An organic fluorescent small-molecule material, a synthesis method and application thereof

Abstract

The application discloses an organic fluorescent small-molecule material, a synthesis method and application, and belongs to the technical field of fluorescent materials. The provided organic fluorescent small-molecule material is formed by connecting a phenothiazine compound and a dioxothiophene compound through a double bond, and a functional group is connected to N of the phenothiazine compound. The above can adjust a large pi conjugated system by adjusting the connecting position and quantity of the phenothiazine group and the dioxothiophene, and enhance the push-pull electron system by adding a modification group at a specific site, so that a series of fluorescent molecules with the characteristics of wavelength adjustable, large stokes shift and high fluorescence quantum yield are designed. The obtained fluorescent molecules are widely applied to fields such as fluorescent labeling, fluorescent imaging and the like, such as gene sequencing, nucleic acid detection, immune detection, immunofluorescence lateral chromatography, flow cytometry fluorescent labeling, surgical navigation and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of light-emitting materials, in particular to an organic fluorescent small-molecule material, a synthesis method and application. BACKGROUND

[0002] The light analysis method has the characteristics of simplicity, intuitiveness, and no need for complex instruments. As a new emerging analysis and detection technology, the fluorescence detection technology is widely used in environmental detection, biological imaging analysis, disease diagnosis and treatment, and is widely concerned by people.

[0003] As a microanalysis technology, the fluorescence detection technology has the advantages of high sensitivity, good selectivity, low detection limit, and small steric hindrance, and overcomes the shortcomings of sample pretreatment, high cost, inability to analyze in real time and online, and visual imaging in traditional analysis methods. It can visually detect single or multiple objects. Small-molecule fluorescent probes, as one of the most important tools in fluorescence detection technology, have the following advantages: high sensitivity in detecting single molecules or multiple molecules, enabling direct communication between humans and molecules, visual spatial resolution and sub-millisecond time resolution, and the ability to enter the body without damaging the body's active environment. After reacting with the target analyte in the body, a change in the fluorescence signal is generated. In the field of fluorescence detection and analysis, developing new small-molecule fluorescent probes with high sensitivity, high selectivity, good light stability, and good biocompatibility is a challenging task that has attracted increasing attention from researchers.

[0004] Small-molecule fluorescent probes have great application potential in environmental analysis, biological labeling, cell and tissue imaging, clinical diagnosis and treatment, etc. due to their adjustable structure, sensitive response, high selectivity, and visual analysis. However, due to the complex nature of biological systems, especially the presence of biological self-absorption and autofluorescence, which can interfere with detection, developing small-molecule fluorescent probes with good optical properties, sensitive response, and high selectivity for biological sample analysis remains a hot and difficult topic for current research.

[0005] Fluorescent probes with long-wavelength emission can effectively avoid the interference of autofluorescence in the body, improve the imaging penetration depth, and avoid damage to tissues caused by short excitation wavelengths. Fluorescent probes with large Stokes shift can reduce fluorescence self-quenching, making the detection results more accurate and reducing the occurrence of false negative signals. In addition, probes with good light stability have strong anti-photobleaching ability and are more suitable for real-time monitoring of substances in the body. Therefore, fluorescent probes with long-wavelength emission, good light stability, and large Stokes shift show great advantages and broad application prospects in the visual imaging analysis of biological bodies.

[0006] Due to unique photoelectric properties, good biocompatibility, modifiability, visualization properties, high detection sensitivity, organic fluorescent small molecule dyes are widely used in organic optoelectronics, biomedical imaging, in vitro detection, environmental monitoring, life science research and clinical surgical navigation and the like.

[0007] Fluorescence technology has become a non-invasive method for detecting analytes, observing cell morphology, structure and understanding physiological processes. Organic small molecule fluorescent probes have become an important part of fluorescence technology in the field of biological sensing due to their controllable synthesis, flexible design scheme and ease of use. At the same time, the imaging ability of organic small molecules has become an important auxiliary means in the current clinical diagnosis and treatment process, and has broad application prospects in the field of biomedicine.

[0008] Fluorescent dyes are an important branch of dye chemistry. In recent decades, fluorescent dyes have made many breakthroughs in materials, biology, medicine, solar energy utilization and other aspects, and have gradually become a key research direction in dye chemistry. Organic fluorescent dyes, as an important fluorescent dye, have a wide variety of structures. But essentially, its structure usually contains a fluorescent emitting nucleus and an auxiliary group that can change the fluorescence wavelength and enhance the fluorescence.

[0009] With the rapid development of disciplines such as analytical chemistry, biological science, life science, and medicine, organic fluorescent dyes have been widely used in biological molecule labeling, enzyme analysis, environmental analysis, cell staining, and clinical examination and diagnosis, and are indispensable fluorescent signal reporters in chemical, biological, environmental science and medical research. Therefore, developing functional organic fluorescent dyes with practical value has become a research topic of great concern. In most widely used commercial organic fluorescent dyes, the defects in their photophysical and photochemical properties cannot meet the needs of current chemical biology research and complex system analysis and detection. Therefore, it is necessary to develop new fluorescent dyes.

