Novel cyanine near-infrared fluorescent dye as well as preparation method and application thereof

Abstract

The invention discloses a novel cyanine near-infrared fluorescent dye as well as a preparation method and application thereof, and belongs to the technical field of small organic molecule fluorescent probes. The method comprises the following steps: dissolving Fisher's aldehyde in an ethanol solution containing sodium hydroxide, placing the solution in a round-bottom flask, raising the temperature of an oil bath to 80 DEG C, dissolving a raw material R in an ethanol solution, placing the solution in a constant-pressure dropping funnel, slowly dropwise adding the solution into the round-bottom flask, carrying out a Knoevenagel reaction, after the reaction is finished, directly carrying out suction filtration on a solid in a reaction flask, and washing with dichloromethane; the novel near-infrared dye platform disclosed by the invention has the advantages of high molar absorption coefficient, low background interference, good biocompatibility and the like, and the dye can be used as an HClO recognition probe under the condition of no modification, so that the purpose of detection is achieved. Meanwhile, the preparation method of the dye provided by the invention is low in cost and obvious in economic and technical effects.

Description

Technical Field

[0001] The invention relates to a novel cyanine near-infrared fluorescent dye and a preparation method and application thereof, and belongs to the field of organic small molecule fluorescent probes. Background Art

[0002] Fluorescent dye detection usually relies on the increase or decrease of fluorescence intensity after the object being detected interacts with the fluorescent dye for analysis. Therefore, the concentration of the dye, the performance of the instrument, the environment, etc. will affect the output of the final fluorescent signal. The survey found that fluorescent probes based on traditional cyanine dyes still have some common shortcomings, such as small Stokes shift, poor photostability, easy self-aggregation in aqueous solution, and easy solvation effect in highly polar solvents. Summary of the invention

[0003] In view of the shortcomings of the above-mentioned traditional cyanine dye fluorescent probes, the present invention designed and synthesized a new cyanine near-infrared fluorescent dye, started with the optimization of the cyanine dye structure, and conducted in-depth research on the relationship between its molecular structure and sensing mechanism. Designing and synthesizing sensors with excellent performance is of great significance for detecting active substances in cells.

[0004] The purpose of the present invention is to provide a novel cyanine near-infrared fluorescent dye, and at the same time provide a preparation method and application of the novel cyanine near-infrared fluorescent dye, so as to solve the problems of insensitive and inaccurate detection of traditional cyanine dyes, short Stokes shift and difficulty in dynamic monitoring of active substances in cells.

[0005] The technical solution adopted by the present invention is:

[0006] The first aspect of the present invention aims to provide a novel cyanine near-infrared fluorescent dye, the structure of which is shown in general formula (I):

[0007]

[0008] In the general formula, R is one of the following independent groups:

[0009]

[0010] The second aspect of the present invention is to provide a method for preparing the fluorescent dye, and the general preparation process is as follows:

[0011]

[0012] The specific preparation steps are as follows:

[0013] S1. Dissolve Fischer's aldehyde in an ethanol solution containing sodium hydroxide in a round-bottom flask, raise the oil bath temperature to 80°C, dissolve the raw material R in the ethanol solution in a constant pressure dropping funnel, and slowly add the solution dropwise to the round-bottom flask to perform a Knoevenagel reaction;

[0014] S2. After the reaction is completed, the solid in the reaction flask is directly filtered and washed with acetonitrile; after the solid is collected, it is dissolved with dichloromethane and then separated by column chromatography to obtain the compound represented by general formula (I).

[0015] As an embodiment of the preparation method of the present invention, in the condensation reaction described in S1, the ratio of raw material R to Fischer's aldehyde is 1:2-3, preferably 1:2.2; the ratio of sodium hydroxide to ethanol solution is 0.01 g / mL-0.03 g / mL, preferably 0.02 g / mL; the raw material R is slowly added using a constant pressure dropping funnel, and the dropwise addition time is 30 min-60 min, preferably 60 min; the condensation reaction time is 3 to 6 hours, and the condensation reaction temperature is 80 to 120 ° C;

[0016] As an embodiment of the preparation method of the present invention, in the post-reaction treatment process of S2, the filtered solid needs to be washed with dichloromethane, and the mother liquor is separated by column chromatography to obtain the compound represented by general formula (I), and the developing solvent for column chromatography separation and purification is dichloromethane / ethyl acetate = 25:1.

