Electron acceptor, fluoroborondipyrromethene fluorescent dye, and preparation method and application thereof

Abstract

The application discloses an electron acceptor, a fluoroborondiphenylamine fluorescent dye and a preparation method and application thereof, wherein, the difluoroboron bridged difluoroboron pseudoindole electron acceptor is easy to synthesize, and can be further condensed with a Fischer aldehyde or a derivative thereof to obtain a series of fluoroborondiphenylamine fluorescent dyes covering far infrared to near infrared, which effectively enriches the types of fluorescent dyes, and the application further provides the fluoroborondiphenylamine fluorescent dye which can be used for lipid droplet fluorescent imaging, and the effects of high-efficiency lipid droplet targeting, high signal-to-noise ratio lipid droplet imaging and multi-color (multi-channel) fluorescent lipid droplet imaging are achieved, wherein, the near-infrared lipid droplet probe has better application prospect in tissue and living body imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescent dyes, in particular to a boron difluoride pseudoindole electron acceptor, a borondifluoro indolenine cyanine fluorescent dye based on the electron acceptor, and a preparation method and application thereof. BACKGROUND

[0002] As a visualization technology, fluorescence imaging has the remarkable characteristics of high sensitivity, non-destructiveness, real-time detection, high spatial and temporal resolution, and has been widely used in biological science and biomedical science. Small molecule organic fluorophores can help to realize the visualization of gene expression, post-translational modification, biomolecule concentration, organelle transport, disease detection, drug development and reaction evaluation, and can also guide tumor surgery through images, so when modern dye chemistry is combined with modern synthetic chemistry, fluorophores are endowed with quite a lot of functions in biological imaging.

[0003] Traditional small molecule fluorescent probes mainly use mature commercial "core" structures as fluorophores, mainly including coumarin, naphthalimide, boron-difluoride complex dipyrromethene (BODIPY), fluorescein, rhodamine and cyanine. Such traditional fluorescent dyes have deficiencies in chemical and optical properties. In recent years, some emerging fluorophores have been continuously developed and rapidly developed.

[0004] However, the types of fluorescent dyes at the present stage are still insufficient, and their photophysical properties need to be further improved. Moreover, the synthesis of existing fluorescent dyes is relatively complex and difficult to promote subsequent functional application. Therefore, it is of great significance to develop multifunctional new fluorescent dyes with better photophysical properties and easy synthesis. SUMMARY

[0005] One object of the present application is to provide a boron difluoro indolenine electron acceptor (BFI). Such electron acceptors based on boron difluoride bridge are not only easy to synthesize, but also can be used to prepare a series of functional borondifluoro indolenine cyanine (BCy) fluorescent dyes covering far infrared to near infrared, effectively enriching the types of fluorescent dyes.

[0006] The above object is achieved by the following technical solutions:

[0007] A boron difluoro indolenine electron acceptor has a structural formula shown in formula I:

[0008]

[0009] In formula I, X is selected from CH or N, R 1selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, hydroxyl, amino, carboxyl and active ester thereof, carboxylate, sulfonic acid, methoxyl, formate, amide, R 2 is selected from the group consisting of hydrogen or halogen.

[0010] In the technical solution, the difluoroboron pseudoindole electron acceptor is a kind of electron acceptor based on difluoroboron bridging, which can be prepared by reacting 2,3,3-trimethyl-3H-pseudoindole, 2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine and its derivatives with boron trifluoride ether under inert gas protection, and halogen-substituted difluoroboron pseudoindole electron acceptor is prepared by halogenation, and the reaction condition is mild and the synthesis path is short. Based on such difluoroboron pseudoindole electron acceptor, fluoroboron porphyrin fluorescent dye can be further obtained by condensation with fisher aldehyde or its derivatives, which not only widens the types of fluorescent dyes, but also has good photophysical properties and wide application value.

[0011] In the technical solution, R 1 The selection of the R group depends on the selection of 2,3,3-trimethyl-3H-pseudoindole, 2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine and its derivatives. From the reaction mechanism, these compounds can all react with boron trifluoride ether to obtain the difluoroboron pseudoindole electron acceptor shown in formula I. Considering the influence of the R 1 group of the electron acceptor on the conjugated system and the influence on the photophysical properties of the fluorescent dye, the R 1 group is preferably hydrogen, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, hydroxyl, amino, carboxyl, sulfonic acid, methoxyl, formate, amide, and the R 2 group can be hydrogen or halogen-substituted. Preferably, the R 2 group is hydrogen, chlorine, bromine, iodine or fluorine.

[0012] In some embodiments, the R 1 group is a substituted or unsubstituted chain alkyl. The chain alkyl can be a straight chain alkyl or a branched chain alkyl, and the number of carbon atoms of the chain alkyl is preferably C1-C2. In one or more embodiments, the chain alkyl is preferably methyl, ethyl or trifluoromethyl.

[0013] In some embodiments, the R 1 group is a substituted or unsubstituted alkenyl. The number of carbon atoms of the alkenyl is preferably C2-C3. In one or more embodiments, the alkenyl is preferably vinyl.

[0014] In some embodiments, the R 1The group is a C2-C4 ynyl group. The number of carbon atoms in the ynyl group is preferably C2-C3. In one or more embodiments, the ynyl group is preferably an acetylene group.

[0015] In some embodiments, R 1 The group can be a substituted or unsubstituted phenyl group.

[0016] In some embodiments, R 1 The functional group can be an activated N-hydroxysuccinimide ester (NHS), 4-nitrophenol ester, pentafluorophenol ester, or other carboxylic acid active esters.

[0017] Furthermore, R is the preferred electron acceptor for difluoroboron pseudoindole. 1 Group, the R 1 Selected from hydrogen, halogen, methyl, ethyl, vinyl, ethynyl, trifluoromethyl, phenyl, hydroxyl, carboxyl and their active esters, carboxylic acid esters, sulfonic acid groups, methoxy groups, and amide groups.

[0018] Furthermore, the difluoroboron pseudoindole electron acceptor is selected from the following compounds:

[0019]

[0020]

[0021] Another objective of this invention is to provide a fluoroboron-based anthocyanin fluorescent dye based on any of the aforementioned difluoroboron pseudoindole electron acceptors. This dye has a short synthetic route, mild reaction conditions, and is easy to prepare. Furthermore, this type of fluoroboron-based anthocyanin fluorescent dye has excellent photophysical properties, which can be finely tuned by controlling the intensity of the electron acceptor and the degree of π-conjugation in the conjugated system. In addition, this type of fluoroboron-based anthocyanin fluorescent dye can also be used for fluorescence imaging of lipid droplets.

[0022] The above objectives are achieved through the following technical solutions:

[0023] A fluoroboron cyanine fluorescent dye having the structural formula shown in Formula II:

[0024]

[0025] In Formula II, X is selected from CH or N, R 1 and R 3 Each group is independently selected from hydrogen, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, hydroxyl, amino, carboxyl and their active esters, sulfonic acid, methoxy, carboxylic acid ester, amide, R 2 Selected from hydrogen or halogen, R 4 Selected from C1 to C 16 Chain alkyl groups, polyethylene glycol groups, C1-C 16alkyl carboxylic acids and active esters, C1-C5 alkyl sulfonic acids and salts thereof, C1-C4 alcohols, n = 1-4.

[0026] In the technical solution, a difluoroboron pseudoindole electron acceptor of formula I is used as an electron acceptor to perform a condensation reaction with fisher's aldehyde or a derivative thereof to obtain a fluoroborophyrin fluorescent dye of formula II.

[0027] In the technical solution, R 1 and R 2 groups are determined by the selected difluoroboron pseudoindole electron acceptor, R 3 and R 4 groups are determined by the selected fisher's aldehyde. Among them, R 3 groups can be the same as R 1 groups, or can be different. R 3 groups are preferably selected from hydrogen, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, hydroxyl, amino, carboxyl, sulfonic acid group, methoxy, formate group, amide group. R 4 is selected from C1-C 16 chain alkyl, polyethylene glycol group, C1-C 16 alkyl carboxylic acids and active esters, C1-C5 alkyl sulfonic acids and salts thereof, C1-C4 alcohols.

[0028] In some embodiments, R 1 groups can be activated N-hydroxysuccinimide ester (NHS), 4-nitrophenol ester, pentafluorophenol ester, and other carboxylic acid active esters.

[0029] Further, the R 1 is selected from hydrogen, halogen, methyl, ethyl, vinyl, ethynyl, trifluoromethyl, phenyl, hydroxyl, carboxyl and active ester thereof, carboxylate group, sulfonic acid group, methoxy, amide group, R 3 is selected from hydrogen or halogen, R 4 is selected from C1-C4 chain alkyl, polyethylene glycol group, C1-C 16 alkyl carboxylic acids and active esters.

