dye 48, or Dye 61, as defined in this application) at the 3' end, and contained MGB (Minor Groove Binder). The PCR characteristics (see Experimental Example 4) and Tm changes (see Experimental Example 3) of the double-labeled oligonucleotides with and without MGB (Minor Groove Binder) were then measured. The measurement results are shown in Table 14 and Figures 9-12. Specifically, dual-labeled oligonucleotides containing MGB are used with MGB-CPGs substituted with Dye 12, Dye 48, or Dye 61 respectively as support for MerMade TMOligonucleotides were synthesized using a 48X DNA synthesizer in a standard manner where only the oxidation step could provide a side reaction element to MGB. The solution was changed to 0.5M CSO [(1S)-(+)-(10-camphorsulfonyl)-oxaziridine], and oligonucleotides were synthesized so that complementary sequences (14mers) to each target were attached as probes, starting from the 3' position. Next, 6-FAM, HEX, TAMRA, or Cy5 was attached to the 5' end, and the phosphor-labeled probes were separated from the stationary phase support using a standard method (NH4OH, 30v / v% in H2O). After separation, only the FLP (Full-Length Product) was separated and dried using RP-HPLC. [Table 13] [Table 14]

[0418] Referring to Table 14, it can be confirmed that the average Tm value for Ex 7-1 to Ex 7-4 is approximately 6°C higher than that for Ex 7-5 to Ex 7-8. Furthermore, referring to Figures 9 to 12, it can be confirmed that the ΔRFU increases and the Ct value decreases for Ex 7-1 to Ex 7-4 compared to Ex 7-5 to Ex 7-8.

[0419] Experimental Example 8. Evaluation of PCR properties of oligonucleotides containing multiple quenchers. Based on the aforementioned experimental examples, the PCR properties (see Experimental Example 4) of oligonucleotides were evaluated using the combinations shown in Table 15 below. Each oligonucleotide had a commercially available fluorescent compound, 6-FAM, labeled at the 5' end, a commercially available quencher, IBFQ (Iowa Black FQ), and a non-fluorescent compound defined in this application, Dye 6, Dye 18, or Dye 60, labeled at the 3' end, with a commercially available quencher, Eclipse® (Elitech group.), or ZEN, added between the oligonucleotides. For Ex 8-5, triple-labeled oligonucleotides sold by Integrated DNA Technologies, Inc. were used as is. The evaluation results are shown in Figure 13. [Table 15]

[0420] Referring to Figure 13, it was confirmed that the background of triple-labeled oligonucleotides Ex 8-1 to Ex 8-3, which contain two quenchers, was reduced by 65%, the ΔRFU was increased by 42%, and they had lower Ct values ​​compared to Ex 8-4, which contains a single quencher (double-labeled oligonucleotide). In other words, it can be confirmed that triple-labeled oligonucleotides containing two quenchers have superior PCR amplification performance compared to double-labeled oligonucleotides containing a single quencher.

[0421] Furthermore, in the case of Ex 8-1 to Ex 8-3, which contain the non-fluorescent compound defined in this application, we were able to confirm that they exhibit PCR amplification performance similar to that of Ex 8-5, a commercially available triple-labeled oligonucleotide.

[0422] Experimental Example 9. Evaluation of the solubility of non-fluorescent compounds Based on the synthesis method of Dye 6 among the synthesis examples, R4 and R in the structure of Dye 6 11 Non-fluorescent compounds in which the group is a hydroxyl group (-OH), and in the structure of Dye 6, R4 and R 11 is sulfonate (-SO3- Non-fluorescent compounds were further synthesized, and their solubility in each solvent shown in Table 16 below was measured. Specifically, an amount equivalent to 2 mg of the non-fluorescent compound was added to 2 ml of solvent, and after standing at 25°C for 30 minutes, it was confirmed whether the non-fluorescent compound was completely dissolved. [Table 16]

[0423] Referring to Table 16, it was confirmed that the non-fluorescent compounds produced by Ex 9-1 and Ex 9-2 were lipophilic, while the non-fluorescent compounds produced by Ex 9-3 were water-soluble.

