阅读说明：本技术 一种检测生物硫醇的近红外荧光探针及其制备方法 (Near-infrared fluorescent probe for detecting biological mercaptan and preparation method thereof ) 是由 刘萍 凤旭凯 李剑利 厍梦尧 陈娇 于 2021-06-17 设计创作，主要内容包括：本发明公开了一种检测生物硫醇的近红外荧光探针及其制备方法,首先,以异佛尔酮和丙二腈为原料,通过Knoevenagel缩合反应合成TEM；以2,4-二羟基苯甲醛和丙酸钠为原料,通过环化和水解反应合成7-羟基-3-甲基-香豆素；之后以7-羟基-3-甲基-香豆素和N-溴代丁二酰亚胺为原料,通过卤化和水解反应合成7-羟基-3-醛基-香豆素；再以TEM和7-羟基-3-醛基-香豆素为原料,经过Knoevenagel缩合反应合成中间体TX-OH；最后以TX-OH和丙烯酰氯为原料,合成荧光探针。该荧光探针对硫醇具有较好的选择性,且不受其它氨基酸的干扰。可用于生物样品中生物硫醇的检测,具有良好的应用前景。(The invention discloses a near-infrared fluorescent probe for detecting biological mercaptan and a preparation method thereof, and the near-infrared fluorescent probe is prepared by synthesizing a TEM (transmission electron microscope) by taking isophorone and malononitrile as raw materials through a Knoevenagel condensation reaction; synthesizing 7-hydroxy-3-methyl-coumarin from 2, 4-dihydroxybenzaldehyde and sodium propionate by cyclization and hydrolysis reaction; then 7-hydroxy-3-methyl-coumarin and N-bromosuccinimide are used as raw materials, and the 7-hydroxy-3-aldehyde-coumarin is synthesized through halogenation and hydrolysis reaction; then TEM and 7-hydroxy-3-aldehyde-coumarin are used as raw materials, and an intermediate TX-OH is synthesized through Knoevenagel condensation reaction; and finally, synthesizing the fluorescent probe by taking TX-OH and acryloyl chloride as raw materials. The fluorescent probe has better selectivity for thiol and is not interfered by other amino acids. Can be used for detecting biological mercaptan in a biological sample and has good application prospect.)

1. A near-infrared fluorescent probe for detecting biological thiol is characterized in that the fluorescent probe has a structural formula shown as the following formula (I):

2. the method for preparing the near-infrared fluorescent probe for detecting the biological thiol as claimed in claim 1, which is implemented by the following steps:

step 1, synthesizing an intermediate TEM by taking isophorone and malononitrile as raw materials through a Knoevenagel condensation reaction;

step 2, synthesizing an intermediate 7-hydroxy-3-methyl-coumarin by cyclization and hydrolysis reaction by taking 2, 4-dihydroxybenzaldehyde and sodium propionate as raw materials;

step 3, synthesizing an intermediate 7-hydroxy-3-aldehyde-coumarin by taking the intermediate 7-hydroxy-3-methyl-coumarin and N-bromosuccinimide obtained in the step 2 as raw materials through halogenation and hydrolysis reactions;

step 4, synthesizing an intermediate TX-OH by taking the intermediate TEM obtained in the step 1 and the step 3 and 7-hydroxy-3-aldehyde-coumarin as raw materials through a Knoevenagel condensation reaction;

and 5, synthesizing the fluorescent probe by using the intermediate TX-OH obtained in the step 4 and acryloyl chloride as raw materials.

3. The method for preparing a near-infrared fluorescent probe for detecting biological thiol according to claim 2, wherein the step 1 specifically comprises:

dissolving isophorone, malononitrile and a catalyst in N, N-dimethylformamide, stirring for 6 hours at 120 ℃ by taking argon as a protective gas, cooling to room temperature after the reaction is finished, injecting the reaction mixed solution into ice water, separating out a brown solid, drying, and separating and purifying by using column chromatography to obtain an intermediate TEM;

the catalyst is a viscous liquid formed by mixing acetic anhydride, glacial acetic acid and piperidine; the molar ratio of isophorone to malononitrile is 1: 1.