[0030] Further, the fluoroborophyrin fluorescent dye is selected from the following compounds:

[0031]

[0032] The present application also provides a preparation method of any one of the aforementioned fluoroborophyrin fluorescent dyes, which adopts the aforementioned condensation reaction of difluoroboron pseudoindole electron acceptor and fisher's aldehyde to prepare fluoroborophyrin fluorescent dye. Specifically, the preparation method comprises the following steps:

[0033] condensing the difluoroboron pseudoindole electron acceptor of Formula I with the compound of Formula III in a mixed solution of a first reaction solvent, a first base, and a first acid to obtain the fluoroboron merocyanine fluorescent dye of Formula II; or

[0034] condensing the difluoroboron pseudoindole electron acceptor of Formula I with the compound of Formula IV in a second reaction solvent, and adding a second base to obtain the fluoroboron merocyanine fluorescent dye of Formula II;

[0035]

[0036] In the present technical solution, there are two preparation methods of the fluoroboron merocyanine fluorescent dye.

[0037] In the first preparation method, the difluoroboron pseudoindole electron acceptor is condensed with the compound of Formula III in a mixed solution of a first reaction solvent, a first base, and a first acid.

[0038] In some embodiments, the first reaction solvent is at least one of dichloromethane, dichloroethane, chloroform, acetone, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, benzene, toluene, n-butanol, isopropanol, ethanol, methanol, chlorobenzene, xylene, mesitylene. In one or more preferred embodiments, the first reaction solvent is preferably toluene or dichloromethane.

[0039] In some embodiments, the first base is at least one of piperidine, pyrrolidine, diethylamine, triethylamine, diisopropylaminoethylamine, pyridine, acetate, carbonate, bicarbonate, potassium tert-butoxide. In one or more preferred embodiments, the first base is piperidine, pyrrolidine.

[0040] In some embodiments, the first acid is at least one of acetic acid, formic acid, propionic acid, butyric acid, methanesulfonic acid, benzoic acid. In some preferred embodiments, the first acid is acetic acid.

[0041] In the second preparation method, after the difluoroboron pseudoindole electron acceptor is dissolved in a second reaction solvent, a second base is added for condensation reaction.

[0042] In some embodiments, the second reaction solvent is at least one of dichloromethane, dichloroethane, chloroform, acetone, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, benzene, toluene, n-butanol, isopropanol, ethanol, methanol, chlorobenzene, xylene, mesitylene. In one or more preferred embodiments, the second reaction solvent is chloroform or dichloromethane.

[0043] In some embodiments, the second base is at least one of triethylamine, diisopropylaminoethylamine, pyridine, acetate, carbonate, bicarbonate, potassium tert-butoxide. In one or more preferred embodiments, the second base is triethylamine, diisopropylaminoethylamine.

[0044] In some embodiments, the reaction temperature of the two condensation reactions is 25-120°C, preferably, the reaction temperature of the condensation reaction is 40-80°C.

[0045] For the fluoroborondipyrromethene fluorescent dye of Formula II, wherein R 1 is a carboxylic acid active ester, the product R 1 is a fluoroborondipyrromethene fluorescent dye of Formula II, wherein R

[0046] In one or more embodiments, the active ester reagent can be N,N'-disuccinimidyl carbonate (DSC), 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate (TSTU), etc. The condensing agent for the condensation reaction of the carboxyl and hydroxyl groups can be EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and its hydrochloride), N,N'-dicyclohexylcarbodiimide (DCC), etc.

[0047] Further, the method for preparing the difluoroboron pseudoindole electron acceptor comprises the following steps:

[0048] dissolving 2,3,3-trimethyl-3H-pseudoindole or its derivative, or 2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine or its derivative in a third reaction solvent, and adding boron trifluoride ether to obtain the difluoroboron pseudoindole electron acceptor of Formula I, wherein R 2 is hydrogen;

[0049] halogenating the difluoroboron pseudoindole electron acceptor of Formula I, wherein R 2 is hydrogen, to obtain the difluoroboron pseudoindole electron acceptor of Formula I, wherein R 2 is halogen.

[0050] In the technical solution, R 2 The difluoroboron pseudoindole electron acceptor of Formula I, wherein R 2 is hydrogen, can be obtained by reacting 2,3,3-trimethyl-3H-pseudoindole or its derivative, or 2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine or its derivative with boron trifluoride ether. The product can be further halogenated to obtain the difluoroboron pseudoindole electron acceptor of Formula I, wherein R 2 is halogen.

[0051] In some embodiments, the third reaction solvent is at least one of acetic anhydride, propionic anhydride, isopropionic anhydride, dichloromethane containing acetic anhydride, dichloroethane, acetone, chloroform, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene, chlorobenzene, xylene. In one or more preferred embodiments, the third reaction solvent is acetic anhydride.

[0052] In some embodiments, the third reaction solvent is at least one of acetic anhydride, propionic anhydride, isopropionic anhydride, dichloromethane containing acetic anhydride, dichloroethane, acetone, chloroform, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene, chlorobenzene, xylene. In one or more preferred embodiments, the third reaction solvent is acetic anhydride. 2 The halogenating agent for halogenating the difluoroborinic pseudoinodo electron acceptor with hydrogen is NCS (N-chlorosuccinimide), NBS (N-bromosuccinimide) or NIS (N-iodosuccinimide), and the fourth reaction solvent for the halogenation reaction is aprotic solvents such as dichloromethane, dichloroethane, chloroform, acetone, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, benzene, toluene, chlorobenzene, xylene, mesitylene, etc.

[0053] In some embodiments, the reaction temperature for reacting 2,3,3-trimethyl-3H-pseudoinodo or its derivative, or 2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine or its derivative with boron trifluoride etherate is 80-150℃, preferably, the reaction temperature is 110-130℃.

[0054] The application also provides applications of any one of the aforementioned fluoroboronic porphyrin fluorescent dyes, in particular, applications of fluoroboronic porphyrin fluorescent dyes in lipid droplet fluorescence imaging.

[0055] Compared with the prior art, the application has the following advantages and beneficial effects:

[0056] 1. The application provides a difluoroborinic pseudoinodo electron acceptor based on boron difluoride bridging, which is easy to synthesize and can be further condensed with fisher aldehyde or its derivative to obtain a series of fluoroboronic porphyrin fluorescent dyes covering far infrared to near infrared, effectively enriching the types of fluorescent dyes;

[0057] 2. The fluoroboronic porphyrin fluorescent dye prepared by the application has excellent photophysical properties, is easy to synthesize and easy to functionalize and modify, and has wide application value;

[0058] 3. The preparation method of the difluoroborinic pseudoinodo electron acceptor and the fluoroboronic porphyrin fluorescent dye provided by the application has a short synthesis path and mild reaction conditions, which is conducive to scale-up production;

[0059] 4. The fluoroboronic porphyrin fluorescent dye of the application can be used for lipid droplet fluorescence imaging, achieving the effects of efficient lipid droplet targeting and high signal-to-noise ratio lipid droplet imaging, as well as the effect of multi-color (multi-channel) fluorescent lipid droplet imaging, wherein the near-infrared lipid droplet probe has better application prospects in tissue and in vivo imaging. BRIEF DESCRIPTION OF DRAWINGS

[0060] The accompanying drawings, which are included to provide a further understanding of the embodiments of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

[0061] Figure 1 Flow chart for the preparation of the fluoroborondiphenylcynine fluorescent dyes in the embodiments of the present application;

[0062] Figure 2 Absorption (10 μΜ, left) and fluorescence spectra (0.5 μΜ, right) of the fluorescent dye BCy 1 in the present application;

[0063] Figure 3 Absorption (10 μΜ, left) and fluorescence spectra (0.5 μΜ, right) of the fluorescent dye BCy 2 in the present application;

[0064] Figure 4 Absorption (10 μΜ, left) and fluorescence spectra (0.5 μΜ, right) of the fluorescent dye BCy 3 in the present application;

[0065] Figure 5 Absorption (10 μΜ, left) and fluorescence spectra (0.5 μΜ, right) of the fluorescent dye BCy 19 in the present application;

[0066] Figure 6 Absorption (10 μΜ, left) and fluorescence spectra (0.5 μΜ, right) of the fluorescent dye BCy 4 in the present application;

[0067] Figure 7 Absorption (10 μΜ, left) and fluorescence spectra (0.5 μΜ, right) of the fluorescent dye BCy 7 in the present application;

[0068] Figure 8 Absorption (10 μΜ, left) and fluorescence spectra (0.5 μΜ, right) of the fluorescent dye BCy 8 in the present application;

[0069] Figure 9 Fluorescent imaging, bright field and merge of the fluorescent dyes BCy 1 and BCy 10 for co-labeling lipid droplets in the embodiments of the present application;

[0070] Figure 10 Fluorescent imaging, bright field and merge of the fluorescent dyes BCy 1 and BCy 11 for co-labeling lipid droplets in the embodiments of the present application;

[0071] Figure 11Fluorescence imaging, Bright Field and Merge of the fluorescent dyes BCy 10, BCy 11 for co-labeling lipid droplets in the specific embodiments of the present application;

[0072] Figure 12 Fluorescence imaging, Bright Field and Merge of the fluorescent dyes BCy 1, BCy 10, BCy 11 for co-labeling lipid droplets in the specific embodiments of the present application;

[0073] Figure 13 Fluorescence imaging and Merge of the fluorescent dyes BCy 1, BCy 10, BCy 11 and the commercial lipid droplet dye Bodipy 493 / 503 for labeling lipid droplets in the specific embodiments of the present application. DETAILED DESCRIPTION

[0074] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be given below in combination with embodiments and drawings, and the schematic embodiments of the present application and the description thereof are only used for explaining the present application, and do not limit the present application.

