Fluorescent dye, and preparation method therefor and use thereof

Abstract

The present application provides a fluorescent dye, and a preparation method therefor and a use thereof. The fluorescent dye has a structure as shown in formula I, is a silicon-based Rhodamine organic dye, has excellent near-infrared fluorescence characteristics, and contains a conjugated ligand group R in the molecular structure, so that the fluorescent dye can coordinate with various transition metals, thereby improving the response between the metals and the silicon-based Rhodamine dye, and an obtained metal complex can fully exert the characteristics of the complex and the characteristics of silicon-based Rhodamine near-infrared light absorption and emission. The fluorescent dye and the metal complex containing same can be used in the fields of photosensitizers, cell staining, photocatalysis, photodynamic therapy and the like, and have wide application prospects. The preparation method for the fluorescent dye provided by the present application is simple, has mild reaction conditions, is suitable for large-scale preparation, has a high yield and few by-products, is easy to purify to obtain pure products, and greatly widens the application range of silicon-based Rhodamine dyes.

Description

A fluorescent dye and its preparation method and application Technical Field

[0001] The present application belongs to the technical field of organic materials, and specifically relates to a fluorescent dye and a preparation method and application thereof. Background Art

[0002] Fluorescence imaging is a visualization imaging technology that uses the luminescence intensity of luminescent probes as the detection signal. It uses fluorescent probes to mark biological structures and utilizes the high sensitivity of fluorescence imaging to visualize and observe cell activities in living organisms. This imaging technology is widely used in the field of life science imaging and detection due to its high sensitivity and minimal trauma.

[0003] Near-infrared fluorescence (650-900nm) has strong tissue penetration ability, is suitable for deep tissue imaging, causes little damage to tissue, and can be clearly distinguished from the background fluorescence of biological tissue, thus avoiding background fluorescence interference. Therefore, it has obvious advantages in the field of bio-optical imaging.

[0004] Rhodamine dyes are widely used organic dyes with excellent photophysical properties, such as high fluorescence quantum yield, high molar extinction coefficient, and good photostability. However, because the UV-visible light absorption and fluorescence emission of ordinary rhodamine are generally between 550-620 nm, the matching visible light lacks the advantages of near-infrared light. Therefore, their application in bioimaging is greatly limited. While retaining its favorable photophysical advantages, structural modification of ordinary rhodamine to red-shift its UV-visible light absorption and fluorescence emission to the near-infrared region is of great research significance.

[0005] The rhodamine structure consists of a central xanthene ring with amino side chains at either end. By extending the conjugated structure of the central heterocycle or modifying the amino side chains, the photophysical properties of rhodamine can be effectively modified. In 2008, Academician Qian Xuhong's team first discovered that replacing the oxygen atom in the central xanthene ring of the fluorophore pyronin with a silicon atom red-shifted its UV-visible absorption and fluorescence emission wavelengths by approximately 90 nm due to the change in the bridging atom, effectively extending the fluorescence emission of rhodamine dyes into the near-infrared region ("A design concept of long-wavelength fluorescent analogs of rhodamine dyes: replacement of oxygen with silicon atom," Meiyan Fu et al., Chemical Communications, 2008, 1780-1782). Tetsuo Nagano's team further red-shifted the fluorescence by modifying the amino side chains at both ends to a more rigid structure ("Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging", Yuichiro Koide et al., Journal of the American Chemical Society, 2012, 34, 5029-5031). However, the existing routes are complex, have low yields, produce many byproducts, and are difficult to purify, limiting the application of silicon-rhodamine dyes in biological imaging.

[0006] Recently, Xu Zhaochao's team redesigned the original synthetic route of silicon-based rhodamine dyes and simplified the design route. However, the reaction conditions used are relatively harsh and suitable for trace preparation. In some cases, cuprous iodide metal catalyst is required ("Systematic study of synthesizing various heteroatom-substituted rhodamines from diaryl ether analogues", Fei Deng et al., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2020, 240, 118466). Therefore, the development of fluorescent dyes with excellent fluorescence properties and simple preparation methods is an urgent problem to be solved in this field.

[0007] Summary of the Invention

[0008] The present application provides a fluorescent dye and its preparation method and application. The fluorescent dye has excellent fluorescence properties and can coordinate with a variety of transition metals. The preparation method is simple, the reaction conditions are mild, the yield is high, and it has broad application prospects.

