peroxidase fluorescent substrate

Abstract

The present application relates to the field of biological detection, and particularly relates to a peroxidase fluorescent substrate. The compound of the present application and peroxidase and peroxide reaction produce an enhanced, optically detectable signal, provide a fluorescent substrate with a larger stokes shift and a higher signal-to-noise ratio, and the fluorescent substrate has good light stability, can be used in TSA detection, and obtain a more optimal imaging effect.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to peroxidase fluorescent substrates. Background Technology

[0002] Immunohistochemistry (IHC) is the process of detecting, locating, and / or quantifying antigens (e.g., proteins) in biological samples using antibodies against specific antigens. A significant advantage of IHC is its ability to precisely identify the location of specific proteins in tissue samples. It is also an effective method for examining the tissue itself. In situ hybridization (ISH) is the process of detecting, locating, and quantifying nucleic acids. Both IHC and ISH can be performed on a variety of biological samples, such as tissues (e.g., fresh-frozen, formalin-fixed, paraffin-embedded) and cytological samples. Regardless of whether the target is a nucleic acid or an antigen, various markers (e.g., chromogenic, fluorescent, luminescent, radioactive) can be used to detect target recognition. To reliably detect, locate, and quantify targets in a clinical setting, amplification of recognition events is necessary, as the ability to reliably detect low-abundance cellular markers is becoming increasingly important for diagnostic purposes. For example, in response to a single antigen detection event, hundreds or thousands of marker molecules are deposited at the labeling site; amplification enhances the ability to detect that recognition event.

[0003] Amplification is often accompanied by adverse events, such as nonspecific signals, which manifest as background signal enhancement. Background signal enhancement can interfere with clinical analysis because it masks weak signals that may be associated with low expression (but are clinically significant). Therefore, while amplification to identify events is desirable, there is a strong need for amplification methods that do not increase background signal. Tyramine signal amplification (TSA) is such a method, also known as catalytic reporter gene deposition (CARD).

[0004] Tyramine signal amplification (TSA) is an enzyme-mediated amplification technique. In the presence of hydrogen peroxide, horseradish peroxidase (HRP) catalyzes the conversion of tyramine into oxygen-reactive free radicals. This intermediate can rapidly covalently bind to proteins or HRP-linked antibodies. A fluorescent dye is then bound to the tyramine molecule, forming a covalent binding site, thereby stably and efficiently enriching the fluorescein around and depositing it onto the target cell, resulting in a geometric amplification of the detection signal. However, the detection sensitivity of the currently used fluorescent substrate TSA-FITC (FITCT) is still relatively low, requiring further improvement. This is mainly because the fluorescence signal intensity of FITC fluorescein itself is insufficient. Other fluoresceins such as Alexa Fluor488, Dylight488, and iFluor488 are available, but these are all imported reagents, resulting in higher costs. Summary of the Invention

[0005] The compound of this invention reacts with peroxidase and peroxide to produce an enhanced, optically detectable signal, providing a fluorescent substrate with a larger Stokes shift and a higher signal-to-noise ratio. Furthermore, this fluorescent substrate exhibits good photostability and can be used in TSA detection to obtain better imaging results.

[0006] On one hand, the present invention provides a peroxidase fluorescent substrate having the structure shown in Formula I: (I); Where R is the dye; L does not exist, or it is a chain consisting of 1-20 atoms; Ring A is aryl, heteroaryl, cycloalkyl, or heterocyclic; Each R 1 Independently hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, cycloalkylalkyl, heterocyclic or heterocyclic alkyl; Each R 2 Independently hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, cycloalkylalkyl, heterocyclic or heterocyclic alkyl; R 3 It is either hydroxyl or amino; n is 0, 1, 2, 3 or 4; m can be 0, 1, 2, 3, or 4.

[0007] In some embodiments, the dye is an acridine, acridine salt, acridine ketone, anthraquinone, azo dye, acridine dye, phthalocyanine, eurygidine dye, saffron dye, indamine, indophenol, oxazine, thiazine, oxazole, thiazole, polythiophene, polypyrrole, pyranone, fluorescein, rhodamine, coumarin, anthocyanin, porphyrin, rhodolol, quinoline, boron fluoride dipyrrole, squaric acid cyanide, perylene diimide, diketopyrrolopyrrole, conjugated polymer, fluorescent protein, quantum dot, or polymer dot.