[0017] The third aspect of the present invention also provides the application of the novel near-infrared fluorescent dye as a fluorescent probe for hypochlorous acid detection.

[0018] (1) The present invention has at least the following beneficial effects:

[0019] The near-infrared fluorescent dye of the present invention has an absorption wavelength greater than 700 nm and an emission wavelength near 800 nm in an acidic environment.

[0020] (2) The near-infrared fluorescent dye of the present invention has good biomembrane permeability and low cytotoxicity.

[0021] (3) The near-infrared fluorescent dye of the present invention can be directly used as a hypochlorous acid fluorescent probe without structural modification. The Stokes shift is greater than 100 nm, which can effectively reduce background signal interference. In addition, this type of probe has the characteristics of high selectivity, high sensitivity (the detection limit can reach the nanomolar order, meeting the detection requirements in vivo), rapid response and good optical stability in response to HClO. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The normalized spectra of ultraviolet absorption and fluorescence emission of the novel cyanine near-infrared fluorescent dye in Example 1 when 1 / 1000 methanesulfonic acid is added to different solvents;

[0023] Figure 2 The normalized spectra of ultraviolet absorption and fluorescence emission of the novel cyanine near-infrared fluorescent dye in Example 2 when 1 / 1000 methanesulfonic acid is added to different solvents;

[0024] Figure 3 The normalized spectra of ultraviolet absorption and fluorescence emission of the novel cyanine near-infrared fluorescent dye in Example 3 when 1 / 1000 methanesulfonic acid is added to different solvents;

[0025] Figure 4 This is the titration spectrum of the novel cyanine near-infrared fluorescent dye identifying hypochlorous acid in Example 1;

[0026] Figure 5 This is the titration spectrum of the novel cyanine near-infrared fluorescent dye identifying hypochlorous acid in Example 2;

[0027] Figure 6 This is the titration spectrum of the novel cyanine near-infrared fluorescent dye identifying hypochlorous acid in Example 3;

[0028] Figure 7 The cytotoxicity spectra of the novel fluorescent dye of Example 1 at different concentrations;

[0029] Figure 8 The cytotoxicity spectra of the novel fluorescent dye of Example 2 at different concentrations;

[0030] Fig. 9 The cytotoxicity spectra of the novel fluorescent dye of Example 3 at different concentrations;

[0031] Fig.10 The normalized spectra of ultraviolet absorption and fluorescence emission of the novel cyanine near-infrared fluorescent dye of Example 4 when 1 / 1000 methanesulfonic acid is added to different solvents;

[0032] Fig.11 The normalized spectra of ultraviolet absorption and fluorescence emission of the novel cyanine near-infrared fluorescent dye of Example 5 when 1 / 1000 methanesulfonic acid is added to different solvents;

[0033] Fig.12 The normalized spectra of ultraviolet absorption and fluorescence emission of the novel cyanine near-infrared fluorescent dye of Example 6 when 1 / 1000 methanesulfonic acid is added to different solvents;

[0034] Fig.13 These are the normalized spectra of ultraviolet absorption and fluorescence emission of the novel cyanine near-infrared fluorescent dye of Example 7 when one thousandth of methanesulfonic acid is added into different solvents. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the present invention clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] In order to better understand the technical solution of the present invention, the following is further described in detail through specific embodiments:

[0039] All reagents used were of analytical grade and purchased directly from Inokai and other reagent companies. Nuclear magnetic resonance spectra were measured using a Bruker spectrometer 400 (MHz); fluorescence spectra were measured using a Hitachi F-4600 spectrofluorophotometer; and cell imaging was measured using a FV1000.

[0040] The present invention provides a novel cyanine near-infrared fluorescent dye, the general structural formula of the novel cyanine near-infrared fluorescent dye is shown in the following formula (I):

[0041]

[0042] The preparation process of the above general formula is as follows:

[0043]

[0044] Wherein R is one of the following independent groups:

[0045]

[0046] The technical solution of the present invention is further described below by way of embodiments.