[0010] In view of this, the present application is proposed. SUMMARY

[0011] The purpose of the present application is to provide an organic fluorescent small molecule material, a synthesis method and an application to overcome the defects of the prior art.

[0012] The present application is implemented as follows:

[0013] The present application provides an organic fluorescent small molecule material, which is formed by connecting a phenothiazine compound and a dioxothiophene compound through a double bond, and a functional group is connected to N of the phenothiazine compound.

[0014] The application provides a preparation method of the organic fluorescent small molecule material, which comprises: bridging a phenothiazine compound and a dioxothiophene compound through a double bond to obtain the organic fluorescent small molecule material.

[0015] The application provides application of the organic fluorescent small molecule material in the fields of fluorescent labeling and fluorescent imaging.

[0016] The application has the following beneficial effects:

[0017] The application provides an organic fluorescent small molecule material, a synthesis method and application, a design principle of the provided organic fluorescent small molecule material combines a large pi conjugated system and a push-pull electron molecular system, phenothiazine is used as an electron donor and a large pi conjugated skeleton, dioxothiophene and a carbon-carbon double bond are used as a conjugated bridging part, and a special modification site is designed at a substitution site of the phenothiazine N. The large pi conjugated system is adjusted by adjusting the connection position and quantity of the phenothiazine group and the dioxothiophene, and the push-pull electron system is enhanced by adding a modification group at a specific site, so that the designed fluorescent molecule has the characteristics of wavelength adjustment, large stokes shift and high fluorescent quantum yield. Therefore, the organic fluorescent small molecule material can be widely applied in the fields of fluorescent labeling and fluorescent imaging, such as the fields of fluorescent labeling and fluorescent imaging, for example, gene sequencing, nucleic acid detection, immune detection, molecular beacon, immunofluorescence lateral flow chromatography, flow cytometry fluorescent labeling, surgical navigation and the like. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0019] Figure 1 The synthesis route map of the organic fluorescent small molecule material provided by the embodiments of the application is shown in the following figure:

[0020] Figure 2 The excitation spectrum diagram of the compound prepared in Embodiment 3 is shown in the following figure:

[0021] Figure 3 The emission spectrum diagram of the compound prepared in Embodiment 3 is shown in the following figure:

[0022] Figure 4 The maximum absorbance standard curve of the compound prepared in Embodiment 3, cy3 and cy5 under the condition of the maximum excitation wavelength of each is shown in the following figure. DETAILED DESCRIPTION

[0023] In order to make the purposes, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If specific conditions are not indicated in the embodiments, conventional conditions or the conditions recommended by manufacturers are adopted. If the manufacturers of the reagents or instruments are not indicated, the reagents or instruments are conventional products that can be purchased in the market.

[0024] The design principle of the current organic fluorescent small molecule material is generally a large pi conjugated system and a push-pull electron molecular system (Intramolecular Charge Transfer, ICT; Twisted Intramolecular Charge Transfer, TICT). The large pi conjugated system generally has a nearly planar molecular structure, including cyanines, BODIPYs, rhodamines, pyrenes, wheelenes, porphyrins, and the like. Compared with the organic dye of the nearly planar pi conjugated system, the push-pull electron molecular system adjusts the delocalization degree of the intramolecular electron by designing an electron donor and an electron acceptor and a conjugated connecting system, so as to control the energy level gap, so as to design the fluorescent molecules in the visible light and near-infrared wavelength range. In the push-pull electron molecular system, the electron donor is generally selected from amino, imino, alkoxy, nitrogen-containing, oxygen-containing, sulfur-containing heterocyclic compounds, ferrocene, and tetrafullerene, and the like, and the cyano, cyanoacetic acid, nitro, carboxyl, sulfonyl, sulfoxide, carbonyl, pyridine salt, benzothiadiazole, and the like are common electron acceptors. The conjugated bridging group is a carbon-carbon triple bond, a carbon-carbon double bond, benzene, stilbene, diphenylacetylene, azobenzene, thiophene, and the like.

[0025] The embodiments of the present application rationally design and synthesize a simple and effective fluorescent dye with a large pi bond conjugation based on the mechanism of intramolecular charge transfer. Through a simple synthesis strategy, a new type of wavelength-adjustable fluorescent dye is constructed, and these dyes are preliminarily applied in the design and development of probes. The spectral performance test shows that the dyes have good fluorescence performance, the excitation wavelength covers the visible light region, the emission wavelength can cover the visible light region, and the near-infrared region can be reached by adjusting the molecular conjugated module. At the same time, the dye has a large molar absorption coefficient, a high fluorescence quantum yield, a large Stokes shift, and a structure easy to modify.

[0026] The organic fluorescent small molecule material, the synthesis method, and the application provided by the embodiments of the present application will be described in detail below.

[0027] In a first aspect, the embodiments of the present application provide an organic fluorescent small molecule material. The organic fluorescent small molecule material is formed by connecting a phenothiazine compound and a dioxothiophene compound through a double bond, and a functional group is connected to N of the phenothiazine compound.

