Dye for constructing high-sensitivity long-wavelength fluorescent probe and preparation method thereof

Abstract

The application provides a dye for constructing a high-sensitivity long-wavelength fluorescent probe and a preparation method thereof, the fluorescent dye parent body is a rhodol dye, a structural formula of the rhodol dye is shown as formula A: formula A is connected with any one of the following structural formulas; wherein R1 is one of H, C1-20 alkyl and a benzene ring; R2 is one of H, F, Cl, Br and I; R3 is one of (n=0-20); R4 is one of (n=0-20). The rhodol dye covers a spectral range of 500-750 nm, is almost completely in an open ring state under physiological conditions, and is completely converted into a non-fluorescent spiro ring state by adopting a methyl or a response site shielding hydroxyl, therefore, the probe constructed based on the dye has extremely low self-background fluorescence; along with activation of the probe, the shielding hydroxyl is gradually released, the non-fluorescent probe is converted into a strong fluorescent dye, thereby the probe is endowed with a significantly improved response activation multiple and extremely high response sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent dye technology and relates to a dye for constructing highly sensitive long-wavelength fluorescent probes and its preparation method. Specifically, it relates to a method for reducing the intrinsic fluorescence of rhodol probes in the visible to near-infrared II region and improving the probe response sensitivity, and provides a series of dye platforms that can be used to design highly sensitive visible to near-infrared II fluorescent probes. Background Technology

[0002] With their advantages of being non-invasive, real-time, and convenient, analyte-responsive fluorescent probes have been widely used to monitor fluctuations in the content or activity of biomolecules in living systems, playing a crucial role in biological and biomedical research (such as signaling pathways). In recent years, to reduce interference from tissue autofluorescence and improve imaging depth, researchers have extended the wavelength of fluorescent dyes by increasing conjugation and introducing heteroatoms, developing a series of long-wavelength fluorescent probes based on these novel dyes. However, many probes often exhibit low response folds (<30-fold), which significantly reduces the sensitivity and accuracy of imaging or detection, a problem particularly pronounced in near-infrared fluorescent probes. The strong intrinsic fluorescence of the probe is the most fundamental reason for its low response fold. Reducing the intrinsic fluorescence of the probe may be an effective method to improve its response sensitivity.

[0003] Over the past three decades, driven by the rapid development of fluorescent probes, rhodol dyes containing functional phenolic hydroxyl groups have received considerable attention. To date, by controlling intramolecular electron density or extending the conjugated π system, the wavelengths of rhodol-like dyes have been extended to the near-infrared II region. Furthermore, due to their simple modification and broad applicability, these dyes have been widely used to design analyte-responsive fluorescent probes. However, introducing a responsive group to shield the phenolic hydroxyl group cannot completely convert rhodol and long-wavelength rhodol-like dyes into the non-fluorescent spironolactone form, resulting in probes built based on these dyes exhibiting high intrinsic fluorescence and low sensitivity. Changing the carboxylic acid of rhodol dye to a hydroxymethyl group can improve this problem, but this modification method is difficult to extend to far-infrared and near-infrared rhodol dyes.

[0004] Therefore, it is of great significance to develop a general method to reduce the intrinsic fluorescence of rhodol probes in the visible to near-infrared II region and improve the probe response sensitivity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a dye for constructing highly sensitive long-wavelength fluorescent probes and a method for preparing the same, wherein the prepared fluorescent probe exhibits low intrinsic fluorescence and high response sensitivity.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A fluorescent dye for constructing highly sensitive visible-near-infrared fluorescent probes, wherein the parent dye is rhodol dye, and the structural formula of the rhodol dye is shown in Formula A:

[0008]

[0009] Connect formula A with any of the following structural formulas;

[0010]

[0011] R1 is one of H, C1-20 alkyl groups, or benzene rings;

[0012] R2 is one of H, F, Cl, Br, and I;

[0013] R3 is One of them (n = 0 to 20);

[0014] R4 is One of them (n = 0 to 20).

[0015] Furthermore, the present invention provides a fluorescent dye for constructing a highly sensitive visible-near-infrared fluorescent probe, wherein the rhodol dye has the structural formula of any one of formulas I to III:

[0016]

[0017] A highly sensitive visible-near-infrared fluorescent probe, constructed based on type I-III rhodol dyes, yields an analyte-responsive fluorescent probe. The preparation method is as follows:

[0018]

[0019] R5-R6 represent the response sites of different analytes, including superoxide anion (O2). - ), hydrogen sulfoxide (HNO), cysteine ​​(Cys), hydrogen peroxide (H2O2), quinone oxidoreductase (hNQO1), cytochrome C (P450) and leucine aminopeptidase (LAP);

[0020] R6 is one of Cl and Br;

[0021] R5 is

[0022]

[0023] Any one of them.