[0075] The terms "first", "second", and the like (e.g. first reaction solvent, second reaction solvent, first base, second base, and the like) used herein are only used for distinguishing corresponding components for the sake of clarity, and are not intended to limit any order or emphasize importance, and the like. In addition, the term "connection" used herein can be direct connection or indirect connection via other groups without special description.

[0076] All raw materials of the present application are not particularly limited in source, and can be purchased on the market or prepared according to conventional methods well known to those skilled in the art. All raw materials of the present application are not particularly limited in purity, and the present application preferably adopts analytical purity or conventional purity requirements in the field of fluorescent dyes. The grade and abbreviation of all raw materials of the present application belong to conventional grade and abbreviation in the art, and each grade and abbreviation is clear and explicit in the field of its relevant use. Those skilled in the art can purchase or prepare by conventional methods according to the grade, abbreviation and corresponding use.

[0077] The expression of the substituent groups of the present application is not particularly limited, and the expression well known to those skilled in the art is adopted. Those skilled in the art can correctly understand the meaning thereof according to the expression based on common sense.

[0078] Preparation of one, difluoroboron pseudoinode electron acceptor

[0079] The 2,3,3-trimethyl-3H-indolizine or its derivative, or 2,3,3-trimethyl-3H- pyrrolo[2,3-B]pyridine or its derivative is added to a third reaction solvent, and under inert gas protection, boron trifluoride ether is added, and the reaction obtains the difluoroboron indolizine electron acceptor of formula I, wherein R 2 is hydrogen; the difluoroboron indolizine electron acceptor of formula I, wherein R 2 is hydrogen, is halogenated to obtain R 2 is halogen.

[0080] In theory, the known 2,3,3-trimethyl-3H-indolizine or its derivative, and 2,3,3- trimethyl-3H-pyrrolo[2,3-B]pyridine or its derivative can be used to synthesize the difluoroboron indolizine electron acceptor, and determine the R 1 group of the difluoroboron indolizine electron acceptor. The following examples only list the preparation of preferred difluoroboron indolizine electron acceptors.

[0081] [Example 1]

[0082]

[0083] The 2,3,3-trimethyl-3H-indolizine (5.0 g, 31.4 mmol) is added to 50 mL of acetic anhydride, and under argon protection, boron trifluoride ether (5 mL, 41 mmol) is added. Then the reaction is stirred at 120°C for 6 hours, and after the solvent is removed by rotary evaporation under vacuum, the product is purified by silica gel column chromatography to obtain 2.8 g of light yellow product BFI 1, with a yield of 35%.

[0084] 1 H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 7.9 Hz, 1H), 7.39-7.30 (m, 2H), 7.26-7.21 (m, 1H), 5.71 (s, 1H), 2.28 (s, 3H), 1.43 (s, 6H).

[0085] 13 C NMR (101 MHz, CDCl3) δ 181.44, 179.57, 143.00, 140.66, 128.68, 125.88, 121.84, 116.21, 116.19, 116.17, 91.17, 91.13, 49.64, 24.49, 23.59.

[0086] [Example 2]

[0087]

[0088] To 2,3,3-trimethyl-3H-indole-5-carboxylic acid (6.1 g, 30 mmol) was added 60 mL of acetic anhydride and under argon protection, boron trifluoride diethyl ether (4.5 mL, 36 mmol) was added. The reaction was then stirred at 120 °C for 8 hours. After the solvent was removed by rotary evaporator under vacuum, the product was purified by silica gel column chromatography to give 3.3 g of light yellow product BFI 2 with a yield of 37%.

[0089] 1 H NMR (400 MHz, Dimethyl sulfoxide-d6) δ 13.05 (s, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 2.31 (s, 3H), 1.49 (s, 6H).

[0090] 13 C NMR (101 MHz, Dimethyl sulfoxide) δ 183.98, 181.88, 167.32, 146.41, 141.81, 131.08, 128.65, 124.42, 115.11, 92.83, 49.98, 24.12, 23.81.

[0091] Example 3

[0092]

[0093] To 1,1,2-trimethyl-1H-benzo[e]indole (5.0 g, 24 mmol) was added 35 mL of acetic anhydride and under argon protection, boron trifluoride diethyl ether (3.8 mL, 31 mmol) was added. The reaction was then stirred at 130 °C for 10 hours. After the solvent was removed by rotary evaporator under vacuum, the product was purified by silica gel column chromatography to give 2.39 g of light yellow product BFI 3 with a yield of 33%.

[0094] 1 H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.93 - 7.86 (m, 2H), 7.58 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.48 (ddd, J = 8.2, 6.8, 1.2 Hz, 1H), 5.81 (s, 1H), 2.32 (s, 3H), 1.69 (s, 6H).

[0095] 13C NMR (101 MHz, CDC13) δ 182.66, 179.10, 140.69, 133.96, 132.36, 130.21, 129.93, 128.47, 127.14, 125.13, 122.17, 115.58, 91.06, 51.25, 24.06, 23.66.

[0096] Example 4

[0097]

[0098] BFI 4 (1.65 g, 45% yield) was obtained as a light yellow solid by stirring the reaction at 120 °C for 8 h, removing the solvent by rotary evaporation under vacuum, and purifying by silica gel column chromatography after adding 5-chloro-2,3,3-trimethylindole (2.5 g, 13 mmol) into 20 mL of acetic anhydride and adding boron trifluoride etherate (2 mL, 16 mmol) under argon protection.

[0099] 1 H NMR (400 MHz, Chloroform-d) δ 7.56 (d, J = 8.2 Hz, 1H), 7.40 - 7.30 (m, 2H), 5.72 (d, J = 2.4 Hz, 1H), 2.30 (s, 3H), 1.45 (s, 6H).

[0100] 13 C NMR (101 MHz, CDC13) δ 181.37, 180.43, 142.43, 141.58, 131.63, 128.87, 122.54, 117.09, 117.07, 117.05, 91.25, 49.81, 24.45, 23.71.

[0101] Example 5

[0102]

[0103] BFI 5 (245 mg, 87% yield) was obtained as a white solid by stirring the reaction at room temperature overnight, removing the solvent by rotary evaporation under vacuum, and purifying by silica gel column chromatography after sequentially adding BFI 1 (750 mg, 3 mmol) and N-chlorosuccinimide (NCS, 479.7 mg, 3.6 mmol) into a reaction flask and then adding 15 mL of chloroform and dimethyl sulfoxide (45 μL, 0.6 mmol).

[0104] 1H NMR (400 MHz, CDC13) δ 7.66 (d, J = 7.8 Hz, 1H), 7.45 - 7.23 (m, 3H), 2.43 (s, 3H), 1.71 (s, 6H).

[0105] 13 C NMR (101 MHz, CDC13) δ 176.63, 176.22, 142.03, 141.65, 128.81, 126.72, 121.53, 116.62, 52.58, 22.10, 21.67.

[0106] Example 6

[0107]

[0108] To a mixture of BFI 2 (596 mg, 2 mmol) and CHCl3(12 mL) and N-methyl-2-pyrrolidone (0.6 mL) was added N-chlorosuccinimide (400 mg, 3 mmol) and dimethyl sulfoxide (38 μL, 0.5 mmol). The reaction was stirred at room temperature for 8 hours. After removing the solvent by rotary evaporator in vacuum, the product BFI 6 was purified by silica gel column chromatography to give 536.3 mg of white solid with a yield of 82%.

[0109] 1 H NMR (400 MHz, dimethyl sulfoxide-d6) δ 13.11 (s, 1H), 8.21 (s, 1H), 8.06 (d, J = 9.9 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 2.47 (s, 3H), 1.73 (s, 6H).

[0110] 13 C NMR (101 MHz, dimethyl sulfoxide) δ 178.95, 178.47, 167.16, 145.30, 142.43, 131.21, 129.56, 124.18, 115.64, 102.10, 52.82, 22.27, 21.77.

[0111] Example 7

[0112]

[0113] To a mixture of BFI 3 (449 mg, 1.5 mmol) and N-chlorosuccinimide (300 mg, 2.25 mmol) was added dimethylsulfoxide (32 L, 0.45 mmol) dissolved in CHCI3(8 mL) and stirred at room temperature for 5 hours. After the completion of the conversion, the solvent was removed by rotary evaporator under vacuum and purified by silica gel column chromatography to obtain 413.6 mg of yellow solid product BFI 7 with a yield of 83%.