[0424] Although several embodiments of the present invention have been described above, any person with ordinary skill in the art can modify or change the present invention in various ways by adding, changing, deleting, or adding components, without departing from the spirit of the invention as described in the claims, and these modifications are also included within the scope of the rights of the present invention.

Claims

1. A non-fluorescent compound represented by the following chemical formula 1, The non-fluorescent compound can have its fluorescence reduced by at least one mechanism selected from static quenching and dynamic quenching. 【Chemistry 1】 Here, Ar is C ５ -C ５０ Ariel, C ２ -C ５０ Heteroaryl or C ５ -C ５０ It is an aliphatic-aromatic mixed ring, The aforementioned Ar may be substituted with at least one R'', R １ ~R ６ 、R′ and R″ are each independently hydrogen, optionally substituted C １ -C ４０ alkyl, optionally substituted C １ -C ４０ heteroalkyl, optionally substituted C ２ -C ４０ alkenyl, optionally substituted C ２ -C ４０ alkynyl, optionally substituted C ３ -C ２０ cycloalkyl, optionally substituted C ３ -C ２０ cycloalkenyl, optionally substituted C ２ -C ２０ heterocycloalkyl, hydroxy, oxide (-O － ), optionally substituted C １ -C ４０ alkoxy, optionally substituted C ３ -C ４０ cycloalkyloxy, optionally substituted C ５ -C ４０ aryloxy, optionally substituted C ２ -C ４０ heteroaryloxy, optionally substituted C ５ -C ５０ aryl, optionally substituted C ２ -C ５０ heteroaryl, optionally substituted C ５ -C ５０ aralkyl, optionally substituted C １ -C ４０ alkylthio, optionally substituted C ５ -C ４０ arylthio, optionally substituted C ３ -C ４０ cycloalkylthio, optionally substituted C ２ -C ４０ Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R ｘ and R ｓ Selected from, R ｘ This is a reactive group, or a group in which at least one of the reactive groups is bonded to a backbone containing a hydrocarbon having 1 to 40 carbon atoms. The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO ２ － ), carboxylates, hydroxyl, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamides, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides. R ｓ This is a carrier molecule, or a group in which at least one of the carrier molecules is bonded to a backbone containing a hydrocarbon having 1 to 40 carbon atoms. R ７ and R ８ These are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, optionally substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, possibly substituted C ５ -C ５０ Alalkyl, R ｘ and R ｓ Selected from, X is CR ２０ R ２１ , NR ２２ , O, S, R ２０ to R ２２ are each independently hydrogen, optionally substituted C １ -C ４０ alkyl, optionally substituted C １ -C ４０ heteroalkyl, optionally substituted C ３ -C ３０ cycloalkyl, optionally substituted C ３ -C ３０ heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, R ｘ and R ｓ are selected from, or combine with each other to form an optionally substituted 5- to 7-membered ring, n is an integer between 0 and 5. R １ ~R ６ Two of these adjacent groups can bond to each other and form a 5- to 7-atom ring, which may be substituted. The non-fluorescent compound represented by the chemical formula 1 contains at least one R ｘ or R ｓ Non-fluorescent compounds that exist.

2. The aforementioned Ar is selected from the following [Ar-1] to [Ar-6], 【Chemistry 2】 Here, The aforementioned [Ar-1] to [Ar-6] may be substituted with at least one R''. * indicates the position of carbon atoms that can condense into the nitrogen-containing pentatomic ring of chemical formula 1. Y is independently CR ２３ R ２４ , NR ２５ , O, S, respectively, and The aforementioned R ２３ ~R ２５ C may be substituted independently of each other. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ３ -C ３０ Cycloalkyl, optionally substituted C ３ -C ３０ Heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, R ｘ and R ｓ The nonfluorescent compound according to claim 1, which forms a 5- to 7-atom ring that may be selected from or bonded to each other and substituted.