[0024] Specifically:

[0025] When the R5 substituent is R5-(1-3), the preparation process of the fluorescent probe is as follows:

[0026] Dyes I-III and diisopropylethylamine were dissolved in dichloromethane. The system temperature was lowered to 0°C, and R5-R6 were added dropwise. Finally, the system temperature was restored to room temperature and reacted for 2-8 hours. The reaction solution was poured into ice water and extracted three times with dichloromethane. The organic phase was dried under reduced pressure, and the residue was separated by column chromatography to obtain the corresponding probes.

[0027] When the R5 substituent is R5-(4-7), the preparation process of the fluorescent probe is as follows:

[0028] Dyes I-III, cesium carbonate, and NaI were dissolved in acetonitrile. The reaction mixture was stirred at room temperature for 10-60 min. Then, R5-R6 were added, and the mixture was stirred at room temperature for 1-24 h. The mixture was then poured into ice water and extracted three times with dichloromethane. The organic phase was evaporated under reduced pressure, and the residue was separated by column chromatography to obtain the corresponding probe.

[0029] The present invention also provides the application of the visible-near-infrared highly sensitive fluorescent probe in the detection of analytes in body fluids.

[0030] This invention successfully modulates the ring-opening and ring-closing balance of rhodol dyes by introducing N,N-dimethylsulfonamide into traditional rhodol and longer-wavelength rhodol derivatives, converting the carboxyl group into an amide that is more easily closed. This allows the dye itself to be in a strongly fluorescent open-ring state in the physiological environment, while the dye with hydroxyl groups shielded by methyl groups is mainly in a non-fluorescent closed-ring state. This significantly increases the fluorescence brightness ratio of rhodol dyes to hydroxymethylated dyes. This method is applicable to the rhodol dye backbone in the visible to near-infrared light regions.

[0031] Compared with existing technologies, the beneficial effects of this invention are as follows:

[0032] (1) The rhodol dye described in this invention has a spectral range covering 500-750 nm. Under physiological conditions, it is almost entirely in a strongly fluorescent open-ring state. However, by using methyl groups or hydroxyl groups to shield the responsive sites, the dye can be completely transformed into a non-fluorescent spirocyclic state. Therefore, the probe constructed based on this type of dye has extremely low background fluorescence. With the activation of the probe, the shielded hydroxyl groups are gradually released, and the non-fluorescent probe is transformed into a strongly fluorescent dye, thereby giving the probe a significantly enhanced response activation factor and extremely high response sensitivity.

[0033] (2) The synthesis method of the visible light-near infrared high-sensitivity fluorescent probe of the present invention is simple, the raw materials are readily available, the product is easy to purify, and the reaction yield is high. Attached Figure Description

[0034] Figure 1 The hydrogen NMR spectrum of S2-1 prepared in Example 1.

[0035] Figure 2 The carbon NMR spectrum of S2-1 prepared in Example 1.

[0036] Figure 3 The proton NMR spectrum of I-1 prepared in Example 1.

[0037] Figure 4 The carbon NMR spectrum of I-1 prepared in Example 1.

[0038] Figure 5 The hydrogen NMR spectrum of S3-1 prepared in Example 2.

[0039] Figure 6 The carbon NMR spectrum of S3-1 prepared in Example 2.

[0040] Figure 7 The hydrogen NMR spectrum of S4-1 prepared in Example 2.

[0041] Figure 8 The carbon NMR spectrum of S4-1 prepared in Example 2.

[0042] Figure 9 The proton NMR spectrum of II-1 prepared in Example 2.

[0043] Figure 10 The hydrogen NMR spectrum of S7-1 prepared in Example 3.

[0044] Figure 11 The carbon NMR spectrum of S7-1 prepared in Example 3.

[0045] Figure 12 The hydrogen NMR spectrum of S8-1 prepared in Example 3.

[0046] Figure 13 The carbon NMR spectrum of S8-1 prepared in Example 3.

[0047] Figure 14 The proton NMR spectrum of III-1 prepared in Example 3.

[0048] Figure 15 The carbon NMR spectrum of III-1 prepared in Example 3.

[0049] Figure 16 The proton NMR spectrum of the III-1-LAP prepared in Example 4.

[0050] Figure 17The carbon NMR spectrum of the III-1-LAP prepared in Example 4.

[0051] Figure 18 The UV-Vis absorption and fluorescence emission spectra of I-1(a) and S2-1(b) prepared in Example 1 in PBS buffer solutions at different pH (4.0-10.0) are shown.

