CN114874249B - Nopinone-based ratio type fluorescent probe for detecting cysteine and preparation method and application thereof - Google Patents
Nopinone-based ratio type fluorescent probe for detecting cysteine and preparation method and application thereof Download PDFInfo
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- XZFDKWMYCUEKSS-UHFFFAOYSA-N 6,6-Dimethylbicyclo[3.1.1]heptan-2-one Chemical compound C1C2C(C)(C)C1CCC2=O XZFDKWMYCUEKSS-UHFFFAOYSA-N 0.000 title claims abstract description 158
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 235000018417 cysteine Nutrition 0.000 title claims abstract description 35
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 9
- -1 (4-acryloyloxy) phenyl Chemical group 0.000 claims abstract description 61
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- 238000001514 detection method Methods 0.000 claims abstract description 15
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- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 claims abstract description 8
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- 238000005882 aldol condensation reaction Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 25
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- JKWMSGQKBLHBQQ-UHFFFAOYSA-N diboron trioxide Chemical compound O=BOB=O JKWMSGQKBLHBQQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- LGQXXHMEBUOXRP-UHFFFAOYSA-N tributyl borate Chemical compound CCCCOB(OCCCC)OCCCC LGQXXHMEBUOXRP-UHFFFAOYSA-N 0.000 claims description 3
- 230000000536 complexating effect Effects 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 238000010898 silica gel chromatography Methods 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 1
- 239000012044 organic layer Substances 0.000 claims 1
- 230000004044 response Effects 0.000 abstract description 5
- 150000001875 compounds Chemical class 0.000 abstract description 3
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- 239000000243 solution Substances 0.000 description 21
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
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- 150000001413 amino acids Chemical class 0.000 description 4
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- 229910021645 metal ion Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001506 fluorescence spectroscopy Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
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- 238000000840 electrochemical analysis Methods 0.000 description 1
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Abstract
The invention is thatDiscloses a nopinone-based ratio type fluorescent probe for detecting cysteine, and a preparation method and application thereof. The invention uses 3-acetyl nopinone as raw material to carry out aldol condensation reaction with p-hydroxybenzaldehyde, then carries out complexation reaction with boron trifluoride-diethyl ether, and finally carries out esterification reaction with acryloyl chloride to obtain the compound 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex. After cysteine is added into THF-PBS solution of the complex, the fluorescence color of the solution is changed from blue to yellow-green under 365nm ultraviolet irradiation, and the detection limit of the cysteine reaches 2.1X10% ‑7 The mol/L response time is 2min, can be used as a nopinone group ratio type fluorescent probe for detecting cysteine, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of fine organic synthesis, and relates to a nopinone-based ratio type fluorescent probe for detecting cysteine, and a preparation method and application thereof.
Background
Cysteine is an amino acid essential to the human body and plays an important role in metabolism, regulation of redox homeostasis, protein synthesis, detoxification, cell signal transduction and the like. However, in vivo cysteine level abnormalities can cause specific diseases such as neurotoxicity, alzheimer's disease, atherosclerosis, parkinson's disease, and the like. Therefore, it is important to develop a cysteine detection method with high sensitivity and selectivity.
There are several detection methods reported so far, including electrochemical analysis, colorimetry and high performance liquid chromatography. However, these detection methods have the disadvantages of complex sample preparation, low sensitivity, long detection time, and the like. In contrast, the fluorescent probe detection method is widely applied because of quick response, high sensitivity, unique selectivity and simple and convenient operation. A novel cysteine specific ratio fluorescent probe is synthesized by using a 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex as a raw material, and no related report exists.
Disclosure of Invention
Aiming at the defects existing in the prior art, the technical problem to be solved by the invention is to provide the nopinone-based ratio type fluorescent probe 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex for detecting cysteine, which can meet the use requirements. Another technical problem to be solved by the present invention is to provide a method for preparing 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex. The invention also solves the technical problem of providing an application of the nopinone group ratio type fluorescent probe for detecting cysteine.