[0047] Example 1

[0048]

[0049] Fischer's aldehyde (931 mg, 5 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of ethanol solution containing 0.2 g of sodium hydroxide was added; 3-heterothiocyclobutanone (441 mg, 10 mmol) was dissolved in 3 mL of ethanol and placed in a constant pressure dropping funnel, and 3-heterothiocyclobutanone was added dropwise under magnetic stirring at 80°C. After 1 hour, the reaction was continued for 5 hours after the addition was completed; the reaction was stopped, and the mixture was concentrated under reduced pressure, leaving a small amount of solvent, cooled and allowed to stand, and solids precipitated. The filtered solids were dissolved in dichloromethane and separated by column chromatography (developing solvent: dichloromethane: ethyl acetate = 25:1) to obtain the target product. HRMS: m / z[M+H] + =454.2079; Theoretical value: [C 29 H 30 N 2 OS] + =454.2069. 1 H NMR (CDCl 3 ,400MHz,ppm): δ7.64(d,J=12.4Hz,2H),7.21(t,J=7.8Hz,2H),7.17(d,J=7.6Hz,2H),6.93 (t,J=7.4Hz,2H),6.71(d,J=8.0Hz,2H),5.00(d,J=12.4Hz,2H),3.21(s,6H),1.60(s,12H); 13 C NMR (CDCl 3 ,100MHz,ppm): δ180.83,164.07,144.71,139.60,135.84,128.17,122.03,121.20,120.49,107.06,91.62,46.80,29.67,28.49.

[0050] The sample prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3 The standard parent solution of M was placed in 3 mL of different solvents (containing 1‰ methanesulfonic acid, MSA) to prepare a main solution with a concentration of 10 μM. The UV spectrum and fluorescence spectrum were tested by UV absorption spectrometer and fluorescence spectrometer respectively to obtain the normalized curves of UV absorption and fluorescence emission of Example 1 in different solvents, see the attached Figure 1 .

[0051] The sample prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3The standard parent solution of 10 μM was added to 3 mL of the detection system to prepare a main solution with a concentration of 10 μM. Different volumes of guest solution were added to the main solution so that the concentration ratio of HClO to dye in the sample bottle was: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50. The fluorescence spectrum was tested by a fluorescence spectrophotometer with an excitation wavelength of 600 nm and a slit width of 5 nm and 10 nm. With the concentration of HClO as the horizontal axis and the fluorescence intensity at 672 nm as the vertical axis, a curve showing the change of fluorescence intensity with HClO concentration was obtained, see attached. Figure 4 .

[0052] The cytotoxicity spectra of the novel fluorescent dye at different concentrations in Example 1 are shown in the attached figure. Figure 7 shown.

[0053] Example 2

[0054]

[0055] Fischer's aldehyde (931.17 mg, 5 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of ethanol solution containing 0.2 g of sodium hydroxide was added; dihydro-2H-tetrahydropyran-3-one (840.12 mg, 10 mmol) was dissolved in 3 mL of ethanol and placed in a constant pressure dropping funnel, and dihydro-2H-tetrahydropyran-3-one was added dropwise under magnetic stirring at 80°C. After 1 hour, the reaction was continued for 5 hours after the addition was completed; the reaction was stopped, and the mixture was concentrated under reduced pressure, leaving a small amount of solvent, cooled and allowed to stand, and solids precipitated. The filtered solids were dissolved in dichloromethane and separated by column chromatography (developing solvent: dichloromethane: ethyl acetate = 25:1) to obtain the target product. HRMS: m / z[M+H] + =482.2392; Theoretical value: [C 31 H 34 N 2 OS+H] + =483.2675. 1 HNMR (CDCl 3,400MHz,ppm): δ8.27(d,J=12.8Hz,2H),7.23-7.17(m,4H),6.94(q,J=5.6Hz,2H),6.72(t,J=6.2Hz,2H),5.55(d, J=12.8Hz,1H),5.38(d,J=13.6Hz,1H),3.24(s,6H),3.07-3.02(m,2H),2.99-2.94(m,2H),1.66(d,J=7.6Hz,12H); 13 C NMR (CDCl 3 ,100MHz,ppm): δ144.80,144.67,139.95,128.04,128.01,127.95,124.24,122.76,122.54,122.08,122.02,3121.2 5,121.02,120.89,108.29,107.05,107.00,93.90,92.51,47.01,46.88,29.69,29.31,29.04,28.91,26.15,24.68.