4. the method for preparing a near-infrared fluorescent probe for detecting biological thiol according to claim 2, wherein the step 2 specifically comprises:

dissolving 2, 4-dihydroxybenzaldehyde, sodium propionate and a catalyst triethylamine in acetic anhydride, heating and refluxing for 12h, injecting water into a reaction liquid, performing suction filtration to obtain a brick red solid, washing, drying, separating and purifying by column chromatography, dissolving the obtained product in dichloromethane, continuously reacting, adding acetic anhydride and pyridine as catalysts, stirring for 24h at room temperature, extracting by using dichloromethane and water, collecting an organic phase, drying by using anhydrous sodium sulfate, filtering, performing reduced pressure rotary evaporation to remove the organic solvent, and separating and purifying by using column chromatography to obtain an intermediate 7-hydroxy-3-methyl-coumarin; the molar ratio of the 2, 4-dihydroxybenzaldehyde to the sodium propionate is 1: 1.

5. the method for preparing a near-infrared fluorescent probe for detecting biological thiol according to claim 2, wherein the step 3 specifically comprises:

using azobisisobutyronitrile as a free radical reaction initiator, dissolving the azodiisobutyronitrile, 7-hydroxy-3-methyl-coumarin and N-bromosuccinimide in carbon tetrachloride, heating and refluxing for 8 hours, decompressing and rotary-steaming to remove an organic solvent, adding sodium acetate to dissolve the sodium acetate in acetic anhydride, heating and refluxing for 12 hours, then adding a hydrochloric acid solution, continuously stirring, cooling to room temperature, carrying out suction filtration, and washing with ice water to obtain a brown solid, namely an intermediate 7-hydroxy-3-aldehyde-coumarin; the molar ratio of 7-hydroxy-3-methyl-coumarin to N-bromosuccinimide is 1: 2.

6. the method for preparing a near-infrared fluorescent probe for detecting biological thiol according to claim 2, wherein in the step 4, the method specifically comprises:

dissolving TEM, 7-hydroxy-3-aldehyde-coumarin and a catalyst in absolute ethyl alcohol, reacting for 4 hours at 50 ℃, cooling to room temperature after the reaction is finished, filtering to obtain red solid precipitate, and repeatedly washing with absolute ethyl alcohol to obtain an intermediate TX-OH; TEM and 7-hydroxy-3-aldehyde-coumarin in a molar ratio of 1:1, the catalyst is piperidine.

7. The method for preparing a near-infrared fluorescent probe for detecting biological thiol according to claim 2, wherein in the step 5, the method specifically comprises:

dissolving TX-OH, acryloyl chloride and a catalyst in dichloromethane, stirring at room temperature, monitoring the reaction process by TLC (thin layer chromatography) until the reaction process is finished, performing reduced pressure spin-drying on the solvent, and performing column chromatography separation and purification to obtain the fluorescent probe; the molar ratio of TX-OH to acryloyl chloride is 1: 2, the catalyst is triethylamine.

Technical Field

The invention belongs to the technical field of analysis and detection, and particularly relates to a near-infrared fluorescent probe for detecting biological thiol and a preparation method of the near-infrared fluorescent probe.

Background

Cysteine (Cys), homocysteine (Hcy), and reduced Glutathione (GSH) are collectively referred to as biological thiols. Being a large number of small molecule sulfhydryl species present in organisms, they play a very important role in balancing redox processes and mitigating damage caused by free radicals and toxins. Studies have shown that fluctuations in biological thiol levels are of great relevance to the development of many human diseases, such as heart disease, dysplasia, skin damage, liver damage, osteoporosis, and alzheimer's disease. Therefore, the detection of Cys/Hcy/GSH in vivo will have a positive and meaningful impact on understanding the development and progression of disease.