[0009] In a first aspect, the present application provides a fluorescent dye having a structure as shown in Formula I:

[0010] In Formula I, R represents a ligand group, which is selected from any one of a substituted or unsubstituted C3-C20 (e.g., C3, C4, C5, C6, C7, C8, C9, C10, C12, C14, C16, C18, etc.) N-containing heteroaryl group, a substituted or unsubstituted C2-C10 (e.g., C3, C4, C5, C6, C7, C8, C9, C10) alkynyl group, or an acetylacetonyl group.

[0011] The substituted substituent in R is selected from any one of halogen, amino, unsubstituted or halogenated C1-C6 (e.g., C1, C2, C3, C4, C5, C6) straight or branched alkyl, or unsubstituted or halogenated C1-C6 (e.g., C1, C2, C3, C4, C5, C6) alkoxy.

[0012] In Formula I, R1, R2, and R3 are each independently selected from any one of C1-C6 (e.g., C1, C2, C3, C4, C5, C6) linear or branched alkyl groups.

[0013] In formula I, R4, R5, R6, and R7 are each independently selected from any one of H, C1-C6 (eg, C1, C2, C3, C4, C5, C6) linear or branched alkyl groups.

[0014] In Formula I, X - Indicates -1 valence anion.

[0015] In formula I, n represents the ring The number of is 1 or 2; when n is 1, it represents the ring is an N hetero five-membered ring; when n is 2, it represents the ring is an N hetero six-membered ring, and 2 They can be the same or different groups, that is, two R6 groups can be the same or different, and two R7 groups can be the same or different. Both represent the attachment site of a group.

[0016] The fluorescent dye provided in the present application has a structure as shown in Formula I. It is a silicon-based rhodamine dye with excellent near-infrared fluorescence properties. The molecular structure contains a conjugated ligand group R, which can coordinate with a variety of transition metals, thereby improving the response between the metal and the silicon-based rhodamine dye. The resulting metal complex has both the properties of transition metals and the properties of small molecule fluorescent dyes, and can give full play to the advantages of both. It is used for cell staining and has broad application prospects.

[0017] The following are preferred technical solutions of this application, but are not intended to limit the technical solutions provided in this application. Through the following preferred technical solutions, the objectives and beneficial effects of this application can be better achieved and realized.

[0018] It should be noted that the C3-C20 N-containing heteroaryl group described in the present application can be an N-containing heteroaryl group of C3, C4, C5, C6, C7, C8, C9, C10, C12, C14, C16, C18, etc., including a monocyclic N-containing heteroaryl group or a condensed-ring N-containing heteroaryl group, illustratively including but not limited to: pyridyl, bipyridyl, terpyridyl, quinolyl, isoquinolyl, pyrimidinyl, pyrazinyl, quinazolinyl, quinoxalinyl or o-phenanthroline, etc.

[0019] The C2-C10 alkynyl group may be a straight chain or branched chain alkynyl group of C2, C3, C4, C5, C6, C7, C8, C9, or C10, and illustratively includes but is not limited to ethynyl, propynyl, or butynyl.

[0020] The halogen includes fluorine, chlorine, bromine or iodine; the "halogen" means that at least one hydrogen in the group is replaced by a halogen (fluorine, chlorine, bromine or iodine).

[0021] The C1-C6 straight chain or branched chain alkyl group can be a straight chain or branched chain alkyl group of C1, C2, C3, C4, C5, or C6, and illustratively includes but is not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, or neohexyl, etc.

[0022] The C1-C6 alkoxy group may be a C1, C2, C3, C4, C5, or C6 straight-chain or branched alkoxy group, which is a monovalent group formed by a C1-C6 straight-chain or branched alkyl group connected to O.

[0023] The X - represents a -1 valence anion, which can be any -1 valence anion, including but not limited to: halogen anions, PF6 - or BF4 - wait.

[0024] In this application, the "substituted or unsubstituted" group may be substituted with one substituent or with multiple (≥2) substituents, and the substitution site of the substituent may be any site permitted by the chemical environment. When there are multiple (≥2) substituents, they may be the same or different substituents.

[0025] Preferably, R1, R2, and R3 are each independently selected from any one of C1-C3 straight-chain or branched alkyl groups, more preferably methyl or ethyl, and more preferably methyl.

[0026] Preferably, R4, R5, R6, and R7 are each independently selected from any one of H, C1-C3 linear or branched alkyl, more preferably H, methyl, or ethyl, and more preferably H or methyl.

[0027] Preferably, the fluorescent dye has a structure as shown in any one of Formula IA, Formula IB or Formula IC:

[0028] Among them, R and X - has the same limitations as in Formula I.