[0056] The sample prepared in Example 2 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3 The standard parent solution of M was placed in 3 mL of different solvents (containing 1‰ methanesulfonic acid, MSA) to prepare a main solution with a concentration of 10 μM. The UV spectrum and fluorescence spectrum were tested by UV absorption spectrometer and fluorescence spectrometer respectively to obtain the normalized curves of UV absorption and fluorescence emission of Example 2 in different solvents, see the attached Figure 2 .

[0057] The sample prepared in Example 2 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3 The standard parent solution of 10 μM was prepared in 3 mL of the detection system to form a main solution with a concentration of 10 μM. Different volumes of guest solution were added to the main solution to make the HClO / ClO - The concentration ratio of the dye is: 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3, 3.3, 3.6, 3.9, 4.2, 4.5, 4.8, 5.4, 6, 6.6. The fluorescence spectrum was tested by a fluorescence spectrophotometer with an excitation wavelength of 540 nm and a slit width of 10 nm and 10 nm. The concentration of HClO was used as the horizontal axis and the fluorescence intensity at 662 nm was used as the vertical axis to obtain a curve of fluorescence intensity changing with HClO concentration, see attached. Figure 5 .

[0058] The cytotoxicity spectra of the novel fluorescent dye at different concentrations in Example 2 are shown in the attached figure. Figure 8 shown.

[0059] Example 3

[0060]

[0061] Fischer's aldehyde (931.17 mg, 5 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of ethanol solution containing 0.2 g of sodium hydroxide was added; tetrahydro-2H-pyran-3-one (840.12 mg, 10 mmol) was dissolved in 3 mL of ethanol and placed in a constant pressure dropping funnel, and dihydro-2H-tetrahydropyran-3-one was added dropwise under magnetic stirring at 80°C. After 1 h, the reaction was continued for 5 h after the addition was completed; the reaction was stopped, and the mixture was concentrated under reduced pressure, leaving a small amount of solvent, cooled and allowed to stand, solids precipitated, and the filtered solids were dissolved in dichloromethane and separated by column chromatography (developing solvent: dichloromethane: ethyl acetate = 25:1) to obtain the target product. 1 H NMR (CDCl 3 ,400MHz,ppm): δ8.18(d,J=13.2Hz,1H),7.37(d,J=12.4Hz,1H),7.21(t,J=8.4Hz,2H) ,7.17(d,J=3.6Hz,2H),6.95(t,J=7.4Hz,1H),6.87(t,J=7.2Hz,1H),6.74(d,J=8.0Hz, 1H),6.65(d,J=8.0Hz,1H),5.61(d,J=13.2Hz,1H),5.36(d,J=13.6Hz,1H),4.21(t,J= 5.6Hz,1H),3.24(s,3H),3.20(s,3H),2.81(t,J=5.6Hz,2H),1.67(s,6H),1.64(s,6H); 13 C NMR (CDCl 3,100MHz,ppm): δ165.21,161.17,146.43,145.18,144.58,139.91,139.78, 134.15,131.41,129.17,128.07,127.92,122.09,121.93,121.70,121.41, 120.34,114.09,114.05,113.99,107.16,106.48,92.30,90.58,90.51,66.14,65.83,47.08,46.40,29.68,29.52,28.96,28.68,26.61,24.67,15.57.

[0062] The sample prepared in Example 3 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3 The standard parent solution of M was placed in 3 mL of different solvent systems and different solvent systems (containing 1‰ methanesulfonic acid, MSA) to prepare a main solution with a concentration of 10 μM. The UV spectrum and fluorescence spectrum were tested by UV absorption spectrometer and fluorescence spectrometer respectively to obtain the normalized curves of UV absorption and fluorescence emission of Example 3 in different solvents. Figure 3 .