3. A non-fluorescent compound represented by the following chemical formula 6, The non-fluorescent compound can have its fluorescence reduced by at least one mechanism selected from static quenching and dynamic quenching. 【Transformation 3】 Here, R １ ~R ６ , R' and R Ｄ These are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, hydroxy, oxide (-O － ), C may be substituted. １ -C ４０ Alkoxy, possibly substituted C ３ -C ４０ Cycloalkyloxy, optionally substituted C ５ -C ４０ Aryloxy, possibly substituted C ２ -C ４０ Heteroaryloxy, possibly substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, possibly substituted C ５ -C ５０ Alalkyl, optionally substituted C １ -C ４０ Alkylthio, optionally substituted C ５ -C ４０ Arylthio, C may be substituted. ３ -C ４０ Cycloalkylthio, optionally substituted C ２ -C ４０ Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R ｘ and R ｓ Selected from, R ｘ This is a reactive group, or a group in which at least one of the reactive groups is bonded to a backbone containing a hydrocarbon having 1 to 40 carbon atoms. The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO ２ － ), carboxylates, hydroxyl, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamides, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides. R ｓ This is a carrier molecule, or a group in which at least one of the carrier molecules is bonded to a backbone containing a hydrocarbon having 1 to 40 carbon atoms. R ７ and R ８ These are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, optionally substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, possibly substituted C ５ -C ５０ Alalkyl, R ｘ and R ｓ Selected from, n is an integer between 0 and 5. o is an integer from 0 to 6, R １ ~R ６ Two of these adjacent groups can bond to each other and form a 5- to 7-atom ring, which may be substituted. The non-fluorescent compound represented by the chemical formula 6 contains at least one R ｘ or R ｓ Non-fluorescent compounds that exist.

4. A non-fluorescent compound represented by the following chemical formula 7, The non-fluorescent compound can have its fluorescence reduced by at least one mechanism selected from static quenching and dynamic quenching. 【Chemistry 4】 Here, R １ ~R ６ And R' are independently hydrogen, or possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, hydroxy, oxide (-O － ), C may be substituted. １ -C ４０ Alkoxy, possibly substituted C ３ -C ４０ Cycloalkyloxy, optionally substituted C ５ -C ４０ Aryloxy, possibly substituted C ２ -C ４０ Heteroaryloxy, possibly substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, possibly substituted C ５ -C ５０ Alalkyl, optionally substituted C １ -C ４０ Alkylthio, optionally substituted C ５ -C ４０ Arylthio, C may be substituted. ３ -C ４０ Cycloalkylthio, optionally substituted C ２ -C ４０ Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R ｘ and R ｓ Selected from, R ｘ This is a reactive group, or a group in which at least one of the reactive groups is bonded to a backbone containing a hydrocarbon having 1 to 40 carbon atoms. The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO ２ － ), carboxylates, hydroxyl, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamides, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides. R ｓ This is a carrier molecule, or a group in which at least one of the carrier molecules is bonded to a backbone containing a hydrocarbon having 1 to 40 carbon atoms. R ７ These are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, optionally substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, possibly substituted C ５ -C ５０ Alalkyl, R ｘ and R ｓ Selected from, X is CR ２０ R ２１ , NR ２２ , O, S, R ２０ ~R ２２ These are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ３ -C ３０ Cycloalkyl, optionally substituted C ３ -C ３０ Heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, R ｘ and R ｓ They may be selected from or bonded to each other to form a 5-7 atom ring, n is an integer between 0 and 5. The a and b in the above chemical formula 7 are R in the following chemical formula 8. ３１ ~R ３５ It condenses with two adjacent ones, 【Transformation 5】 R ３１ ~R ３５ Of these, those that do not condense with a and b of the above chemical formula 7 are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, hydroxy, oxide (-O － ), C may be substituted. １ -C ４０ Alkoxy, possibly substituted C ３ -C ４０ Cycloalkyloxy, optionally substituted C ５ -C ４０ Aryloxy, possibly substituted C ２ -C ４０ Heteroaryloxy, possibly substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, possibly substituted C ５ -C ５０ Alalkyl, optionally substituted C １ -C ４０ Alkylthio, optionally substituted C ５ -C ４０ Arylthio, C may be substituted. ３ -C ４０ Cycloalkylthio, optionally substituted C ２ -C ４０ Heteroarylthio, optionally substituted acylamino, acyloxy, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, nitrile, acetal, ketal, optionally substituted amino, thioamide, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, sulfonate, thiocarbonylaminoisonitrile, cyanate, imide, ether, thioether, R ｘ and R ｓ Selected from, R ３１ and R ３５ Of these, two adjacent groups that do not condense with a and b of chemical formula 7 can bond to each other to form a 5- to 7-atom ring, which may be substituted. Q is hydrogen, and C may be substituted. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, optionally substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, possibly substituted C ５ -C ５０ Alalkyl, R ｘ and R ｓ Selected from, The non-fluorescent compound represented by the chemical formula 7 contains at least one R ｘ or R ｓ Non-fluorescent compounds that exist.