[0052] Figure 19 The UV-Vis absorption and fluorescence emission spectra of II-1(a) and S4-1(b) prepared in Example 2 in PBS buffer solutions at different pH (4.0-10.0) are shown.

[0053] Figure 20 The UV-Vis absorption and fluorescence emission spectra of III-1(a) and S8-1(b) prepared in Example 3 in PBS buffer solutions at different pH (4.0-10.0) are shown.

[0054] Figure 21 The UV absorption and fluorescence spectra of III-1-LAP prepared in Example 4 changed with the increase of LAP addition. Detailed Implementation

[0055] The specific embodiments of the present invention will be further illustrated below, but the specific embodiments of the present invention are not limited to the following embodiments.

[0056] The synthetic route for fluorescent dye I is as follows:

[0057]

[0058] Includes the following steps:

[0059] Step 1: 3-Disubstituted aminophenol and 2-(2-hydroxy-4-methoxy-5-substituted phenyl)benzoic acid are dissolved in methanesulfonic acid and heated to 90-140℃ for 4-10 hours. The reaction mixture is then poured into ice water, and an inorganic acid is added. The large amount of solid precipitate that forms is filtered, washed, dried, and then separated by column chromatography to obtain dye intermediate S1. R1 is one of H, C1-20 alkyl, or benzene ring; R2 is one of H, F, Cl, Br, or I; and the inorganic acid is one of sulfuric acid, hydrochloric acid, or perchloric acid.

[0060] Step 2: Dye intermediate S1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in dichloromethane and reacted overnight at room temperature. The solvent is removed under reduced pressure, and the residue is separated by column chromatography to obtain dye intermediate S2.

[0061] Step 3: Dissolve dye intermediate S2 in dichloromethane, lower the reaction temperature to 0°C, slowly add boron tribromide, and after the addition is complete, react at room temperature for 2-7 h. Add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain fluorescent dye I.

[0062] The synthetic route for fluorescent dye II is as follows:

[0063]

[0064] Includes the following steps:

[0065] Step 1: 5-Substituted-8-methoxy-1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinoxaline is dissolved in methanesulfonic acid and heated to 90-140℃ for 4-10 h. The reaction mixture is then poured into ice water, and an inorganic acid is added. The precipitated solid is filtered, washed, and dried, followed by column chromatography to obtain dye intermediate S3, where R2 is one of H, F, Cl, Br, or I, and R3 is... One of the following (n = 0-20), wherein the inorganic acid is one of sulfuric acid, hydrochloric acid, and perchloric acid;

[0066] Step 2: Dye intermediate S3, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane and reacted overnight at room temperature. The solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain dye intermediate S4.

[0067] Step 3: Dissolve dye intermediate S4 in dichloromethane, lower the reaction temperature to 0°C, slowly add boron tribromide, and after the addition is complete, react at room temperature for 2-7 hours. Add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain fluorescent dye II.

[0068] The synthetic route for fluorescent dye III is as follows:

[0069]

[0070] Includes the following steps:

[0071] Step 1: Dye intermediates S5 and S6 are dissolved in acetic anhydride, potassium acetate is added, and the mixture is reacted at 60°C for 2-6 hours. The reaction mixture is then poured into ice water, filtered, washed with water, and the filter residue is dried. Column chromatography is then performed to separate the residue and obtain dye intermediate S7. R2 is one of H, F, Cl, Br, and I; R4 is... One of them (n = 0-20);

[0072] Step 2: Dye intermediate S7, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane and reacted overnight at room temperature. The solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain dye intermediate S8.

[0073] Step 3: Dissolve dye intermediate S8 in dichloromethane, lower the reaction temperature to 0°C, slowly add boron tribromide, and after the addition is complete, react at room temperature for 2-7 hours. Add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain fluorescent dye III.

[0074] The following description, through specific embodiments and accompanying drawings, provides further details.

[0075] Example 1

[0076] Synthesis of type I rhodol dyes:

[0077]

[0078] Step 1: 3-Diethylaminophenol (12 mmol) and 2-(2-hydroxy-4-methoxy-5-fluorophenyl)benzoic acid (12 mmol) were dissolved in methanesulfonic acid (10 mL), heated to 90 °C and reacted for 4 h. The reaction mixture was then poured into ice water, and 1 mL of perchloric acid was added. The large amount of solid precipitate that precipitated was filtered, washed, dried, and then separated by column chromatography to obtain dye intermediate S1-1.