[0063] The sample prepared in Example 3 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3 The standard parent solution of 10 μM was prepared in 3 mL of the detection system to form a main solution with a concentration of 10 μM. Different volumes of guest solution were added to the main solution to make the HClO / ClO - The concentration ratios of the dye are: 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.4, 3, 3.6, 4.2, 4.8, 5.4, 6. The fluorescence spectrum was tested by a fluorescence spectrophotometer with an excitation wavelength of 460nm and a slit width of 10nm, 10nm. The concentration of HClO was used as the horizontal axis and the ratio of the fluorescence intensity at 517nm and 677nm was used as the vertical axis to obtain a curve of fluorescence intensity changing with HClO concentration, see attached. Figure 6 .

[0064] The cytotoxicity spectra of the novel fluorescent dye at different concentrations in Example 3 are shown in the attached figure. Fig. 9 shown.

[0065] Example 4

[0066]

[0067] Fischer's aldehyde (931.17 mg, 5 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of ethanol solution containing 0.2 g of sodium hydroxide was added; ethylthioacetone (710.03 mg, 6 mmol) was dissolved in 3 mL of ethanol and placed in a constant pressure dropping funnel, and diethylthioacetone was added dropwise under magnetic stirring at 80°C. After 1 h, the reaction was continued for 5 h after the addition was completed; the reaction was stopped, concentrated under reduced pressure, leaving a small amount of solvent, cooled and allowed to stand, solid precipitated, the filtered solid was dissolved in dichloromethane, and separated by column chromatography (developing solvent: dichloromethane: ethyl acetate = 25:1) to obtain the target product. 1 H NMR (CDCl 3 ,400MHz,ppm): δ8.61(d,J=12.4Hz,1H),8.17(s,1H),7.23-7.17(m,5H),6.97-6.91(m,2H),6.76-6.70(m,2H),6.20(d,J=1 2.8Hz,1H),5.66(d,J=13.2Hz,1H),3.29(s,3H),3.22(s,3H),2.69(q,J=7.6Hz,2H),1.65(s,12H),1.24(t,J=7.4Hz,3H,); 13 C NMR (100 MHz, CDCl 3 ,ppm): δ183.43,165.85,159.77,148.77,144.99,144.04,139.61,139.37,128.45,127.91,127.66,124.44,121.82 ,121.63,121.59,119.87,107.22,106.06,103.65,92.34,89.77,69.99,47.01,45.94,29.54,29.21,23.50,23.12.

[0068] The sample prepared in Example 4 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3 The standard parent solution of M was placed in 3 mL of different solvents (containing 1‰ methanesulfonic acid, MSA) to prepare a main solution with a concentration of 10 μM. The ultraviolet spectrum and fluorescence spectrum were tested by ultraviolet absorption spectrometer and fluorescence spectrometer respectively to obtain the normalized curves of ultraviolet absorption and fluorescence emission of Example 4 in different solvents, as shown in the attached Fig.10 .

[0069] Example 5

[0070]

[0071] Fischer's aldehyde (931.17 mg, 5 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of ethanol solution containing 0.2 g of sodium hydroxide was added; (phenylthio)acetone (997.44 mg, 6 mmol) was dissolved in 3 mL of ethanol and placed in a constant pressure dropping funnel, and di(phenylthio)acetone was added dropwise under magnetic stirring at 80°C. After 1 h, the reaction was continued for 5 h after the addition was completed; the reaction was stopped, and the mixture was concentrated under reduced pressure, leaving a small amount of solvent, cooled and allowed to stand, solids precipitated, and the filtered solids were dissolved in dichloromethane and separated by column chromatography (developing solvent: dichloromethane: ethyl acetate = 25:1) to obtain the target product. 1 H NMR (400 MHz, CDCl 3 , ppm): δ8.94 (d, J = 12.8Hz, 1H), 8.20 (t, J = 13.2Hz, 1H), 7.10 (t, J = 7.2Hz, 3H); 7 .06(d,J=3.2Hz,1H),7.00(t,J=7.2Hz,1H),6.94(t,J=7.2Hz,1H),6.88(d,J=8.0 Hz,1H),6.77(d,J=7.6Hz,1H),6.72(d,J=7.6Hz,1H),6.10(d,J=13.2Hz,1H),5.5 7(d,J=13.2Hz,1H),3.24(s,5H),2.00(d,J=8.0Hz,6H),1.72(s,6H),1.6(s,6H).