[0114] 1 H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.90 (q, J = 8.8 Hz, 2H), 7.62 (t, J = 8.3 Hz, 1H), 7.52 (t, J = 8.1 Hz, 1H), 2.46 (s, 3H), 1.97 (s, 6H).

[0115] 13 C NMR (101 MHz, CDCI3) δ 177.69, 175.68, 139.50, 132.88, 130.42, 130.05, 127.72, 127.30, 125.48, 122.34, 115.54, 101.46, 54.18, 21.70, 20.72.

[0116] Example 8

[0117]

[0118] To a mixture of 5-iodo-2,3,3-trimethylindole (3.7 g, 13 mmol) was added 20 mL of acetic anhydride and then boron trifluoride etherate (2 mL, 16 mmol) under argon protection. The reaction was then stirred at 120 °C for 5 hours. After the completion of the conversion, the solvent was removed by rotary evaporator under vacuum and purified by silica gel column chromatography to obtain 2.09 g of yellow solid product BFI 8 with a yield of 42%.

[0119] 1 H NMR (400 MHz, Chloroform-d) δ 7.69 (dd, J = 8.3, 1.2 Hz, 1H), 7.64 (d, J = 1.6 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 5.71 (d, J = 2.4 Hz, 1H), 2.29 (s, 3H), 1.43 (s, 6H).

[0120] 13C NMR (101 MHz, CDC13) δ 181.01, 180.74, 143.01, 142.80, 137.70, 131.18, 117.91, 91.17, 89.94, 49.65, 24.47, 23.79.

[0121] Example 9

[0122]

[0123] BFI 9 (375 mg, 1.5 mmol) and NCS (333.3 mg, 2.5 mmol) were added to a mixture of dimethyl sulfoxide (30 μL, 0.3 mmol) and CHCI3(8 mL) and stirred at room temperature for 10 h. After removal of the solvent in vacuo using a rotary evaporator, the product BFI 10 was purified by column chromatography on silica gel to give 247 mg of product in 58% yield.

[0124] 1 H NMR (400 MHz, Chloroform-d) δ 8.49 (d, J = 4.7 Hz, 1H), 7.66 (d, J = 7.4 Hz, 1H), 7.17 (dd, J = 7.4, 5.1 Hz, 1H), 5.81 (s, 1H), 2.35 (s, 3H), 1.48 (s, 6H).

[0125] 13 C NMR (101 MHz, CDC13) δ 183.29, 182.77, 157.64, 149.02, 133.42, 130.31, 120.55, 91.49, 47.90, 24.35, 24.08.

[0126] Example 10

[0127]

[0128] BFI 9 (375 mg, 1.5 mmol) and NCS (333.3 mg, 2.5 mmol) were added to a mixture of dimethyl sulfoxide (30 μL, 0.3 mmol) and CHCI3(8 mL) and stirred at room temperature for 10 h. After removal of the solvent in vacuo using a rotary evaporator, the product BFI 10 was purified by column chromatography on silica gel to give 247 mg of product in 58% yield.

[0129] 1H NMR (400 MHz, Chloroform-d) δ 8.52 (dd, J = 5.1, 1.6 Hz, 1H), 7.66 (dd, J = 7.5, 1.6 Hz, 1H), 7.23 (dd, J = 7.5, 5.0 Hz, 1H), 2.50 (s, 3H), 1.76 (s, 6H).

[0130] 13 C NMR (101 MHz, CDCl3) δ 180.58, 177.78, 156.60, 149.34, 134.31, 130.07, 121.31, 101.76, 50.74, 22.38, 21.74.

[0131] 19 F NMR (377 MHz, Chloroform-d) δ -127.04 - -149.51 (m).

[0132] Example 11

[0133]

[0134] BFI 1 (100 mg, 0.4 mmol) was dissolved in dichloroethane (DCE, 4 mL), then NBS (142 mg, 0.8 mmol) was added and stirred at 60 °C for 1 hour. After removing the solvent by rotary evaporator under vacuum, 80 mg of product BFI 11 was obtained by silica gel column chromatography with a yield of 47%.

[0135] 1 H NMR (400 MHz, Chloroform-d) δ 7.69 (d, J = 7.9 Hz, 1H), 7.43 - 7.37 (m, 1H), 7.36 - 7.29 (m, 2H), 2.51 (s, 3H), 1.75 (s, 6H).

[0136] 13 C NMR (101 MHz, CDCl3) δ 177.34, 176.85, 141.83, 128.78, 126.77, 121.55, 116.59, 53.18, 24.02, 22.29.

[0137] Example 12

[0138]

[0139] BFI 1 (249 mg, 1 mmol) was added to DCE (10 mL) and N-iodosuccinimide (NIS, 450 mg, 2 mmol) was added. The reaction was stirred at 60 °C for 30 min. Then washed with sodium thiosulfate, extracted with dichloromethane and purified by silica gel column chromatography to give 242 mg of product BFI 12 with a yield of 65%.

[0140] 1 H NMR (400 MHz, Chloroform-d) δ 7.68 (d, J = 7.9 Hz, 1H), 7.40 - 7.33 (m, 1H), 7.32 - 7.27 (m, 2H), 2.63 (s, 3H), 1.73 (s, 6H).

[0141] 13 C NMR (101 MHz, CDCl3) δ 179.80, 177.45, 142.05, 141.53, 128.73, 126.80, 121.62, 116.46, 53.57, 28.21, 22.67.

[0142] Example 13

[0143]

[0144] BFI 6 (32.7 mg, 0.1 mmol) and tert-butyl N-(3-hydroxypropyl)carbamate (21 mg, 0.12 mmol) were dissolved in anhydrous dichloromethane (1 mL) and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride diisopropylethylamine (EDCI, 24.7 mg, 0.13 mmol) was added. The resulting mixture was stirred at room temperature for 5 h and purified by silica gel column chromatography to give 41.1 mg of product BFI 13 with a yield of 85%.

[0145] 1 H NMR (400 MHz, Chloroform-d) δ 8.11 - 8.06 (m, 1H), 8.01 (d, J = 1.5 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 4.80 (s, 1H), 4.42 (td, J = 6.2, 1.3 Hz, 2H), 3.29 (q, J = 6.5 Hz, 2H), 2.47 (d, J = 1.4 Hz, 3H), 2.03 - 1.94 (m, 2H), 1.75 (s, 6H), 1.44 (s, 9H).

[0146] 13C NMR (101 MHz, CDC13) δ 178.64, 178.01, 165.94, 155.92, 145.89, 141.75, 130.98, 128.35, 123.12, 116.17, 116.14, 116.12, 102.17, 79.35, 62.84, 52.53, 37.45, 29.32, 28.41, 22.01.

[0147] Preparation of fluoroborondipyrromethene fluorescent dyes

[0148] The synthetic route for preparing the fluoroboradipyrromethene fluorescent dye using the difluoroboron pseudoindole electron acceptor has two kinds, one is that the difluoroboron pseudoindole electron acceptor and the compound of formula III are added into a mixed solution composed of a first reaction solvent, a first base and a first acid to carry out a condensation reaction to obtain the fluoroboradipyrromethene fluorescent dye of formula II; the other is that the difluoroboron pseudoindole electron acceptor and the compound of formula IV are dissolved in a second reaction solvent, and a second base is added to carry out a condensation reaction to obtain the fluoroboradipyrromethene fluorescent dye of formula II.

[0149]

[0150]

Example 14

[0151]

[0152] BFI 1 (50.0 mg, 0.2 mmol) and 1,3,3-trimethyl-2-(formylmethylene) indolinium (52.3 mg, 0.26 mmol) were added to a mixed solution of toluene (2 mL), piperidine (60 μL) and acetic acid (30 μL) and dissolved at room temperature under argon protection. Then stirred at 100°C for 3 hours, after removing the solvent under reduced pressure, purified by silica gel column chromatography to obtain 78.5 mg of product BCy1, with a yield of 91%.

[0153] 1 H NMR (400 MHz, CDC13) δ 8.19 - 8.08 (m, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 7.0 Hz, 1H), 7.25 - 7.19 (m, 2H), 7.14 (t, J = 7.9 Hz, 1H), 6.98 (td, J = 7.4, 0.9 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H), 5.88 (d, J = 13.9 Hz, 1H), 5.57 - 5.49 (m, 2H), 3.25 (s, 3H), 1.65 (s, 6H), 1.43 (s, 6H).

[0154] 13C NMR (101 MHz, CDC13) δ 179.49, 172.20, 162.08, 145.37, 144.40, 143.73, 140.90, 140.68, 139.30, 128.52, 127.91, 127.87, 124.74, 122.65, 121.75, 121.70, 120.85, 117.89, 115.52, 106.76, 97.04, 91.14, 49.08, 46.41, 29.28, 28.31, 28.27, 25.23.