[0008] In some embodiments, the dye is AF350, AF405, AF425, AF430, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF640, AF647, AF660, AF680, CF405M, CF450, CF488A, CF532, CF543, CF555, CF568, CF594, CF620R, CF633, CF640R, CF647, CF660C, CF680, CF680R, CF750, CF770, CF790, or CF820.

[0009] In some embodiments, ring A is C 6-10 Aryl, 5-10 heteroaryl, C 3-6 Cycloalkyl or 5-7 membered heterocyclic groups.

[0010] In some embodiments, ring A is phenyl, naphthyl, pyrrolyl, furanyl, thiophene, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrazinyl, pyranonel, indolyl, benzofuranyl, benzothiophene, quinolinyl, isoquinolinyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydrothiophene, tetrahydropyranyl, piperidinyl, tetrahydrothiophene, dioxanecycloyl, piperazinyl, hexahydropyrazinyl, or morpholinyl.

[0011] In some embodiments, each R 1 and R 2 Independently hydroxyl, amino, halogen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy, C 1-6 Alkylamino, C 3-6 cycloalkyl, C 3-6 cycloalkyl C 1-6 Alkyl, 5-7 membered heterocyclic or 5-7 membered heterocyclic C 1-6 alkyl.

[0012] In some embodiments, each R 1 and R 2 Independently, it is hydroxyl, amino, F, Cl, Br, methyl, ethyl, propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, methylamino, ethylamino, propylamino, isopropylamino, butylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclopropylethyl, cyclopentylmethyl, oxecyclopropyl, azicyclopropyl, oxecyclobutyl, azicyclobutyl, tetrahydrofuranyl, tetrahydropyrrolyl, piperidinyl, tetrahydropyranyl, oxecyclopropylmethyl, azicyclopropylmethyl, oxecyclobutylmethyl, azicyclobutylmethyl, oxecyclopropylethyl, azicyclopropylethyl, oxecyclobutylethyl, or azicyclobutylethyl.

[0013] In some embodiments, L is absent, or is a chain consisting of 1-20 atoms; L serves a connecting function, and its selection depends on the size of the dye molecule. If the dye molecule is too large, L is selected from a relatively long chain; if the dye molecule is small and hinders the interaction site, L may also be selected from a relatively short chain or L may not be required.

[0014] In some embodiments, the chain consisting of 1-20 atoms is selected from: , , , , , , , ; t is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is selected from 1, 2, 3, or 4; Each q is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0015] In some embodiments, the fluorescent substrate of the present invention has the structure shown in Formula II: (II) R, R 1 R 2 R 3 A, n, and m have the definitions described herein.

[0016] L 1 It is a C1-10 straight-chain carbon or -CH2-(CH2OCH2) p -CH2-; p is selected from 1, 2, 3 or 4.

[0017] In some embodiments, the structure shown in Formula I has one of the following structures: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11).

[0018] On the other hand, the present invention provides a reagent composition comprising the peroxidase fluorescent substrate or its stereoisomer described herein.

[0019] On the other hand, the present invention provides the use of the peroxidase fluorescent substrate of the present invention in the preparation of fluorescent detection reagents or cell imaging reagents.

[0020] The compounds disclosed herein can be used as fluorescent substrates, enabling efficient fluorescence detection and application in TSA (Transmission of Substances) detection. The fluorescent substrates provided herein improve the imaging quality of low-abundance targets through tyrosine signal amplification technology, exhibiting relatively higher detection sensitivity and efficient detection of low-expression biomarkers, demonstrating significant advantages. The absorption wavelength range covered by the fluorescent substrates is 400-600 nm, with a preferred range of 488-594 nm. In practical applications, fluorescent substrates with different absorption wavelengths can be selected according to different detection conditions and requirements.

[0021] The beneficial effects achieved by this disclosure are as follows: The peroxidase fluorescent substrate or its stereoisomer in this invention achieves highly sensitive fluorescence detection, which can be widely used to detect cells with low expression of specific antigens. Compared with traditional fluorescent secondary antibody methods and TSA-FITCT methods, it has higher detection sensitivity and detection stability.