In order to solve the technical problems, the invention adopts the following technical scheme:
the nopinone-based ratio fluorescent probe for detecting cysteine is a 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex, and the structural formula is as follows:
the preparation method of the 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex comprises the following steps:
1) Performing aldol condensation reaction on the 3-acetyl nopinone and parahydroxyben-zaldehyde to obtain 3- (3- (4-hydroxyphenyl) acryloyl) nopinone;
2) Complexing 3- (3- (4-hydroxyphenyl) acryloyl) nopinone with boron trifluoride-diethyl ether to obtain 3- (3- (4-hydroxyphenyl) acryloyl nopinone boron difluoride complex;
3) Esterifying the 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex with acryloyl chloride to obtain a 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex;
the specific reaction formula is:
in the step 1), 3-acetyl nopinone and p-hydroxybenzaldehyde are subjected to aldol condensation reaction to obtain 3- (3- (4-hydroxyphenyl) acryloyl) nopinone, which comprises the following specific steps:
(1) 10 to 20mmol of 3-acetyl nopinone, 5 to 10mmol of diboron trioxide, 10 to 20mmol of p-hydroxybenzaldehyde and 10 to 20mmol of tributyl borate are sequentially added into 100 to 200mL of ethyl acetate to react for 30 to 60 minutes at the temperature of 60 to 80 ℃, then 5 to 30 mu L of n-butylamine is added dropwise, and the reaction is continued for 15 to 20 hours. Cooling the reaction solution to 60 ℃, adding 0.4M hydrochloric acid to adjust the pH of the solution to neutral, and stirring for 30-60 min;
(2) After the completion of the reaction, distilled water was added to wash the reaction solution. The obtained organic solution is dried by anhydrous sodium sulfate, filtered and distilled to obtain a crude product of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone;
(3) After purification of the crude 3- (3- (4-hydroxyphenyl) acryloyl) nopinone by silica gel chromatography (petroleum ether/ethyl acetate=10:1, v/v), 3- (3- (4-hydroxyphenyl) acryloyl) nopinone was obtained as a pale yellow solid.
In the step 2), 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and boron trifluoride-diethyl ether are subjected to complexation reaction to obtain 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex, and the specific steps are as follows:
(1) 2-4 mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and 3-6 mmol of boron trifluoride-diethyl ether are added into 30-60 mL of dichloromethane to react for 12h at 40 ℃;
(2) After the reaction is completed, the reaction solution is cooled to room temperature, after methylene dichloride is removed by reduced pressure distillation, the reaction solution is recrystallized and dried in normal hexane to obtain a pale yellow solid 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex.
In the step 3), the 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex and acryloyl chloride are subjected to esterification reaction to obtain the 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex, which comprises the following specific steps:
(1) Adding 2-4 mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex, 4-8 mmol of triethylamine and 5-10 mmol of acryloyl chloride into 20-40 mL of anhydrous dichloromethane, and reacting for 5h at room temperature;
(2) Distilling the reaction solution to remove the solvent to obtain a crude 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex product;
(3) The crude product of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex was purified by a silica gel column (petroleum ether/ethyl acetate=10:1, v/v) to give 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex.
The 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex is applied to detection of cysteine.
The 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex is a nopinone group ratio type fluorescent probe for detecting cysteine, can specifically identify the cysteine in aqueous solution, and changes the fluorescence of the solution from blue to yellow-green under 365nm ultraviolet irradiation.
The beneficial effects are that: compared with the prior art, the invention utilizes nopinone as the initial raw material to prepare the novel 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex, can specifically identify cysteine, changes the fluorescence of the solution from blue to yellow-green under 365nm ultraviolet irradiation, and has the advantages of simple synthesis, specific identification, high sensitivity, rapid response and the like as a fluorescent probe for detecting the cysteine, wherein the detection limit of the cysteine reaches 2.1x10 - 7 mol/L, response time is 2min, and has good application prospect.
Drawings
FIG. 1 is a graph of fluorescence spectra of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex in effect with different concentrations of cysteine;
FIG. 2 is a fluorescence spectrum of the effect of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex with various metal ions, anions and amino acids.
Detailed Description
The invention will be further illustrated with reference to specific examples.