[0079] Step 2: Dye intermediate S1-1 (1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2 mmol) and 4-dimethylaminopyridine (0.2 mmol) were dissolved in dichloromethane (20 mL), reacted overnight at room temperature, the solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain dye intermediate S2-1;

[0080] Nuclear magnetic resonance (NMR) Figure 1 and Figure 2 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1H NMR(400MHz,Chloroform-d)δ7.93(d,J=7.4Hz,1H),7.55(m,2H),7.05(d,J=7.2Hz,1H),6.79(d,J=7.3Hz,1H),6.52(d,J=8.8H z,1H),6.47–6.35(m,2H),6.30(dd,J=8.8,2.3Hz,1H),3.89(s,3H),3.32(q,J=6.8Hz,4H),2.74(s,6H),1.14(t,J=6.9Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ167.12,153.25,149.22,149.06,148.83,146.90,134.99,129.28,128.81,128.14,124.79 ,123.86,114.06,113.86,111.28,108.03,104.82,101.47,97.65,68.31,56.26,44.45,37.99,12.66.MALDI-TOF / MS,m / z calc.for C 27 H 28 FN3O5S[M+1]calc 526.17, found 526.28.

[0081] Step 3: Dissolve dye intermediate S2-1 (1 mmol) in dichloromethane (10 mL), lower the reaction temperature to 0 °C, and slowly add boron tribromide (99%, 3 mmol). After the addition is complete, react at room temperature for 2 h, add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain dye I-1.

[0082] Nuclear magnetic resonance (NMR) Figure 3 and Figure 4 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1H NMR(400MHz,Methanol-d4 containing 20%chloroform-d)δ7.86(d,J=9.0Hz,1H),7.58(t,J=7.0Hz,1H),7.51(t,J=7.4Hz,1H),7.01(d,J=7.6Hz,1H),6.68(dd,J=7.6, 2.6Hz,1H),6.42(dd,J=8.8,2.2Hz,1H),6.32(s,1H),6.30–6.23(m,2H),3.30–3.24(m,4H),2.65(s,6H),1.08(t,J=6.9Hz,6H). 13 C NMR(100MHz,Methanol-d4 containing 20%chloroform-d)δ168.51,154.24,150.07,149.97,147.43,147.28,136.06,130.15,129.54,128.70,125. 65,124.42,114.71,114.50,111.26,108.84,105.82,98.56,69.82,45.20,38.30,12.91.MALDI-TOF / MS,m / z calc.for C 26 H 26 FN3O5S[M+1]calc 512.16, found 512.27.

[0083] Example 2

[0084] Synthesis of type II rhodol dyes:

[0085]

[0086] Step 1: 5-Trifluoroethyl-8-methoxy-1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinoxaline (12 mmol) and 2-(2-hydroxy-4-methoxy-5-phenyl)benzoic acid (12 mmol) were dissolved in methanesulfonic acid (10 mL) and heated to 90 °C for 4 h. The reaction was then poured into ice water and perchloric acid (1 mL) was added. The large amount of solid precipitate that precipitated was filtered, washed, dried, and separated by column chromatography to obtain dye intermediate S3-1.

[0087] Nuclear magnetic resonance (NMR) Figure 5 and Figure 6 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1H NMR (400MHz, Methanol-d4) δ8.34(s,1H),7.82(m,2H),7.40(t,J=6.0Hz,1H),7.32(s,1H),7.24(t,J=8.8Hz,1H),7.06(d,J=8.8Hz,1H),6.89(d,J =3.0Hz,1H),6.33–6.23(m,1H),3.98(s,4H),3.86(m,4H),3.73(q,J=9.8 Hz,1H),3.24–3.13(m,1H),2.29(s,2H),2.20–2.07(m,1H),1.67(m,1H). 13 C NMR(100MHz,Methanol-d4)δ167.20,157.75,157.01,156.81,156.49,150.17,135.75,133.93,133.80,132.51,131.54,131.44,130.79 ,130.71,128.18,125.35,118.81,117.58,105.25,101.19,96.90,59.64,57.32,53.14,52.93,50.56,30.74,23.84.MALDI-TOF / MS,m / z calc.for C 28 H 24 F3N2O4[M]calc 509.17,found 509.23.

[0088] Step 2: Dye intermediate S3-1 (1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2 mmol) and 4-dimethylaminopyridine (0.2 mmol) were dissolved in dichloromethane (20 mL), reacted overnight at room temperature, the solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain dye intermediate S4-1;

[0089] Nuclear magnetic resonance (NMR) Figure 7 and Figure 8 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1H NMR(400MHz,Chloroform-d)δ7.94(d,J=7.3Hz,1H),7.53(m,6.6Hz,2H),7.05(t,J =7.9Hz,1H),6.70(s,1H),6.64(d,J=8.6Hz,1H),6.51(d,J=10.7Hz,1H),6.24(d,J =5.3Hz,1H),5.84(d,J=7.6Hz,1H),3.81(s,3H),3.62(m,1H),3.45–3.30(m,5H),2 .98–2.87(m,1H),2.72(s,6H),2.13–2.03(m,2H),1.96(m,1H),1.67–1.59(m,1H). 13 C NMR(100MHz,Chloroform-d)δ167.52,160.65,153.73,146.94,146.79,137.22,134.85,129.19,129.11,128.79,128.25,124.89,123. 75,111.86,110.66,109.52,105.45,100.62,97.82,97.54,55.59,54.59,53.55,47.19,38.05,30.07,27.13,23.31.MALDI-TOF / MS,m / z calc.for C 30 H 29 F3N4O5S[M]calc 614.18,found 614.25.