[0022] Detailed explanation: Certain embodiments of the present invention will now be described in detail. The present invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials described herein can be used to practice the present invention. The present invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0023] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0024] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0025] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0026] In the following content, all numbers disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number with a value of N is disclosed, any numbers with values ​​of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction.

[0027] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.

[0028] When a substituent is described using a conventional chemical formula written from left to right, it also includes chemically equivalent substituents obtained when the structural formula is written from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0029] The term "alkyl" refers to a hydrocarbon group selected from straight-chain and branched saturated hydrocarbon groups, having 1-18, 1-12, 1-6, or 1-3 carbon atoms. Examples of alkyl groups include methyl, ethyl, 1-propyl or n-propyl ("n-Pr"), 2-propyl or isopropyl ("i-Pr"), 1-butyl or n-butyl ("n-Bu"), 2-methyl-1-propyl or isobutyl ("i-Bu"), 1-methylpropyl or sec-butyl ("s-Bu"), and 1,1-dimethylethyl or tert-butyl ("t-Bu"). Other examples of alkyl groups include 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

[0030] The term "alkenyl" refers to a hydrocarbon group selected from straight-chain and branched groups having one or more carbon-carbon double bonds, having 2-18, 2-12, 2-6, or 2-4 carbon atoms. Typical examples include vinylidene (-CH=CH-), propenidene (-CH=CHCH2- and -CH2CH=CH-), n-butenyl and 3-methyl-2-pentenyl, hexenyl, heptenyl, octeneyl, nonenyl, and decenyl, etc.

[0031] The term "alkynyl" refers to a hydrocarbon group selected from straight-chain and branched groups having one or more carbon-carbon triple bonds, having 2-18, 2-12, 2-6, or 2-4 carbon atoms. Typical examples include ethynyl, 1-propynyl, and 2-propynyl.

[0032] The term "alkoxy" refers to the aforementioned alkyl groups having a specific number of carbon atoms and connected by oxygen bridges. C1-6 alkoxy groups include C1, C2, C3, C4, C5, and C6 alkoxy groups. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and sec-pentoxy.

[0033] The term "alkanoylamino" refers to the aforementioned alkyl groups having a specific number of carbon atoms and being linked by nitrogen bridges. C1-6 alkanoylamino groups include C1, C2, C3, C4, C5, and C6 alkanoylamino groups. Examples of alkoxy groups include, but are not limited to: methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, sec-butylamino, tert-butylamino, n-pentanamino, and sec-pentanamino.

[0034] The term "aryl" refers to a group selected from the following: 5- and 6-membered carbocyclic aromatic rings, such as phenyl; bicyclic systems (such as 7-12-membered bicyclic systems) in which at least one ring is a carbocyclic ring and is aromatic, such as naphthalene, indane, and 1,2,3,4-tetrahydroquinoline; and tricyclic systems (such as 10-15-membered tricyclic systems) in which at least one ring is a carbocyclic ring and is aromatic, such as fluorene.

[0035] The term "heteroaryl" refers to a group comprising a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 π electrons in a cyclic arrangement) having a ring carbon atom and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (referred to as "5-14 membered heteroaryl"), or groups derived therefrom. In heteroaryls containing one or more nitrogen atoms, the bonding point can be a carbon atom or a nitrogen atom, depending on the valence. Heteroaryl polycyclic systems may contain one or more heteroatoms in one or two rings. "Heteroaryl" includes cyclic systems in which a heteroaryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the bonding point is located on the heteroaryl ring; in such cases, the ring membership number still refers to the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems fused with one or more aryl groups as defined above, where the linker is located on either the aromatic or heteroaryl ring. In such cases, the ring membership number refers to the total number of ring members in the fused polycyclic (aryl / heteroaryl) system. For polycyclic heteroaryl groups where one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the linker can be located on either ring, i.e., a ring containing a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl). Unless otherwise stated, each heteroaryl instance is independently unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl"). Specific examples of heteroaryl ring systems include substituted or unsubstituted thiophene, benzothiophene, naphthothiophene, furanyl, pyranyl, isobenzofuranyl, benzoxazolyl, benzothiophene, xanthonyl, phenanthonyl, pyrroleyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, purinyl, quinolinyl, phthalazinyl, naphridinyl, quinazolinyl, cinnamyl, carbazolyl, phenanthridine, acridineyl, phenazinyl, thiazolyl, oxazolyl, furanyl, phenoxazinyl, or tetrazolyl.