[0029] Preferably, R is selected from any one of the following groups:

[0030] Among them, the dotted line represents the attachment site of the group;

[0031] R' is independently selected from any one of H, halogen, amino, unsubstituted or halogenated C1-C6 (e.g., C1, C2, C3, C4, C5, C6) linear or branched alkyl, or unsubstituted or halogenated C1-C6 (e.g., C1, C2, C3, C4, C5, C6) alkoxy.

[0032] Preferably, the R' is selected from any one of H, halogen, amino, C1-C4 linear or branched alkyl, more preferably H or methyl.

[0033] Preferably, the X - PF6 - or BF4 - .

[0034] Preferably, the fluorescent dye is selected from any one of the following compounds:

[0035] Preferably, the ultraviolet-visible absorption peak of the fluorescent dye is 650-750 nm, for example, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm or 740 nm, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0036] Preferably, the fluorescence emission peak of the fluorescent dye is 700-800 nm, for example, it can be 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm or 790 nm, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0037] In a second aspect, the present application provides a method for preparing the fluorescent dye according to the first aspect, the preparation method comprising the following steps:

[0038] (1) reacting a first compound of formula II with a silicon-containing compound of formula III in the presence of an alkyl lithium reagent to obtain a second compound of formula IV;

[0039] (2) reacting the second compound with an aldehyde compound having a structure represented by Formula V to obtain a third compound having a structure represented by Formula VI;

[0040] (3) reacting the third compound with chlorobenzoquinone to obtain a fourth compound having a structure represented by Formula VII;

[0041] (4) The fourth compound and M + X - Reaction to obtain the fluorescent dye with the structure shown in Formula I;

[0042] The reaction formula of the preparation method is as follows:

[0043] Among them, R, R1, R2, R3, R4, R5, R6, R7, X - and n have the same defined ranges as in Formula I;

[0044] Hal1 and Hal2 are each independently selected from any one of the halogens, more preferably Cl or Br.

[0045] M + Indicates a +1 valent cation.

[0046] The preparation method of the fluorescent dye provided in this application includes four steps. The synthetic route is simple, the reaction conditions are milder and more universal, and it can be prepared in large quantities. The total yield of the reaction can be ≥60%, and can reach more than 65%. The yield is high and it is easy to purify to obtain a pure product. It can be applied on a large scale to the design and synthesis of similar silicon-based rhodamine containing a coordinating structure.

[0047] Preferably, the Hal1 is Br.

[0048] Preferably, the Hal2 is Cl.

[0049] Preferably, the alkyl lithium reagent includes n-butyl lithium and / or tert-butyl lithium, more preferably n-butyl lithium.

[0050] Preferably, the molar ratio of the alkyl lithium reagent to the first compound is 1:(1-1.8), for example, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7 or 1:7.75.

[0051] Preferably, the molar ratio of the silicon-containing compound to the first compound is 1:(1-2), for example, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8 or 1:1.9, etc.

[0052] Preferably, the reaction in step (1) is carried out in the presence of an organic solvent.

[0053] Preferably, the organic solvent in step (1) comprises any one or a combination of at least two of tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, acetone, butanone, and cyclohexanone, and tetrahydrofuran and / or acetone are further preferred.

[0054] Preferably, based on the amount of the first compound being 1 mmol, the amount of the organic solvent is 1-10 mL, for example, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL or 9 mL, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0055] Preferably, the temperature of the reaction in step (1) is -80°C-0°C, for example, it can be -78°C, -75°C, -70°C, -65°C, -60°C, -55°C, -50°C, -45°C, -40°C, -35°C, -30°C, -25°C, -20°C, -15°C, -10°C, -5°C or -1°C, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of brevity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0056] Preferably, the reaction time of step (1) is 2-12 hours, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of brevity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0057] Preferably, the reaction method of step (1) comprises: first mixing the first compound with an organic solvent, cooling the mixture to -80°C-0°C, then adding an alkyl lithium reagent thereto, and then slowly adding (dropwise adding) the silicon-containing compound under stirring. After all the compounds are added, the mixture is reacted at -80°C-0°C for 2-12 hours until the reaction is completed.

[0058] Preferably, after the reaction in step (1) is completed, the steps of quenching, extraction, solvent removal and purification are further included.

[0059] Preferably, the quenching reagent comprises ethanol.

[0060] Preferably, the molar ratio of the second compound to the aldehyde compound is 1:(1-1.2), for example, 1:1.02, 1:1.05, 1:1.08, 1:1.1, 1:1.12, 1:1.15 or 1:1.18.