Fluorescent probe molecules generally consist of a fluorophore, a linker arm, and a recognition group. Fluorescent probe molecules generally emit at a shorter wavelength and are generally interfered by the background of the biological sample itself, resulting in false positive signals. And the near infrared (650-900 nm) fluorescence emission can effectively reduce the interference factor. Therefore, the development of near-infrared fluorescent probes will greatly promote the accuracy of biological sample detection.

Disclosure of Invention

The invention aims to provide a near-infrared fluorescent probe for detecting biological thiol, and the fluorescent probe molecule has good selectivity and anti-interference capability on detection of the biological thiol.

The invention also aims to provide a preparation method of the near-infrared fluorescent probe for detecting the biological thiol.

The invention adopts the technical scheme that a near-infrared fluorescent probe for detecting biological mercaptan has a structural formula shown as the following formula (I):

the invention adopts another technical scheme that a preparation method of a near-infrared fluorescent probe for detecting biological mercaptan is implemented according to the following steps:

step 1, synthesizing an intermediate TEM by taking isophorone and malononitrile as raw materials through a Knoevenagel condensation reaction;

step 2, synthesizing an intermediate 7-hydroxy-3-methyl-coumarin by cyclization and hydrolysis reaction by taking 2, 4-dihydroxybenzaldehyde and sodium propionate as raw materials;

step 3, synthesizing an intermediate 7-hydroxy-3-aldehyde-coumarin by taking the intermediate 7-hydroxy-3-methyl-coumarin and N-bromosuccinimide obtained in the step 2 as raw materials through halogenation and hydrolysis reactions;

step 4, synthesizing an intermediate TX-OH by taking the intermediate TEM obtained in the step 1 and the step 3 and 7-hydroxy-3-aldehyde-coumarin as raw materials through a Knoevenagel condensation reaction;

and 5, synthesizing the fluorescent probe by using the intermediate TX-OH obtained in the step 4 and acryloyl chloride as raw materials.

The present invention is also characterized in that,

in the step 1, the method specifically comprises the following steps:

dissolving isophorone, malononitrile and a catalyst in N, N-dimethylformamide, stirring for 6 hours at 120 ℃ by taking argon as a protective gas, cooling to room temperature after the reaction is finished, injecting the reaction mixed solution into ice water, separating out a brown solid, drying, and separating and purifying by using column chromatography to obtain an intermediate TEM;

the catalyst is a viscous liquid formed by mixing acetic anhydride, glacial acetic acid and piperidine; the molar ratio of isophorone to malononitrile is 1: 1.

in the step 2, the method specifically comprises the following steps:

dissolving 2, 4-dihydroxybenzaldehyde, sodium propionate and a catalyst triethylamine in acetic anhydride, heating and refluxing for 12h, injecting water into a reaction liquid, performing suction filtration to obtain a brick red solid, washing, drying, separating and purifying by column chromatography, dissolving the obtained product in dichloromethane, continuously reacting, adding acetic anhydride and pyridine as catalysts, stirring for 24h at room temperature, extracting by using dichloromethane and water, collecting an organic phase, drying by using anhydrous sodium sulfate, filtering, performing reduced pressure rotary evaporation to remove the organic solvent, and separating and purifying by using column chromatography to obtain an intermediate 7-hydroxy-3-methyl-coumarin; the molar ratio of the 2, 4-dihydroxybenzaldehyde to the sodium propionate is 1: 1.

in step 3, the method specifically comprises the following steps:

using azobisisobutyronitrile as a free radical reaction initiator, dissolving the azodiisobutyronitrile, 7-hydroxy-3-methyl-coumarin and N-bromosuccinimide in carbon tetrachloride, heating and refluxing for 8 hours, decompressing and rotary-steaming to remove an organic solvent, adding sodium acetate to dissolve the sodium acetate in acetic anhydride, heating and refluxing for 12 hours, then adding a hydrochloric acid solution, continuously stirring, cooling to room temperature, carrying out suction filtration, and washing with ice water to obtain a brown solid, namely an intermediate 7-hydroxy-3-aldehyde-coumarin; the molar ratio of 7-hydroxy-3-methyl-coumarin to N-bromosuccinimide is 1: 2.

