阅读说明：本技术 一种检测生物硫醇新型荧光探针及其制备方法和应用 (Novel fluorescent probe for detecting biological thiol and preparation method and application thereof ) 是由 崔京南 何鑫 田镇豪 刘涛 何深贵 赵鑫 田艺浓 于 2019-10-30 设计创作，主要内容包括：一种检测生物硫醇新型荧光探针的制备方法及应用,其属于生物硫醇分析检测技术领域。探针采用蒽酰亚胺为荧光母体,丙烯基为反应基团与生物硫醇反应,通过探针的荧光变化实现生物硫醇的检测。荧光探针与半胱氨酸反应仅5 min即可达到最大响应值,在601 nm处荧光强度显著增强121倍；检测下限为1.0 x10<Sup>-5</Sup>mol/L；荧光探针和反应后的产物,在pH>6.5时荧光强度不受pH变化影响。检测时,观察到溶液颜色从无色至紫色,裸眼能直接判断有无生物硫醇存在。探针检测谷胱甘肽和同型半胱氨酸具有类似的效果。因此,所述荧光探针可用于检测生物硫醇,具有响应快速、灵敏度高、可在生理pH下工作的特点,在生物分子检测领域具有广阔的应用前景。(A preparation method and application of a novel fluorescent probe for detecting biological mercaptan belong to the technical field of biological mercaptan analysis and detection. The probe adopts anthracene imide as a fluorescent parent, propenyl as a reactive group to react with biological mercaptan, and the detection of the biological mercaptan is realized through the fluorescent change of the probe. The maximum response value can be reached only by 5min of reaction between the fluorescent probe and cysteine, and the fluorescence intensity at 601nm is obviously enhanced by 121 times; lower limit of detection is 1.0x10 ‑5 mol/L; fluorescent probes and products of the reaction, at pH>The fluorescence intensity at 6.5 was not affected by pH change. During detection, the color of the solution is observed to be from colorless to purple, and the existence of the biological thiol can be directly judged by naked eyes. Probes detect glutathione and homocysteine with similar effect. Therefore, the fluorescent probe can be used for detecting biological thiol,the method has the characteristics of quick response, high sensitivity and capability of working under physiological pH, and has wide application prospect in the field of biomolecule detection.)

1. A fluorescent probe for detecting biological thiol is characterized in that the molecular formula of the probe is C₂₃H₁₉NO₄The specific structural formula is as follows:

2. the method for preparing a fluorescent probe for detecting bio-thiol according to claim 1, comprising the steps of:

(1) under the protection of nitrogen, dissolving 9-bromoanthracene and anhydrous aluminum trichloride in carbon disulfide, dropwise adding an oxalyl chloride solution under an ice bath condition, reacting for 30 minutes, removing the ice bath, and stirring at normal temperature for 5-6 hours; after the reaction is completed, 50ml of 1M HCl solution is gradually dripped; cooling to room temperature, pouring the reaction solution into water, filtering, collecting solid, and finally purifying by using a silica gel chromatographic column to obtain a compound 6, wherein the compound 6 has the following structural formula:

(2) dissolving the compound 6 obtained in the step (1) in methanol under the protection of nitrogen, adding potassium peroxymonosulfonate, heating to reflux, stirring for reaction for 48 hours, cooling to room temperature, pouring the reaction liquid into water, filtering, collecting solids, and finally purifying by using a silica gel chromatographic column to obtain a compound 5, wherein the structural formula of the compound 5 is as follows:

(3) under the protection of nitrogen, dissolving the compound obtained in the step (2) in ethanol, dropwise adding n-butylamine solution, heating to reflux, stirring for 8-12h, cooling to room temperature, spin-drying the solvent, and purifying by using a silica gel chromatography column to obtain a compound 4, wherein the structural formula of the compound 4 is as follows:

(4) under the protection of nitrogen, dissolving the compound obtained in the step (3) in methanol, adding sodium methoxide, heating to reflux, stirring for 1-2h, cooling to room temperature, spin-drying the solvent, and purifying by using a silica gel chromatography column to obtain a compound 3, wherein the structure of the compound 3 is as follows:

(5) under the protection of nitrogen, dissolving the compound obtained in the step (4) in hydroiodic acid, heating to 132 ℃, reacting for 8-12h, cooling to room temperature after the reaction is finished, adding a 2M sodium hydroxide solution, adjusting the pH value to 4-5, filtering, and purifying by using a silica gel chromatography column to obtain a compound 2, wherein the compound 2 has the following structure:

(6) and (3) under the protection of nitrogen, dissolving the compound obtained in the step (5) in dichloromethane, dropwise adding 1ml of triethylamine solution under ice bath, stirring for 30min, dropwise adding acryloyl chloride solution, stirring for 1-2h, pouring the reaction solution into water after complete reaction, extracting with dichloromethane, collecting an organic phase, and finally purifying by using a silica gel chromatographic column to obtain the probe 1.