[0061] Preferably, the reaction in step (2) is carried out in an acidic solution.

[0062] Preferably, the acidic solution in step (2) comprises hydrochloric acid.

[0063] Preferably, the concentration of the hydrochloric acid is 1-3 mol / L, for example, it can be 1.2 mol / L, 1.5 mol / L, 1.8 mol / L, 2 mol / L, 2.2 mol / L, 2.5 mol / L or 2.8 mol / L, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0064] Preferably, based on the amount of the second compound being 1 mmol, the amount of hydrochloric acid used is 10-30 mL, for example, 12 mL, 15 mL, 18 mL, 20 mL, 22 mL, 25 mL or 28 mL, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0065] Preferably, the reaction temperature in step (2) is 50-100°C, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range, and the reflux temperature is further preferred.

[0066] Preferably, the reaction time of step (2) is 2-24 hours, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of brevity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0067] Preferably, the molar ratio of the third compound to chloranil is 1:(1.5-5), for example, it can be 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, 1:3.2, 1:3.5, 1:3.8, 1:4, 1:4.2, 1:4.5 or 1:4.8, etc.

[0068] Preferably, the reaction in step (3) is carried out in the presence of an organic solvent.

[0069] Preferably, the organic solvent in step (3) comprises any one of dichloromethane, acetonitrile, and chlorobenzene, or a combination of at least two thereof, more preferably dichloromethane.

[0070] Preferably, based on the amount of the third compound being 1 mmol, the amount of the organic solvent is 1-20 mL, for example, 2 mL, 5 mL, 8 mL, 10 mL, 12 mL, 15 mL or 18 mL, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0071] Preferably, the reaction temperature in step (3) is 15-40°C, for example, it can be 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C or 38°C, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range, and room temperature / normal temperature is further preferred.

[0072] Preferably, the reaction time of step (3) is 0.5-3h, for example, it can be 0.6h, 0.8h, 1h, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h or 2.8h, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0073] Preferably, the M + Selected from Na + , K + or NH4 + Any one of .

[0074] Preferably, the M + X -It is any one or a combination of at least two selected from sodium hexafluorophosphate, potassium hexafluorophosphate, ammonium hexafluorophosphate, sodium tetrafluoroborate, potassium tetrafluoroborate, and ammonium tetrafluoroborate, and ammonium hexafluorophosphate is more preferred.

[0075] Preferably, the fourth compound and M + X - The molar ratio is 1:(1.5-2.5), for example, it can be 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3 or 1:2.4, etc.

[0076] Preferably, the reaction in step (4) is carried out in a solvent.

[0077] Preferably, the solvent in step (4) comprises water and / or acetonitrile, and more preferably a combination of water and acetonitrile.

[0078] Preferably, the temperature of the reaction in step (4) is 15-40°C, for example, it can be 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C or 38°C, as well as specific values ​​between the above points. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific points included in the range, and room temperature / normal temperature is further preferred.

[0079] Preferably, the reaction time of step (4) is 0.5-3h, for example, it can be 0.6h, 0.8h, 1h, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h or 2.8h, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, this application no longer exhaustively lists the specific point values ​​included in the range.

[0080] In a third aspect, the present application provides a use of the fluorescent dye described in the first aspect in a metal complex, a photosensitizer, a fluorescent probe or a fluorescent imaging agent.

[0081] Illustratively, the metal complex comprises the fluorescent dye and a transition metal coordinated to the R group in the fluorescent dye; the transition metal comprises any one or a combination of at least two of iridium, ruthenium, rhodium, rhenium, platinum, and osmium. The metal complex can enhance the response between the metal and the silicon-based rhodamine dye, effectively leveraging the advantages and properties of both, thereby obtaining a metal complex with near-infrared absorption and emission.

[0082] Preferably, the fluorescent dye and the metal complex can be used as photosensitizers in the fields of OLED, photocatalysis, photodynamic therapy, etc., and have very broad application prospects.

[0083] Compared with the prior art, this application has the following beneficial effects:

[0084] (1) The fluorescent dye provided by the present application has a molecular structure as shown in Formula I, which is a silicon-based rhodamine organic dye with excellent near-infrared fluorescence properties, and can achieve the effect of near-infrared light absorption and emission. At the same time, the molecular structure of the fluorescent dye contains a conjugated ligand group R, which can be coordinated with a variety of transition metals, thereby improving the response between the metal and the silicon-based rhodamine dye, and the obtained metal complex can give full play to the characteristics of the complex and the characteristics of silicon-based rhodamine near-infrared light absorption and emission. The fluorescent dye and the metal complex containing it can be used in the fields of photosensitizer, cell staining, photocatalysis or photodynamic therapy, and have broad application prospects.