[0155] Example 15

[0156]

[0157] BFI 1 (50.0 mg, 0.2 mmol) and Fischer aldehyde (36.3 mg, 0.25 mmol) were dissolved in a mixture of toluene (2 mL), piperidine (60 μL) and acetic acid (30 μL) under argon protection. The mixture was then stirred at 110 °C for 2 hours. After removing the solvent under reduced pressure, 56.1 mg of product BCy 2 was obtained by silica gel column chromatography with a yield of 95%.

[0158] 1 H NMR (400 MHz, Chloroform-d) δ 7.70 (dd, J = 14.4, 11.8 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 6.4 Hz, 1H), 7.32 - 7.27 (m, 2H), 7.22 - 7.14 (m, 3H), 6.93 (t, J = 7.4 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 6.18 (dd, J = 13.8, 11.8 Hz, 1H), 5.97 (d, J = 14.4 Hz, 1H), 5.63 (s, 1H), 5.47 (d, J = 12.4 Hz, 1H), 3.18 (s, 3H), 1.61 (s, 6H), 1.44 (s, 6H).

[0159] 13 C NMR (101 MHz, CDC13) δ 179.49, 172.20, 162.08, 145.37, 144.40, 143.73, 140.90, 140.68, 139.30, 128.52, 127.91, 127.87, 124.74, 122.65, 121.75, 121.70, 120.85, 117.89, 115.52, 106.76, 97.04, 91.14, 49.08, 46.41, 29.28, 28.31, 28.27, 25.23.

[0160] Example 16

[0161]

[0162] BFI 2 (43.9 mg, 0.15 mmol) and 1,3,3-trimethyl-2- (formylmethylidene) indolin (42 mg, 0.2 mmol) were dissolved in a mixture of toluene (2 mL), piperidine (40 μL) and acetic acid (20 μL) under argon protection. The mixture was then stirred at 90 °C for 3 hours. After removing the solvent under reduced pressure, most of the impurities were removed by silica gel column chromatography to obtain BCy20.

[0163] BCy 20 (about 0.15 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), and diisopropylethylamine (49 μL, 0.3 mmol) was added. The mixture was stirred at room temperature for 5 hours, and after removing the solvent under vacuum using a rotary evaporator, the product was purified by silica gel column chromatography to obtain 62.7 mg of blue product BCy 3 with a yield of 75%.

[0164] 1 H NMR (400 MHz, Chloroform-d) δ 8.26 (t, J = 13.4 Hz, 1H), 8.15 (dd, J = 8.4, 1.8 Hz, 1H), 8.00 (d, J = 1.7 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.29 - 7.22 (m, 3H), 7.04 (t, J = 7.3 Hz, 1H), 6.85 - 6.80 (m, 1H), 5.89 (d, J = 13.6 Hz, 1H), 5.62 (d, J = 13.0 Hz, 1H), 5.60 (s, 1H), 3.30 (s, 3H), 2.92 (s, 4H), 1.67 (s, 6H), 1.46 (s, 6H).

[0165] 13 C NMR (101 MHz, CDC13) δ 178.95, 174.45, 169.44, 167.82, 161.77, 150.12, 143.66, 143.33, 140.80, 139.87, 132.48, 128.04, 124.04, 122.34, 121.96, 119.61, 114.62, 114.15, 107.82, 96.75, 91.36, 48.29, 47.55, 29.70, 28.71, 25.81, 25.72.

[0166] Example 17

[0167] Example 17 The procedure for synthesizing BCy 20 was the same as that for Example 16, except that intermediate BCy 20 (about 0.15 mmol) and N-hydroxysuccinimide (NHS) were dissolved in anhydrous dichloromethane (2 mL), and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride diisopropylethylamine (EDCI, 57 mg, 0.3 mmol) was added. The resulting mixture was stirred at room temperature for 5 hours, and purified by silica gel column chromatography to give 65.1 mg of blue product BCy 3 in 77% yield.

[0168] [Example 18]

[0169]

[0170] BFI 6 (65.4 mg, 0.2 mmol) and 1,3,3-trimethyl-2-(formylmethylidene) indolinium (52.3 mg, 0.26 mmol) were dissolved in a mixture of toluene (3 mL), piperidine (60 μL), and acetic acid (30 μL) under argon protection. The mixture was then stirred at 60 °C for 5 hours. After removing the solvent under reduced pressure, most of the impurities were removed by silica gel column chromatography to give BCy 4.

[0171] BCy 4 (about 0.2 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), and diisopropylethylamine (49 μL, 0.3 mmol) was added. The resulting mixture was stirred at room temperature for 5 hours, and then the solvent was removed. Purification by silica gel column chromatography gave 103.9 mg of blue product BCy 5 in 85% yield.

[0172] 1 H NMR (400 MHz, CDC13) δ 8.33 (t, J = 13.4 Hz, 1H), 8.04 (dd, J = 8.4, 1.7 Hz, 1H), 7.89 (d, J = 1.7 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.27 - 7.20 (m, 2H), 7.03 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 8.1 Hz, 1H), 6.41 (d, J = 13.4 Hz, 1H), 5.76 (d, J = 13.3 Hz, 1H), 3.31 (s, 3H), 2.84 (s, 4H), 1.67 (s, 6H), 1.61 (s, 6H).

[0173] 13C NMR (101 MHz, CDC13) δ 172.61, 170.20, 170.13, 169.41, 161.67, 149.43, 146.74, 143.26, 141.37, 140.21, 132.41, 128.20, 123.67, 123.25, 122.07, 119.78, 114.51, 109.62, 108.51, 100.11, 98.59, 51.08, 48.14, 30.01, 28.71, 25.71, 22.94.

[0174] Example 19

[0175]

[0176] BFI 6 (65.4 mg, 0.2 mmol) and Fischer aldehyde (59.0 mg, 0.26 mmol) were dissolved in a mixture of toluene (3 mL), piperidine (60 μL) and acetic acid (30 μL) under argon protection. The mixture was then stirred at 50 °C for 2 hours. After removing the solvent under reduced pressure, most of the impurities were removed by silica gel column chromatography to obtain BCy 6.

[0177] BCy 6 (about 0.2 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), and diisopropylethylamine (49 μL, 0.3 mmol) was added. The mixture was then stirred at room temperature for 5 hours, and after removing the solvent, it was purified by silica gel column chromatography to obtain 98.7 mg of blue product BCy 7 with a yield of 78%.

[0178] 1 H NMR (400 MHz, CDC13) δ 8.12 (dd, J = 8.4, 1.7 Hz, 1H), 8.01 - 7.89 (m, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.55 (t, J = 13.1 Hz, 2H), 7.30 - 7.21 (m, 3H), 7.03 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 6.54 (d, J = 13.9 Hz, 1H), 6.34 (t, J = 12.0 Hz, 1H), 5.63 (d, J = 12.8 Hz, 1H), 3.29 (s, 3H), 2.92 (s, 4H), 1.75 (s, 7H), 1.64 (s, 6H).

[0179] 13C NMR (101 MHz, Dimethyl sulfoxide) δ 168.86, 165.28, 164.61, 161.10, 156.84, 146.57, 144.31, 141.61, 138.99, 136.86, 134.99, 127.63, 123.35, 118.97, 118.63, 117.49, 117.16, 115.61, 110.18, 107.32, 103.02, 96.25, 93.78, 46.67, 42.54, 24.94, 23.50, 20.96, 18.05.

[0180] Example 20

[0181]

[0182] To a mixture of BFI 6 (49 mg, 0.15 mmol) and merocyanine (99 mg, 0.2 mmol) in dichloromethane (2 mL) was added diisopropylethylamine (59 μL, 0.33 mmol) under argon. The mixture was then stirred at 50 °C for 90 min. After cooling to room temperature, N,N'-disuccinimidyl carbonate (76 mg, 0.3 mmol) and diisopropylethylamine (33 μL, 0.2 mmol) were added to the solution. The mixture was stirred at room temperature for 12 h, and then the solvent was removed. Purification by silica gel column chromatography gave 45.5 mg of product BCy 8 in 46% yield.

[0183] 1 H NMR (400 MHz, Chloroform-d) δ 8.14 (dd, J = 8.4, 1.7 Hz, 1H), 8.00 (d, J = 1.7 Hz, 1H), 7.83 (dd, J = 14.1, 12.1 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.25 - 7.16 (m, 3H), 7.01 (dd, J = 13.9, 11.6 Hz, 1H), 6.95 (t, J = 7.4 Hz, 1H), 6.73 (d, J = 7.8 Hz, 1H), 6.65 (d, J = 14.1 Hz, 1H), 6.41 (t, J = 13.0 Hz, 1H), 6.22 (dd, J = 13.7, 11.7 Hz, 1H), 5.53 (d, J = 12.5 Hz, 1H), 3.22 (s, 3H), 2.92 (s, 4H), 1.75 (s, 6H), 1.62 (s, 6H).