[0090] Step 3: Dissolve dye intermediate S4-1 (1 mmol) in dichloromethane (10 mL), lower the reaction temperature to 0 °C, slowly add boron tribromide (3 mmol), after the addition is complete, react at room temperature for 2 h, add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain dye II-1.

[0091] Nuclear magnetic resonance (NMR) Figure 9 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1H NMR(400MHz,Chloroform-d)δ8.00–7.84(m,1H),7.63–7.42(m,2H),7.03(m,1H),6.67(m,1H),6.52(m,2H),6.22(s,1H),5.81(s,1H) ),3.60–3.51(m,1H),3.36(m,4H),3.06–2.99(m,2H),2.75(m,6H),2.14–2.01(m,2H),1.97(s,1H),1.42(s,1H).MALDI-TOF / MS,m / z calc.forC 29 H 27 F3N4O5S[M+1]calc 601.17, found 601.26. Example 3

[0092] Synthesis of type III rhodol dyes:

[0093]

[0094] Step 1: S5-1 (12 mmol) and S6-1 (12 mmol) were dissolved in acetic anhydride (20 mL), potassium acetate (36 mmol) was added, and the mixture was reacted at 60 °C for 4 h. The reaction mixture was poured into ice water, filtered, washed with water, and the filter residue was dried. The residue was then separated by column chromatography to obtain the dye intermediate S7-1.

[0095] Nuclear magnetic resonance (NMR) Figure 10 and Figure 11 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1 H NMR(400MHz,Methanol-d4 containing 20%Chloroform-d)δ8.78(d,J=14.8Hz,1H),8.24(d,J=7.8Hz,1H),7.76(t,J =7.5Hz,1H),7.65(t,J=7.7Hz,1H),7.58(d,J=7.4Hz,1H),7.47(t,J=8.8Hz,2 H),7.40(t,J=7.1Hz,1H),7.24(d,J=7.1Hz,2H),6.42(dd,J=12.7,9.6Hz,2H ),4.04(s,3H),3.82(s,3H),2.68(t,J=5.3Hz,2H),2.33(m,2H),1.84(s,8H). 13C NMR(100MHz,Methanol-d4 containing 20%Chloroform-d)δ177.66,167.39,161.73,151.51,150.96,149.94,148.51 ,146.97,145.51,142.33,141.81,135.41,132.99,131.32,130.39,129.43,12 8.80,126.83,124.77,122.27,116.09,114.97,112.08,111.40,103.09,101.0 1,56.51,50.44,31.59,27.25,27.21,27.00,23.75,19.92.MALDI-TOF / MS,m / z calc.forC 34 H 31 FNO4[M]calc 536.22, found 536.31.

[0096] Step 2: Dye intermediate S7-1 (1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2 mmol), and 4-dimethylaminopyridine (0.2 mmol) were dissolved in dichloromethane (20 mL), reacted overnight at room temperature, the solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain dye intermediate S8-1;

[0097] Nuclear magnetic resonance (NMR) Figure 12 and Figure 13 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1 H NMR(400MHz,Chloroform-d)δ7.89(t,J=7.2Hz,1H),7.62(t,J=7.4Hz,1H),7.52(t,J=7.3Hz,1H ),7.45(d,J=13.8Hz,1H),7.16(dd,J=9.2,5.5Hz,3H),6.84(t,J=7.2Hz,1H),6.72(d,J=7.3Hz, 1H),6.61(d,J=7.7Hz,1H),6.31(d,J=11.4Hz,1H),5.36(d,J=12.5Hz,1H),3.95(s,3H),3.14(s ,3H),2.85(s,6H),2.72–2.63(m,1H),2.45–2.35(m,1H),2.12–1.90(m,2H),1.73–1.68(m,8H). 13C NMR(100MHz,Chloroform-d)δ167.34,158.04,151.68,149.38,148.82,148.35,146 .98,145.49,139.01,134.84,133.23,129.39,128.79,127.89,124.29,123.62,121. 69,120.15,119.50,113.67,110.86,105.91,102.15,101.32,92.31,71.15,56.43,4 6.00,45.65,38.16,29.32,28.59,25.52,23.72,22.12.MALDI-TOF / MS,m / zcalc.for C 36 H 36 FN3O5S[M+1]calc 642.24, found 642.39.

