MicroRNA response type xanthene-hemicyanine fluorescent dye as well as bi-component probe system, synthetic method and application of microRNA response type xanthene-hemicyanine fluorescent dye

Abstract

The invention discloses a microRNA (Ribonucleic Acid) response type xanthene-hemicyanine fluorescent dye as well as a two-component probe system, a synthesis method and application thereof. The structure of the fluorescent dye is shown as a general formula I, and the fluorescent dye can be connected with a targeting ligand (such as short-chain RNA) through carboxyl. The two-component probe system is composed of two short-chain RNAs which are respectively connected with the dye and a biological orthogonal reaction partner (such as tetrazine), a biological orthogonal reaction is triggered through target microRNA-21 mediated hybridization, and the near-infrared fluorescence and photosensitive activity of the dye are specifically activated. The dye and the probe system can realize fluorescence imaging with high signal-to-noise ratio for overexpressed microRNA-21 in tumors, and generate active oxygen under laser irradiation for photodynamic therapy, thereby realizing integration of diagnosis and treatment. The dye and the probe system provided by the invention have the advantages of high biological safety, simplicity and convenience in preparation, high specificity and the like, and have wide application prospects in early diagnosis and treatment of malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of fine chemical technology, specifically relating to a microRNA-responsive xanthene-hemicyanine, its two-component probe system, synthesis method, and its application. Background Technology

[0002] In the past decade or so, the incidence and mortality rates of malignant tumors have both remained high, making it one of the major public health problems seriously threatening human health. Developing new methods for early detection and effective treatment of tumors is essential to improving patients' quality of life and reducing mortality. Therapeutic approaches are a novel biomedical technology that combines disease diagnosis and monitoring with drug therapy, simultaneously possessing multiple functions such as tumor monitoring, anti-tumor treatment, and efficacy evaluation. MicroRNAs (miRNAs), short non-coding RNAs, have become promising biomarkers for cancer diagnosis, prognosis, and treatment monitoring. Therefore, developing therapeutic probes that respond to microRNAs will be of great significance for the early diagnosis and effective treatment of malignant tumors.

[0003] Near-infrared fluorescent probe-based imaging technology can significantly reduce background interference from autofluorescence in organisms, possesses deep tissue penetration, and exhibits low phototoxicity, thus attracting widespread attention in early tumor detection research. Photodynamic therapy, as an emerging tumor treatment strategy, relies on photosensitizers to convert ambient oxygen into reactive oxygen species under illumination, thereby inducing tumor cell death through multiple mechanisms. This therapy shows great potential in clinical applications due to its minimally invasive nature, low side effects, and broad anti-tumor applicability. Developing fluorescent molecules that respond to tumor-highly expressed microRNAs and can be used for photodynamic therapy could enable simultaneous visualization and precise treatment of tumors in vivo, providing an innovative path for building an integrated tumor diagnosis and treatment platform. However, the performance of such probes largely depends on the stability of pre-linked reactive groups in complex biological environments. Insufficient stability can lead to non-specific "background reactions" before the probe reaches the target, resulting in a decreased signal-to-noise ratio, increased imaging background, and potentially unintended phototoxicity to normal tissues, greatly increasing the difficulty and uncertainty of research and development, which severely limits its potential for clinical translation. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a microRNA-responsive oxanthracene-hemicyanine near-infrared fluorescent dye, a two-component probe system, its preparation method, and its applications, aiming to solve the technical problems of difficulty in early tumor diagnosis and lack of effective treatment methods. To achieve the above objectives, this invention employs the following technical solutions:

[0005] First, this invention provides an oxanthracene-hemicyanine that can be used in the microRNA-21 response, with the following general structural formula:

[0006] In general formula I, R1 is selected from any one of alkylcarboxyl or benzoic acid groups with 3-10 carbons, more preferably from one of alkylcarboxyl or benzoic acid groups with 3-6 carbons; more specifically, R1 is selected from one of the following groups: The following groups are used: propionic acid group (—(CH2)2COOH), butylcarboxyl group (—(CH2)3COOH), pentylcarboxyl group (—(CH2)4COOH), hexylcarboxyl group (—(CH2)5COOH), and benzoic acid group (—C6H4COOH, usually referring to para- or unsubstituted benzoyloxymethylene forms, depending on the linkage site); among them, the hexylcarboxyl group (i.e., —(CH2)5COOH derived from hexabromohexanoic acid) is explicitly used in Example 1.5 and is a preferred specific example. Further explanation: Here, "alkylcarboxyl group" should be understood as an ω-carboxyl-substituted straight-chain or branched alkyl group, i.e., the general formula —(CH2). n COOH (n = 2-9), corresponding to C3-C 10 Carboxylic acid residues. In the context of this invention, the actual chain attached to the indole nitrogen atom is a side chain formed by the N-alkylation of a haloalkane carboxylic acid (such as 6-bromohexanoic acid), therefore R¹ is essentially —(CH2). m COOH (m = 2-9), preferably m = 2-5 (i.e. C3-C6 alkylcarboxyl groups).

[0007] R2 is selected from one of vinyl (—CH=CH2), ethynyl (—C≡CH), and azide (—N3), more preferably from vinyl; R3 is selected from one of hydrogen, fluorine, chlorine, bromine, and iodine, and more preferably from one of hydrogen, iodine, and bromine; X is selected from one of oxygen, sulfur, selenium, and tellurium; more preferably, it is selected from one of oxygen and sulfur. Y is selected from either oxygen (O) or imino (NH).

[0008] In a preferred embodiment, R1 is -(CH2)5COOH (hexanoyl carboxyl), R2 is an azide group (-N3), R3 is iodine (I) at the 5-position of indole, and X and Y are both oxygen (O). They respectively constitute the oxanthracene fluorescent core and form a rigid planar structure to enhance the fluorescence quantum yield. This combination has been successfully used in Example 1 to prepare the near-infrared diagnostic probe OCTz, which has microRNA-21 responsiveness, fluorescence "on" effect, and reactive oxygen species (ROS) generation capability.