[0028] The embodiment of the present application provides a kind of organic fluorescent small molecule material, its design principle combines big π conjugated system and push-pull electron molecular system, use phenothiazine as electron donor and big π conjugated skeleton, dioxythiophene and carbon-carbon double bond as conjugated bridging part, and special modification site is designed at phenothiazine N substitution site.The above can be adjusted by adjusting the connection position and quantity of phenothiazine group and dioxythiophene to adjust big π conjugated system, and increase modification group at specific site to enhance push-pull electron system, design a series of small molecule fluorescent materials with big molar absorption coefficient, high fluorescence quantum yield, big Stokes shift, structure is easy to modify etc. characteristics relying on the above structure.

[0029] In optional embodiments, a functional group is further connected to the aromatic ring of the phenothiazine compound and / or the dioxythiophene compound;

[0030] Preferably, the organic fluorescent small molecule material is formed by connecting the phenothiazine compound shown in general formula I and the dioxythiophene compound shown in general formula II through double bond;

[0031]

[0032] Wherein, R 2 is any one of O, S and N; when R 2 is N, R 4 exists;

[0033] R 1 , R 3 , R 4 and R 5 are functional groups;

[0034] More preferably, the functional group is a water-soluble group, a fat-soluble group, an electron-donating group, a labeling group or a specific targeting group.

[0035] In optional embodiments, R 1 , R 3 , R 4 and R 5 are each independently selected from maleimide, carboxyl, alkyl, sulfonic acid group, polyethylene glycol group, alkoxy, phenolic hydroxyl, aniline group, amino, phenolic hydroxyl, dimethylamino, aryl, tetraphenylstyryl, acyl, aldehyde, amido, nitrile, nitro, halogen formyl, quaternary amine group, polypeptide, triphenylphosphine imine, peptide receptor antagonist, o-dicarbonyl, phenol, morpholine, morphine, borate, hemicyanine derivative, quaternary amine salt and MKT-077 derivative.

[0036] In the above organic fluorescent small molecule material, R 2 is any one of O, S and N; when R 2 is N, R 4R 1 and R 4 is a water-soluble group, a fat-soluble group, a labeling group, an electron-withdrawing group or an electron-donating group or a specific targeting group; R 3 and R 5 is a water-soluble group, a fat-soluble group, a labeling group, an electron-donating group, an electron-withdrawing group or a specific targeting group. Among them, the labeling group, the water-soluble group, the fat-soluble group, the electron-withdrawing group, the electron-donating group or the specific targeting group are known in the art, such as the water-soluble group includes but is not limited to sulfonic acid group, polyethylene glycol group, the fat-soluble group includes but is not limited to polyalkyl chain, the labeling group includes but is not limited to maleimide, amino, carboxyl, activated ester, the electron-withdrawing group includes but is not limited to alkoxy, aniline group, amino, phenolic hydroxyl, dimethylamino, aryl, tetraphenyl ethylene group, the electron-donating group includes but is not limited to acyl, aldehyde group, carboxyl, amido, sulfonic acid group, nitrile group, nitro group, halogen form group and quaternary amine group, and the specific targeting group includes but is not limited to polypeptide, triphenyl phosphonium salt imine, release peptide receptor antagonist, o-dicarbonyl, phenol, morpholine, morphine, borate, hemicyanine derivative, quaternary amine salt and MKT-077 derivative.

[0037] In an optional embodiment, the organic fluorescent small molecule material is one of the following compounds:

[0038]

[0039]

[0040] In an optional embodiment, the organic fluorescent small molecule material has a fluorescence quantum yield of 2%-70%, an excitation wavelength of 350-800 nm and an emission wavelength of 400-1000 nm.

[0041] The organic fluorescent small molecule material provided by the embodiments of the present application provides a new fluorophore skeleton. Compared with existing fluorophore skeletons such as cyanines, BODIPYs and rhodamines, the phenothiazine-π-dioxothiophene fluorophore skeleton has the characteristics of adjustable wavelength, special functional modification site and large Stokes shift.

[0042] (1) Wavelength adjustable

[0043] The organic fluorescent small molecule material provided by the embodiments of the present application mainly adjusts the large π conjugated system by adjusting the connection position and number of phenothiazine groups and dioxothiophene, and increases the modification group at a specific site to enhance the push-pull electron system and enhance the intermolecular charge delocalization, so as to realize the reduction of energy level gap and cause the red shift of wavelength.

[0044] The design principle combines the large pi conjugated system and the push-pull electron molecular system, has the wavelength adjustable property, realizes the flexible adjustable of 400-750nm waveband, which is the defect of the existing fluorescent group skeleton such as BODIPY class, rhodamine class. While the florescent molecule of the flower cyan class can adjust the fluorescent wavelength, but the conjugated bridge unit is the superimposed carbon-carbon double bond, and the stability is poor, and the stability of the phenothiazine-pi-dioxothiophene fluorescent skeleton is better than that of the carbon-carbon double bond.