in step 4, the method specifically comprises the following steps:

dissolving TEM, 7-hydroxy-3-aldehyde-coumarin and a catalyst in absolute ethyl alcohol, reacting for 4 hours at 50 ℃, cooling to room temperature after the reaction is finished, filtering to obtain red solid precipitate, and repeatedly washing with absolute ethyl alcohol to obtain an intermediate TX-OH; TEM and 7-hydroxy-3-aldehyde-coumarin in a molar ratio of 1:1, the catalyst is piperidine.

In step 5, the method specifically comprises the following steps:

dissolving TX-OH, acryloyl chloride and a catalyst in dichloromethane, stirring at room temperature, monitoring the reaction process by TLC (thin layer chromatography) until the reaction process is finished, performing reduced pressure spin-drying on the solvent, and performing column chromatography separation and purification to obtain the fluorescent probe; the molar ratio of TX-OH to acryloyl chloride is 1: 2, the catalyst is triethylamine.

The invention has the advantages that the near-infrared fluorescent probe is constructed by a simple organic synthesis means to realize the simultaneous detection of three kinds of biological mercaptan, and an on-off signal response mechanism is presented by the change of the fluorescence intensity. In the detection process, the fluorescent probe molecule shows good selectivity and anti-interference capability, has a proper pH application range, and provides a certain application potential for further detection under physiological conditions.

Drawings

FIG. 1 is a fluorescence emission spectrum of probe molecules (10. mu. mol/L) in a mixed solution of different organic solvents and water (5:5, V: V);

FIG. 2 shows fluorescence emission spectra of probe molecules (10. mu. mol/L) and Cys responses in different organic solvent and water (5:5, V: V) mixed solutions;

FIG. 3 shows DMSO and H₂O fluorescence emission spectra of response of probe molecules (10 mu mol/L) and Cys in the solution with different volume ratios;

FIG. 4 is DMSO: H₂A graph of the change in fluorescence of probe molecules (10. mu. mol/L) in O (5:5, V: V) solution in response to Cys at different pH;

FIG. 5 is DMSO: H₂Fluorescence emission spectra of probe molecules (10. mu. mol/L) in O (5:5, V: V) solution in selective response to biological thiol;

FIG. 6 is DMSO: H₂Histogram of fluorescence intensity of competition response of probe molecules (10. mu. mol/L) in O (5:5, V: V) solution with Cys and other different amino acids;

FIG. 7 is DMSO: H₂Histogram of fluorescence intensity of competitive responses of probe molecules (10. mu. mol/L) in O (5:5, V: V) solution with Hcy and other different amino acids;

FIG. 8 is DMSO: H₂Histogram of fluorescence intensity of competitive responses of probe molecules (10. mu. mol/L) in O (5:5, V: V) solution with GSH and other different amino acids;

FIG. 9 is DMSO: H₂Responding to fluorescence emission spectra of probes in O (5:5, V: V) solution and Cys with different concentrations,

FIG. 10 shows the increase in fluorescence intensity before and after the probe response (F-F)₀) A linear fit to Cys concentration;

FIG. 11 is DMSO: H₂Responding to ultraviolet absorption spectrums by a probe in an O (5:5, V: V) solution and Cys with different concentrations;

FIG. 12 is DMSO: H₂Fluorescence emission spectra of probes in O (5:5, V: V) solution and different concentrations of Hcy response;

FIG. 13 shows the increase in fluorescence intensity before and after the probe response (F-F)₀) A linear fit to Hcy concentration;

FIG. 14 is DMSO: H₂Responding to ultraviolet absorption spectrums by probes in O (5:5, V: V) solution and Hcy with different concentrations;

FIG. 15 is DMSO: H₂In O (5:5, V: V) solutionThe probe responds to fluorescence emission spectra with GSH with different concentrations;

FIG. 16 shows the increase in fluorescence intensity before and after the probe response (F-F)₀) A linear fit to GSH concentration;

FIG. 17 is DMSO: H₂Probes in O (5:5, V: V) solution respond to ultraviolet absorption spectra with GSH of different concentrations.