[0184] 13C NMR (101 MHz, CDC13) δ 174.71, 169.85, 169.34, 162.54, 161.53, 149.78, 149.36, 148.75, 144.23, 141.85, 140.10, 139.35, 132.37, 127.98, 126.13, 124.63, 123.76, 121.76, 121.23, 120.93, 115.38, 115.12, 101.63, 98.04, 51.73, 46.62, 29.43, 28.32, 25.72, 22.65.

[0185] Example 21

[0186]

[0187] BFI 5 (56.6 mg, 0.2 mmol) and 1,3,3-trimethyl-2-(formylmethylidene) indolin (52.3 mg, 0.26 mmol) were dissolved in a mixture of toluene (2 mL), piperidine (60 μL) and acetic acid (30 μL) under argon protection. The mixture was then stirred at 50 °C for 2 hours. After removing the solvent under reduced pressure, 78.5 mg of product BCy10 was obtained by silica gel column chromatography with a yield of 91%.

[0188] 1 H NMR (400 MHz, CDC13) δ 8.28 (t, J = 13.3 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.27 - 7.22 (m, 3H), 7.17 (d, J = 7.7 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.48 (d, J = 13.6 Hz, 1H), 5.72 (d, J = 13.1 Hz, 1H), 3.30 (s, 3H), 1.71 (s, 6H), 1.67 (s, 6H).

[0189] 13 C NMR (101 MHz, CDC13) δ 173.37, 168.51, 167.65, 143.96, 143.71, 143.23, 141.25, 139.91, 128.48, 128.02, 124.64, 122.27, 121.95, 121.29, 115.21, 110.48, 107.79, 99.17, 97.54, 51.71, 47.52, 29.69, 28.73, 22.82.

[0190] Example 22

[0191]

[0192] BFI 5 (34.0 mg, 0.12 mmol) and Fischer aldehyde (36.3 mg, 0.16 mmol) were dissolved in a mixed solution of toluene (2 mL), piperidine (40 μL), and acetic acid (20 μL) under argon protection. The mixture was then stirred at 50 °C for 2 hours, desolventized under reduced pressure, and purified by silica gel column chromatography (PE:EA 11:1–9:1) to give 56.1 mg of product BCY 11, with a yield of 95%.

[0193] 1H NMR(400MHz,Chloroform-d)δ7.83(dd,J=14.1,12.0Hz,1H),7.61(d,J=7.8Hz,1H),7.40(d,J=13.2Hz,1H),7.32(td,J=7.6,1.4Hz,1H),7.30–7.16(m,5H ),6.97(td,J=7.4,0.9Hz,1H),6.74(d,J=7.8Hz,1H),6.54(d,J=14.2Hz,1H) ,6.29(t,1H),5.54(d,J=12.6Hz,1H),3.23(s,3H),1.72(s,6H),1.62(s,6H).

[0194] 13 C NMR (101MHz, CDCl3) δ174.26,168.13,163.73,148.70,144.25,143.39,143.06,141.62,139.59,128.68,128.0 8,125.33,123.40,121.93,121.50,115.75,113.32,107.28,100.40,97.77,52.12,46.89,29.56,28.39,22.80.

[0195]

Example 23

[0196]

[0197] BFI 7 (50 mg, 0.15 mmol) and 1,3,3-trimethyl-2-(formylmethylene)indoline (40.3 mg, 0.2 mmol) were dissolved in a mixed solution of toluene (2 mL), piperidine (50 μL), and acetic acid (25 μL) under argon protection. The solution was then stirred at room temperature for 2 hours. After solvent removal under reduced pressure, the solution was purified by PE:EA (11:1–9:1) silica gel column chromatography to give 68.5 mg of product BCY 12, in 89% yield.

[0198] 1 H NMR (400 MHz, CDC13) δ 8.32 (t, J = 13.4 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.89 (s, 2H), 7.59 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.33 - 7.26 (m, 2H), 7.06 (t, J = 7.4 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 13.6 Hz, 1H), 5.77 (d, J = 13.1 Hz, 1H), 3.34 (s, 3H), 2.03 (s, 6H), 1.71 (s, 6H).

[0199] 13 C NMR (101 MHz, CDC13) δ 174.70, 168.16, 167.47, 143.75, 143.69, 140.78, 139.91, 133.41, 132.08, 130.01, 129.95, 128.07, 128.01, 126.79, 124.32, 122.21, 122.20, 121.95, 115.31, 110.62, 107.74, 99.01, 97.57, 53.41, 47.50, 29.68, 28.74, 21.30.

[0200] Example 24

[0201]

[0202] BFI 7 (50.0 mg, 0.15 mmol) and Fischer aldehyde (45.4 mg, 0.2 mmol) were dissolved in a mixed solution of toluene (2 mL), piperidine (50 μL) and acetic acid (25 μL) under argon protection. Then the mixture was stirred at 50 °C for 2 hours. After removing the solvent under reduced pressure, 79.0 mg of product BCy 13 was obtained by silica gel column chromatography with a yield of 96%.

[0203] 1H NMR (400 MHz, Chloroform-d) δ 8.04 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.93 - 7.81 (m, 3H), 7.60 (t, J = 7.0 Hz, 1H), 7.52 - 7.41 (m, 2H), 7.29 - 7.22 (m, 2H), 7.00 (t, J = 7.4 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.62 (d, J = 14.2 Hz, 1H), 6.34 (t, J = 9.0 Hz, 1H), 5.57 (d, J = 12.5 Hz, 1H), 3.26 (s, 3H), 2.03 (s, 6H), 1.66 (s, 6H).

[0204] 13 C NMR (101 MHz, CDC13) δ 175.38, 167.64, 163.48, 148.30, 144.17, 143.05, 140.49, 139.49, 133.98, 132.31, 130.12, 129.98, 127.98, 127.95, 126.91, 124.63, 123.37, 122.28, 121.81, 121.35, 115.42, 113.35, 107.13, 100.16, 97.65, 53.68, 46.75, 29.44, 28.28, 21.20.

[0205] Example 25

[0206]

[0207] To a solution of BFI 5 (112 mg, 0.225 mmol) and merocyanine (42.5 mg, 0.15 mmol) in dichloromethane was added diisopropylethylamine (32 μL). The reaction mixture was stirred at 50 °C for 90 min. After cooling to room temperature, purification by flash chromatography afforded 32 mg of product BCy 14 in 42% yield.

[0208] 1H NMR (400 MHz, Chloroform-d) δ 7.72 (dd, J = 14.3, 11.9 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.38 - 7.29 (m, 1H), 7.28 (d, J = 6.2 Hz, 2H), 7.26 - 7.17 (m, 3H), 7.21 - 7.07 (m, 3H), 6.96 - 6.84 (m, 2H), 6.69 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 14.6 Hz, 2H), 6.43 - 6.32 (m, 1H), 6.25 - 6.13 (m, 1H), 5.46 (d, J = 12.3 Hz, 1H), 3.18 (s, 3H), 1.72 (s, 7H), 1.61 (s, 6H).

[0209] 13 C NMR (101 MHz, CDC13) δ 174.75, 167.56, 160.88, 147.05, 146.57, 144.53, 142.76, 141.70, 139.20, 137.62, 128.64, 127.87, 126.34, 125.63, 124.63, 121.69, 121.39, 120.61, 116.24, 115.97, 106.64, 101.03, 97.47, 52.20, 46.25, 29.28, 28.34, 22.57.

[0210] Example 26

[0211]

[0212] BFI 9 (17.0 mg, 0.06 mmol) and 1,3,3-trimethyl-2-(formylidene) indolinium (16.0 mg, 0.08 mmol) were dissolved in a mixture of toluene (1 mL), piperidine (20 μL) and acetic acid (10 μL) under argon protection. The mixture was then stirred at 50 °C for 2 hours. After removing the solvent under reduced pressure, the product BCy 15 was obtained by purification on a silica gel column with dichloromethane:EA (20:1) as eluent, with a yield of 20.7 mg, 74%.

[0213] 1H NMR (400 MHz, Chloroform-d) δ 8.48 - 8.34 (m, 2H), 7.51 (dd, J = 7.4, 1.6 Hz, 1H), 7.37 - 7.28 (m, 2H), 7.11 (t, J = 7.7 Hz, 1H), 7.03 (dd, J = 7.4, 5.1 Hz, 1H), 6.91 (d, J = 7.9 Hz, 1H), 6.51 (d, J = 13.4 Hz, 1H), 5.81 (d, J = 13.3 Hz, 1H), 3.39 (s, 3H), 1.74 (s, 6H), 1.69 (s, 6H).

[0214] 13 C NMR (101 MHz, CDC13) δ 172.16, 170.79, 169.78, 158.02, 148.78, 146.59, 143.48, 140.31, 134.04, 129.39, 128.25, 123.15, 122.19, 118.99, 110.04, 108.47, 99.60, 98.43, 49.53, 48.18, 30.06, 28.84, 22.79.