[0009] A second aspect of the present invention is to provide a two-component probe system for specifically recognizing target microRNA, based on the above-mentioned oxanthracene-hemicyanine near-infrared fluorescent dye.

[0010] The two-component probe system comprises a first probe component and a second probe component. The first probe component is formed by covalently linking an oxanthracene-hemicyanine (as shown in Formula I) to a first targeting ligand via its R1 carboxyl group. The first targeting ligand is a short-chain RNA capable of specifically hybridizing to the first complementary region of the target microRNA molecule. The second probe component comprises a second reactive group capable of undergoing a bioorthogonal reaction with the R2 group of the first probe component. The second reactive group is covalently linked to the second targeting ligand via an amide or ester bond. The second targeting ligand is a short-chain RNA capable of specifically hybridizing to the second complementary region of the target microRNA molecule. The first and second complementary regions are adjacent in sequence to the target microRNA molecule. The short-chain RNA is modified with 2'-O-methylribonucleotide.

[0011] When the target microRNA molecule is present, it simultaneously forms a ternary hybridization complex with the first and second target ligands through base complementary pairing, which brings the first probe component and the second probe component closer together, thereby triggering a bioorthogonal reaction between the R2 position group and the second reactive group, and thus activating the near-infrared fluorescence emission and photosensitivity of the oxanthracene-hemicyanine fluorescent dye.

[0012] In a preferred embodiment, the target microRNA is microRNA-21.

[0013] In a further preferred embodiment, the first targeting ligand is a short-chain RNA (5'-UGAUAAGCUA-3') with the nucleotide sequence shown in SEQ ID NO: 2, which is capable of complementary hybridization with the 3' end region of the mature human microRNA-21 (SEQ ID NO: 1); the second targeting ligand is a short-chain RNA (5'-UCAACAUCAGU-3') with the nucleotide sequence shown in SEQ ID NO: 3, which is capable of complementary hybridization with the 5' end region of the mature human microRNA-21 (SEQ ID NO: 1). The first and second targeting ligands are covalently linked by reacting their terminal modified amino groups with the carboxyl groups of the dye or the activated ester groups of the second reactive group to form amide bonds. The bioorthogonal reaction is a reverse electron-demanding Diels-Alder reaction, and the second reactive group is a tetrazine or a tetrazine derivative. This system achieves high specificity and high signal-to-noise ratio detection and treatment through the proximity-driven effect mediated by microRNA-21.

[0014] In the most preferred embodiment, the R2-position group is a vinyl group, and the second reactive group is 2,5-dioxopyrrolidone-1-yl5-oxo-5-((6-(6-(pyridin-2-yl)-1,2,4,5-tetraazine-3-yl)pyridin-3-yl)amino)valerate, which are paired via an inverse electron-demanding Diels-Alder reaction; Furthermore, the R2-position group and the second reactive group can also be selected from other bioorthogonal reaction pairs. For example, the R2-position group can be an azide group, and the corresponding second reactive group is dibenzocyclooctyn-propionic acid or its salt, which are paired via an azide-yne ​​cycloaddition reaction.

[0015] A third aspect of this application is to protect a method for synthesizing the above-mentioned oxanthracene-hemicyanine fluorescent dye, comprising the following steps: (1) At 0-30℃, POCl3 and DMF are slowly added dropwise to an organic solvent as reaction raw materials, wherein the volume ratio of POCl3 to DMF is 1:1-6, and the mixture is stirred at room temperature for 0.5-3 h. Cyclohexanone is then added, wherein the volume ratio of POCl3 to cyclohexanone is 1:0.3-1.5. After reacting for 8-15 h, the mixture is neutralized, and the solvent is evaporated to obtain intermediate product S-1. (2) At 10-50 °C, the phenol / thiophenol compounds shown in S-1 and S-2 are dissolved in a polar solvent and reacted under inorganic base catalysis for 10-30 h. After extraction, concentration and purification, intermediate product S-3 containing R2 and X substitution is obtained. The compound of S-2 is preferably 2-hydroxy-4-methoxybenzaldehyde. (3) In an organic solvent, at 60-120°C, the indole derivative shown in formula YI is reacted with an R1-substituted haloalkanes (N-alkylating agent) for 12-24 h, and recrystallized to obtain the quaternary ammonium salt shown in Y-2 containing an N-R1-substituted side chain; wherein the molar ratio of compound Y-1 to the R1-substituted haloalkanes is 1:1-10; more preferably 1:5-8, and the R1-substituted haloalkanes are preferably hexabromohexanoic acid; (4) In an organic solvent, at 60-120℃, the quaternary ammonium salt shown in Y-2 and compound S-3 are dissolved in the organic solvent and undergo a condensation reaction under the catalysis of an organic base. After recrystallization and purification, the oxane-hemicyanine near-infrared fluorescent dye shown in general formula I is obtained.

[0016]

[0017] In the above-mentioned technical solution, preferably, in step (1), the organic solvent is selected from one of dichloromethane, chloroform, acetone, and tetrahydrofuran; For the technical solution described above, preferably, in step (2) above, the polar solvent is selected from DMF, DMSO, methanol, and ethanol; For the technical solution described above, preferably, in step (2) above, the inorganic base is selected from any one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium acetate, and sodium ethoxide; For the above-described technical solution, preferably, in step (3) above, the organic solvent is selected from any one or a mixture of several of benzene, toluene, o-dichlorobenzene, and DMF; the recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone, and propanol. For the above-described technical solution, preferably, in step (4) above, the solvent used for recrystallization is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, water, ethyl acetate, diethyl ether, acetone, and propanol. For the technical solution described above, preferably, in step (4) above, the organic solvent is selected from any one or a mixture of several of ethanol, acetic acid, acetic anhydride, and DMF.