[0045] (2) Special functional modification site

[0046]

[0047] Among them, R 2 is any one of O, S, N; when R 2 is N, R 4 exists;

[0048] R 1 and R 4 are water-soluble groups, lipid-soluble groups, electron-donating groups or specific targeting groups, labeling groups;

[0049] R 3 and R 5 are water-soluble groups, lipid-soluble groups, electron-donating groups, electron-withdrawing groups or specific targeting groups, labeling groups.

[0050] In an optional embodiment, R 1 and R 4 are each independently selected from maleimide, carboxyl, alkyl, sulfonic acid group, polyethylene glycol group, alkoxy, phenolic hydroxyl, aniline group, amino, dimethylamino, aryl, tetraphenylstyryl, polypeptide, triphenylphosphonium imine, peptide receptor antagonist, o-dicarbonyl, phenol, morpholine, morphine, borate, hemicyanine derivative, quaternary ammonium salt and MKT-077 derivative;

[0051] R 3 and R 5 are each independently selected from maleimide, alkoxy, aniline group, amino, phenolic hydroxyl, dimethylamino, aryl, tetraphenylstyryl, acyl, aldehyde, carboxyl, amido, sulfonic acid group, nitrile group, nitro group, halogen formyl group, quaternary ammonium group, polypeptide, triphenylphosphonium imine, peptide receptor antagonist, o-dicarbonyl, phenol, morpholine, morphine, borate, hemicyanine derivative, quaternary ammonium salt and MKT-077 derivative.

[0052] (3) Large stokes shift

[0053] The organic fluorescent small molecule material provided by the embodiment of the present application has a carbon-carbon double bond as a pi bridging unit between phenothiazine and dioxothiophene. In the phenothiazine-pi-dioxothiophene fluorescent molecular skeleton, the carbon-carbon double bond causes the molecule to twist between the electron donor (phenothiazine group) and dioxothiophene due to the twisted molecular plane structure, the energy consumption of the molecule during the excited state lifetime is increased before returning to the ground state, and the fluorescent emission spectrum is greatly red-shifted.

[0054] Therefore, the fluorescent molecule provided by the embodiment of the present application has the above fluorescent skeleton design, a large Stokes shift, a small overlap between the excitation spectrum and the emission spectrum, and a large Stokes shift of 100 nm-220 nm. The existing BODIPY, rhodamine skeleton, and cyanine fluorescent molecules have a Stokes shift of only 20-50 nm, and the excitation spectrum and the emission spectrum overlap greatly, which can cause self-fluorescence absorption and affect the imaging resolution. The large Stokes shift can reduce the self-absorption of the fluorescent molecule during excitation and emission, has a high signal-to-noise ratio, and has great advantages in imaging. The organic fluorescent molecule with a large Stokes shift also has application requirements in scenes requiring quantitative testing such as fluorescent test strips and gene sequencing.

[0055] In summary, the organic fluorescent small molecule material provided by the embodiment of the present application is designed based on the above structure, a series of organic fluorescent small molecule materials are realized, the wavelength of the commonly used visible light fluorescent band 400 nm-650 nm and the red light and near-infrared band 650-780 nm can be adjusted, the fluorescent quantum yield is 2%-70%, and the molar extinction coefficient is 20000-300000. In addition, by expanding the conjugated system and designing groups on the molecule to increase the electron push-pull ability to enhance the intramolecular electron delocalization to reduce the energy level gap, the dye can also emit light in the near-infrared region.

[0056] In a second aspect, the embodiment of the present application provides a preparation method of the above-mentioned organic fluorescent small molecule material, which comprises: bridging a phenothiazine compound and a dioxothiophene compound by a double bond to obtain an organic fluorescent small molecule material.

[0057] In an optional embodiment, the following steps are included: introducing an aldehyde group in the conjugated module by using a Vilsmeier-Haack reaction; introducing another conjugated module by using a Wittig reaction or a Horner-Wadsworth-Emmons reaction through double bond construction; and constructing multiple groups of organic small molecules by using the Vilsmeier-Haack reaction and the Wittig reaction or the Horner-Wadsworth-Emmons reaction multiple times, wherein the conjugated module is a phenothiazine compound or a dioxothiophene compound.

[0058] In an alternative embodiment, the following steps are included: under inert gas protection, the phenothiazine compound and the dioxothiophene compound are added into an organic solvent containing a base, the reaction is stirred, the reaction is quenched by adding water, extraction is performed using an organic solvent, and then column chromatography is used to separate the solid;

[0059] Preferably, the base is at least one of K2CO3, NaH and potassium tert-butoxide, and the amount of the base is 1-20 times the molar equivalent of the total amount of the raw materials;

[0060] Preferably, the reaction temperature is 20-80℃, and the reaction time is 3-24h;

[0061] Preferably, the organic solvent used in the reaction is at least one of DMF, THF, DMSO, toluene, isopropanol, dichloromethane and dichloroethane, the organic solvent used in the extraction is at least one of ethyl acetate and dichloromethane, and the organic solvent used in the column chromatography is a petroleum ether / ethyl acetate system or a petroleum ether / dichloromethane system.

[0062] In a third aspect, the embodiments of the present application provide an application of the above-mentioned organic fluorescent small-molecule material in the field of fluorescent labeling and fluorescent imaging.