[0036] The term "cycloalkyl" refers to a hydrocarbon group selected from saturated and partially unsaturated cyclic hydrocarbon groups, including monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups. For example, the cycloalkyl group may have 3-12, 3-8, 3-6, 3-4, or 5-6 carbon atoms. As another example, the cycloalkyl group may be a monocyclic group having 3-12, 3-8, or 3-6 carbon atoms. Examples of monocyclic cycloalkyl groups include: cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohexyl-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Examples of bicyclic cycloalkyl groups include bicyclic systems having 7-12 ring atoms arranged in the following configurations: [4,4], [4,5], [5,5], [5,6], and [6,6] ring systems, or bridged bicyclic systems such as bicyclic [2.2.1]heptane, bicyclic [2.2.2]octane, and bicyclic [3.2.2]nonane. The rings may be saturated or have at least one double bond (i.e., partially unsaturated), but are not fully conjugated and are not aromatic (as defined herein).

[0037] The term "heterocyclic group" refers to a monocyclic, bicyclic, or tricyclic group containing a carbon atom and one, two, three, or four independent cyclic heteroatoms or heteroatom groups selected from N, O, and S, which may be saturated, partially unsaturated, or non-aromatic rings.

[0038] The terms “halogen” or “halogenated” refer to fluorine, chlorine, bromine, or iodine, either on their own or as part of another substituent.

[0039] The term "hydroxyl group" is -OH.

[0040] The term "amino" is -NH2.

[0041] The term “dye”, either by itself or as part of another group, refers to an aryl, heteroaryl, or conjugated compound whose longest absorption peak is longer than 400 nm, or whose longest fluorescence peak is longer than 400 nm, or whose longest emission peak is longer than 400 nm.

[0042] Specific examples of the fluorescent dye optionally include substituted or unsubstituted ATTO 465, ATTO 488, ATTO495, ATTO 514, ATTO 532, ATTO 550, ATTO 565, ATTO 590, ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO740, 5-carboxy-2,7-Dichlorofluorescein, 5-FAM, 5-Carboxynaphthalenefluorescein, 5-ROX, 6-TAMRA, 6-Carboxyrhodamine 6G, 6-JOE, 6-FAM, 6-ROX, Bodipy 492 / 515, Bodipy 493 / 503, Bodipy 500 / 510, Bodipy 505 / 515, Bodipy 530 / 550, Bodipy 542 / 563, Bodipy 558 / 568, Bodipy 564 / 570, Bodipy 576 / 589, Bodipy 581 / 591, Bodipy 630 / 650-X, Bodipy 650 / 665-X, Bodipy 665 / 676, BodipyFl, Bodipy R6G SE, Bodipy TMR, Bodipy TR, AF350, AF405, AF425, AF430, AF488, AF532, AF546, AF555, AF568, AF594, AF63 3. AF640, AF647, AF660, AF680, CF405M‌, ‌CF450‌, ‌CF488A, ‌CF532‌, ‌CF543, CF 555, CF568, CF594‌, CF620R, CF 633, ‌CF640R, ‌CF647, ‌CF660C, CF680‌, ‌CF680R, CF750‌, ‌CF770, ‌CF790‌, ‌CF82 0‌, CL-NERF, CMFDA, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, DDAO, DiA, DiD, DiI, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755, DyLight 800, DiO, DiR, DM-NERF, DsRed, DTAF, DY-490, DY-495, DY- 505, DY-530, DY-547, DY-548, DY-549, DY-549P1, DY-550, DY-554, DY-555, DY-556, DY-560, DY-590, DY-59 1. DY-594, DY-605, DY-610, DY-615, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, DY-647, DY- 648,DY-649, DY-649P1, DY-650, DY-651, DY-652, DY-654, DY-675, DY-676, DY-677, DY-678, DY -679, DY-679P1, DY-680, DY-681, DY-682, DY-700, DY-701, DY-703, DY-704, DY-730, DY-73 1. DY-732, DY-734, DY-749, DY-750, DY-751, DY-752, DY-754, DY-776, DY-777, DY-778, DY -780, DY-781, DY-782, DY-800, DY-831, Eosin, Erythrosine, FITC, Fluo-3, Fluo-4, Fluor-Ruby, Fluor X, FM 1-43, FM 4-46, iFluor 488, iFluor 555, iFluor 594, iFluor 647, iFluor 680, iFluor 700, iFluor 750, iFluor 780, Lyso Tracker Green, Lyso Tracker Yellow, Mitotracker Green, Mitotracker Orange, Mitotracker Red, NBD, Oregon Green 488, Oregon Green 514, PKH26, PKH67, Halogen, GFP, YFP, RFP, PE, APC, PerCP, Brilliant Violet 421, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 750, Brilliant Violet 785, BUV 395, BUV 4936, SuperBright 436, SuperBright 600, SuperBright 645, SuperBright 702 or SuperBright 780. ,