[0085] (2) The preparation method of the fluorescent dye provided in this application is simple and easy, the reaction conditions are mild, it is suitable for large-scale preparation, the yield is high, there are few by-products, and it is easy to purify to obtain pure products, which greatly promotes the application range of silicon-based rhodamine dyes and expands their research in the biological field. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIG1 is a hydrogen nuclear magnetic spectrum of the fluorescent dye bpy-Si-Rho provided in Example 1.

[0087] FIG2 is a carbon-NMR spectrum of the fluorescent dye bpy-Si-Rho provided in Example 1.

[0088] FIG3 is a high-resolution mass spectrum of the fluorescent dye bpy-Si-Rho provided in Example 1.

[0089] FIG4 is a diagram of the UV-visible absorption and fluorescence spectra of the fluorescent dye bpy-Si-Rho provided in Example 1.

[0090] FIG5 is a cell imaging diagram of the fluorescent dye bpy-Si-Rho provided in Example 1.

[0091] FIG6 is a graph showing the UV-visible absorption and fluorescence spectra of the fluorescent dye terpy-Si-Rho provided in Example 2. DETAILED DESCRIPTION

[0092] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0093] As used herein, the terms "comprises," "including," "having," "containing" or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0094] "Optionally," "optionally," or "either" means that the subsequently described matter or event can or cannot occur, and that the description includes instances where the event occurs and instances where it does not.

[0095] In this application, features specified as "first," "second," "third," or "fourth" may explicitly or implicitly include one or more of such features, and are used to distinguish and describe features in no particular order or importance. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0096] In the following specific embodiments of the present application, the raw materials for which the preparation methods are not provided are commercially available chemicals and can be purchased through market channels.

[0097] Example 1

[0098] A fluorescent dye bpy-Si-Rho, the molecular structure is The preparation method is as follows:

[0099] (1) Under nitrogen, 3.448 g of compound 1 was added to a dry two-necked flask containing 60 mL of anhydrous tetrahydrofuran. The two-necked flask was then placed in a dry ice / acetone bath at -78°C. After the temperature stabilized, 9.74 mL of a 2 mol / L n-butyllithium solution was taken with a syringe and slowly added dropwise to the system over 20 minutes while stirring vigorously. After 20 minutes, the temperature was kept stable at -78°C. A solution of 1.02 mL of dichlorodimethylsilane dissolved in 10 mL of anhydrous tetrahydrofuran was taken with a syringe and slowly added dropwise to the reaction system over 30 minutes. The reaction was continued at -78°C for 6 hours. After that, 1 mL of ethanol was slowly added to the reaction system to quench the unreacted active substance and stirring was continued for 10 minutes. 100 mL of water was first added to the reaction solution, and then the solution was extracted with 300 mL of dichloromethane three times. The organic phase was collected and the solvent was removed using a rotary evaporator to obtain the crude product. Separation and purification were performed using a silica gel column with an eluent of dichloromethane and petroleum ether in a ratio of 1:10 to obtain 2.49 g of oily compound 2 with a yield of 95.5%.

[0100] (2) 2.0 g of compound 2 obtained in step (1) and 1.34 g of 4-methyl-4'-formaldehyde-2,2'-bipyridine were added to 100 mL of 2 mol / L hydrochloric acid solution to form a suspended turbid liquid. The reaction solution was heated to reflux for 12 h. After the temperature dropped to room temperature, saturated sodium bicarbonate solution was added, and the mixture was extracted three times with 300 mL of dichloromethane. The organic phase was collected and the solvent was removed by rotary evaporation to obtain a crude compound 3.

[0101] (3) The crude compound 3 obtained in step (2) was dissolved in 30 mL of dichloromethane, 3 g of tetrachlorobenzoquinone was added and stirred at room temperature for 1 h, and then the solvent was removed to obtain a crude compound 4.

[0102] (4) The crude compound 4 obtained in step (3) was dissolved in 30 mL of acetonitrile, and 3 mL of saturated ammonium hexafluorophosphate solution was added dropwise and stirred at room temperature for 1 h. Finally, the solvent was removed to obtain the final crude product. Separation and purification were performed using a silica gel column with acetonitrile / water in a ratio of 20:1 as the eluent to obtain 2.45 g of the pure target product bpy-Si-Rho, with a yield of 65.2%, as a green solid.