[0063] In an alternative embodiment, the organic fluorescent small-molecule material is applied to gene sequencing, immune detection, nucleic acid detection, molecular beacon, immunofluorescence test strip, flow cytometry and surgical navigation.

[0064] The features and performances of the present application are further described in detail below in combination with embodiments.

[0065] The present application provides a novel organic fluorescent small-molecule material, which has the following structure:

[0066] The present application combines a large π conjugated system and a push-pull electron molecular system, uses phenothiazine, dioxothiophene and carbon-carbon double bond to construct a large π conjugated skeleton, and designs a special modification site at the N substitution site of the phenothiazine. In addition, some functional groups such as water-soluble groups, fat-soluble groups, electron-donating groups, electron-withdrawing groups, labeling groups or specific targeting groups can be designed on the aromatic ring of the phenothiazine and / or dioxothiophene as a part of energy level gap control.

[0067] Meanwhile, the embodiments of the present application also provide a preparation method of the above-mentioned novel organic fluorescent small-molecule material, which can be seen from Figure 1 which comprises the following steps:

[0068] (1) introducing an aldehyde group into the conjugated module by using a Vilsmeier-Haack reaction;

[0069] (2) using Wittig reaction or Horner-Wadsworth-Emmons reaction, introducing another conjugated module by construction of double bond;

[0070] (3) using Vilsmeier-Haack reaction and Wittig reaction or Horner-Wadsworth-Emmons reaction for multiple times, various kinds of the organic fluorescent small molecule materials can be constructed.

[0071] The conjugated modules include, but are not limited to, phenothiazine, thiophene and the like in the art. The above conjugated modules can be combined arbitrarily without violating the common sense in the art, that is, the preferred embodiments of the present application are obtained. The overall process of the embodiments of the present application is simple, and is a green, efficient and environment-friendly route which can be applied to large-scale production.

[0072] As a preferred mode provided by the embodiments of the present application, the preparation method of the novel organic fluorescent small molecule material includes the following steps:

[0073] Under inert gas protection, the phenothiazine compound and the dioxothiophene compound are added into an organic solvent containing a base, stirred and reacted, quenched by adding water, extracted by using an organic solvent, and then separated by column chromatography to obtain a solid;

[0074] Preferably, the base is at least one of K2CO3, NaH and potassium tert-butoxide, and the amount of the base is 1-20 times of the molar equivalent of the total amount of raw materials;

[0075] Preferably, the reaction temperature is 20-80℃, and the reaction time is 3-24h;

[0076] Preferably, the organic solvent used in the reaction is at least one of DMF, THF, DMSO, toluene, isopropanol, dichloromethane and dichloroethane, the organic solvent used in the extraction is at least one of ethyl acetate and dichloromethane, and the organic solvent used in the column chromatography is petroleum ether / ethyl acetate system or petroleum ether / dichloromethane system.

[0077] Embodiment 1

[0078]

[0079] Synthesis procedure: In a reaction flask, under inert gas protection, at 0°C, add EPE and 60 ml DMF, add 4-(3-formyl-10H-phenothiazin-10-yl)butyl)sodium(III) sulfite with 1:1 molar equivalent, then add 1:4 molar equivalent of base (NaH), stir at 80°C for 12 h. Climb the board to analyze the completion of the reaction. Add water to quench the reaction, extract with ethyl acetate, and purify the product by column chromatography with ethyl acetate / petroleum ether eluent system.

[0080] Another conjugated module can be introduced by the construction of a double bond using the Wittig reaction or the Horner-Wadsworth-Emmons reaction. In the above reaction, EPE is not limited to thienyl compounds, and phenothiazine compounds can also be modified into Wittig / Hormor reagents; the aldehyde compound generated by the Vilsmeier-Haack reaction is not limited to phenothiazine compounds, and thienyl compounds can also be used.

[0081] The hydrogen spectrum data thereof is as follows:

[0082] 1H NMR (400 MHz, DMSO) δ 7.31 (d, J = 9.9 Hz, 1H), 7.23-7.15 (m, 2H), 7.16-7.09 (m, 1H), 7.02 (dd, J = 15.9, 9.2 Hz, 2H), 6.99-6.89 (m, 2H), 6.72 (d, J = 16.2 Hz, 1H), 6.51 (s, 1H), 4.33-4.13 (m, 4H), 3.85 (s, 2H), 2.45 (t, J = 7.2 Hz, 2H), 1.85-1.62 (m, 4H).

[0083] Example 2

[0084]

[0085] Synthesis procedure: In a reaction flask, under inert gas protection, at 0°C, add PT-E and 10 times molar equivalent of DMF and 10 times molar equivalent of phosphorus oxychloride, add reaction solvent dichloroethane or tetrahydrofuran, stir at 85°C for 12 h. Climb the board to analyze the completion of the reaction. Add water to quench the reaction, adjust the pH to about neutral with sodium hydroxide, extract, and purify the product by column chromatography.