Example 1
A method for preparing 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex, which has the following reaction formula:
the method comprises the following specific steps:
1) Preparation of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone:
10mmol of 3-acetyl nopinone, 5mmol of diboron trioxide, 10mmol of p-hydroxybenzaldehyde and 10mmol of tributyl borate are added into 100mL of ethyl acetate in sequence to react for 30min at 80 ℃, then 30 mu L of n-butylamine is added dropwise, and the reaction is continued for 20h. Cooling the reaction solution to 60 ℃, adding 0.4M hydrochloric acid to adjust the pH of the solution to be neutral, and stirring for 30min; after the completion of the reaction, the reaction solution was washed with distilled water. Drying the organic solution with anhydrous sodium sulfate, filtering and distilling to obtain a crude 3- (3- (4-hydroxyphenyl) acryloyl) nopinone product; the crude 3- (3- (4-hydroxyphenyl) acryloyl) nopinone product was chromatographed on silica gel column (petroleum ether/ethyl acetate=10/1, v/v) to give 3- (3- (4-hydroxyphenyl) acryloyl) nopinone as a pale yellow solid in 67%. 1 H NMR(600MHz，DMSO-d 6 )δ：14.75(s，1H)，9.95(s，1H)，7.56(d，J＝8.2Hz，2H)，7.42(d，J＝15.7Hz，1H)，6.80(d，J＝8.3Hz，2H)，6.70(d，J＝15.7Hz，1H)，2.72(dd，J＝15.3，3.3Hz，1H)，2.65-2.61(m，1H)，2.57(dt，J＝10.6，5.9Hz，1H)，2.41(t，J＝5.5Hz，1H)，2.31-2.27(m，1H)，1.35(d，J＝10.0Hz，1H)，1.32(s，3H)，0.85(s，3H)； 13 C NMR(150MHz，DMSO-d 6 )δ：208.69，168.26，159.83，138.86，130.45，126.82，116.27，115.86，104.24，54.54，39.54，39.51，27.76，26.20，26.02，21.73；HRMS(m/z)：[M+H] + calcd for C 18 H 20 O 3 +H + ，285，1491；found，285.1494。
2) Preparation of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex:
2mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and 3mmol of boron trifluoride-diethyl etherate were added to 30mL of dichloromethane and reacted at 40℃for 12h; after the reaction is completed, the reaction solution is cooled to room temperature, methylene dichloride is removed through reduced pressure distillation, and then the reaction solution is recrystallized and dried in normal hexane to obtain a pale yellow solid 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex, and the yield is 52%. 1 H NMR(600MHz，DMSO-d 6 )δ：10.42(s，1H)，7.93(d，J＝15.4Hz，1H)，7.83-7.76(m，2H)，6.96(d，J＝15.4Hz，1H)，6.90-6.83(m，2H)，2.83(dd，J＝15.2，3.2Hz，1H)，2.69(tt，J＝8.5，4.2Hz，2H)，2.63(t，J＝5.4Hz，1H)，2.37(tt，J＝5.7，3.0Hz，1H)，1.39(d，J＝7.6Hz，4H)，0.85(s，3H)； 13 C NMR(150MHz，DMSO-d 6 )δ：198.25，175.34，161.63，147.34，132.32，128.34，125.55，116.17，113.57，104.25，49.87，40.06，38.99，28.07，25.19，25.02，21.07；HRMS(m/z)：[M+H] + calcd for C 18 H 19 BF 2 O 3 +H + ，333.1474；found，333.1469。
3) Preparation of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex:
2mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex, 4mmol of triethylamine and 5mmol of acryloyl chloride were added to 20mL of anhydrous dichloromethane, and the mixture was reacted at room temperature for 5 hours. The reaction solution was distilled to remove the solvent, thereby obtaining a crude 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex. Crude 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex product through siliconPurification by column chromatography (petroleum ether/ethyl acetate=10/1, v/v) afforded 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex in 56% yield. 1 H NMR(600MHz，DMSO-d 6 )δ：8.03-7.98(m，3H)，7.35-7.30(m，2H)，7.21(d，J＝15.6Hz，1H)，6.57(dd，J＝17.3，1.2Hz，1H)，6.43(dd，J＝17.3，10.4Hz，1H)，6.19(dd，J＝10.4，1.2Hz，1H)，2.89(dd，J＝15.4，3.2Hz，1H)，2.76-2.68(m，3H)，2.39(tt，J＝5.9，2.9Hz，1H)，1.43(d，J＝9.8Hz，1H)，1.40(s，3H)，0.87(s，3H)； 13 C NMR(150MHz，DMSO-d 6 )δ：201.08，175.04，164.31，153.17，145.69，134.54，132.60，131.48，127.93，122.99，118.45，105.84，50.66，40.56，39.34，28.36，25.64，25.40，21.58；HRMS(m/z)：[M+Na] + calcd for C 18 H 19 BF 2 O 3 +Na + ，409.1399；found，409.1399。
Example 2
3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex was dissolved in (ph=7.2, 10mM, tetrahydrofuran/water, 5/5 (v/v)) to formulate 1×10 -5 M, cysteine was dissolved in PBS buffer to prepare solutions with concentrations of 0, 5, 10, 25, 40, 55, 70, 85, 100, 120, 150, 200. Mu.M. Fluorescence emission spectra of different concentrations of cysteine versus 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex were measured on a fluorescence spectrophotometer using fluorescence spectrometry, as shown in figure 1. The result shows that under 365nm ultraviolet light irradiation, the blue fluorescence of the solution gradually disappears and the green fluorescence gradually increases along with the gradual increase of the concentration of cysteine in the aqueous solution. Thus, the compound can be used as a fluorescent probe for sensitively detecting cysteine, and the detection limit reaches 2.1X10 -7 mol/L, response time was 2min.