Detailed Description

The present invention will be described in detail with reference to the following detailed description and accompanying drawings.

The invention relates to a near-infrared fluorescent probe for detecting biological mercaptan, which has a structural formula shown as the following formula (I):

the invention relates to a preparation method of a near-infrared fluorescent probe for detecting biological mercaptan, which is implemented according to the following steps:

step 1, synthesizing an intermediate TEM (transmission electron microscope) shown as a following formula (II) by taking isophorone and malononitrile as raw materials through a Knoevenagel condensation reaction;

the method specifically comprises the following steps: dissolving isophorone, malononitrile and a catalyst in N, N-dimethylformamide, stirring for 6 hours at 120 ℃ by taking argon as a protective gas, cooling to room temperature after the reaction is finished, injecting the reaction mixed solution into ice water, separating out a brown solid, drying, and separating and purifying by using column chromatography to obtain an intermediate TEM;

the catalyst is a viscous liquid formed by mixing acetic anhydride, glacial acetic acid and piperidine; the molar ratio of isophorone to malononitrile is 1: 1;

step 2, synthesizing an intermediate 7-hydroxy-3-methyl-coumarin shown in the following formula (III) by using 2, 4-dihydroxybenzaldehyde and sodium propionate as raw materials through cyclization and hydrolysis reaction;

the method specifically comprises the following steps: dissolving 2, 4-dihydroxybenzaldehyde, sodium propionate and a catalyst triethylamine in acetic anhydride, heating and refluxing for 12h, injecting water into a reaction liquid, separating out a solid, performing suction filtration to obtain a brick red solid, repeatedly washing ice water, drying, performing column chromatography separation and purification, dissolving the obtained product in dichloromethane, continuously reacting, adding acetic anhydride and pyridine as catalysts, stirring for 24h at room temperature, extracting with dichloromethane and water, collecting an organic phase, drying with anhydrous sodium sulfate, filtering, performing reduced pressure rotary evaporation to remove the organic solvent, and performing column chromatography separation and purification to obtain an intermediate 7-hydroxy-3-methyl-coumarin;

the molar ratio of the 2, 4-dihydroxybenzaldehyde to the sodium propionate is 1: 1;

step 3, taking the intermediates 7-hydroxy-3-methyl-coumarin and N-bromosuccinimide (NBD) obtained in the step 2 as raw materials, and synthesizing the intermediate 7-hydroxy-3-aldehyde-coumarin shown in the following formula (IV) through halogenation and hydrolysis reaction;

the method specifically comprises the following steps: taking Azobisisobutyronitrile (AIBN) as a free radical reaction initiator, dissolving the Azobisisobutyronitrile (AIBN), 7-hydroxy-3-methyl-coumarin and N-bromosuccinimide in carbon tetrachloride, heating and refluxing for 8 hours, carrying out reduced pressure rotary evaporation to remove an organic solvent, then adding sodium acetate to dissolve the sodium acetate in acetic anhydride, heating and refluxing for 12 hours, then adding a hydrochloric acid solution, continuously stirring, cooling to room temperature, carrying out suction filtration, and repeatedly washing with ice water to obtain a brown solid, namely an intermediate 7-hydroxy-3-aldehyde-coumarin;

the molar ratio of 7-hydroxy-3-methyl-coumarin to N-bromosuccinimide is 1: 2;

step 4, taking the intermediate TEM obtained in the steps 1 and 3 and the 7-hydroxy-3-aldehyde-coumarin as raw materials, and synthesizing an intermediate TX-OH shown as the following formula (V) through a Knoevenagel condensation reaction;

the method specifically comprises the following steps: dissolving TEM, 7-hydroxy-3-aldehyde-coumarin and a catalyst in absolute ethyl alcohol, reacting for 4 hours at 50 ℃, cooling to room temperature after the reaction is finished, filtering to obtain red solid precipitate, and repeatedly washing with absolute ethyl alcohol to obtain an intermediate TX-OH;