[0018] For the technical solution described above, preferably, in step (4) above, the organic base is selected from any one of triethylamine, pyridine and DIPEA.

[0019] The fourth aspect of this application is to protect the use of a class of oxanthracene-hemisocyanine near-infrared fluorescent dyes, their photosensitizers, their activation products, or the said two-component probe system in the preparation of any of the following products: a) Reagents used for cell imaging; b) Reagents used for microRNA labeling; c) Fluorescent probes; d) Reagents used to detect microRNA; e) Photosensitizers for photodynamic therapy of tumors; f) Reagents for detecting intracellular reactive oxygen species; g) Therapeutic reagents for the specific killing of cancer cells; h) Detection kit for specific recognition of microRNA-21.

[0020] In a further preferred embodiment, the active ingredient in the photosensitizer for tumor photodynamic therapy or the reagent for photodynamic therapy using intracellular reactive oxygen species is the R2=H product generated by the removal of the R2-position group from the microRNA-responsive oxanthracene-hemicyanine fluorescent dye as described above after a bioorthogonal reaction.

[0021] More preferably, in the application described above, the fluorescent probe or detection kit comprises a product in which the carboxyl group of a microRNA-responsive oxanthracene-hemicyanine fluorescent dye, as described above, is activated by an N-hydroxysuccinimide or similar active imide group.

[0022] In a specific embodiment, the activated product generated by the oxanthracene-hemicyanine near-infrared fluorescent dye in response to target microRNA, as described in this invention, is the direct executor of photodynamic therapy. Its characteristic feature is that the R2 group in general formula I is removed after a bioorthogonal reaction and replaced by a hydrogen atom (H), as shown in general formula II below:

[0023] This fluorescent dye, used as a near-infrared fluorescent probe in the field of biological staining, is generated after recognizing specific microRNAs. It uses the carboxyl group of the dye molecule as the active group, and generates an amide bond through a condensation reaction with the RNA chain modified with amino groups at the terminal position, thereby achieving the purpose of connecting short RNA chains.

[0024] For the technical solutions described above, preferably, the microRNA labeling reagent or the two-component probe system is constructed by covalently linking the dye described in this invention with short-chain RNA or modified ribonucleotides; Specifically, the dye described in this invention is combined with a tetrazine molecule through the aforementioned two-component design to construct a two-component probe system for recognizing sequence-specific microRNAs (such as microRNA-21).

[0025] For the technical solution described above, preferably, the application is for non-diagnostic and non-therapeutic purposes.

[0026] More preferably, the non-diagnostic and non-therapeutic applications include the preparation of the product for scientific research, in vitro sample testing, or drug screening.

[0027] For the technical solution described above, preferably, the product prepared from the dye has a fluorescence emission wavelength of 700-760 nm when applied.

[0028] Compared with the prior art, the beneficial effects of this application are as follows: 1. Compounds 1 and 2 prepared in the embodiments of this application have been verified by spectral testing to have an absorption wavelength of up to 700 nm and a maximum fluorescence emission wavelength of 720 nm, which are located in the near-infrared region and are well-suited for long-wavelength fluorescence detection scenarios. They can effectively avoid interference from biological background fluorescence, have strong tissue penetration, and cause minimal light damage to living organisms.

[0029] 2. The dye of the present invention introduces an active carboxyl group at a reasonable position. Without affecting the spectral properties of the compound, the carboxyl group can be successfully used as an active site and can be linked to short RNA sequences, thereby realizing sequence-specific microRNA detection.

[0030] 3. The microRNA-responsive xanthane-hemicyanine near-infrared fluorescent dye prepared in this invention can exhibit enhanced near-infrared fluorescence response to the overexpressed microRNA-21 sequence in tumor tissue, and can recover fluorescence through a bioorthogonal reaction, unaffected by complex environments. Under near-infrared light irradiation, it can exhibit significant phototoxicity to tumor cells, making it an excellent diagnostic and therapeutic probe.

[0031] 4. The microRNA-responsive xanthate-hemicyanine near-infrared fluorescent dye prepared in this invention exhibits good biosafety. MTT assays showed that, even under light-free conditions, with an OCTz probe concentration as high as 7.5 μM, the survival rate of HepG-2 cells remained above 90%. Figure 9 This indicates that it has low dark toxicity and is suitable for in vivo application.

[0032] 5. The method of the present invention has the advantages of simple operation, low cost, speed and sensitivity, and is easy to promote and apply.

[0033] 6. The probe of this invention can effectively activate fluorescence and generate reactive oxygen species in cells, enabling selective imaging and killing of cancer cells, and demonstrating good potential for integrated diagnosis and treatment.

[0034] More importantly, this probe system achieves high specificity recognition of microRNA-21 through a bioorthogonal reaction mediated by two-component RNA hybridization, effectively avoiding the problem of single-stranded probes being easily degraded by nucleases or non-specifically bound, and significantly improving the detection signal-to-noise ratio and treatment accuracy. Attached Figure Description

