CN114874249A - 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 163
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 235000018417 cysteine Nutrition 0.000 title claims abstract description 33
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 11
- -1 (4-acryloyloxy) phenyl Chemical group 0.000 claims abstract description 60
- OKZIUSOJQLYFSE-UHFFFAOYSA-N difluoroboron Chemical compound F[B]F OKZIUSOJQLYFSE-UHFFFAOYSA-N 0.000 claims abstract description 42
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 claims abstract description 28
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 claims abstract description 16
- HFBMWMNUJJDEQZ-UHFFFAOYSA-N acryloyl chloride Chemical compound ClC(=O)C=C HFBMWMNUJJDEQZ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 7
- 238000005882 aldol condensation reaction Methods 0.000 claims abstract description 4
- 238000005886 esterification reaction Methods 0.000 claims abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 18
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 10
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 239000012295 chemical reaction liquid Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- JKWMSGQKBLHBQQ-UHFFFAOYSA-N diboron trioxide Chemical compound O=BOB=O JKWMSGQKBLHBQQ-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000003208 petroleum Substances 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 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
- 238000001914 filtration Methods 0.000 claims description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- LGQXXHMEBUOXRP-UHFFFAOYSA-N tributyl borate Chemical compound CCCCOB(OCCCC)OCCCC LGQXXHMEBUOXRP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012044 organic layer Substances 0.000 claims 1
- 239000012266 salt solution Substances 0.000 claims 1
- 229920006395 saturated elastomer Chemical class 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 9
- 230000004044 response Effects 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 150000001450 anions Chemical class 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000011734 sodium Substances 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
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000029412 cell redox homeostasis Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
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Abstract
The invention discloses a nopinone-based ratio type fluorescent probe for detecting cysteine, and a preparation method and application thereof. The method comprises the steps of carrying out aldol condensation reaction on 3-acetyl nopinone serving as a raw material and p-hydroxybenzaldehyde, then carrying out complex reaction on the 3-acetyl nopinone and p-hydroxybenzaldehyde, and finally carrying out esterification reaction on the 3-acetyl nopinone and acryloyl chloride to obtain a 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 the irradiation of 365nm ultraviolet light, and the detection limit of cysteine reaches 2.1 multiplied by 10 ‑7 mol/L, response time of 2min, can be used as a nopinone-based 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 essential amino acid in human body, and plays an important role in metabolism, regulation of redox homeostasis, protein synthesis, detoxification, cell signal transduction, and the like. However, abnormal cysteine levels in the body can cause specific diseases such as neurotoxicity, Alzheimer's disease, atherosclerosis, Parkinson's disease, etc. Therefore, it is important to develop a cysteine detection method with high sensitivity and selectivity.
Several detection methods have been reported, including electrochemical analysis, colorimetry, and high performance liquid chromatography. However, these detection methods have the disadvantages of complicated sample preparation, low sensitivity, long detection time, and the like. In contrast, the fluorescent probe detection method is widely applied due to fast reaction, high sensitivity, unique selectivity and simple operation. The novel cysteine ratio fluorescent probe is synthesized by using a 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex as a raw material, and related reports are not available temporarily.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a nopinone-based ratio type fluorescent probe 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex for detecting cysteine, which can meet the use requirement. The invention aims to solve another technical problem of providing a preparation method of a 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex. The invention also aims to solve a technical problem of providing an application of the nopinone-based ratio type fluorescent probe for detecting cysteine.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the nopinone-based ratio type fluorescent probe for detecting cysteine is a 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex, and the structural formula of the probe 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 3-acetyl nopinone and p-hydroxybenzaldehyde to obtain 3- (3- (4-hydroxyphenyl) acryloyl) nopinone;
2) carrying out complex reaction on 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and boron trifluoride-diethyl ether to obtain a 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex;
3) carrying out esterification reaction on the 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex and acryloyl chloride to obtain a 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex;
the specific reaction formula is as follows:
in the step 1), the 3-acetyl nopinone and p-hydroxybenzaldehyde are subjected to aldol condensation reaction to obtain 3- (3- (4-hydroxyphenyl) acryloyl) nopinone, and the specific steps are as follows:
(1) sequentially adding 10-20 mmol of 3-acetyl nopinone, 5-10 mmol of boron trioxide, 10-20 mmol of p-hydroxybenzaldehyde and 10-20 mmol of tributyl borate into 100-200 mL of ethyl acetate, reacting at 60-80 ℃ for 30-60 min, dropwise adding 5-30 mu L of n-butylamine, and continuing to react for 15-20 h. Cooling the reaction liquid to 60 ℃, adding 0.4M hydrochloric acid to adjust the pH value of the solution to be neutral, and stirring for 30-60 min;
(2) after the reaction was completed, distilled water was added to wash the reaction solution. Drying the obtained organic solution by using anhydrous sodium sulfate, filtering and distilling to obtain a crude product of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone;
(3) the crude 3- (3- (4-hydroxyphenyl) acryloyl) nopinone was purified by silica gel chromatography (petroleum ether/ethyl acetate 10: 1, v/v) to give 3- (3- (4-hydroxyphenyl) acryloyl) nopinone as a pale yellow solid.