TEM and 7-hydroxy-3-aldehyde-coumarin in a molar ratio of 1:1, the catalyst is piperidine;

and 5, synthesizing the fluorescent probe TX by taking the intermediate TX-OH obtained in the step 4 and acryloyl chloride as raw materials, wherein the structural formula is shown as the formula (I).

The method specifically comprises the following steps: dissolving TX-OH, acryloyl chloride and a catalyst in dichloromethane, stirring at room temperature, monitoring the reaction process by TLC (thin layer chromatography) until the reaction process is finished, performing reduced pressure spin-drying on the solvent, and performing column chromatography separation and purification to obtain the fluorescent probe;

the molar ratio of TX-OH to acryloyl chloride is 1: 2, the catalyst is triethylamine.

The principle of the fluorescence probe prepared by the method for detecting the biological mercaptan is as follows:

in DMSO, H₂In the O (5:5, V: V) mixed solution, the probe solution itself hardly emits a fluorescent signal. When the biological thiol molecules are added into the solution, the fluorescence intensity is obviously improved at 488nm excitation wavelength and 718nm, and an 'on-off' response mechanism of a fluorescence signal is presented. And the color of the solution turned yellow to dark blue under "naked eye" observation.

Examples

The invention relates to a preparation method of a near-infrared fluorescent probe for detecting biological mercaptan, which is implemented according to the following steps:

the synthetic route is as follows:

step 1, synthesizing an intermediate TEM (transmission electron microscope) shown as a following formula (II) by taking isophorone and malononitrile as raw materials through a Knoevenagel condensation reaction;

the method specifically comprises the following steps: in a 250mL three-necked flask, under argon atmosphere, was added 0.2g of acetic anhydride, 0.4mL of glacial acetic acid, and 1.8mL of piperidine, followed by 16.5mL (110mmol) of isophorone, 6.6g (110mmol) of malononitrile, and the mixture was dissolved in 55mL of N, N-dimethylformamide and stirred at 120 ℃ for 6 hours. After the reaction was completed, the reaction mixture was cooled to room temperature. Pouring the reaction mixed solution into ice water, separating out a brown solid, drying, and separating and purifying by using column chromatography, wherein an eluent is petroleum ether and dichloromethane is 1:1(v: v), so as to obtain a yellow solid;

step 2, synthesizing an intermediate 7-hydroxy-3-methyl-coumarin shown in the following formula (III) by using 2, 4-dihydroxybenzaldehyde and sodium propionate as raw materials through cyclization and hydrolysis reaction;

the method specifically comprises the following steps: 6.0g of 2, 4-dihydroxybenzaldehyde and 9.0g of sodium propionate were added to a 100mL round-bottom flask and dissolved in 15mL of propionic anhydride, 6mL of triethylamine was slowly added dropwise to the flask using a dropping funnel, and the mixture was heated under reflux for 12 hours to react, whereby the color of the solution changed from yellow to black. 30mL of water is injected into the reaction solution, and then solid is separated out; and (5) carrying out suction filtration to obtain a brick red solid, repeatedly washing with ice water and drying. Separating and purifying by column chromatography, wherein an eluent is dichloromethane and methanol which are 30:1, and obtaining white solid;

step 3, taking the intermediates 7-hydroxy-3-methyl-coumarin and N-bromosuccinimide (NBD) obtained in the step 2 as raw materials, and synthesizing the intermediate 7-hydroxy-3-aldehyde-coumarin shown in the following formula (IV) through halogenation and hydrolysis reaction;