3. The method for preparing a fluorescent probe for detecting biological thiol as claimed in claim 2, wherein the molar ratio of 9-bromoanthracene to oxalyl chloride in step (1) is 1: 2-3, and the eluent for column chromatography is petroleum ether with a volume ratio of 10: 1: ethyl acetate;

in the step (2), the molar ratio of 6-bromoaceanthrylene-1, 2-dione to potassium peroxymonosulfonate is 1: 4-5, and an eluent for column chromatography is dichloromethane with a volume ratio of 1.5: 1: petroleum ether;

in the step (3), the molar ratio of 6-bromo-1, 2-dicarboxy anthracene anhydride to n-butylamine is 1: 1.2-1.5, and an eluent for column chromatography is dichloromethane with the volume ratio of 2: 1: petroleum ether;

in the step (4), the molar ratio of the N-N-butyl-6-bromo-anthraimide to the sodium methoxide is 1: 5-6, and an eluent used for column chromatography is dichloromethane with the volume ratio of 1: petroleum ether;

in the step (5), the molar ratio of the N-N-butyl-6-methoxy-anthracene imide to the hydroiodic acid is 1: 1-1.2, and an eluent used for column chromatography is dichloromethane with the volume ratio of 20: 1: methanol;

in the step (6), the molar ratio of the N-N-butyl-6-hydroxy-anthracene imide to the acryloyl chloride is 1: 0.8-1, and an eluent used for column chromatography is petroleum ether with a volume ratio of 5: 1: and (3) ethyl acetate.

4. The use of the novel fluorescent probe for detecting biological thiol as claimed in claim 1, wherein the biological thiol fluorescent probe is mixed with a buffer solution of phosphate buffer and dimethyl sulfoxide, and the test solution is added to detect the presence or absence of biological thiol by using the change of fluorescence intensity at two different emission wavelengths.

5. The use of the novel fluorescent probe for detecting biological thiols as claimed in claim 4, wherein: when the solution to be detected does not contain biological thiol, the maximum excitation wavelength of the reaction solution is 492nm, and after the biological thiol is added, the maximum excitation wavelength is 549nm and the maximum fluorescence emission wavelength is 601 nm.

6. The use of the novel fluorescent probe for detecting biological thiols according to claim 4, wherein the biological thiol comprises any one of cysteine, homocysteine, and glutathione.

Technical Field

The invention relates to a novel fluorescent probe for detecting biological thiol as well as a preparation method and application thereof, belonging to the technical field of preparation of biological thiol probes.

Background

Biological thiols such as Glutathione (GSH), Cysteine (CYS), Homocysteine (HCY) play an important role in human physiological activities, and when cysteine levels in the human body are abnormal, symptoms such as slow growth, loose skin, weakness and the like are caused. Excessive homocysteine concentration in humans is likely to cause cardiovascular disease and alzheimer's disease. Glutathione is the largest small molecule biological thiol (1-10mM) in cells, plays an important role in maintaining the redox dynamic balance of cells, and is related to cancer if the concentration of the glutathione is abnormal in a human body, so that the detection of the biological thiol is of great significance to the human body.

At present, main methods for detecting the biological mercaptan comprise an electrochemical method, an elemental analysis method, a high performance liquid chromatography and a mass spectrometry method, but the detection methods have the defects of more complex sample preparation process, more damage to samples in the detection process, expensive instruments, long detection time and the like. In recent years, the fluorescent probe method is widely applied to the field of analysis and detection, and has the advantages of good selectivity, high sensitivity, rapidness, simplicity and the like, but the currently reported biological thiol fluorescent probe has the defects, for example, the probes reported in patents CN106046012A and CN108947994A have longer reaction time, namely 50min and 30min respectively; in the paper, X.Dai, Q.H.Wu, P.C.Wang, J.Tian, Y.Xu, S.Q.Wang, J.Y.Miao, B.X.ZHao, Biosens Bioelctron 2014,59,35-39. the reaction time of the fluorescence probe for detecting the biological thiol is reported to be 90 min; the paper J.Zhou, S.xu, X.Dong, W.ZHao, Q.Zhu, Dyes and Pigments 2019,167,157 and 163. the reported reaction time for detecting the biological thiol fluorescent probe is 10 min; the probe reported in patent CN107721922A is greatly affected by pH. Therefore, the invention of the biological thiol fluorescent probe which has the advantages of quick response, good selectivity, high sensitivity and little influence by pH under physiological conditions is very important.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a biological thiol fluorescent probe which has good selectivity, high sensitivity, rapidness, convenience and little influence of pH under physiological conditions, and also provides a preparation method and application of the biological thiol fluorescent probe.