[0035] Figure 1 This is the high-resolution mass spectrum of compound 1; Figure 2 The mass spectrum of compound 1 after activation and attachment of short RNA chains (RNA-OCy); Figure 3 This is the mass spectrum of RNA-Tz-Py; Figure 4 Figure 4a shows the absorption spectra of the compounds in methanol, where: the absorption spectra of compound 1 and its activated dye D-1 are shown. Figure 4 b is the absorption spectrum of compound 2 and its corresponding dye D-2; Figure 5Figure 5a shows the fluorescence spectra of the compounds in methanol, where: Figure 5a shows the fluorescence spectra of compound 1 and its activated dye D-1 (left-right comparison); Figure 5b shows the fluorescence spectra of compound 2 and its corresponding dye D-2 (left-right comparison). Figure 6 This is the degradation absorption spectrum of dye D-1 activated by compound 1 under light irradiation; Figure 7 This is the degradation absorption spectrum of compound 1 under light irradiation for DPBF; Figure 8 This is the UV-Vis absorption response diagram of microRNA-21 to the oxanthracene-hemicyanine near-infrared fluorescent probe OCTz synthesized in Example 1. Figure 9 These are images of the near-infrared fluorescent probe OCTz prepared by compound 1 at different times of cell uptake; Figure 10 This is an MTT assay diagram of the near-infrared fluorescent probe OCTz prepared by compound 1; Figure 11 This is a diagram showing the production of reactive oxygen species in cells by the probe OCTz; Figure 12 This is a cell liveness and death imaging image of the probe OCTz. The three columns on the left are tumor cells (HePG2 cells), and the three columns on the right are normal cells (HOEC). Detailed Implementation

[0036] The present invention will now be described in further detail.

[0037] Unless otherwise stated, the terms used herein have the following meanings.

[0038] The term "halogen" as used in this article includes fluorine, chlorine, bromine, and iodine.

[0039] The term "alkyl" as used in this invention includes straight-chain alkyl and branched-chain alkyl.

[0040] Z is used in this article - This refers to negative ions, which can be any suitable negative ion, including inorganic and organic negative ions. Examples include, but are not limited to, halide ions and ClO4. - F6 - F4 - H3COO - CF3COO - or OTs - The anion Z - The choice of [aspect name] does not affect the core photosensitivity of the compound of this invention, but mainly serves to adjust the solubility or crystallinity of the compound.

[0041] Those skilled in the art should understand that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Various equivalent substitutions, modifications, or combinations may be made to the present invention without departing from its spirit and scope, and all such substitutions, modifications, or combinations should be included within the scope of protection of the present invention.

[0042] In one embodiment, the compounds of the present invention may be used as derivatives of the compound of Formula I. These derivatives include, but are not limited to, conjugates formed by covalently linking with a target molecule, a water-soluble group, a bioorthogonal reactive group, or a nucleic acid chain, and such conjugation does not affect its ability to generate reactive oxygen species under light irradiation or its response to microRNA.

[0043] The instruments and equipment used in the embodiments are as follows: In the column chromatography process of this invention, 200-300 mesh and 100-200 mesh silica gels purchased from Qingdao Meigao Group Co., Ltd. were used. Silica gel for column chromatography and 20-40 mesh analytical grade quartz sand purchased from Tianjin Tianda Chemical Reagent Factory were used. The reversed-phase purification chromatograph was a CHEETAH rapid purification preparative chromatograph manufactured by Bona Electronics.

[0044] Dye absorption and emission spectra were obtained using an Agilent Cary 60 UV-Vis spectrophotometer and a CaryEclipse fluorescence spectrophotometer. All spectral measurements were performed at room temperature in dimethyl sulfoxide (DMSO) or phosphate-buffered saline (PBS, pH 7.4) at a concentration of approximately 10 μM.

[0045] The nucleic acid sequence information involved in this invention is as follows: SEQ ID NO: 1: Human microRNA-21 mature sequence (5'-UAGCUUAUCAGACUGAUGUUGA-3'); SEQ ID NO: 2: First complementary short RNA sequence (Seq A: 5' NH2-*U*G*A*U*A*A*G*C*U*A-3', * indicates 2'-O-methyl modification); SEQ ID NO: 3: Second complementary short RNA sequence (Seq B: 5'-*U*C*A*A*C*A*U*C*A*G*U-NH) 2- 3', * represents 2'-O-methyl modification).

[0046] It is understood that, within the two-component probe system concept described below, the bioorthogonal reaction pair is not limited to the most preferred specific molecules described below. The selection of the R2-position group and the second reactive group is based on mature and efficient bioorthogonal reaction principles. For example: When the R2-position group is selected from vinyl or ethynyl, the second reactive group can be selected from tetrazine compounds that can undergo an electron-demanding Diels-Alder reaction, including but not limited to phenyltetrazine, pyridyltetrazine and other aryltetrazine derivatives and their activated forms.

[0047] When the R2-position group is an azide group, the second reactive group may also be selected from alkyne-containing compounds that can undergo an azide-alkyne cycloaddition reaction with it, such as terminal alkynes.

[0048] The specific tetrazine molecule used in the embodiments of this invention (2,5-dioxopyrrolidone-1-yl5-oxo-5-((6-(6-(pyridin-2-yl)-1,2,4,5-tetraazine-3-yl)pyridin-3-yl)amino)valeric acid) is a typical aryl tetrazine derivative that can undergo a rapid, reverse-electron-demand Diels-Alder reaction with vinyl groups. All of the aforementioned reactive groups are known in the art and can be obtained commercially or synthesized using known methods.

[0049] Example 1: Preparation of Compound 1 Structural formula of compound 1

[0050] Example 1.1

[0051] Prepare a 50 mL round-bottom flask, cool it to 0°C, add 7.9 mL of DMF (64 mmol), and dropwise add 7.7 mL of phosphorus oxychloride (82 mmol). Stir at room temperature for 15 min. Then, while maintaining the temperature at 0°C, add 5 g (50.9 mmol) of cyclohexanone dropwise and stir at room temperature for 2 hours. Quench the reaction mixture with ice (200 g) and slowly add solid sodium bicarbonate until pH = 7. Separate the organic layer and extract the aqueous layer with ethyl acetate (200 mL). Dry the collected organic matter on anhydrous magnesium sulfate, filter, and concentrate under vacuum to give a yellow oil (6.25 g, 84%) of compound 1.1 as the crude product.