[0103] The fluorescent dye bpy-Si-Rho provided in this embodiment was tested as follows:

[0104] 1. Structural Characterization of the Target Product bpy-Si-Rho

[0105] Structural characterization, including H-NMR, C-NMR, and high-resolution mass spectrometry, confirmed that the synthetic route was feasible and the target compound was correct. The results are as follows:

[0106] The H NMR spectrum of bpy-Si-Rho is shown in Figure 1. The H NMR spectrum data are: 1 H NMR (400MHz, CD3CN-d3): δ (ppm) = 8.94 (d, J = 4.3Hz, 1H), 8.66 (d, J = 5.0Hz, 1H), 8.44 (s, 1H), 8.28 (s, 1H), 7.60 (d, J = 4.7Hz, 1H), 7. 44(d,J=4.2Hz,1H),7.12(s,2H),6.72(s,2H),3.80(t,J=7.9Hz,4H),3.21(s,6H),2.90(t,J=7.7Hz,4H),2.61(s,3H),0.57(s,6H).

[0107] The NMR carbon spectrum of bpy-Si-Rho is shown in Figure 2, and the NMR carbon spectrum data are: 13 C NMR (101MHz, CD3CN-d3): δ (ppm) = 160.22, 156.92, 155.03, 151.43, 151.08, 150.81, 150.10, 149.5 9,133.45,132.60,127.40,126.64,125.73,123.50,122.09,115.37,54.50,33.11,25.89,20.97.

[0108] The high-resolution mass spectrum of bpy-Si-Rho is shown in Figure 3. The high-resolution mass spectrum data are: HRMS (ESI): theoretical molecular parent ion mass: C 32 H 33 N4SiPF6[M-PF6 - ] + m / z=501.24690; detection data: [M-PF6 - ] + m / z=501.24686.

[0109] 2. UV-visible absorption and fluorescence spectra

[0110] The luminescence properties of the fluorescent dye bpy-Si-Rho provided in this embodiment were tested using an ultraviolet-visible spectrophotometer (Cary 60 UV-vis spectrophotometer, Agilent) and a fluorescence spectrometer (FS5 fluorescence spectrometer, Edinburgh Instruments). The resulting ultraviolet-visible absorption and fluorescence spectra are shown in Figure 4. As can be seen from Figure 4, the fluorescent dye bpy-Si-Rho has an ultraviolet absorption peak at 700 nm and a fluorescence emission wavelength of about 728 nm, indicating that it has the effect of being able to achieve near-infrared light absorption and emission.

[0111] 3. Cell Imaging

[0112] Cancer cells 4T1 were cultured in 8-well chamber cell culture slides and cultured in a cell culture incubator for 24 hours. Then, 1 μL of a dimethyl sulfoxide (DMSO) solution (1 mM) of the fluorescent dye bpy-Si-Rho was added to the wells of the cell culture slides. After further incubation for 30 minutes, the cells were observed using a confocal laser scanning microscope (Leica TCS SP8 microscope) with excitation and emission wavelengths of 640 nm and 700 nm, respectively.

[0113] FIG5 is a cell imaging diagram of the fluorescent dye bpy-Si-Rho. As can be seen from FIG5 , the fluorescent dye bpy-Si-Rho can stain mitochondria and emit a strong red fluorescent signal.

[0114] Comparative Preparation Example 1

[0115] The general synthesis method of the fluorescent dye bpy-Si-Rho is as follows:

[0116] (1) 2.28 g of compound 1 and 1.07 g of 4-methyl-4'-formaldehyde-2,2'-bipyridine were added to 100 mL of 2M hydrochloric acid solution, heated under reflux, and reacted for 12 h. The mixture was then cooled to room temperature and the pH was adjusted to 7 with saturated NaHCO3 solution. The mixture was extracted with 150 mL of dichloromethane three times. The organic phase was collected and the crude intermediate was removed by dissolution. The crude intermediate was purified by silica gel column to obtain compound 2D as a light yellow solid (2.87 g) with a yield of 95%.

[0117] (2) 2.0 g of compound 2D and 30 mL of anhydrous tetrahydrofuran / ether (volume ratio 1:1) were added to a dry, nitrogen-filled 100 mL three-necked flask. 7.9 mL of 1 M n-butyl lithium was then added dropwise at -78°C and stirred for 30 min. At this temperature, 10 mL of a solution containing 0.58 mL of dichlorodimethylsilane was slowly added. After reacting at this temperature for 6 h, the system was quenched with 1 mL of 1 M hydrochloric acid to quench the unreacted active substance and continued to stir for 10 min. The pH was adjusted to 7 with saturated NaHCO3 and extracted three times with 150 mL of dichloromethane. The organic phase was collected and the solution was removed to obtain the crude intermediate compound 3.