[0043] The term "TSA" refers to the highly sensitive detection of a target analyte achieved through the catalytic action of tyrosinase, which deposits a fluorescein-conjugated tyrosine substrate around the analyte and amplifies the signal. In this invention, specific antibodies are first screened for the target analyte and labeled with horseradish peroxidase. Simultaneously, fluorescein is linked to p-hydroxyphenylethylamine. Through horseradish peroxidase catalysis, p-hydroxyphenylethylamine is linked to tyrosine residues of the protein, enriching the fluorescein around the target analyte and achieving highly sensitive detection.

[0044] The term "quantum yield" refers to the ratio of the number of fluorescent photons emitted after a fluorescent substance absorbs light to the number of photons of the absorbed excitation light, used to express the luminescence efficiency of a sample. The magnitude of the fluorescence quantum yield is primarily determined by the structure and properties of the compound, and is also related to environmental factors. The fluorescence quantum yield only reflects the luminescence efficiency of the fluorescent substrate in that solution. The intensity of the fluorescence signal detected in a sample is related to both the characteristics of the fluorophore and the amount of fluorophore labeled in the sample.

[0045] Abbreviation: PPh3: Triphenylphosphine TBSCl: tert-butyldimethylchlorosilane DMF: N,N-dimethylformamide t-BuOK: Potassium tert-butoxide DCM: Dichloromethane -OTBS: tert-butyldimethylsilyloxy TBAF: Tetrabutylammonium fluoride TBTU: O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate TSTU: O-(N-succinimide)-N,N,N',N'-tetramethylurea tetrafluoroborate DIPEA: N,N-Diisopropylethylamine CF3COOH: Trifluoroacetic acid Et3N: Triethylamine THF: Tetrahydrofuran NaOH: Sodium hydroxide MeOH: Methanol Attached Figure Description

[0046] Figure 1 Immunofluorescence staining of rat lung tissue with α-Actin. (A) Staining group of compound 1; (B) Staining group of control compound. Detailed Implementation

[0047] I. Synthesis of Compounds Example 1: Synthesis of Compound 1:

[0048] Step 1: Synthesis of Compounds 1-2 Methyl 4-bromomethylphenylacetate (11.50 g, 47.32 mmol) and triphenylphosphine (12.40 g, 47.32 mmol) were weighed and added to toluene (100 mL). The mixture was refluxed at 120 °C for about 5 hours. After cooling to room temperature, the mixture was filtered, and the filter cake was washed with a small amount of ethyl acetate. The filtrate was concentrated under reduced pressure to give compounds 1-2 (white solid, 15.7 g, yield 78.07%).

[0049] LC-MS [M-Br] + = 425.17.

[0050] Step 2: Synthesis of compounds 1-4 Weigh 10.00 g (58.14 mmol) of 6-hydroxy-2-naphthaldehyde and 11.86 g (174.41 mmol) of imidazole into DMF. After cooling in an ice-water bath for 30 min, add TBSCl (13.14 g, 87.02 mmol) in portions. After the addition is complete, react at room temperature for about 24 hours. Add 250 mL of water and extract with ethyl acetate (150 mL x 2). Wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure, purify by silica gel column chromatography, elute with petroleum ether:ethyl acetate = 95:5, and concentrate to give compounds 1-4 (orange-red oily liquid, 12.8 g, yield 76.98%).

[0051] LC-MS [M+H] + = 287.14.