Example 3
3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex was formulated as 1×10 in PBS buffer (ph=7.2, 10mM, tetrahydrofuran/water, 5/5 (v/v)) -5 Dissolution of MThe solution is prepared by dissolving different metal ions, anions and amino acids in PBS buffer solution to obtain a solution with a concentration of 1×10 -4 M. Fluorescence emission spectra of different metal ions, anions, and amino acids versus 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex were measured on a fluorescence spectrophotometer using fluorescence spectrometry, as shown in fig. 2. The results show that the addition of cysteine can make the blue fluorescence of the aqueous solution system disappear and the green fluorescence be enhanced, and other analytes such as Hcy, GSH, thr, arg, tyr, leu, val, trp, gly, ala and Na are added + ，Cu 2+ ，Ni 2+ ，Mg 2+ ，Cd 2+ ，Ca 2+ ，I - ，HSO 3 - ，HPO 4 2- ，SO 3 2- ，NO 3 - ，NO 2 - ，HCO 3 - ，CO 3 2- The fluorescence spectrum of the solution was not significantly changed as observed in the comparative reference. Thus, the compound can be used as a fluorescent probe for specifically recognizing cysteine.
Claims (7)
1. A nopinone-based ratio-type fluorescent probe for detecting cysteine, which is characterized by having the structural formula:
2. the method for preparing nopinone-based ratio fluorescent probe for detecting cysteine according to claim 1, comprising the steps of:
1) Performing aldol condensation reaction on the 3-acetyl nopinone and parahydroxyben-zaldehyde to obtain 3- (3- (4-hydroxyphenyl) acryloyl) nopinone;
2) Complexing 3- (3- (4-hydroxyphenyl) acryloyl) nopinone with boron trifluoride-diethyl ether to obtain 3- (3- (4-hydroxyphenyl) acryloyl nopinone boron difluoride complex;
3) Esterifying the 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex with acryloyl chloride to obtain a 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex;
the specific reaction formula is:
3. the method for preparing nopinone-based ratio fluorescent probe for cysteine detection according to claim 2, wherein the specific steps of step 1) are as follows:
(1) Sequentially adding 10-20 mmol of 3-acetyl nopinone, 5-10 mmol of diboron trioxide, 10-20 mmol of p-hydroxybenzaldehyde and 10-20 mmol of tributyl borate into 100-200 mL of ethyl acetate, reacting for 30-60 min at 70-90 ℃, then dropwise adding 5-30 mu L of n-butylamine, and continuing reacting for 15-20 h; cooling the reaction solution to 60 ℃, adding 0.4M hydrochloric acid to adjust the pH of the solution to neutral, and stirring for 30-60 min;
(2) After the reaction is finished, 50-100 mL of distilled water is added and stirred for 5-10 min, the separated organic layer is washed to be neutral by distilled water and saturated saline water, and then dried, filtered and distilled by anhydrous sodium sulfate to obtain a crude product of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone;
(3) The crude 3- (3- (4-hydroxyphenyl) acryloyl) nopinone product was purified by silica gel chromatography to give 3- (3- (4-hydroxyphenyl) acryloyl) nopinone as a pale yellow solid.
4. The method for preparing nopinone-based ratio fluorescent probe for cysteine detection according to claim 2, wherein the specific steps of step 2) are as follows:
(1) 2-4 mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and 3-6 mmol of boron trifluoride-diethyl ether are added into 30-60 mL of dichloromethane to react for 12h at 40 ℃;
(2) After the reaction is completed, the reaction solution is cooled to room temperature, after methylene dichloride is removed by reduced pressure distillation, the reaction solution is recrystallized and dried in normal hexane to obtain a pale yellow solid 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex.
5. The method for preparing nopinone-based ratio fluorescent probe for cysteine detection according to claim 2, wherein the specific steps of step 3) are as follows:
(1) Adding 2-4 mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex, 4-8 mmol of triethylamine and 5-10 mmol of acryloyl chloride into 20-40 mL of anhydrous dichloromethane, and reacting for 5h at room temperature;
(2) Distilling the reaction solution to remove the solvent to obtain a crude 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex product;
(3) Crude product of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex was separated by silica gel column, petroleum ether/ethyl acetate=10/1, v/v to obtain 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex as white solid.
6. Use of the nopinone-based ratio fluorescent probe for cysteine detection according to claim 1 for the detection of cysteine; the detection cysteine is not used for diagnosing and treating diseases.
7. The use according to claim 6, wherein the solution fluorescence changes from blue to yellow-green under 365nm ultraviolet light irradiation.
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