In order to solve the technical problems, the invention adopts the following technical scheme:

a fluorescent probe for detecting biological thiol is characterized in that the molecular formula of the probe is C₂₃H₁₉NO₄The specific structural formula is as follows:

the biological thiol fluorescent probe takes cysteine as an example, and the response time to cysteine is 5 min. The response time is the time required for the thiol probe to act on the aqueous solution containing cysteine and observe the peak value of the fluorescence spectrum to reach the maximum by adopting a fluorescence spectrometer.

The biological thiol fluorescent probe can resist Gly, Arg, Trp, Lys, Trp and Na⁺，K⁺，Mg²⁺，Ca²⁺，Fe²⁺，Cu²⁺，ClO^-，NO₃ ^-，CO₃ ^2-，SO₄ ^2-，HCO₃ ^-The selection specificity is good.

As a general inventive concept, the present invention also provides a method for preparing the above novel fluorescent probe for detecting bio-thiol, comprising the following steps:

(1) under the protection of nitrogen, dissolving 20mmol of 9-bromoanthracene and 25mmol of anhydrous aluminum trichloride in carbon disulfide, dropwise adding 7.68ml of oxalyl chloride solution under the ice bath condition, reacting for 30 minutes, removing the ice bath, and stirring at normal temperature for 5-6 hours; after the reaction is completed, 50ml of 1M HCl solution is gradually dripped; cooling to room temperature, pouring the reaction solution into water, filtering, collecting solid, and finally purifying by using a silica gel chromatographic column to obtain a compound 6, wherein the compound 6 has the following structural formula:

(2) under the protection of nitrogen, dissolving 3.82mol of the compound 6 obtained in the step (1) in methanol, adding 19.1mmol of potassium peroxymonosulfonate, heating to reflux, stirring for reaction for 48 hours, cooling to room temperature, pouring the reaction liquid into water, filtering, collecting solid, and finally purifying by using a silica gel chromatographic column to obtain the compound 1, wherein the compound 5 has the following structural formula:

(3) under the protection of nitrogen, dissolving 1.98mmol of the compound obtained in the step (2) in ethanol, dropwise adding 2.38mmol of n-butylamine solution, heating to reflux, stirring for 8-12h, cooling to room temperature, spin-drying the solvent, and purifying by using a silica gel chromatographic column to obtain a compound 4, wherein the structural formula of the compound 4 is as follows:

(4) under the protection of nitrogen, dissolving 0.523mmol of the compound obtained in the step (3) in methanol, adding 3.139mmol of sodium methoxide, heating to reflux, stirring for 1-2h, cooling to room temperature, spin-drying the solvent, and purifying by using a silica gel chromatography column to obtain a compound 3, wherein the structure of the compound 3 is as follows:

(5) under the protection of nitrogen, dissolving 0.3mmol of the compound obtained in the step (4) in 10ml of hydroiodic acid, heating to 132 ℃, reacting for 8-12h, cooling to room temperature after the reaction is finished, adding a 2M sodium hydroxide solution, adjusting the pH value to 4-5, filtering, and purifying by using a silica gel chromatography column to obtain a compound 2, wherein the compound 2 has the following structure:

(6) and (3) under the protection of nitrogen, dissolving 0.157mmol of the compound obtained in the step (5) in dichloromethane, dropwise adding 1ml of triethylamine solution under ice bath, stirring for 30min, dropwise adding 0.309mmol of acryloyl chloride solution, stirring for 1-2h, pouring the reaction solution into water after complete reaction, extracting with dichloromethane, collecting an organic phase, and finally purifying by using a silica gel chromatographic column to obtain the probe 1.

In the step (1), the molar ratio of 9-bromoanthracene to oxalyl chloride is 1: 2-3, and an eluent for column chromatography is petroleum ether with a volume ratio of 10: 1: and (3) ethyl acetate.

In the step (2), the molar ratio of 6-bromoaceanthrylene-1, 2-dione to potassium peroxymonosulfonate is 1: 4-5, and an eluent for column chromatography is dichloromethane with a volume ratio of 1.5: 1: petroleum ether.

In the step (3), the molar ratio of 6-bromo-1, 2-dicarboxy anthracene anhydride to n-butylamine is 1: 1.2-1.5, and an eluent for column chromatography is dichloromethane with the volume ratio of 2: 1: petroleum ether.