5. The nonfluorescent compound contains at least one R ｘ or at least one R ｓ If present, at least one R present in the nonfluorescent compound ｘ or at least one R ｓ The non-fluorescent compound according to any one of claims 1 to 4, characterized in that it is protected with a protecting group.

6. The nonfluorescent compound according to any one of claims 1 to 4, characterized in that it is possible to reduce fluorescence in the 550 to 900 nm wavelength range by dynamic quenching or to reduce fluorescence in the 300 to 900 nm wavelength range by static quenching.

7. The nonfluorescent compound according to any one of claims 1 to 4, characterized in that it is possible to reduce fluorescence in the 550 to 900 nm wavelength range by fluorescence resonance energy transfer, or to reduce fluorescence in the 300 to 900 nm wavelength range by ground state quenching.

8. The non-fluorescent compound according to any one of claims 1 to 4, characterized in that the non-fluorescent compound is a nucleic acid labeling compound.

9. A conjugate comprising a non-fluorescent compound as a quencher according to any one of claims 1 to 4.

10. The conjugate according to claim 9, wherein the conjugate is a nucleotide conjugate.

11. The conjugate according to claim 9, wherein the conjugate is a probe or a primer.

12. Quencher comprising a non-fluorescent compound according to any one of claims 1 to 4; and Phosphor; A conjugate that includes this.

13. A first quencher comprising a non-fluorescent compound according to any one of claims 1 to 4; A second quencher selected from azo, coumarin, cyanine, bolus, furosein, rhodamine, pyrene, carbopyronine, benzo[c,d]indole, oxazine, xanthene, thioxanthene, acridine and derivatives thereof; and Phosphor; A conjugate that includes this.

14. The second quencher is dabcyl, Eclipse ＴＭ , Black Hole Quencher ＴＭ BHQ0 (BHQnova), Black Hole Quencher ＴＭ BHQ1, Black Hole Quencher ＴＭ BHQ2, Black Hole Quencher ＴＭ BHQ3, BlackBerry ＴＭ Quencher 650 (BBQ650 ＴＭ ), Iowa Black ＴＭ FQ (IABkFQ), SFC ＴＭ Q1, SFC ＴＭ Q2, Iowa Black ＴＭ RQ-n1, Iowa Black ＴＭ RQ n2 and Iowa Black ＴＭ RQSp, TAMRA, Deep Dark Quencher I (DDQ I), Deep Dark Quencher II (DDQ II), QXL ＴＭ 520, QXL ＴＭ 570, QXL ＴＭ 610, QXL ＴＭ 670, IRQXL ＴＭ , IRDye ＴＭ , QC-1, QSY ＴＭ QSY ＴＭ 2. The conjugate according to claim 13, which is at least one selected from BMN-Q460, BMN-Q535, BMN-Q1, BMN-Q2, BMN-Q590, BMN-Q620, and BMN-Q651.

15. The conjugate according to claim 12, wherein the phosphor is at least one selected from coumarin, cyanine, bolus, furosein, rhodamine, pyrene, carbopyronine, oxazine, xanthene, thioxanthene, acridine, and / or derivatives thereof.