[0086] As above, the inert gas is nitrogen or argon; the reaction solvent is preferably DMF, dichloromethane, tetrahydrofuran, dichloroethane; the extraction organic solvent is preferably ethyl acetate, dichloromethane; the column chromatography organic solvent is preferably petroleum ether / ethyl acetate system, petroleum ether / dichloromethane system.

[0087] The hydrogen spectrum data thereof is as follows:

[0088] 1H NMR (400 MHz, CDC13) δ 9.70 (d, J = 2.5 Hz, 1H), 7.52 - 7.42 (m, 2H), 7.41 - 7.34 (m, 2H), 7.33 - 7.21 (m, 2H), 7.05 - 7.00 (m, 1H), 6.93 - 6.84 (m, 1H), 6.79 - 6.69 (m, 1H), 4.40 - 4.35 (m, 4H), 3.30 - 3.21 (m, 3H).

[0089] Example 3

[0090]

[0091]

[0092] Synthesis step: in a reaction bottle, under the condition of 0°C, inert gas protection, EPE and PT-E-Q were put into a round-bottom flask with a molar ratio of 1:1, DMF was used as the solvent, 30 ml, NaH was added, and stirred for 12 h. The reaction was analyzed by climbing plate, and the reaction was quenched by adding water, extracted, and the purified product was separated by column chromatography.

[0093] As above, the inert gas is nitrogen or argon; the reaction solvent is preferably DMF, THF, the extraction organic solvent is preferably ethyl acetate, dichloromethane; the column chromatography organic solvent is preferably petroleum ether / ethyl acetate system, petroleum ether / dichloromethane system.

[0094] The hydrogen spectrum data thereof are as follows:

[0095] 1H NMR (400 MHz, DMSO) δ 7.39 - 7.32 (m, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.19 - 7.14 (m, 1H), 7.10 - 7.04 (m, 1H), 6.97 (t, J = 7.2 Hz, 3H), 6.91 (d, J = 8.7 Hz, 1H), 6.79 (t, J = 12.6 Hz, 2H), 6.75 - 6.68 (m, 1H), 6.54 (s, 1H), 4.38 - 4.26 (m, 8H), 3.32 (s, 3H).

[0096] The excitation and emission spectrum thereof is shown in Figure 2 and Figure 3(standardized by visible light region 400-650 nm, 700-900 nm near infrared region I, 900-1700 nm near infrared region II), the compound prepared in Example 3 was prepared into a solution with a concentration of 2.5-25 μM using DMSO as a solvent, and the test instrument was used for testing. As can be seen from the figure, the excitation spectrum is in the visible light region, the maximum excitation wavelength is 447 nm, the emission spectrum starts from 500 nm, the maximum is 800 nm, and extends to the near infrared region, and the maximum emission wavelength is 607 nm.

[0097] The standard curve of the maximum absorbance of the compound prepared in Example 3, cy3 and cy5 under the condition of the respective maximum excitation wavelength is shown in Figure 4 (Using cy3 with a molar absorption coefficient of 150,000 and cy5 with a molar absorption coefficient of 250,000 as a comparison), based on the Lambert-Beer law, it is calculated that the molar absorption coefficient of the compound prepared in Example 3 is 102,000.

[0098] Fluorescence quantum yield determination method: testing agency: Shenzhen Testing Center of Zhongke Baiji, testing method: integral sphere method for determining fluorescence quantum yield, testing instrument: steady-state transient fluorescence spectrometer, FLS1000 / FSS, produced by Edinburgh, UK. Test results: the fluorescence quantum yield of the prepared compound is 46.5%.

[0099] Example 4

[0100]

[0101] Synthesis steps: in a reaction bottle, PT-E-Q is dissolved in toluene at room temperature, and then 3 times the molar equivalent of methyl(triphenylphosphorane) acetate is added. Then heat to reflux at 120°C for 12h. After analyzing the reaction completion by TLC, cool to room temperature, extract, and column chromatography to obtain the purified product.

[0102] As above, the inert gas is nitrogen or argon; the reaction solvent is preferably toluene, THF, the extraction organic solvent is preferably ethyl acetate, dichloromethane; the column chromatography organic solvent is preferably petroleum ether / ethyl acetate system, petroleum ether / dichloromethane system.

[0103] The hydrogen spectrum data thereof are as follows:

[0104] 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 15.6 Hz, 1H), 7.25-7.19 (m, 2H), 7.19-7.09 (m, 2H), 6.98 (dd, J = 20.1, 9.0 Hz, 2H), 6.85-6.72 (m, 3H), 6.14 (d, J = 15.7 Hz, 1H), 4.31 (q, J = 5.1 Hz, 4H), 3.78 (d, J = 5.9 Hz, 3H), 3.39 (s, 3H).

[0105] Example 5

[0106]

[0107] Synthesis procedure: add aldehyde group at ortho position of PT-EE thiophene sulfur using VH-reaction, then react EPE with aldehyde group using wittig reaction to get PT-EEE. Refer to Example 1-3 for detailed synthesis method.