[0052] Step 3: Synthesis of compounds 1-5 Compounds 1-2 (11.50 g, 27.06 mmol) were weighed and dissolved in dichloromethane (10 mL). The mixture was purged with nitrogen three times and cooled to -80 °C. Potassium tert-butoxide (7.6 g, 67.73 mmol) was added, followed by the addition of a dichloromethane solution of compound 1-4 (7.74 g, 27.06 mmol). The mixture was then reacted at -80 °C for approximately 24 hours. The pH was adjusted to 6 by adding 1 mol / L dilute hydrochloric acid at 0 °C. Water (5 V) was added, and the mixture was extracted with dichloromethane (3 V x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with petroleum ether:ethyl acetate = 98:2. The purified compound 1-5 was then concentrated to obtain a white solid (3.5 g, yield 47.49%).

[0053] LC-MS [M+H] + = 433.21.

[0054] Step 4: Synthesis of compounds 1-6 Compounds 1-5 (2.10 g, 4.86 mmol) were weighed and added to THF (5 mL), followed by TBAF (1.30 g, 4.86 mmol). The reaction was allowed to proceed at room temperature for approximately 24 hours. The mixture was then extracted with water (5 V) and ethyl acetate (3 V x 2), washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate = 80:20, and concentrated to give compounds 1-6 (pale yellow solid, 1.45 g, yield 93.82%).

[0055] LC-MS [M+H] + = 319.13.

[0056] Step 5: Synthesis of compounds 1-7 Compounds 1-6 (1.8 g, 5.66 mmol) were weighed and added to 15 mL of methanol. An aqueous solution of sodium hydroxide (0.60 g, 15.00 mmol) (1.5 mL) was added dropwise. After the addition was complete, the mixture was reacted at room temperature for approximately 24 hours. The mixture was concentrated under reduced pressure, and 10 mL of pure water was added. The pH was adjusted to 4 by adding 1 mol / L dilute hydrochloric acid. The mixture was extracted with ethyl acetate (3 V x 2), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compounds 1-7 (a grayish-white solid, 1.7 g, yield 98.80%).

[0057] LC-MS [M+H] + = 305.11.

[0058] Step 6: Synthesis of compounds 1-8 Weigh compounds 1-7 (1.8 g, 5.92 mmol) and add DMF (15 mL). Under ice-water bath cooling, add TsTu (2.14 g, 7.11 mmol), DIPEA (2.29 g, 17.75 mmol), and mono-Boc ethylenediamine. After the addition is complete, react at room temperature for about 24 hours.

[0059] Post-treatment: Water (5 V) was added and extracted with ethyl acetate (3 V x 2); the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by silica gel column chromatography, eluted with dichloromethane:methanol = 95:5, and concentrated to give compounds 1-8 (pale yellow oil, 2.35 g, yield 88.90%).

[0060] LC-MS [M+H] + = 447.22.

[0061] Step 7: Synthesis of compounds 1-9 Compounds 1-8 (3.35 g, 7.50 mmol) were weighed and added to dichloromethane (30 mL). Trifluoroacetic acid (6 mL) was added under ice-water bath cooling. After the addition was complete, the reaction was carried out at room temperature for about 24 hours. The mixture was concentrated under reduced pressure and extracted with saturated sodium bicarbonate (5 V) and dichloromethane (3 V x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compounds 1-9 (grayish-white solid, 1.56 g, yield 60.11%).

[0062] LC-MS [M+H] + = 347.17.

[0063] Step 8: Synthesis of Compound 1 Weigh 5-AF488-NHS (CAS: 1374019-99-4, 120 mg, 0.19 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (38 mg, 0.38 mmol), followed by compounds 1-9 (66 mg, 0.19 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Post-treatment: Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 1 (orange solid, 24 mg, yield 15.00%).

[0064] LC-MS [M+H] + = 863.16; 1 H NMR (600 MHz, deuterated water) δ 7.72 (q, J = 8.2 Hz, 2H), 7.36 (d, J = 8.8Hz, 1H), 7.16 (s, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.80 (s, 1H), 6.77 – 6.72 (m, 4H), 6.70 (d, J = 9.1 Hz, 1H), 6.59 (d, J = 9.3Hz, 2H), 6.43 (d, J = 9.3 Hz, 2H), 6.14 (d, J = 12.3 Hz, 1H), 6.02 (d, J =12.3 Hz, 1H), 3.39 (s, 2H), 3.34 – 3.31 (m, 2H), 3.27 (s, 2H).