16. The aforementioned phosphor is 6-FAM ＴＭ , TET ＴＭ JOE ＴＭ , VIC (registered trademark), HEX ＴＭ , NED ＴＭ PET (registered trademark), ROX ＴＭ TAMRA ＴＭ , TET ＴＭ Texas Red (registered trademark), SUN, MAX, ABY, JUN, LIZ, TAZ, CAL Fluor Gold (registered trademark) 540, CAL Fluor Orange (registered trademark) 560, CAL Fluor Red (registered trademark) 590, CAL Fluor Red (registered trademark) 610, CAL Fluor Red (registered trademark) 635, Cy (registered trademark) (cyanine) 3, Cy (registered trademark) 3.5, Cy (registered trademark) 5, Cy (registered trademark) 5.5, Cy (registered trademark) 7, Cy (registered trademark) 7.5, Quasar (registered trademark) 570, Quasar (registered trademark) 670, Quasar (registered trademark) 705, Rhodamine Green ＴＭ , Rhodamine Red ＴＭ LightCycle® Cyan 500, LightCycle® Red 610, LightCycle® Red 640, LightCycle® Red 670, LightCycle® Red 705, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Alexa Fluor® dies 350, 405, 488, 532, 546, 555, 568, 594, 610, 647, 680 and Alexa Fluor® 750, BODIPY® dies, Epoch Blue®, AMCA, Marina Blue®, Pacific Blue ＴＭ , Pacific Green ＴＭ , Pacific Orange ＴＭ , Yakima Yellow ＴＭ , ATTO dies 390, 425, 465, 488, 495, 514, 520, 532, Rho6G, 542, 550, 565, Rho3B, Rho11, Rho12, Thio12, Rho10 1, 590, 594, Rho13, 610, 620, Rho14, 633, 643, 647, 647N, 655, Oxa12, 665, 680, 700, 725, and ATTO 740, Oyster 645, SFC ＴＭ -V, SFC ＴＭ -N, SFC ＴＭ 574, SFC ＴＭ 647, SFC ＴＭ - C610, SFC ＴＭ 620, SFC ＴＭ 670, SFC ＴＭ 705, Chamel ＴＭ 560, Chamel ＴＭ 610, Chamel ＴＭ 670 and Chamel ＴＭ The conjugate according to claim 15, which is at least one selected from 705.

17. The conjugate according to claim 12, further comprising a minor groove binder (MGB).

18. A nucleic acid detection composition comprising the conjugate described in claim 12.

19. A quencher comprising a non-fluorescent compound according to any one of claims 1 to 4; Support; and A connecting portion that connects the quencher and the support; A support for nucleic acid detection, including the following:

20. The support for nucleic acid detection according to claim 19, wherein the support is glass, cellulose, nylon, acrylamide gel, dextran, polystyrene, or resin.

21. (a) A step of preparing a reaction mixture comprising a target nucleic acid, reagents necessary for amplifying the target nucleic acid, and the conjugate described in claim 12; (b) a step of amplifying the target nucleic acid in the reaction mixture; and (c) A step of measuring the fluorescence intensity of the reaction mixture; A nucleic acid detection method, including the following.

22. The aforementioned step (b) is, (b-1) The step in which the conjugate hybridized to the target nucleic acid is extended by polymerase; (b-2) The step of separating the quencher and fluorophor of the conjugate from the target nucleic acid by the exonuclease activity of the polymerase; and (b-3) The step in which the phosphor released from the quencher emits fluorescence; The nucleic acid detection method according to claim 21, including the following:

23. The nucleic acid detection method according to claim 21, further comprising step (d) of measuring the amount of amplification of a target nucleic acid from the fluorescence intensity measured in step (c).

24. The target nucleic acid is used in strand substitution amplification (SDA), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time Polymerase Chain Reaction), allele-specific polymerase chain reaction (Allele-specific Polymerase Chain Reaction), and ligase chain reaction (LCR). Reaction, Rolling Circle Amplification (RCA), Isothermal Multiple Displacement Amplification (IMDA), Recombinase Polymerase Amplification (RPA), Self-Sustained Sequence Replication (3SR), Single Primer Isothermal Amplification (SPIA), Multiple Displacement Amplification (MDA) Amplification, Whole Genome Amplification (WGA), Cross-priming Amplification (CPA), RNA Signal-Mediated Amplification of RNA Technology (SMART), Transcription-Mediated Amplification (TMA), Nucleic Acid Sequence-Based Amplification (NASBA), Loop-Mediated Isothermal Amplification (LAMP) Amplification, and helicase-dependent amplification (HDA).The nucleic acid detection method according to claim 21, wherein the sample is amplified by a method selected from Amplification.

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