[0108] The hydrogen spectrum data thereof is as follows:

[0109] 1 H NMR (400 MHz, DMSO) δ 7.36 (d, J = 4.9 Hz, 1H), 7.22 (t, J = 7.7 Hz, 2H), 7.17 (d, J = 6.8 Hz, 1H), 7.08 (d, J = 16.2 Hz, 1H), 6.99 - 6.93 (m, 3H), 6.91 (d, J = 8.4 Hz, 2H), 6.75 (dd, J = 8.8, 3.4 Hz, 2H), 6.70 (s, 1H), 5.32 (s, 1H), 4.39 - 4.12 (m, 12H), 3.27 (s, 3H).

[0110] Example 6

[0111]

[0112] Synthesis procedure: introduce aldehyde group in conjugated module using Vilsmeier-Haack reaction, introduce another conjugated module by double bond construction using Wittig reaction or Horner-Wadsworth-Emmons reaction; refer to Example 1-3 for detailed synthesis method.

[0113] The hydrogen spectrum data thereof is as follows:

[0114] 1 H NMR (400 MHz, CD2Cl2) δ 7.28 (s, 4H), 7.25 (s, 2H), 7.16 (d, J = 22.3 Hz, 2H), 7.01 (d, J = 16.2 Hz, 1H), 6.94 (s, 1H), 6.87 (d, J = 10.6 Hz, 2H), 6.80 (s, 3H), 6.75 (d, J = 16.1 Hz, 2H), 6.20 (s, 1H), 4.24 (d, J = 22.7 Hz, 4H), 3.38 (s, 7H).

[0115] Example 7

[0116]

[0117] Synthetic procedure: introduce an aldehyde group in the conjugated module using Vilsmeier-Haack reaction, introduce another conjugated module by double bond construction using Wittig reaction or Horner-Wadsworth-Emmons reaction; refer to Example 1-3 for specific synthetic methods.

[0118] Its hydrogen spectrum data are as follows:

[0119] 1 H NMR (400 MHz, CDC13) δ 7.23 (m, 2H), 7.15 (dd, J = 16.7, 9.1 Hz, 6H), 7.03 - 6.98 (m, 1H), 6.94 (dd, J = 14.8, 7.2 Hz, 2H), 6.79 (ddd, J = 29.9, 14.1, 4.8 Hz, 6H), 6.71 - 6.61 (m, 1H), 6.52 (t, J = 11.9 Hz, 1H), 6.33 - 6.25 (m, 1H), 4.25 (dd, J = 19.8, 14.3 Hz, 8H), 3.40 (d, J = 14.9 Hz, 6H).

[0120] Example 8

[0121]

[0122] Synthetic procedure: introduce an aldehyde group in the conjugated module using Vilsmeier-Haack reaction, introduce another conjugated module by double bond construction using Wittig reaction or Horner-Wadsworth-Emmons reaction; refer to Example 1-3 for specific synthetic methods.

[0123] Its hydrogen spectrum data are as follows:

[0124] 1 H NMR (400 MHz, CDC13) δ 7.23 (m, 2H), 7.15 (dd, J = 16.7, 9.1 Hz, 6H), 7.03 - 6.98 (m, 1H), 6.94 (dd, J = 14.8, 7.2 Hz, 2H), 6.79 (ddd, J = 29.9, 14.1, 4.8 Hz, 6H), 6.71 - 6.61 (m, 1H), 6.52 (t, J = 11.9 Hz, 1H), 6.33 - 6.25 (m, 1H), 4.25 (dd, J = 19.8, 14.3 Hz, 8H), 3.40 (d, J = 14.9 Hz, 6H).

[0125] Example 9

[0126]

[0127] Synthesis procedure: EPE and PT-BQ were put into a round bottom flask with a 1:1 molar ratio, DMF as solvent, and base (such as K2CO3, NaH, potassium tert-butoxide, etc.) was added, and stirred for 12 hours. Water was added to quench the reaction, and then extracted with organic solvent (ethyl acetate, dichloromethane, etc. commonly used solvents). Then column chromatography was used to separate to obtain yellow solid.

[0128] The hydrogen spectrum data thereof are as follows:

[0129] 1 H NMR (400 MHz, CDC13) δ 9.80 (d, J = 3.6 Hz, 1H), 7.66 (dd, J = 8.4, 1.9 Hz, 1H), 7.61 (d, J = 1.8 Hz, 1H), 7.24 (s, 1H), 7.22 - 7.18 (m, 1H), 7.02 (d, J = 16.2 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.76 (dd, J = 20.4, 12.4 Hz, 2H), 6.21 (s, 1H), 4.26 (ddd, J = 8.0, 6.3, 3.5 Hz, 4H), 3.43 (d, J = 6.5 Hz, 3H).

[0130] Example 10

[0131]

[0132] Synthesis procedure: The method refers to Example 4, Example 9.

[0133] The hydrogen spectrum data thereof are as follows:

[0134] 1 H NMR (400 MHz, CDC13) δ 9.80 (d, J = 3.6 Hz, 1H), 7.66 (dd, J = 8.4, 1.9 Hz, 1H), 7.61 (d, J = 1.8 Hz, 1H), 7.24 (s, 1H), 7.22 - 7.18 (m, 1H), 7.02 (d, J = 16.2 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.76 (dd, J = 20.4, 12.4 Hz, 2H), 6.21 (s, 1H), 4.26 (ddd, J = 8.0, 6.3, 3.5 Hz, 4H), 3.43 (d, J = 6.5 Hz, 3H).