[0065] Example 2: Synthesis of Compound 8:

[0066] Weigh AF430-NHS (CAS: 467233-94-9, 100 mg, 0.17 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (34 mg, 0.33 mmol), followed by compounds 1-9 (66 mg, 0.19 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Post-treatment: Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 8 (grayish-white solid, 35 mg, yield 24.77%).

[0067] LC-MS [M+H] + = 832.28.

[0068] Example 3: Synthesis of Compound 9:

[0069] Weigh AF555-NHS (CAS: 407627-69-4, 110 mg, 0.12 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (24 mg, 0.24 mmol), followed by compounds 1-9 (45 mg, 0.13 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 9 (red solid, 46 mg, yield 33.04%).

[0070] LC-MS [M+H] + = 1161.33.

[0071] Example 4: Synthesis of Compound 10:

[0072] Weigh AF647-NHS (CAS: 1620475-28-6, 110 mg, 0.12 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (24 mg, 0.24 mmol) and NAP (45 mg, 0.13 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 10 (blue solid, 51 mg, yield 35.82%).

[0073] LC-MS [M+H] + = 1187.34 Example 5: Synthesis of Compound 11:

[0074] Weigh AF568-NHS (CAS: 247145-38-6, 100 mg, 0.13 mmol) and add it to DMF (3 mL). While cooling in an ice-water bath, add triethylamine (26 mg, 0.26 mmol), followed by compounds 1-9 (48 mg, 0.14 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Concentrate under reduced pressure, prepare and purify, and lyophilize to obtain compound 11 (red solid, 44 mg, yield 33.11%).

[0075] LC-MS [M+H] + = 1023.29.

[0076] II. Fluorescent Staining Test Example of fluorescent staining: Immunofluorescence staining of compound 1 and control compound 1. Experimental Materials Experimental sample: Paraffin-embedded sections of rat lung tissue.

[0077] Experimental reagents: ethanol, xylene, sodium citrate antigen retrieval solution (pH 6.0), HRP Blocking Buffer (#BUF0102), Antibody Dilution & Blocking Buffer (#BUF0103), DAPI (#BUF0105), Tyramide LUMO Buffer (#BUF0101), and anti-fluorescence quenching mounting medium.

[0078] Antibodies: α-Actin (#23660-1-AP), HRP-polymer conjugated Secondary Antibody (Goat Anti-Rabbit IgG) (#BUF0106).

[0079] Tyramine dyes: Compound 1, control compound.

[0080] Compare the structures of the compounds:

[0081] 2. Experimental Procedure (1) Dewaxing and hydration: Immerse the tissue sections in 100% xylene I and II for 10 minutes each; then immerse in 100% ethanol I and II for 10 minutes each; finally immerse in 95%, 85%, 75%, and 50% ethanol for 5 minutes each. Rinse the sections with ddH2O for 5 minutes, repeating 3 times.

[0082] (2) Antigen retrieval: Immerse the tissue sections in sodium citrate antigen retrieval solution (pH 6.0), microwave on high (100% power) for 10 minutes, then microwave on medium (50% power) for 15 minutes. After cooling to room temperature, rinse the sections with 1×PBS for 5 minutes. Repeat 3 times.

[0083] (3) Tissue permeability: Add an appropriate amount of cell permeability solution to cover the tissue, incubate at room temperature for 10-15 minutes, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times.

[0084] (4) Quenching endogenous HRP: Add an appropriate amount of HRP Blocking Buffer to cover the tissue, incubate at room temperature for 10-15 minutes, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times.

[0085] (5) Non-specific blocking: Add an appropriate amount of Antibody Dilution & Blocking Buffer to cover the tissue and incubate at room temperature for 30 minutes. (6) Peroxidase labeling: Add an appropriate amount of antibody dilution solution to cover the tissue and incubate overnight at 4°C. After the primary antibody incubation is complete, allow the sections to return to room temperature, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times. Then add HRP-polymer conjugated secondary antibody (Goat Anti-Rabbit IgG) to cover the tissue, incubate at room temperature for 30-60 minutes, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times.