[0072] The sample prepared in Example 5 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3 The standard parent solution of M was placed in 3 mL of different solvents (containing 1‰ methanesulfonic acid, MSA) to prepare a main solution with a concentration of 10 μM. The ultraviolet spectrum and fluorescence spectrum were tested by ultraviolet absorption spectrometer and fluorescence spectrometer respectively to obtain the normalized curves of ultraviolet absorption and fluorescence emission of Example 5 in different solvents, as shown in the attached Fig.11 .

[0073] Example 6

[0074]

[0075] Fischer's aldehyde (931.17 mg, 5 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of ethanol solution containing 0.2 g of sodium hydroxide was added; tetrahydrothiophene-3-one (610.45 mg, 6 mmol) was dissolved in 3 mL of ethanol and placed in a constant pressure dropping funnel, and tetrahydrothiophene-3-one was added dropwise under magnetic stirring at 80°C. After 1 h, the reaction was continued for 5 h after the addition was completed; the reaction was stopped, and the mixture was concentrated under reduced pressure, leaving a small amount of solvent, cooled and allowed to stand, solids precipitated, and the filtered solids were dissolved in dichloromethane and separated by column chromatography (developing solvent: dichloromethane: ethyl acetate = 25:1) to obtain the target product. 1 H NMR (400 MHz, CDCl 3 ,ppm): δ7.93(s,1H),7.80(s,1H),7.23(d,J=2.8Hz,1H);7.21(d,J=2.0Hz,1H)),7.7 9(d,J=3.6Hz,1H);7.17(d,J=3.2Hz,1H),6.99(t,J=7.4Hz,1H,),6.92(t,J=7.4Hz,1H ),6.78(d,J=6.8Hz,1H),6.70(d,J=8.0Hz,1H),5.30(t,J=6.60Hz,1H),5.21(d,J=12 .8Hz,1H),3.98(d,J=2.0Hz,2H),3.27(s,3H),3.22(s,3H),1.67(s,6H),1.65(s,6H); 13 C NMR (100 MHz, CDCl 3 ,ppm): δ187.56,165.60,162.26,144.63,144.14,139.62,139.50,132.58,127.99,127.87,127.72,124.41,123.23,12 1.83,121.71,121.44,120.51,107.11,106.53,94.23,92.80,46.94,46.42,29.50,29.34,28.60,28.49,28.33,28.20.

[0076] The sample prepared in Example 6 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3 The standard parent solution of M was placed in 3 mL of different solvents (containing 1‰ methanesulfonic acid, MSA) to prepare a main solution with a concentration of 10 μM. The UV spectrum and fluorescence spectrum were tested by UV absorption spectrometer and fluorescence spectrometer respectively to obtain the normalized curves of UV absorption and fluorescence emission of Example 6 in different solvents, see the attached Fig.12 .

[0077] Example 7

[0078]

[0079] Fischer's aldehyde (931.17 mg, 5 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of ethanol solution containing 0.2 g of sodium hydroxide was added; tetrahydrofuran-3-one (520.33 mg, 6 mmol) was dissolved in 3 mL of ethanol and placed in a constant pressure dropping funnel, and tetrahydrofuran-3-one was added dropwise under magnetic stirring at 80°C. After 1 h, the reaction was continued for 5 h after the addition was completed; the reaction was stopped, and the mixture was concentrated under reduced pressure, leaving a small amount of solvent, cooled and allowed to stand, solids precipitated, and the filtered solids were dissolved in dichloromethane and separated by column chromatography (developing solvent: dichloromethane: ethyl acetate = 25:1) to obtain the target product. 1 H NMR (400 MHz, CDCl 3 ,ppm): δ7.70(d,J=13.2Hz,1H),7.23(d,J=2.4Hz,1H);7.21(d,J=2.4Hz,1H)-7.16( d,J=2.8Hz,2H)7.14(d,J=2.8Hz,2H),6.98(t,J=7.4Hz,1H,),6.86(t,J=7.4Hz,1H) ,6.76(t,J=9.6Hz,2H),6.63(d,J=7.6Hz,1H),5.5(d,J=12.8Hz,1H),5.13(d,J=2.0 Hz,2H),5.05(d,J=13.2Hz,1H),3.24(s,3H),3.18(s,3H),1.66(s,6H),1.64(s,6H); 13 C NMR (100 MHz, CDCl 3 ,ppm): δ183.43,165.85,159.77,148.77,144.99,144.04,139.61,139.37,128.45,127.91,127.66,124.44,121.82 ,121.63,121.59,119.87,107.22,106.06,103.65,92.34,89.77,69.99,47.01,45.94,29.54,29.21,23.50,23.12.