[0215] Example 27

[0216]

[0217] BFI 9 (17.1 mg, 0.06 mmol) and merocyanine (17.1 mg, 0.06 mmol) were dissolved in 0.8 mL of dichloromethane, and then 15.6 μL of DIEPA (0.09 mmol) was added. The reaction mixture was stirred at 50 °C for 50 min. Purification by PE:EA (4:1-2:1) silica gel column chromatography yielded 22.5 mg of product BCy 16 with a yield of 72%.

[0218] 1H NMR (400 MHz, CDC13) δ 8.44 (d, J = 6.6 Hz, 1H), 7.84 (dd, J = 13.9, 12.3 Hz, 1H), 7.54 (d, J = 8.9 Hz, 1H), 7.21 (dd, J = 8.9, 6.2 Hz, 3H), 7.08 (dd, J = 7.4, 5.1 Hz, 1H), 7.01 - 6.90 (m, 2H), 6.72 (d, J = 7.9 Hz, 1H), 6.64 (d, J = 14.2 Hz, 1H), 6.38 (dd, J = 14.0, 12.0 Hz, 1H), 6.20 (dd, J = 13.6, 11.9 Hz, 1H), 5.50 (d, J = 12.4 Hz, 1H), 3.21 (s, 3H), 1.73 (s, 6H), 1.62 (s, 6H).

[0219] 13 C NMR (101 MHz, CDC13) δ 174.32, 170.35, 162.13, 157.41, 149.68, 149.04, 148.75, 144.31, 139.49, 139.33, 134.33, 129.52, 127.93, 126.16, 124.62, 121.75, 121.05, 119.83, 115.37, 106.95, 100.95, 97.86, 50.08, 46.53, 29.39, 28.35, 22.35.

[0220] Example 28

[0221]

[0222] BFI 9 (22.7 mg, 0.08 mmol) and aldehyde (22.7 mg, 0.1 mmol) were dissolved in a mixture of toluene (2 mL), piperidine (30 μL) and acetic acid (15 μL) under argon protection. The mixture was then stirred at 50 °C for 2 hours. The solvent was removed under reduced pressure and purified by silica gel chromatography with PE:EA (4:1-2:1) to give 35.3 mg of product BCy 17 with a yield of 90%.

[0223] 1H NMR (400 MHz, Chloroform-d) δ 8.42 (dd, J = 5.0, 1.6 Hz, 1H), 7.99 (dd, J = 13.7, 12.4 Hz, 2H), 7.56 - 7.48 (m, 2H), 7.23 (d, J = 7.5 Hz, 2H), 7.06 - 6.99 (m, 2H), 6.79 (d, J = 7.8 Hz, 1H), 6.53 (d, J = 14.0 Hz, 1H), 6.31 (t, J = 12.8 Hz, 1H), 5.60 (d, J = 12.8 Hz, 1H), 3.27 (s, 3H), 1.73 (s, 6H), 1.63 (s, 6H).

[0224] 13 C NMR (101 MHz, CDC13) δ 173.28, 170.68, 165.57, 157.76, 151.57, 149.02, 146.11, 143.93, 139.82, 134.22, 129.50, 128.16, 123.22, 122.16, 122.01, 119.49, 112.20, 107.74, 100.38, 98.33, 49.91, 47.30, 29.74, 28.34, 22.62.

[0225] Example 29

[0226]

[0227] BFI 9 (14.2 mg, 0.05 mmol) and merocyanine (37.5 mg, 0.08 mmol) were dissolved in 0.5 mL dichloromethane, and 14 μL DIEPA (0.08 mmol) was added. The reaction mixture was stirred at 50 °C for 50 min. After cooling, it was passed through a column. Spotted on plates D:E = 15:1, passed through a column, D:E = 12:1 to give product BCy 18, 14.7 mg, 55% yield.

[0228] 1 H NMR (400 MHz, Chloroform-d) δ 8.43 - 8.27 (m, 2H), 7.49 (d, J = 7.3 Hz, 1H), 7.30 - 7.17 (m, 2H), 7.09 (t, J = 7.3 Hz, 1H), 7.04 - 6.93 (m, 1H), 6.87 (d, J = 7.7 Hz, 1H), 6.46 (d, J = 13.3 Hz, 1H), 5.81 (d, J = 13.3 Hz, 1H), 3.82 - 3.59 (m, 2H), 1.72 (s, 6H), 1.67 (s, 10H), 1.46 - 1.19 (m, 8H), 0.90 (d, J = 6.2 Hz, 3H).

[0229] 13 C NMR (101 MHz, CDC13) δ 171.78, 170.54, 169.29, 157.99, 148.67, 146.70, 142.85, 140.37, 133.87, 129.23, 128.12, 123.05, 122.10, 118.78, 109.40, 108.65, 99.36, 98.25, 49.34, 48.20, 43.30, 31.48, 28.77, 26.81, 26.71, 22.70, 22.52, 14.00.

[0230] Example 30

[0231]

[0232] BFI 2 (43.9 mg, 0.15 mmol) and aldehyde (40.8 mg, 0.18 mmol) were dissolved in a mixture of toluene (2 mL), piperidine (40 μL) and acetic acid (20 μL) under argon protection. The mixture was then stirred at 80 °C for 3 hours. After removing the solvent under reduced pressure, most of the impurities were removed by silica gel column chromatography to obtain the product.

[0233] BCy 21 (about 0.15 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were first dissolved in anhydrous dichloromethane (2 mL), and then diisopropylethylamine (49 μL, 0.3 mmol) was added. The mixture was stirred at room temperature for 6 hours, and then the solvent was removed. Purification by silica gel column chromatography yielded 65.5 mg of product BCy 19 with a yield of 93%.

[0234] 1 H NMR (400 MHz, Chloroform-d) δ 8.15 (dd, J = 8.4, 1.7 Hz, 1H), 8.01 (d, J = 1.6 Hz, 1H), 7.81 (dd, J = 14.2, 11.9 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.42 (t, J = 13.1 Hz, 1H), 7.25 - 7.17 (m, 2H), 6.97 (t, J = 7.4 Hz, 1H), 6.74 (d, J = 7.8 Hz, 1H), 6.22 (t, J = 13.2 Hz, 1H), 5.98 (d, J = 14.2 Hz, 1H), 5.68 (s, 1H), 5.53 (d, J = 12.6 Hz, 1H), 3.23 (s, 3H), 2.92 (s, 4H), 1.62 (s, 6H), 1.47 (s, 6H).

[0235] 13 C NMR (101 MHz, CDC13) δ 179.82, 174.22, 169.41, 163.81, 161.67, 149.75, 147.98, 144.09, 143.49, 140.96, 139.45, 132.45, 127.98, 124.09, 122.53, 121.81, 121.47, 120.19, 116.86, 115.02, 107.21, 97.57, 92.09, 48.64, 46.79, 29.45, 28.26, 25.72, 25.54.

[0236] Photophysical properties testing of fluoroborondipyrromethene fluorescent dyes

[0237] The stock solutions of BCy 1, 2, 3, 4, 7, 8, 12 dyes prepared in the examples were added to dichloromethane, tetrahydrofuran, methanol, dimethyl sulfoxide, and their optical properties, including the maximum absorption wavelength (λ Abs ), molar absorption coefficient (ε), maximum emission wavelength (λ em ), Stokes shift (Δλ), quantum yield, brightness (φ x ε) were measured by UV-Vis spectrophotometer or fluorescence spectrometer.

[0238] The absorption and fluorescence spectra of BCy fluorescent dyes in dichloromethane, tetrahydrofuran, methanol, dimethyl sulfoxide are shown in Figures 2 to 8 The optical properties of BCy fluorescent dyes in dichloromethane and dimethyl sulfoxide are shown in Table 1:

[0239] Table 1

[0240]

[0241] In combination Figures 2 to 8 , and Table 1, it can be seen that all BCy dyes show strong fluorescence. The absorbance (maximum absorption wavelength) and fluorescence maximum (maximum emission wavelength) of BCy 1 are 564 nm and 602 nm, respectively, with a Stokes shift of 38 nm, and its molar absorption coefficient and quantum yield are 0.92 x 10 5 M -1 cm -1 and 0.26, respectively. The maximum absorption peak of BCy 2 is at 593 nm, with a fluorescence quantum yield of 0.33 and a molar absorption coefficient of 0.76 x 10 5 M -1 cm -1 BCy 4 and BCy 7 have the most significant fluorescence and the highest molar absorption coefficient, and have ultra-high brightness, up to 1.0 x 10 5 cm -1M -1 Meanwhile, the absorption spectrum becomes more sharp, and the red shift of the maximum emission peak is larger. In addition, BCy 8 also exhibits excellent photophysical properties, with an emission wavelength of more than 800 nm.