[0052] Example 1.2

[0053] The compound from Example 1.1 (1 g, 6.94 mmol), 2-hydroxy-4-methoxybenzaldehyde (0.88 g, 5.78 mmol), and CsCO3 (5.65 g, 17.35 mmol) were dissolved in DMF (40 mL). The solution was stirred at room temperature for 16 h. The reaction mixture was filtered, and the filtrate was filtered again after adding 200 mL of distilled water to give compound 1.2 as a yellow solid (1.09 g, 78%).

[0054] Example 1.3

[0055] Intermediate compound 1.2 (1 g, 2.19 mmol) was dissolved in anhydrous dichloromethane and stirred at 0 °C under a nitrogen atmosphere. BBr3 (2.2 mL, 22.6 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. The reaction was quenched with water and extracted with dichloromethane. The organic phase was washed three times with water, dried over anhydrous magnesium sulfate, and filtered to give compound 1.3.

[0056] Example 1.4

[0057] 1,5-cyclooctadiene iridium chloride dimer (76 mg, 0.1 mmol) and sodium carbonate (640 mg, 6 mmol) were dissolved in toluene, and compound 1.3 (110 mg, 0.5 mmol) and vinyl acetate (2.15 g, 25 mmol) were added. The mixture was then stirred at 100 °C for 6 h under a nitrogen atmosphere. After the reaction was completed, a white solid compound 1.4 (70 mg, 0.28 mmol) was given in 55% yield.

[0058] Example 1.5

[0059] 5-Iodo-2,3,3-trimethyl-3H-indole (1 g, 3.5 mmol) and 6-bromohexanoic acid (5 g, 25 mmol) were added to 20 mL of acetonitrile and refluxed under nitrogen for 24 h. The reaction was then stopped. 50 mL of ethyl acetate was added, and the mixture was filtered to give 1.5% of the target compound (800 mg, 2 mmol, Y = 57.1%).

[0060] Manufacturing compound 1 Compounds 1.4 (70 mg, 0.28 mmol) and 1.5 (200 mg, 0.5 mmol) were dissolved in 10 mL of acetic anhydride at 100 °C. 0.2 mL of triethylamine was added to catalyze the reaction, and the mixture was stirred for 2 h before stopping. After the reaction solution cooled to room temperature, it was added dropwise to 150 mL of ethyl acetate for recrystallization. The crude product was purified by HPLC to give compound 1 (106 mg, 0.14 mmol, Y = 50%). Its high-resolution mass spectra are shown below. Figure 1 As shown, the prepared compound 1 has the expected molecular weight and structural characteristics, and the target compound was successfully synthesized.

[0061] Manufacturing activated oxane-hemicyanine near-infrared fluorescent dye D-1

[0062] Compound 1.3 (60 mg, 0.26 mmol) and compound 1.5 (200 mg, 0.5 mmol) were dissolved in 10 mL of acetic anhydride at 100 °C. 0.2 mL of triethylamine was added to catalyze the reaction. After stirring for 2 h, the reaction was stopped. After the reaction solution cooled to room temperature, it was added dropwise to 150 mL of ethyl acetate for recrystallization. The crude product was purified by HPLC to give compound D-1 (96 mg, 0.16 mmol, Y = 61.5%).

[0063] Manufacturing activated compound 1

[0064] At room temperature, N,N,N',N'-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (0.025 g, 0.074 mmol) was added to 3 mL of a DMF solution of compound 1 (47 mg, 0.062 mmol), followed by the addition of triethylamine (0.18 mL). The reaction was allowed to proceed for 2 h, and then the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The precipitate was purified by HPLC to obtain activated compound 1 (0.045 g, 0.052 mmol, Y = 83.9%).

[0065] Comparative Example 1: Preparation of Compound 2 The structural formula of compound 2:

[0066] Comparative Example 1.1

[0067] Compounds 1.5 (500 mg, 1.25 mmol) and 2 chlorine 3 (hydroxymethyl)cyclohexyl 1 Enal (110 mg, 0.64 mmol, 0.5 eq) and anhydrous sodium acetate (130 mg, 1.59 mmol) were dissolved in anhydrous ethanol (20 mL). The reaction was carried out overnight at 80 °C under a nitrogen atmosphere. After the reaction was complete, the reaction solution was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The residue was subjected to silica gel column chromatography (DCM / MeOH = 100 / 1). 20 / 1) gave a green solid as compound 2.1 (345 mg, 0.36 mmol, yield 57.6%).

[0068] Comparative Example 1.2

[0069] 3 Nitrophenol (123 mg, 0.88 mmol, 3.5 eq) and potassium carbonate (120 mg, 0.88 mmol, 3.5 eq) were dissolved in... In anhydrous acetonitrile (20 mL), the mixture was stirred at room temperature for 30 min. Compound 2.1 (233 mg, 0.25 mmol, 1.0 eq) was dissolved in acetonitrile (5 mL). The solvent was added dropwise to the above reaction solution, and the mixture was stirred at room temperature for 4 hours. After removing the solvent under reduced pressure, the residue was dissolved in a distillate solution. In chloromethane, potassium carbonate is removed by washing with water. After removing the solvent under reduced pressure, the residue is then dissolved in anhydrous methanol. The next step of the reaction is then carried out. A solution containing dissolved stannous chloride dihydrate (395 mg, 1.75 mmol, 7.0 eq) and hydrochloric acid (3 mL) is added to the solution. Add 5 mL of alcohol solution dropwise to the above reaction solution and stir overnight at 70°C. After the reaction solution cools to room temperature, dissolve it in saturated sodium carbonate solution. The reaction mixture was neutralized in liquid. The organic phase was collected with dichloromethane, washed with water, and the solvent was removed under reduced pressure. The residue was subjected to silica gel column chromatography (DCM / ). MeOH = 50 / 1 (10 / 1) The blue solid compound 2.2 (73 mg, 0.12 mmol, yield 48.0%) was obtained. This compound is also the photosensitizer structure of compound 2 in response to the substrate.