[0086] (7) TSA staining: Dilute 200× tyramine dye to 1× with Tyramide LUMO Buffer. Add an appropriate amount of 1× tyramine dye to cover the tissue and incubate at room temperature for 10-15 minutes. Rinse the sections with 1× PBS for 5 minutes, repeat 3 times.

[0087] (8) DAPI staining: Add an appropriate amount of DAPI to cover the tissue, rinse the section with ddH2O for 5 minutes, and repeat 3 times.

[0088] (9) Mounting: Add anti-fluorescence quenching mounting medium to mount the slide.

[0089] (10) Microscopic imaging: Select the FITC fluorescence channel for imaging and taking pictures.

[0090] 3. Experimental Results Figure 1 It can be seen that the compound 1 staining group (A) has a higher signal-to-noise ratio and stronger sensitivity than the control compound staining group (B).

Claims

1. A peroxidase fluorescent substrate having the structure shown in Formula I: （I）； in, R is a dye; L does not exist, or it is a chain consisting of 1-20 atoms; Ring A is aryl, heteroaryl, cycloalkyl, or heterocyclic; Each R 1 Independently hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, cycloalkylalkyl, heterocyclic or heterocyclic alkyl; Each R 2 Independently hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, cycloalkylalkyl, heterocyclic or heterocyclic alkyl; R 3 It is either hydroxyl or amino; n can be 0, 1, 2, 3, or 4; m can be 0, 1, 2, 3, or 4.

2. The peroxidase fluorescent substrate according to claim 1, wherein, The dyes are acridines, acridine salts, acridine ketones, anthraquinones, azo dyes, acridine dyes, phthalocyanines, eurygidine dyes, saffron dyes, indamines, indophenols, oxazines, thiazines, oxazoles, thiazoles, polythiophenes, polypyrroles, pyranones, fluorescein, rhodamines, coumarins, anthocyanins, porphyrins, rhodolols, quinolines, boron fluoride dipyrroles, squaric acid cyanides, perylene diimides, diketopyrrolopyrroles, conjugated polymers, fluorescent proteins, quantum dots, or polymer dots.

3. The fluorescent substrate according to claim 1, wherein, The dye is AF350, AF405, AF425, AF430, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF640, AF647, AF660, AF680, CF405M, CF450, CF488A, CF532, CF543, CF555, CF568, CF594, CF620R, CF633, CF640R, CF647, CF660C, CF680, CF680R, CF750, CF770, CF790, or CF820.

4. The peroxidase fluorescent substrate according to claim 1 or 2, wherein, Ring A is C 6-10 Aryl, 5-10 heteroaryl, C 3-6 Cycloalkyl or 5-7 membered heterocyclic groups.

5. The peroxidase fluorescent substrate according to claim 1 or 2, wherein, L does not exist, or it is a chain of 1-20 atoms; a chain of 1-20 atoms is selected from: 、 、 、 、 、 、 、 ； t is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is selected from 1, 2, 3, or 4; Each q is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

6. The peroxidase fluorescent substrate according to claim 1 or 2, wherein, Each R 1 and R 2 Independently hydroxyl, amino, halogen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkoxy, C 1-6 Alkylamino, C 3-6 cycloalkyl, C 3-6 cycloalkyl C 1-6 Alkyl, 5-7 membered heterocyclic or 5-7 membered heterocyclic C 1-6 alkyl.

7. The peroxidase fluorescent substrate according to claim 1 or 2, wherein, The fluorescent substrate has the structure shown in Formula II: (II), L 1 It is a C1-10 straight-chain carbon or -CH2-(CH2OCH2) p -CH2-; p is selected from 1, 2, 3 or 4.

8. The peroxidase fluorescent substrate according to claim 1 or 2, wherein, The structure shown in Equation I has one of the following structures: （1） （2）、 （3）、 （4）、 （5）、 （6）、 （7）、 （8）、 （9）、 （10）、 （11）。 9. A reagent composition comprising the peroxidase fluorescent substrate or its stereoisomer as described in any one of claims 1-8.

10. Use of the peroxidase fluorescent substrate according to any one of claims 1-8 in the preparation of fluorescent detection reagents or cell imaging reagents.

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