[0098] Step 3: Dissolve dye intermediate S8-1 (1 mmol) in dichloromethane (10 mL), lower the reaction temperature to 0 °C, slowly add boron tribromide (3 mmol), after the addition is complete, react at room temperature for 2 h, add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain dye III-1.

[0099] Nuclear magnetic resonance (NMR) Figure 14 and Figure 15 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1 H NMR(400MHz,Chloroform-d)δ7.95(d,J=6.6Hz,1H),7.65–7.55(m,2H),7.51(t ,J=7.2Hz,1H),7.17(q,J=7.3Hz,3H),6.87(d,J=6.9Hz,2H),6.65(d,J=4.1Hz, 1H),6.33(d,J=10.7Hz,1H),5.40(d,J=12.7Hz,1H),3.18(s,3H),2.85(s,6H), 2.66(d,J=15.2Hz,1H),2.45–2.35(m,1H),2.06(d,J=12.4Hz,2H),1.67(s,8H). 13C NMR(100MHz,Chloroform-d)δ170.14,167.78,158.29,152.85,149.37,147.58, 146.61,139.49,138.98,134.39,133.36,130.77,129.34,127.91,124.85,123. 66,121.96,120.12,119.40,112.89,111.72,105.91,105.18,103.82,92.86,91 .96,45.72,38.24,34.86,29.90,28.66,25.26,21.97,14.34.MALDI-TOF / MS,m / z calc.for C 35 H 34 FN3O5S[M+1]calc 628.22, found 628.38.

[0100] Example 4

[0101] Synthesis of highly sensitive fluorescent probes based on type III dyes:

[0102]

[0103] III-1 (1 mmol), cesium carbonate (3 mmol), and sodium iodide (3 mmol) were dissolved in acetonitrile (10 mL). The mixture was stirred at room temperature for 30 min. Tert-butyl (1.5 mmol) carbamate (4-(bromoethylphenyl)amino)-5-methyl-1-oxohexanoyl-3-yl)carbamate was added to the mixture, and the mixture was stirred overnight at room temperature. The mixture was then poured into ice water and extracted three times with dichloromethane. The organic phase was dried and concentrated. The residue was redissolved in dichloromethane (0.5 mL), and trifluoroacetic acid (0.5 mL) was added. The mixture was stirred at room temperature for 20 min. The organic phase was removed under reduced pressure, and the residue was separated by column chromatography to obtain probe III-1-LAP.

[0104] Nuclear magnetic resonance (NMR) Figure 16 and Figure 17 The structure of the compound was analyzed by MALDI-TOF mass spectrometry. 1H NMR(400MHz,Chloroform-d)δ7.89(d,J=7.5Hz,1H),7.68(d,J=8.1Hz,3H),7.60(t,J=7.4Hz,1H),7.53–7.49(m, 1H),7.41(d,J=8.1Hz,3H),7.22–7.13(m,3H),6.85(d,J=4.8Hz,1H),6.77(d,J=7.2Hz,1H),6.62(d,J=7.2Hz,1H) ,6.31(d,J=11.2Hz,1H),5.42–5.27(m,1H),5.12(q,J=11.6Hz,2H),3.82–3.62(m,1H),3.21–3.07(m,2H),2.83(s ,6H),2.68–2.61(m,1H),2.43–2.31(m,2H),2.02(d,J=16.0Hz,2H),1.67(s,9H),1.54–1.47(m,2H),1.25(s,6H). 13 CNMR(100MHz,Chloroform-d)δ167.19,162.18,159.60,157.52,156.26,149.47,148.45,147.64,14 7.51,147.07,138.01,135.21,134.59,131.53,130.02,129.18,128.59,127.65,125.02,124.19,12 4.00,121.66,119.74,113.61,113.41,111.19,110.58,108.55,108.02,105.68,103.04,71.10,53. 78,37.97,32.21,29.71,29.07,26.40,24.88,23.45,23.17,21.65,14.14,11.45.MALDI-TOF / MS,m / z calc.for C 48 H 52 FN5O6S[M+1]calc 845.36,found846.51.

[0105] Example 5

[0106] Absorption and emission spectra of the dye in PBS buffer at pH 4.0–10.0:

[0107] The UV-Vis absorption and fluorescence emission spectra of the rhodol dye I-1 and the hydroxymethylated dye S2-1 prepared in Example 1 are shown. (UV-Vis absorption spectra were measured using a Shimadzu UV-1800 spectrophotometer, and fluorescence emission spectra were measured using an Edinburgh fluorescence spectrometer.)