[0070] Manufacturing compound 2

[0071] (4E)-trans-cycloocten-ol (25 mg, 0.2 mmol) and pyridine (100 μL) were dissolved in dichloromethane (20 mL). Under a nitrogen atmosphere at 0 °C, a dichloromethane solution containing p-nitrobenzyl chloride (70 mg, 0.35 mmol) was added dropwise to the reaction solution while stirring. The reaction was continued at 0 °C with stirring for 4 h, then heated to room temperature and stirred overnight. After the reaction was complete, the solvent was removed under reduced pressure to obtain a yellow viscous liquid, which served as the crude intermediate (approximately 34 mg). This crude product could be rapidly added to the next reaction without purification. The intermediate (34 mg, 0.12 mmol) and N,N Diisopropylethylamine (DIPEA, 50 μL) was dissolved in a mixed solution of dichloromethane and DMF and stirred for 30 min at 0 °C under a nitrogen atmosphere. Then, a dichloromethane solution containing compound 2 (73 mg, 0.12 mmol) was added dropwise to the reaction mixture, and the temperature was raised to 35 °C, and the reaction was allowed to proceed overnight. The reaction mixture was treated with water and extracted with dichloromethane. The organic phase was washed three times with water, the solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography (DCM / MeOH = 15 / 1). 10 / 1) yielded a blue solid compound, compound 2 (15 mg, 0.02 mmol, yield 16.6%).

[0072] Manufacturing activated compound 2

[0073] At room temperature, N,N,N',N'-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (0.025 g, 0.074 mmol) was added to 3 mL of a DMF solution of compound 2 (47 mg, 0.062 mmol), followed by the addition of triethylamine (0.18 mL). The reaction was allowed to proceed for 2 h, and then the reaction was stopped. The reaction solution was poured into ethyl acetate, centrifuged at 9000 rpm, and the precipitate was collected. The precipitate was purified by HPLC to obtain activated compound 2 (0.04 g, 0.046 mmol, Y = 74.2%).

[0074] Example 1 Solution experiments were conducted on compounds 1 and 2 to perform biological orthogonal reactions. Compound 1 and compound 2 were each prepared into 100 nM working solutions. 100 μL of each working solution was placed in a tetrazine solution of equal concentration and incubated at 37 °C. The absorption and fluorescence intensity of the two systems were then measured. The experimental results are as follows: Figure 4 and 5As shown, both compounds 1 and 2 absorb in the near-infrared region. Furthermore, compound 2 exhibits a sustained increase in fluorescence intensity over time in the absence of a target, indicating a rapid background reaction rate; in contrast, compound 1 maintains a consistently low fluorescence intensity with almost no significant recovery, demonstrating an extremely weak background reaction. Therefore, compound 1 demonstrates superior stability under substrate-free conditions, avoiding damage to normal tissues and making it more suitable for subsequent photodynamic therapy applications. Thus, compound 1 is the preferred probe.

[0075] Example 2 Test of the ability of compound 1 and activated dye D-1 to generate reactive oxygen species under light. In the experiment, DPBF was added to a cuvette containing 3 mL of DCM, and the absorbance was adjusted to approximately 1.0. Then, compound 1 and the activated dye D-1 were added separately to adjust the absorbance of the photosensitizer at 700 nm to between 0.2 and 0.4. Subsequently, at 700 nm (3 mW / cm²), the absorbance was measured... 2 Under irradiation, the absorbance at 415 nm was observed every 120 s using a UV-Vis spectrophotometer.

[0076] The results are as follows Figure 6 and 7 As shown, at the same concentration, compound 1 significantly reduced the photodegradation rate of DPBF compared to D-1. This result confirms that the singlet oxygen production capacity of compound 1 is effectively suppressed compared to D-1. Therefore, the negligible fluorescence emission and suppression of D-1 are significant advantages of compound 1. 1 The ability to generate O2 keeps the photosensitizer in an "OFF" state before activation.

[0077] Therapeutic probe compound 1's recognition response to microRNA-21 Two short amino-modified RNA sequences complementary to the target microRNA were designed. The 5' amino ends of Seq A and Seq B were designed to be complementary to the target microRNA-21. The first sequence (Seq A) was 5'-U GAUAAGCUA-3', and the second sequence (Seq B) was 5'-UCAACA UC AG U-3'. U, A, G, and C are ribonucleotides, and to ensure probe stability, the entire chain was modified with a 2'-O-methyl group. Compound 1 and the commercial tetrazine molecule 2,5-dioxopyrrolidone-1-yl5-oxo-5-((6-(6-(pyridin-2-yl)-1,2,4,5-tetraazine-3-yl)pyridin-3-yl)amino)valerate were respectively linked to the ends of the two sequences to form RNA-OCy and RNA-Tz-Py. These were purified and aliquoted by HPLC (High-performance liquid chromatography) and designated as probe OCTz. Its high-resolution mass spectrometry, such as Figure 2 and Figure 3 As shown, the prepared probe has the expected molecular weight and structural characteristics, and the target compound was successfully synthesized.

[0078] The two components of the OCTz probe, RNA-OCy and RNA-Tz-Py, were both kept at a concentration of 1 μM and mixed with the microRNA-21 template in a buffer solution (100 μL). After reacting at 37℃ for 2 h, the fluorescence spectrum and fluorescence intensity at the maximum emission wavelength were measured using a multi-mode microplate reader and an ultra-micro fluorescence spectrometer, respectively. Each experiment was repeated three times, and significance analysis was performed. The response results to microRNA-21 are as follows: Figure 8 As shown, the probe OCTz has a good response to the target, and the background fluorescence is very low when only RNA-OCy is present, which ensures the protection of normal tissues and the effective killing of cancer cells during photodynamic therapy.