the method specifically comprises the following steps: a50 mL round bottom flask was charged with 0.190g of 7-hydroxy-3-methyl-coumarin dissolved in 20mL of dichloromethane, 2mL of acetic anhydride and 3 drops of pyridine were added, stirred at room temperature for 24h, and the progress of the reaction was monitored by TLC until the reaction was complete. Extraction was performed using dichloromethane and water, and the organic phase was collected and dried using anhydrous sodium sulfate. Filtering, performing reduced pressure rotary evaporation to remove the organic solvent, and separating and purifying by using column chromatography, wherein an eluent is pure dichloromethane, so as to obtain a white solid product, namely 7-acetoxyl-3-aldehyde-coumarin;

step 4, taking the intermediate TEM obtained in the steps 1 and 3 and the 7-hydroxy-3-aldehyde-coumarin as raw materials, and synthesizing an intermediate TX-OH shown as the following formula (V) through a Knoevenagel condensation reaction;

the method specifically comprises the following steps: under argon, 0.30g (1.6mmol) of 7-hydroxy-3-aldehyde-coumarin and 0.372g (2.0mmol) of TEM were dissolved in 10mL of absolute ethanol, 100. mu.L of piperidine was then added, and the reaction mixture was reacted overnight at 50 ℃. After the reaction was completed, the reaction mixture was cooled to room temperature. Filtering to obtain red solid precipitate, and repeatedly washing with anhydrous ethanol;

step 5, synthesizing a fluorescent probe TX by taking the intermediate TX-OH obtained in the step 4 and acryloyl chloride as raw materials, wherein the structural formula is shown as the formula (I);

the method specifically comprises the following steps: in N₂Under protection, 0.10g (0.28mmol) of TX-OH and 0.045mg (0.50mmol) of acryloyl chloride were dissolved in 5mL of anhydrous dichloromethane, and 100. mu.L of anhydrous triethylamine was added. Stir at rt and monitor the progress to completion by TLC. Removing the organic solvent by a rotary evaporator under reduced pressure, and separating and purifying by column chromatography, wherein an eluent is n-hexane and ethyl acetate which are 3:1 to obtain an orange-yellow solid;

wherein the product is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.38(s,1H),7.64(d,J＝8.5Hz,1H),7.49(d,J＝16.2Hz,1H),7.28(d,J＝1.9Hz,1H),7.20-7.00(m,2H),6.73(s,1H),6.53-6.38(m,1H),6.31(dd,J＝17.2,10.3Hz,1H),6.14-6.02(m,1H),2.36(s,4H),0.89(s,6H).

¹³C NMR(400MHz,DMSO-d₆)δ170.54,164.15,159.65,155.24,153.93,153.60,140.88,135.00,133.47,130.83,130.37,127.79,124.71,122.89,119.66,117.87,114.13,113.34,110.49,78.31,42.77,38.43,32.22,27.90.

MS:([M+Na]⁺)；Calcd for C₂₅H₂₀N₂O₄:435.1315；Found:435.1268.

probe test solvent screening

Based on the examples, the fluorescence emission performance of the probe TX (10 μmol/L) in different mixed solutions of organic solvent and water was tested. As shown in fig. 1, by comparing the fluorescence emission intensity in different organic solvents. It was found that when the organic solvent was chosen to be DMSO, the probe itself had a small background fluorescence emission. Further, as shown in fig. 2, the fluorescence emission performance of probe TX (10 μmol/L) after responding to Cys (5.0equiv.) in various organic solvent and water mixed solutions was tested. When the organic solvent was DNSO, a significant increase in fluorescence signal was exhibited.