In the step (4), the molar ratio of the N-N-butyl-6-bromo-anthraimide to the sodium methoxide is 1: 5-6, and an eluent used for column chromatography is dichloromethane with the volume ratio of 1: petroleum ether.

In the step (5), the molar ratio of the N-N-butyl-6-methoxy-anthracene imide to the hydroiodic acid is 1: 1-1.2, and an eluent used for column chromatography is dichloromethane with the volume ratio of 20: 1: methanol.

In the step (6), the molar ratio of the N-N-butyl-6-hydroxy-anthracene imide to the acryloyl chloride is 1: 0.8-1, and an eluent used for column chromatography is petroleum ether with a volume ratio of 5: 1: and (3) ethyl acetate.

The synthetic route of the biological thiol fluorescent probe is as follows:

the compound 1 is a probe.

The fluorescent probe can detect biological thiol (cysteine for example) in water environment by using a fluorescence spectrometer.

The above application, in particular, includes:

and observing the fluorescence spectrum change of the water environment to be detected before and after adding the biological thiol fluorescent probe, wherein the excitation wavelength of fluorescence is 549 nm.

The fluorescence spectrum change is as follows: change of a fluorescence peak value at 601nm in a fluorescence spectrum; if the peak value is increased, it indicates that the test solution contains mercaptan. Preferably, the fluorescence spectrum is tested using a fluorescence spectrometer.

The application specifically comprises the following steps:

(1) dissolving the probe 1 in DMSO to prepare a probe mother solution;

(2) adding the probe mother liquor into the solution to be detected;

testing the fluorescence spectrum in the liquid to be tested by using a fluorescence spectrometer, wherein the change of a fluorescence peak value at 601nm is shown, and if the peak value is enhanced, the cysteine is contained in the test liquid; wherein the excitation wavelength of the fluorescence spectrometer is 549 nm.

Firstly, cysteine in the aqueous solution can cause the change of the fluorescence spectrum of the fluorescent probe, so that the content of the cysteine in the solution can be judged by observing the change condition of the fluorescence spectrum in a fluorescence spectrometer, thereby carrying out quantitative detection; the lower limit of detection is 1.0x10^-5mol/L。

Compared with the prior art, the invention has the advantages that:

1. the novel fluorescent probe for detecting the biological thiol is a fluorescent probe taking an allyl group as an identification unit, and practices show that the fluorescent probe molecule shows higher selectivity and sensitivity when detecting the biological thiol.

2. The novel fluorescent probe for detecting the biological thiol has extremely fast response to the cysteine, can reach the maximum fluorescence intensity after reacting for 5min, is not influenced by pH in a physiological environment, and has the lower limit of detection of the cysteine of 10x10^{- 6}mol/L。

3. The novel fluorescent probe for detecting the biological thiol can resist Gly, Arg, Trp, Lys, Trp and Na⁺，K⁺,Mg²⁺，Ca²⁺，Fe²⁺，Cu²⁺，ClO^-，NO₃ ^-，CO₃ ^2-，SO₄ ^2-，HCO₃ ^-The selection specificity is good.

Drawings

FIG. 1 is a drawing of example 1, Compound 6¹H NMR spectrum

FIG. 2 shows Compound 5 of example 1¹H NMR spectrum.

FIG. 3 is a drawing of Compound 4 of example 1¹H NMR spectrum.

FIG. 4 is a drawing of Compound 3 of example 1¹H NMR spectrum.

FIG. 5 is a drawing of Compound 2 of example 1¹H NMR spectrum.

FIG. 6 shows a probe 1 of example 1¹H NMR spectrum.

FIG. 7 shows a probe 1 of example 1¹³C NMR spectrum.

FIG. 8 is a TOF-MS spectrum of probe 1 of example 1.

FIG. 9 shows the absorption spectra before and after the reaction of Probe 1 with CYS in example 2.

FIG. 10 is a fluorescence emission spectrum before and after the reaction of Probe 1 of example 3 with CYS, GSH, HCY.

FIG. 11 is a graph showing the change in fluorescence at 601nm of probe 1 of example 4 after addition of different concentrations of CYS.

FIG. 12 is a graph showing the change in fluorescence at 601nm before and after reaction of Probe 1 of example 5 with CYS at different pH.

FIG. 13 is a graph showing the time-dependent change in fluorescence intensity at 601nm before and after addition of the compound of Probe 1 of example 6 to CYS.

FIG. 14 shows the fluorescence spectrum at 601nm of probe 1 of example 7 after reaction with other amino acids and common cations and anions.

Detailed Description