[0079] Example 4: Therapeutic probes for tumor cell uptake. After counting the prepared HepG2 cell suspension using a hemocytometer, take 1×10⁻⁶ cells. 4Cells were seeded into confocal cell culture dishes, and 2 mL of fresh culture medium was added. The dishes were then incubated at 37°C for 24 h. After washing three times with PBS, 2 mL of Opti-MEM medium containing OCTz (0.2 μM) was added to the confocal cell culture dishes, and timing was started. The confocal cell culture dishes were then placed on a 60x oil immersion laser confocal scanning microscope, and fluorescence images of the cells were taken every 15-30 minutes. The uptake was detected based on the relative fluorescence intensity of the images. The excitation wavelength for fluorescence imaging was 640 nm, and the emission and reception bands were 670 nm to 750 nm. The results are as follows: Figure 9 As shown, maximum uptake can be achieved within 2 hours, proving that the probe is taken up by cells and activates fluorescence.

[0080] Example 5: Reactive Oxygen Species Generation Capacity Test With a density of approximately 1×10 4 HepG-2 cells / mL were added to glass-bottomed culture dishes and cultured at 37°C for 24 h. After incubation with 2 μM OCTz for 2 h, 1 μL of DCFH-DA (10 mM) was added and incubated for 20 min. Cells not incubated with OCTz or only exposed to light served as the control group. All cells in the culture dish were incubated at 700 nm (100 mW / cm²). 2 The cells were exposed to light for 3 minutes. Finally, the generation of reactive oxygen species in each group was observed using a laser confocal scanning microscope. The results are as follows: Figure 11 As shown, after responding to microRNA-21, the photosensitizer molecule changed from an "OFF" state with weak fluorescence and low ROS to an "ON" state with strong fluorescence and high reactive oxygen species, verifying the feasibility of OCTz as an activatable photosensitizer.

[0081] Example 6: Therapeutic probes used for photodynamic killing of tumor cells 3-(4,5)-Dimethylthiophene azo(-2-yl)-3,5-diphenyltetrazolium (MTT) was used to detect cell viability. HepG-2 cells were seeded in 96-well plates at a density of 1 × 10⁻⁶ cells per well. 5 Cells / mL were cultured in 100 μL of DMEM medium containing 10% fetal bovine serum. After 24 h of cell growth, 100 μL of different concentrations of OCTz or TCOTz (0–7.5 μM) were added to the DMEM medium. The non-illuminated group was cultured for 12 h, while the illuminated group was incubated with OCTz or TCOTz for 2 h before being exposed to 700 nm light (100 mW / cm²). 2After irradiation for 5 min, and subsequent incubation for 12 h, the culture medium was removed. 100 μL of medium containing MTT (0.5 mg / mL) was added to each well, and the cells were incubated at 37°C for 4 h in a 5% CO2 humidified incubator. The MTT-containing medium was then gently poured off, and the formazan crystals generated in each well were dissolved in 200 μL of DMSO. The absorbance of the solution was measured using a microplate reader, and cell viability was calculated. The experimental results are as follows: Figure 10 As shown, the probe has a good killing effect on HepG2 cells and has very little dark toxicity.

[0082] Example 7: Comparison of the killing effect of therapeutic probes on tumor cells and normal cells. Tumor cells (HePG2) and normal cells (HOEC) were seeded separately in cell culture dishes (cell density approximately 1×10⁻⁶). 4 Cells / mL) were cultured overnight, incubated at 37°C with 2 μM OCTz for 2 h, and then exposed to 700 nm light (100 mW / cm²). 2 Cells were irradiated with 2 μM OCTz solution for 5 min and then cultured for another 12 h. Simultaneously, untreated blank cells and cells incubated with 2 μM OCTz without illumination served as control groups. After these different treatments, the fluorescent probes Calcein-AM and Propidium iodide (PI) were added to culture dishes and incubated for 30 min. Cells were then imaged using a 10x lens laser confocal scanning microscope. Results are as follows: Figure 12 As shown, the control group cells treated only with light emitted bright green fluorescence, demonstrating the safety of the light conditions used. The control group cells treated only with photosensitizer (dark group) also emitted bright green fluorescence, indicating that OCTz has biosafety and biocompatibility. PDT (700 nm, 100 mW / cm²) 2 After treatment (5 min), normal cells still showed bright green fluorescence (columns 4-6 in the light-illuminated group), while tumor cells exhibited red PI fluorescence (columns 1-3 in the light-illuminated group), indicating that OCTz does indeed have the ability to specifically kill cancer cells. These results further validate that microRNA-21 is the key to OCTz's selective killing of tumor cells, and that this activatable photosensitizer can distinguish between tumor cells and normal cells, thereby minimizing the side effects of PDT.

[0083] Finally, it should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Any substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this invention, based on the technical solutions and inventive concepts of the present invention, are within the protection scope of this invention.

Claims

1. A microRNA-responsive xanthene-hemicyanine fluorescent dye, characterized in that, The structure is shown in general formula I: wherein R1 is selected from any one of alkylcarboxyl with 3-10 carbons or benzoic acid group; R2 is selected from any one of vinyl group, ethynyl group, azido group; R3 is selected from any one of hydrogen, fluorine, chlorine, bromine, iodine; X is selected from any one of oxygen, sulfur, selenium, tellurium; Y is selected from any one of oxygen atom, imino group.

2. A two-component probe system comprising a microRNA-responsive xanthene-hemicyanine fluorescent dye according to claim 1, characterized in that, comprising a first probe component and a second probe component: the first probe component is covalently linked to a first targeting ligand through the carboxyl group at R1 of the microRNA-responsive xanthene-hemicyanine fluorescent dye according to claim 1, the first targeting ligand is a short-chain RNA capable of specifically hybridizing with a first complementary segment of the target microRNA molecule; the second probe component comprises a second reactive group capable of undergoing a bio-orthogonal reaction with the R2 group of the first probe component, the second reactive group is covalently linked to a second targeting ligand through an amide bond or an ester bond, the second targeting ligand is a short-chain RNA capable of specifically hybridizing with a second complementary segment on the sequence of the target microRNA molecule; wherein the first complementary segment and the second complementary segment are adjacent on the sequence of the target microRNA molecule; the short-chain RNA comprises a 2'-O-methyl ribonucleotide modification; when the target microRNA molecule is present, it simultaneously forms a ternary hybridization complex with the first targeting ligand and the second targeting ligand through base complementary pairing, so that the first probe component and the second probe component are spatially fixed and close, thereby triggering the bio-orthogonal reaction between the R2 group and the second reactive group, and further activating the near-infrared fluorescence emission and photosensitive activity of the microRNA-responsive xanthene-hemicyanine fluorescent dye.