CysCys (5.0equiv.) responses were tested for probe TX (10. mu. mol/L) in the presence of mixed solvents DMSO and water at different volume ratios, as shown in FIG. 3. It was found that when the probe TX was in DMSO: H₂The fluorescent material has better fluorescence emission under a mixed solvent system with the O ratio of 1: 1.

pH range test for probe application

In DMSO, H₂The effect of probes on Cys response in O (5:5, V: V) solution at different pH (3-11) ranges was tested, as shown in FIG. 4. The following are found: the fluorescence of the probe per se can obviously increase in a strong alkaline solvent, but the fluorescence intensity value tends to be in a stable state in neutral and alkaline solutions after response. Therefore, the probe is suitable for testing with a pH value ranging from 7 to 9.

Selective testing of probes

In DMSO, H₂O (5:5, V: V) solution, the fluorescence emission spectra of each amino acid added to probe TX (10. mu. mol/L) were compared, including: 100 μmol/L, 1) Thr; 2) ser; 3) phe; 4) met; 5) lys; 6) leu; 7) ile; 8) his; 9) arg; 10) ala; 11) val; 12) tyr, and 20. mu. mol/L, 13) Cys; 14) hcy; 15) GSH. As shown in FIG. 5, in addition to the three thiol analytes, no significant increase in fluorescence intensity was observed at 488nm for the other amino acids. The result shows that the probe TX shows a good selective detection effect on the three biological thiols.

Anti-interference capability test of probe

The interfering atmosphere was created by adding 100. mu. mol/L of each amino acid of the above examples to 10. mu. mol/L of probe TX. Thereafter, the change in the fluorescence spectrum was measured after adding 20. mu. mol/LCys, Hcy and GSH as analytes. As shown in FIGS. 6, 7 and 8, the fluorescence responses of the other various amino acids (100. mu. mol/L, bars in the grid) and the fluorescence intensities after addition of Cys, Hcy and GSH (20. mu. mol/L, bars in the black) were compared, respectively. The fluorescence emission intensity of the probe TX shows obvious change at 718nm under the excitation wavelength of 488nm, and the result shows that the recognition of the three biological thiols by the probe TX in the presence of other various amino acids is hardly influenced.

Titration experiment of the Probe

As shown in FIG. 9, the probe TX solution itself had no significant fluorescence emission signal under 488nm wavelength excitation. However, when Cys is present, the solution color is shifted. And, it showed a great increase in the value of fluorescence intensity at 710 nm. In addition, when the analyte reached 20. mu. mol/L, the fluorescence intensity was increased by about 5 times. Meanwhile, from the ultraviolet-visible absorption spectrum 10, there was a tendency similar to the increase of fluorescence at 525 nm. As shown in FIGS. 11-14, the probe showed Cys-like behavior for Hcy and GSH.

Further, as shown in FIGS. 15-17, probes TX respond with Cys with a net increase in fluorescence intensity (F-F)₀) Shows better linear correlation with Cys concentration of 0-10 mu mol/L (wherein, F₀And F are fluorescence intensity values before and after the probe reacts with Cys, Hcy, and GSH, respectively). Regression equation for probe TX is y-30.17 +79.58x(R²0.9810). At the same time, probes TX and Hcy (GSH) respond to net increase in fluorescence intensity (F-F)₀) Has better linear correlation with the concentration of Hcy (GSH) between 0 and 4 mu mol/L, and the regression equation is that y is 48.14+205.51x (R)²＝0.9772)(y＝53.36+192.51x(R²＝0.9734))。

The fluorescent probe disclosed by the invention is used for detecting biological thiol by constructing a probe TX based on a dicyan isophorone framework with strong electron pulling capability, and the fluorescence emission wavelength of the fluorescent probe is red-shifted to an NIR region by conjugated connection of a coumarin group. At the excitation wavelength of 488nm, the probe has almost no fluorescence. After the biological thiol reacts with the probe molecules, the fluorescence intensity at 718nm is obviously enhanced. And, the net increase in fluorescence intensity before and after the reaction (F-F)₀) And the concentration of the biological thiol shows better linear correlation in a certain range. The probe TX has good selectivity to thiol and is not interfered by other amino acids. Can be used for detecting biological mercaptan in a biological sample and has good application prospect.