[0118] (3) Compound 3 was dissolved in 40 mL of dichloromethane, and 3.6 g of tetrachlorobenzoquinone was added. The mixture was stirred at room temperature for 2 h, and the solvent was removed to obtain a crude product of compound 4.

[0119] (4) 20 mL of acetonitrile was added to the crude compound 4 to dissolve the product. 1 mL of saturated aqueous ammonium hexafluorophosphate was added and the mixture was stirred at room temperature for 1 h. The solid was filtered to remove the solid. The filtrate was retained and the solvent was removed to obtain a green crude product. The crude product was purified by reverse-phase preparative high-performance liquid chromatography (C18 column, acetonitrile and water gradient elution) to obtain pure green solid bpy-Si-Rho with an overall yield of ≤10%.

[0120] Compared with the synthetic route of Preparation Example 1, the yield is low, the yield is ≤10%, and many by-products are produced, especially in step (2). A large amount of by-products are produced. The structure of the confirmed by-products is shown below:

[0121] These by-products have similar polarity to the products and are difficult to purify.

[0122] It can be seen that the preparation method of the fluorescent dye provided in the present application has the characteristics of simple synthesis route, mild reaction conditions, high yield, few by-products and easy purification.

[0123] Example 2

[0124] A fluorescent dye terpy-Si-Rho, the molecular structure is The preparation method is as follows:

[0125] (1) Compound 2 was prepared by the same method as step (1) of Example 1.

[0126] (2) 2.0 g of compound 2 obtained in step (1) and 1.61 g of 4'-formyl-2,2':6',2"-terpyridine were added to 100 mL of 2 mol / L hydrochloric acid solution to form a suspended turbid liquid. The reaction solution was heated to reflux for 12 h. After the temperature dropped to room temperature, saturated sodium bicarbonate solution was added, and the mixture was extracted with 300 mL of dichloromethane three times. The organic phase was collected and the solvent was removed by rotary evaporation to obtain a crude compound 3-2.

[0127] (3) The crude compound 3-2 obtained in step (2) was dissolved in 30 mL of dichloromethane, 3 g of tetrachlorobenzoquinone was added and stirred at room temperature for 1 h, and then the solvent was removed to obtain the crude compound 4-2.

[0128] (4) The crude compound 4-2 obtained in step (3) was dissolved in 30 mL of acetonitrile, and 3 mL of saturated ammonium hexafluorophosphate solution was added dropwise and stirred at room temperature for 1 h. Finally, the solvent was removed to obtain the final crude product. Separation and purification were performed using a silica gel column with acetonitrile / water in a ratio of 20:1 as the eluent to obtain 2.58 g of the pure target product terpy-Si-Rho, with a yield of 59.3%, as a green solid.

[0129] The fluorescent dye terpy-Si-Rho provided in this embodiment was tested as follows:

[0130] 1. Structural Characterization of the Target Product terpy-Si-Rho

[0131] Structural characterization, including H-NMR, C-NMR, and high-resolution mass spectrometry, confirmed that the synthetic route was feasible and the target compound was correct. The results are as follows:

[0132] The H-NMR spectrum data are: 1 H NMR (400MHz, CDCl3-d): δ (ppm) 8.80 (d, J = 8.0Hz, 2H), 8.70 (d, J = 4.1Hz, 2H), 8.39 (s, 2H), 7.99 (t, J = 7.7Hz, 2H), 7. 55–7.39(m,2H),6.93(s,2H),6.80(s,2H),3.81(t,J=8.0Hz,4H),3.24(s,6H),2.90(t,J=7.7Hz,4H),0.60(s,6H).

[0133] The NMR carbon spectrum data are: 13C NMR (101MHz, CDCl3-d)): δ (ppm) = 162.22, 156.90, 155.38, 154.90, 150.92, 150.70, 148.9 9,137.74,133.31,132.79,127.90,124.66,121.84,121.09,114.76,54.69,33.69,26.37.

[0134] 2. UV-visible absorption and fluorescence spectra

[0135] The luminescence properties of the fluorescent dye terpy-Si-Rho were tested using the same method as in Example 1. The obtained UV-visible absorption and fluorescence spectra are shown in FIG6 . As can be seen from FIG6 , the fluorescent dye terpy-Si-Rho has an UV absorption peak at 702 nm and a fluorescence emission wavelength of about 730 nm, indicating that it has the effect of achieving near-infrared light absorption and emission.