[0080] The sample prepared in Example 7 was dissolved in dimethyl sulfoxide (DMSO) and prepared into 5×10 -3 M mother solution for later use; take 6 μL of 5×10 -3The standard parent solution of M was placed in 3 mL of different solvents (containing 1‰ methanesulfonic acid, MSA) to prepare a main solution with a concentration of 10 μM. The UV spectrum and fluorescence spectrum were tested by UV absorption spectrometer and fluorescence spectrometer respectively to obtain the normalized curves of UV absorption and fluorescence emission of Example 7 in different solvents, as shown in the attached Fig.13 .

[0081] The preparation method provided by the invention has low cost, high yield and obvious technical effect.

[0082] Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the scope of protection of the present invention, and the given embodiments do not fully cover all the options of the technical solution of the present invention, which are only used for understanding the technical solution of the present invention. All the options of the technical solution of the present invention, such as the raw material R, the dosage, and the process conditions in the preparation method, can achieve the purpose of the present invention and achieve the same technical effect as the above-mentioned embodiment. It should be further explained that any person skilled in the art can make some possible changes and modifications without departing from the concept of the present application, so the scope of protection of the present application should be defined by the claims of the present application.

Claims

1. A novel cyanine near-infrared fluorescent dye, characterized in that: The structure of the novel cyanine near-infrared fluorescent dye is shown in general formula (I): In the general formula, R is one of the following independent groups:

2. The method for preparing a novel cyanine near-infrared fluorescent dye according to claim 1, characterized in that: The general preparation process is as follows: The specific preparation steps are as follows: S1. Dissolve Fischer's aldehyde in an ethanol solution containing sodium hydroxide in a round-bottom flask, raise the oil bath temperature to 80°C, dissolve the raw material R in the ethanol solution in a constant pressure dropping funnel, and slowly add the solution dropwise to the round-bottom flask to perform a Knoevenagel reaction; S2. After the reaction is completed, the solid in the reaction flask is directly filtered and washed with acetonitrile; after the solid is collected, it is dissolved with dichloromethane and then separated by column chromatography to obtain the compound represented by general formula (I).

3. The preparation method according to claim 2, characterized in that: In the condensation reaction described in S1, the ratio of the raw material R to the Fischer aldehyde is 1:2-3; In the condensation reaction described in S1, the ratio of the ethanol solution containing sodium hydroxide is 0.01 g / mL to 0.03 g / mL; In the condensation reaction described in S1, the raw material R is slowly added using a constant pressure dropping funnel, and the addition time is 30min-60min; In S1, the condensation reaction time is 3 to 6 hours, and the condensation reaction temperature is 80 to 120°C.

4. The preparation method according to claim 1 or 3, characterized in that: In the condensation reaction described in S1, the ratio of the raw material R to the Fischer aldehyde is 1:2.2; In the condensation reaction described in S1, the ratio of the ethanol solution containing sodium hydroxide is 0.02 g / mL; In the condensation reaction described in S1, the raw material R is slowly added using a constant pressure dropping funnel, and the addition is completed in 60 minutes.

5. The preparation method according to claim 2, characterized in that: During the post-reaction treatment of S2, the filtered solid needs to be washed with acetonitrile, and the collected solid is separated by column chromatography to obtain the compound represented by general formula (I). The developing solvent for column chromatography separation and purification is dichloromethane / ethyl acetate = 25:

1.

6. The use of a novel cyanine near-infrared fluorescent dye according to claim 1, characterized in that: Used to construct fluorescent probes.

7. The use according to claim 6, characterized in that: Application detection as a fluorescent probe for hypochlorous acid.

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