[0135] Example 11

[0136]

[0137] Synthetic procedure: introduce an aldehyde group in the conjugated module by Vilsmeier-Haack reaction, introduce another conjugated module by construction of a double bond using Wittig reaction or Horner-Wadsworth-Emmons reaction; the specific synthetic method refers to Example 1-Example 3.

[0138] The hydrogen spectrum data thereof is as follows:

[0139] 1 H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.16 (dd, J = 13.6, 7.5 Hz, 6H), 7.01 (d, J = 8.5 Hz, 1H), 6.93 (t, J = 7.4 Hz, 2H), 6.81 (d, J = 7.9 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 4.29 (s, 8H), 3.36 (d, J = 13.0 Hz, 3H).

[0140] Example 12

[0141]

[0142] Synthetic procedure: introduce an aldehyde group in the conjugated module by Vilsmeier-Haack reaction, introduce another conjugated module by construction of a double bond using Wittig reaction or Horner-Wadsworth-Emmons reaction; the specific synthetic method refers to Example 1-Example 4.

[0143] The hydrogen spectrum data thereof is as follows:

[0144] 1 H NMR (400 MHz, DMSO) δ 7.56 (d, J = 15.6 Hz, 1H), 7.37 (d, J = 6.8 Hz, 2H), 7.22 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 7.08 (d, J = 16.1 Hz, 1H), 7.03-6.88 (m, 4H), 6.77 (dd, J = 16.0, 6.7 Hz, 2H), 6.07 (d, J = 15.6 Hz, 1H), 4.46-4.27 (m, 8H), 3.73-3.64 (m, 3H), 3.32 (s, 3H).

[0145] The performance of the synthesized small molecule fluorescent dyes was tested, and part of the test results are shown in Table 1 below:

[0146] Table 1

[0147]

[0148]

[0149] In summary, the embodiment of the present application provides an organic fluorescent small molecule material, a synthesis method and an application, a simple and effective fluorescent dye with a large pi conjugated bond is reasonably designed and synthesized based on the mechanism of intramolecular charge transfer, the organic fluorescent small molecule material combines a large pi conjugated system and a push-pull electron molecular system, a new type of wavelength adjustable fluorescent dye is constructed through a simple synthesis strategy, and the dyes are preliminarily applied in the design and development of probes. Spectral performance tests show that the dyes have good fluorescence performance, the dyes have good near-infrared fluorescence performance, including near-infrared region emission, a large molar absorption coefficient, a high fluorescence quantum yield, a large Stokes shift, and the structure is easy to modify.

[0150] The above only for the preferred embodiment of the present application, and is not used to limit the present application, for those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An organic fluorescent small-molecule compound characterized in that, The organic fluorescent small-molecule compound is one of the following compounds: 2.The organic fluorescent small-molecule compound according to claim 1, characterized in that, The organic fluorescent small-molecule compound has a fluorescence quantum yield of 2%-70%, an excitation wavelength of 350-800 nm, and an emission wavelength of 400-1000 nm.

3. A method for preparing the organic fluorescent small-molecule compound according to any one of claims 1-2, characterized in that, The method comprises the following steps: An aldehyde group is introduced into a conjugated module by a Vilsmeier-Haack reaction; Another conjugated module is introduced by a double bond construction by a Wittig reaction or a Horner-Wadsworth-Emmons reaction; and the organic fluorescent small-molecule compound is constructed by multiple Vilsmeier-Haack reactions and Wittig reactions or Horner-Wadsworth-Emmons reactions, wherein the conjugated module is a phenothiazine compound or a dioxothiophene compound.

4. The production method according to claim 3, characterized by, The method comprises the following steps: The phenothiazine compound and the dioxothiophene compound are added into an organic solvent containing a base under inert gas protection, and stirred to react, then quenched by water, extracted by an organic solvent, and separated by column chromatography to obtain a solid.

5. The preparation method according to claim 4, characterized in that, The base is at least one of K2CO3, NaH, and potassium tert-butoxide, and the amount of the base is 1-20 times of a molar equivalent of the total amount of raw materials.

6. The production method according to claim 4, characterized by, The reaction temperature is 20-80°C, and the reaction time is 3-24 h.

7. The preparation method according to claim 4, characterized in that, The organic solvent used in the reaction is at least one of DMF, THF, DMSO, toluene, isopropanol, dichloromethane, and dichloroethane, the organic solvent used in the extraction is at least one of ethyl acetate and dichloromethane, and the organic solvent used in the column chromatography separation is a petroleum ether / ethyl acetate system or a petroleum ether / dichloromethane system.

8. Use of the organic fluorescent small-molecule compound according to any one of claims 1-2 or prepared by the method according to any one of claims 3-7 in the preparation of an organic fluorescent small-molecule material.

Images