[0136] The applicant declares that while the above-described embodiments illustrate the fluorescent dyes, their preparation methods, and applications, this application is not limited to the aforementioned process steps, nor does it imply that implementation of this application requires reliance on these process steps. Persons skilled in the art should understand that any improvements to this application, equivalent substitutions for the raw materials used, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

Claims

1. A fluorescent dye having the structure shown in Formula I: Wherein, R represents a ligand group selected from any one of substituted or unsubstituted C3-C20 N-containing heteroaryl, substituted or unsubstituted C2-C10 alkynyl, or acetylacetonyl; The substituents of the substitution in R are selected from any one of halogen, amino, unsubstituted or halogenated C1-C6 straight-chain or branched-chain alkyl, or unsubstituted or halogenated C1-C6 alkoxy; R1, R2, and R3 are each independently selected from any one of C1-C6 straight-chain or branched-chain alkyl; R4, R5, R6, and R7 are each independently selected from H or any one of C1-C6 straight-chain or branched-chain alkyl; X - represents a -1 valent anion; and n is 1 or 2.

2. The fluorescent dye according to claim 1, wherein, The fluorescent dye has a structure represented by any one of Formula IA, Formula IB or Formula IC: wherein, R and X - have the same defined ranges as in formula I.

3. The fluorescent dye according to claim 1 or 2, wherein, R is selected from any one of the following groups: wherein, the dotted line represents the connection site of the group; Wherein, each R' is independently selected from H, halogen, amino, unsubstituted or halogenated C1-C6 straight-chain or branched-chain alkyl, or unsubstituted or halogenated C1-C6 alkoxy; preferably any one of H, halogen, amino, C1-C4 straight-chain or branched-chain alkyl; more preferably H or methyl.

4. The fluorescent dye according to any one of claims 1 - 3, wherein, The said X - is PF6 - or BF4 - .

5. A method for preparing the fluorescent dye according to any one of claims 1 - 4, comprising the following steps: (1) Reacting a first compound having the structure shown in Formula II with a silicon-containing compound having the structure shown in Formula III in the presence of an alkyllithium reagent to obtain a second compound having the structure shown in Formula IV; (2) The second compound reacts with the aldehyde compound having the structure shown in Formula V to obtain a third compound having the structure shown in Formula VI; (3) The third compound reacts with tetrachlorobenzoquinone to obtain a fourth compound having the structure shown in Formula VII; and (4) The fourth compound reacts with M + X - to obtain the fluorescent dye having the structure shown in Formula I; The reaction formula of the preparation method is as follows: wherein, R, R1, R2, R3, R4, R5, R6, R7, X - and n have the same defined ranges as in Formula I; Hal1 and Hal2 are each independently selected from any one of halogen; and M + represents a +1 valence cation.

6. The preparation method according to claim 5, wherein, The alkyllithium reagent includes n-butyllithium and / or tert-butyllithium, preferably n-butyllithium; Preferably, the molar ratio of the alkyllithium reagent to the first compound is 1:(1-1.8); Preferably, the molar ratio of the silicon-containing compound to the first compound is 1:(1-2); Preferably, the temperature of the reaction in step (1) is -80°C - 0°C; Preferably, the reaction time in step (1) is 2 - 12 h.

7. The preparation method according to claim 5 or 6, wherein, The molar ratio of the second compound to the aldehyde compound is 1:(1-1.2); Preferably, the reaction in step (2) is carried out in an acidic solution; Preferably, the acidic solution includes hydrochloric acid; Preferably, the temperature of the reaction in step (2) is 50 - 100°C; Preferably, the reaction time in step (2) is 2 - 24 h.

8. The preparation method according to any one of claims 5 - 7, wherein, The molar ratio of the third compound to tetrachlorobenzoquinone is 1:(1.5-5); Preferably, the temperature of the reaction in step (3) is 15 - 40°C; Preferably, the reaction time in step (3) is 0.5 - 3 h.

9. The preparation method according to any one of claims 5 - 8, wherein, The said M + is selected from Na + , K + or NH4 + ; any one of them Preferably, the molar ratio of the fourth compound to M + X - is 1:(1.5 - 2.5); Preferably, the temperature of the reaction in step (4) is 15 - 40°C; Preferably, the reaction time in step (4) is 0.5 - 3 h.

10. Use of a fluorescent dye according to any one of claims 1 - 4 in a metal complex, a photosensitizer, a fluorescent probe or a fluorescence imaging agent.

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