3. The two-component probe system of claim 2, wherein, the target microRNA molecule is microRNA-21.

4. The two-component probe system according to claim 3, characterized in that: comprising a first probe component and a second probe component: the first probe component is covalently linked to a first targeting ligand through the carboxyl group at R1 of the microRNA-responsive xanthene-hemicyanine fluorescent dye according to claim 1, the first targeting ligand is a short-chain RNA with a nucleotide sequence of 5'-UGAUAAGCUA-3' (SEQ ID NO: 2), which is capable of complementary hybridization with the 3' end segment of human microRNA-21 mature body (SEQ ID NO: 1); the second probe component comprises a tetrazine or tetrazine derivative capable of undergoing a reverse electron demand Diels-Alder reaction with the R2 group of the first probe component, the tetrazine or tetrazine derivative is covalently linked to a second targeting ligand through an amide bond or an ester bond, the second targeting ligand is a short-chain RNA with a nucleotide sequence of 5'-UCAACAUCAGU-3' (SEQ ID NO: 3), which is capable of complementary hybridization with the 5' end segment of human microRNA-21 mature body (SEQ ID NO: 1).

5. The two-component probe system according to claim 2 or 3, characterized in that: The R2group in the first probe component and the second reactive group in the second probe component are selected from any one of the following groups: (a) the R2group is an azido group, and the second reactive group is dibenzo cyclooctyne-propionic acid or a salt thereof, which are paired by azido-yne cycloaddition reaction; (b) the R2group is selected from vinyl or ethynyl, and the second reactive group is selected from 2,5-dioxopyrrolidin-1-yl 5-oxo-5-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)pentanoic acid or a salt thereof, which are paired by inverse electron demand Diels-Alder reaction.

6. The method of synthesis of microRNA-responsive xanthene-hemicyanine fluorescent dyes according to claim 1, wherein, The method comprises the following steps: (1) At 0-30℃, POCl3 and DMF are slowly added dropwise into an organic solvent as reaction raw materials, wherein the volume ratio of POCl3 to DMF is 1:1-6, stirring at room temperature for 0.5-3 h, then adding cyclohexanone, wherein the volume ratio of POCl3 to cyclohexanone is 1:0.3-1.5; after the reaction for 8-15 h, neutralizing the reaction, evaporating the solvent to obtain an intermediate product S-1; (2) At 10-50℃, S-1 and a phenol / thiophenol compound represented by formula S-2 are dissolved in a polar solvent, and after the reaction for 10-30 h under the catalysis of an inorganic base, extraction, concentration and purification are performed to obtain an intermediate product S-3 containing R2 and X substitution; (3) At 60-120℃, an indole derivative represented by formula Y-I is reacted with an R1-substituted haloalkane in an organic solvent for 12-24 h, and recrystallization is performed to obtain a quaternary ammonium salt represented by Y-2 containing an N-R1-substituted side chain; wherein the molar ratio of compound Y-1 to the R1-substituted haloalkane is 1:1-10; (4) At 60-120℃, the quaternary ammonium salt represented by Y-2 is dissolved in an organic solvent with compound S-3 in an organic solvent, and condensation reaction occurs under the catalysis of an organic base, and recrystallization purification is performed to obtain a microRNA-responsive xanthene-hemicyanine fluorescent dye represented by general formula I.

7. The method of synthesis of claim 6, wherein: In step (1), the organic solvent is selected from one of dichloromethane, chloroform, acetone and tetrahydrofuran; In step (2), the polar solvent is selected from one of DMF, DMSO, methanol and ethanol; and the inorganic base is selected from any one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium acetate and sodium ethoxide; In step (3), the organic solvent is selected from any one of benzene, toluene, o-dichlorobenzene and DMF, or a mixed solvent of any combination of two or more thereof; and the recrystallization solvent is selected from any one of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone and propanol, or a mixed solvent of any combination of two or more thereof; In step (4), the organic solvent is selected from any one of ethanol, acetic acid, acetic anhydride and DMF, or a mixed solvent of any combination of two or more thereof; In step (4), the organic base is selected from any one of triethylamine, pyridine and DIPEA.

8. Use of the microRNA-responsive xanthene-hemicyanine fluorescent dye of claim 1, or the two-component probe system of claim 2 in the preparation of any one of the following products: a) reagents for cell imaging; b) reagents for microRNA labeling; c) fluorescent probes; d) reagents for detecting microRNA; e) photosensitizers for tumor photodynamic therapy; f) reagents for photodynamic of intracellular reactive oxygen species generation; g) theranostic reagents for cancer cell specific killing; h) detection kits for specific recognition of microRNA-21.

9. The use according to claim 8, wherein the active ingredient in the photosensitizers for tumor photodynamic therapy or the reagents for photodynamic of intracellular reactive oxygen species generation is the product of R2 = H after the biological ortho reaction of the microRNA-responsive xanthene-hemicyanine fluorescent dye of claim 1 at the R2 position group.

10. The use according to claim 8, wherein the fluorescent probes or detection kits comprise the product after the carboxyl group of the microRNA-responsive xanthene-hemicyanine fluorescent dye of claim 1 is activated by N-hydroxysuccinimide or similar active imide groups.

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