[0108] The rhodol dye I-1 and hydroxymethylated dye S2-1 prepared in Example 1 were prepared into a 1 mM stock solution using DMSO solvent. Then, 5 μM of the stock solution was taken and diluted to 1 mL with 20 mM PBS buffer corresponding to different pH values ​​to prepare a 5 μM test solution.

[0109] Dye S2-1 exhibits almost no absorption or emission peaks in buffered solvents with pH ranges of 4.0-10.0. Figure 18 b); Dye I-1 exhibited specific absorption peaks at 520 nm and 555 nm, respectively, and the absorption and emission intensities of the dye gradually increased with increasing pH, reaching maximum absorption and emission intensities at pH 7.5. Figure 18 a) At pH 7.5, the emission intensity ratio of the two dyes reached 1200 times.

[0110] The UV-Vis absorption and fluorescence emission spectra of the rhodol dye II-1 and the hydroxymethylated dye S4-1 prepared in Example 2 were obtained. (UV-Vis absorption spectra were measured using a Shimadzu UV-1800 spectrophotometer, and fluorescence emission spectra were measured using an Edinburgh fluorescence spectrometer.)

[0111] The rhodol dye II-1 and hydroxymethylated dye S4-1 prepared in Example 2 were prepared into a 1 mM stock solution using DMSO solvent. Then, 5 μM of the stock solution was taken and diluted to 1 mL with 20 mM PBS buffer corresponding to different pH values ​​to prepare a 5 μM test solution.

[0112] Dye S4-1 exhibits extremely low absorption and emission in buffered solvents with pH ranges of 4.0-10.0. Figure 19 b); Dye II-1 exhibited specific absorption peaks at 550 nm and 610 nm, respectively, and with increasing pH, the absorption and emission intensities of the dye gradually increased, reaching maximum absorption and emission intensities at pH 7.5. Figure 19 a) At pH 7.5, the emission intensity ratio of the two dyes reached 300 times.

[0113] The UV-Vis absorption and fluorescence emission spectra of the rhodol dye III-1 and the hydroxymethylated dye S8-1 prepared in Example 3 are shown. (UV-Vis absorption spectra were measured using a Shimadzu UV-1800 spectrophotometer, and fluorescence emission spectra were measured using an Edinburgh fluorescence spectrometer.)

[0114] The rhodol dye III-1 and hydroxymethylated dye S8-1 prepared in Example 3 were prepared into a 1 mM stock solution using DMSO solvent. Then, 5 μM of the stock solution was taken and diluted to 1 mL with 20 mM PBS buffer corresponding to different pH values ​​to prepare a 5 μM test solution.

[0115] Dye S8-1 exhibits extremely low absorption and emission in buffered solvents with pH 4.0–10.0. Figure 20 b); Dye III-1 exhibited specific absorption peaks at 680 nm and 710 nm, respectively, and the absorption and emission intensities of the dye gradually increased with increasing pH, reaching maximum absorption and emission intensities at pH 7.5. Figure 20 a) At pH 7.5, the emission intensity ratio of the two dyes reached 370 times.

[0116] Example 6

[0117] Photophysical property testing of probe III-1-LAP developed based on III-1 dye:

[0118] The III-1-LAP prepared in Example 4 was prepared into a 1 mM stock solution using DMSO. 5 μL of the stock solution was taken and diluted to 1 mL with 20 mM PBS at pH 7.4. 0–100 U / L of LAP was added to the solution, and the solution was incubated in a shaker at 37 °C for 1 h. Subsequently, the absorption and emission spectra of the probe were measured.

[0119] The results are as follows Figure 21 As shown, the probe exhibited extremely low absorption and emission intensities without the addition of LAP. With the gradual addition of LAP, the probe showed significant absorption and emission maxima at 680 nm and 710 nm, respectively, reaching their maximum at a LAP concentration of 50 U / L. At a LAP enzyme concentration of 1 U / L, the probe's fluorescence increased by 26.4 times, and at 50 U / L, the fluorescence increased by 287 times. The detection limit for LAP was 0.004 U / L. These results demonstrate that this novel dye platform endows the probe with extremely high response sensitivity and can be used to construct ultra-sensitive fluorescent probes.

[0120] This application was supported by the National Natural Science Foundation of China (Grant No. 22325401) and the Hunan Provincial Science and Technology Program (Grant No. 2021RC4021).