[0242] Fluoroboronic cyanine fluorescent dyes not only have excellent photophysical properties, but also their photophysical properties can be fine-tuned by controlling the strength of the electron acceptor and the degree of π conjugation in the conjugated system. As shown in the ultraviolet and fluorescence spectra and fluorescence data table, when increasing the electron-withdrawing group (ester group and chlorine atom), both the absorption and fluorescence wavelengths are red-shifted, and the Stokes shift and peak width are reduced. For example, the maximum absorption wavelength and the maximum fluorescence wavelength of BCy 2 in dimethyl sulfoxide are 593 nm and 700 nm, and the Stokes shift is 107 nm; after introducing an ester group on the R 1 group of BCy 2, the maximum absorption wavelength and the maximum fluorescence wavelength of BCy 19 are 641 nm and 706 nm, and the Stokes shift is 65 nm. Finally, on the basis of BCy 19, an electron-withdrawing chlorine atom is introduced to obtain BCy 7, with a maximum absorption wavelength and a maximum fluorescence wavelength of 717 nm and 742 nm, and a Stokes shift of 25 nm.

[0243] Use of fluoroborondipyrromethene fluorescent dyes for lipid droplets

[0244] The BCy dyes prepared in the present application have the characteristics of high fat solubility in chemical structure, can pass through the cell membrane well, and therefore have good lipid droplet targeting ability. In addition, since the BCy dyes can produce orange to near-infrared fluorescence, they can be used for multi-color (multi-channel) high signal-to-noise ratio lipid droplet imaging.

[0245] Take BCy 1, BCy 10 and BCy 11 as examples for multi-channel imaging of lipid droplets.

[0246] A549 cells were incubated with 200 nM fluoroboronic cyanine fluorescent dyes BCy 1, BCy 10 in a confocal dish for 20 min, and then imaged with a confocal microscope. The excitation wavelength λ ex = 520 nm, and the emission wavelength λ em = 540-595 nm for BCy 1; the excitation wavelength λ ex = 560 nm, and the emission wavelength λ em = 595-650 nm for BCy 10; the scale bar is 20 μm. The imaging results are shown in Figure 9 .

[0247] A549 cells were incubated with 200 nM fluoroboronic cyanine fluorescent dyes BCy 1, BCy 11 in a confocal dish for 20 min, and then imaged with a confocal microscope. The excitation wavelength λ ex = 520 nm, and the emission wavelength λem = 540-595 nm; BCy 11 excitation wavelength λ ex = 620 nm, emission wavelength λ em = 650-710 nm; scale bar is 20 μm. The imaging results are shown in Figure 10

[0248] A549 cells were co-incubated with 200 nM fluoroboronic acid derivative fluorochrome BCy 10, BCy 11 in a confocal dish for 20 min, and then imaged with a confocal microscope. Among them, BCy 10 excitation wavelength λ ex = 570 nm, emission wavelength λ em = 580-630 nm; BCy 11 excitation wavelength λ ex = 640 nm, emission wavelength λ em = 650-710 nm; scale bar is 20 μm. The imaging results are shown in Figure 11

[0249] A549 cells were co-incubated with 200 nM fluoroboronic acid derivative fluorochrome BCy 1, BCy 10 and BCy 11 in a confocal dish for 20 min, and then imaged with a confocal microscope. Among them, BCy 1 excitation wavelength λ ex = 520 nm, emission wavelength λ em = 540-595 nm; BCy 10 excitation wavelength λ ex = 560 nm, emission wavelength λ em = 595-640 nm; BCy 11 excitation wavelength λ ex = 620 nm, emission wavelength λ em = 650-730 nm; scale bar is 20 μm. The imaging results are shown in Figure 12

[0250] A549 cells were co-incubated with fluoroboronic acid derivative fluorochrome BCy 1 (200 nM), BCy 10 (1 μM), BCy 11 (1 μM) and commercial specific lipid droplet dye Bodipy 493 / 503 (5 μM) in a confocal dish for 20 min, and then imaged with a confocal microscope. Among them, Bodipy 493 / 503 excitation wavelength λ ex = 488 nm, emission wavelength λ em = 495-550 nm; BCy 1 excitation wavelength λ ex = 520 nm, emission wavelength λ em = 560-650 nm; BCy 10 excitation wavelength λ ex = 560 nm, emission wavelength λ em = 595-680 nm; BCy 11 excitation wavelength λ​​​ex = 620 nm, emission wavelength λ em = 650-750 nm; scale bar, 10 μm. Imaging results are shown in Figure 13 Figure 1

[0251] Thus, the present application realizes multi-color high signal-to-noise ratio and high specificity imaging of lipid droplets.

[0252] The above detailed description further explains the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the protection scope of the present application.​

Claims

1. A difluoroboron pseudoindole electron acceptor characterized in that, having a structural formula of Formula I: In formula I, X is selected from CH or N, R 1 is selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, hydroxy, amino, carboxyl, carboxylate, sulfonic acid, methoxy, amide, R 2 is selected from the group consisting of hydrogen or halogen.

2. The difluoroboroindolizine electron acceptor according to claim 1, wherein said R 1 selected from the group consisting of hydrogen, halogen, methyl, ethyl, ethenyl, ethynyl, trifluoromethyl, phenyl, hydroxyl, carboxyl, carboxylate, sulfonic acid, methoxyl, amide.

3. A difluoroborane pseudoindole electron acceptor characterized by, The difluoroboron pseudoindole electron acceptor is selected from the group consisting of compounds of:

4. A fluoroboronic fluorochrome characterized in that, having a structural formula of Formula II: In formula II, X is selected from CH or N, R 1 and R 3 each independently is selected from hydrogen, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, hydroxyl, amino, carboxyl, sulfonic acid, methoxyl, carboxylate, amide, R 2 is selected from hydrogen or halogen, R 4 is selected from C1-C 16 chained alkyl, polyethylene glycol group, C1-C 16 alkyl carboxylic acid, C1-C5 alkyl sulfonic acid and salts thereof, C1-C4 alcohol, n = 1-4.

5. The fluoroborondipyrromethene fluorescent dye of claim 4, wherein The R 1 selected from hydrogen, halogen, methyl, ethyl, ethenyl, ethynyl, trifluoromethyl, phenyl, hydroxy, carboxy, sulfonic acid, methoxy, formic acid ester, amide, R 3 selected from hydrogen or halogen, R 4 selected from C1-C4 chain alkyl, polyethylene glycol group, C1-C 16 alkyl carboxylic acid.

6. A fluoroborondipyrromethene fluorescent dye characterized by, The fluoroborate-fluorochrome is selected from the group consisting of the following compounds:

7. A method for the preparation of a fluoroborondipyrromethene fluorescent dye, characterized in that, The fluoroboroderivative fluorescein dye of any one of claims 4-6 is prepared using the difluoroboron pseudoindole electron acceptor of any one of claims 1-3, the method comprising the steps of: condensing the difluoroboron pseudoindole electron acceptor of Formula I with a compound of Formula III in a mixture solution of a first reaction solvent, a first base, and a first acid to obtain the fluoroboroderivative fluorescein dye of Formula II; or condensing the difluoroboron pseudoindole electron acceptor of Formula I with a compound of Formula IV in a second reaction solvent, adding a second base to obtain the fluoroboroderivative fluorescein dye of Formula II; Formula III: Formula IV: wherein R 3 selected from the group consisting of hydrogen, halogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, phenyl, hydroxy, amino, carboxy, sulfonic acid, methoxy, carboxylate, amide, R 4 selected from the group consisting of C1-C 16 chain alkyl, polyethylene glycol group, C1-C 16 alkyl carboxylic acid, C1-C5 alkyl sulfonic acid and salts thereof, C1-C4 alcohol, n = 1-4.

8. A method of preparing a fluoroborondiphenylene chromophore fluorescent dye according to claim 7, characterized in that, The method for preparing the difluoroboron pseudoindole electron acceptor comprises the steps of: dissolving 2,3,3-trimethyl-3H-indolium or 2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine in a third reaction solvent and adding boron trifluoride etherate to yield R 2 is a difluoroboronic indolium electron acceptor; halogenated to give a difluoroboratabenzazoline electron acceptor of Formula I wherein R 2 is hydrogen. The difluoroboratabenzazoline electron acceptor of Formula I wherein R 2 is halogen.

9. The method for preparing a fluorinated boron cyanine fluorescent dye according to claim 7, characterized in that, The first reaction solvent or the second reaction solvent is at least one of dichloromethane, dichloroethane, chloroform, acetone, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, benzene, toluene, n-butanol, isopropanol, ethanol, methanol, chlorobenzene, xylene, mesitylene; the first base or the second base is at least one of piperidine, pyrrolidine, diethylamine, triethylamine, diisopropylaminoethylamine, pyridine, acetate, carbonate, bicarbonate, potassium tert-butoxide; and the first acid is at least one of acetic acid, formic acid, propionic acid, butyric acid, methanesulfonic acid, benzoic acid.

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