[0121] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A dye for constructing highly sensitive long-wavelength fluorescent probes, wherein the parent dye is rhodol dye, characterized in that, The structural formula of the rhodol dye is shown in Formula A: Formula A Connect formula A with any of the following structural formulas; Wherein, R1 is ethyl; R2 is either H or F; R3 is ; R4 is a methyl group.

2. The dye for constructing a highly sensitive long-wavelength fluorescent probe according to claim 1, characterized in that, The preparation process of type I fluorescent dyes is as follows: Step 1: 3-Disubstituted aminophenol and 2-(5-substituted (R2)-2-hydroxy-4-methoxybenzoyl)benzoic acid are dissolved in methanesulfonic acid and heated to 90-140°C for 4-10 h. The reaction mixture is then poured into ice water, and an inorganic acid is added. The large amount of solid precipitate that forms is filtered, washed, dried, and separated by column chromatography to obtain dye intermediate S1, wherein R1 is ethyl; R2 is H or F; and the inorganic acid is one of sulfuric acid, hydrochloric acid, or perchloric acid. Step 2: Dye intermediate S1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in dichloromethane and reacted overnight at room temperature. The solvent is removed under reduced pressure, and the residue is separated by column chromatography to obtain dye intermediate S2. Step 3: Dissolve dye intermediate S2 in dichloromethane, lower the reaction temperature to 0°C, slowly add boron tribromide, and after the addition is complete, react at room temperature for 2-7 h, add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain fluorescent dye I.

3. The dye for constructing a highly sensitive long-wavelength fluorescent probe according to claim 1, characterized in that, The preparation process of type II fluorescent dyes is as follows: Step 1: 5-Substituted-8-methoxy-1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinoxaline and 2-(5-Substituted(R2)-2-hydroxy-4-methoxybenzoyl)benzoic acid are dissolved in methanesulfonic acid and heated to 90-140°C for 4-10 h. The reaction is then poured into ice water, and an inorganic acid is added. The large amount of solid precipitate that has formed is filtered, washed, dried, and separated by column chromatography to obtain dye intermediate S3, wherein R2 is H or F. R3 is The inorganic acid is one of sulfuric acid, hydrochloric acid, and perchloric acid. Step 2: Dye intermediate S3, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane and reacted overnight at room temperature. The solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain dye intermediate S4. Step 3: Dissolve dye intermediate S4 in dichloromethane, lower the reaction temperature to 0°C, slowly add boron tribromide, and after the addition is complete, react at room temperature for 2-7 h, add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain fluorescent dye II.

4. The dye for constructing a highly sensitive long-wavelength fluorescent probe according to claim 1, characterized in that, The preparation process of type III fluorescent dyes is as follows: Step 1: Dye intermediates S5 and S6 are dissolved in acetic anhydride, potassium acetate is added, and the mixture is reacted at 60°C for 2-6 h. The reaction mixture is then poured into ice water, filtered, washed with water, and the filter residue is dried. The residue is then separated by column chromatography to obtain dye intermediate S7; wherein R2 is H or F; and R4 is methyl. Step 2: Dye intermediate S7, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane and reacted overnight at room temperature. The solvent was removed under reduced pressure, and the residue was separated by column chromatography to obtain dye intermediate S8. Step 3: Dissolve dye intermediate S8 in dichloromethane, lower the reaction temperature to 0°C, slowly add boron tribromide, and after the addition is complete, react at room temperature for 2-7 h, add ultrapure water to quench the reaction, extract three times with dichloromethane, evaporate the organic phase under reduced pressure, and separate the residue by column chromatography to obtain fluorescent dye III.

5. A highly sensitive visible-near-infrared fluorescent probe, characterized in that, Based on the dye construction described in claim 1, fluorescent probes based on type I-III rhodol dyes are obtained, and their preparation methods are as follows: R5 is: R6 is Br; R5-R6 are response sites for different analytes, including superoxide anion (O2). - ), hydrogen sulfoxide (HNO), cysteine ​​(Cys), hydrogen peroxide (H2O2), quinone oxidoreductase (hNQO1), cytochrome C (P450) and leucine aminopeptidase (LAP).

6. The method for preparing the visible-near-infrared highly sensitive fluorescent probe according to claim 5, characterized in that, The preparation process of the fluorescent probe is as follows: Dyes I-III, cesium carbonate, and NaI were dissolved in acetonitrile. The reaction mixture was stirred at room temperature for 10-60 min. Then, R5-R6 were added, and the mixture was stirred at room temperature for 1-24 h. The mixture was then poured into ice water and extracted three times with dichloromethane. The organic phase was evaporated under reduced pressure, and the residue was separated by column chromatography to obtain the corresponding probe.

7. The application of the visible-near-infrared high-sensitivity fluorescent probe according to claim 5 in the detection of analytes in body fluids.

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