In the step 2), the 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and boron trifluoride-diethyl ether are subjected to complex reaction to obtain a 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex, which comprises the following specific steps:
(1) adding 2-4 mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and 3-6 mmol of boron trifluoride-diethyl ether into 30-60 mL of dichloromethane, and reacting for 12h at 40 ℃;
(2) and after the reaction is finished, cooling to room temperature, distilling the reaction liquid under reduced pressure to remove dichloromethane, recrystallizing in normal hexane, and drying to obtain a light 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, and the specific steps 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 liquid to remove the solvent to obtain a crude product of the 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex;
(3)3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex the crude product was purified by 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 the detection of cysteine.
The 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex is a nopinone-based ratio type fluorescent probe for detecting cysteine, can specifically identify the cysteine in an aqueous solution, and changes the fluorescence of the solution from blue to yellow green under the irradiation of 365nm ultraviolet light.
Has the advantages that: compared with the prior art, the novel 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex is prepared by using nopinone as an initial raw material, cysteine can be specifically identified, the fluorescence of the solution is changed from blue to yellow green under the irradiation of 365nm ultraviolet light, and the fluorescent probe for detecting cysteine has the advantages of simplicity in synthesis, specificity in identification, high sensitivity, quick response and the like, wherein the detection limit of cysteine reaches 2.1 multiplied by 10 - 7 mol/L, response time of 2min, and good application prospect.
Drawings
FIG. 1 is a graph of the fluorescence spectrum of the action of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex with different concentrations of cysteine;
FIG. 2 is a fluorescence spectrum of the action of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex with different metal ions, anions and amino acids.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1
The preparation method of the 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex has the following reaction formula:
the method comprises the following specific steps:
1) preparation of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone:
adding 10mmol of 3-acetyl nopinone, 5mmol of boron trioxide, 10mmol of p-hydroxybenzaldehyde and 10mmol of tributyl borate into 100mL of ethyl acetate in sequence, reacting at 80 ℃ for 30min, then dropwise adding 30 mu L of n-butylamine, and continuing to react for 20 h. Cooling the reaction solution to 60 ℃, adding 0.4M hydrochloric acid to adjust the pH value of the solution to be neutral, and stirring for 30 min; after completion of the reaction, the reaction solution was washed with distilled water. Drying the organic solution by using anhydrous sodium sulfate, filtering and distilling to obtain a crude product of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone; the crude 3- (3- (4-hydroxyphenyl) acryloyl) nopinone was chromatographed on silica gel (petroleum ether/ethyl acetate 10/1, v/v) to give 3- (3- (4-hydroxyphenyl) acryloyl) nopinone as a pale yellow solid in 67% yield. 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:
adding 2mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and 3mmol of boron trifluoride-diethyl ether into 30mL of dichloromethane, and reacting at 40 ℃ for 12 h; after the reaction is finished, cooling to room temperature, removing dichloromethane from the reaction solution through reduced pressure distillation, recrystallizing in normal hexane, and drying to obtain a light yellow solid 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex with the yield of 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 reacted at room temperature for 5 hours. And distilling the reaction liquid to remove the solvent to obtain a crude product of the 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex. The crude product of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex was purified by silica gel column (petroleum ether/ethyl acetate: 10/1, v/v) to give 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- ((1)4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex was dissolved in (pH 7.2, 10mM, tetrahydrofuran/water, 5/5(v/v)) to prepare 1 × 10 -5 M, cysteine was dissolved in PBS buffer to prepare solutions of 0, 5, 10, 25, 40, 55, 70, 85, 100, 120, 150, and 200. mu.M, respectively. Fluorescence emission spectra of cysteine versus 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex at different concentrations were measured on a fluorescence spectrophotometer by fluorescence spectrometry, as shown in fig. 1. The result shows that under 365nm ultraviolet irradiation, with the gradual increase of the concentration of cysteine in the aqueous solution, the blue fluorescence of the solution gradually disappears, and the green fluorescence gradually increases. Thus, the compound can be used as a fluorescent probe for sensitively detecting cysteine, and the detection limit reaches 2.1 multiplied by 10 -7 mol/L and response time of 2 min.
Example 3
The 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex was dissolved in PBS buffer (pH 7.2, 10mM, tetrahydrofuran/water, 5/5(v/v)) to prepare 1 × 10 -5 M solution, dissolving different metal ions, anions and amino acids in PBS buffer solution to obtain 1 × 10 solution -4 A solution of M. Fluorescence emission spectra of different metal ions, anions and amino acid pairs of 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex are measured on a fluorescence spectrophotometer by a fluorescence spectrum titration method, and are shown in figure 2. The results show that the addition of cysteine can eliminate the blue fluorescence of the aqueous system and enhance the green fluorescence, while the addition of other analytes such as Hcy, GSH, Thr, Arg, Tyr, Leu, Val, Trp, Gly, Ala, Na + ，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 does not change significantly when observed by an isocontrast reference. Thus, the compound canSo as to be used as a fluorescent probe for specifically recognizing cysteine.
Claims (7)
1. A nopinone-based ratio-type fluorescent probe for detecting cysteine is characterized in that the structural formula is as follows:
2. the method of claim 1, wherein the method comprises the steps of:
1) performing aldol condensation reaction on 3-acetyl nopinone and p-hydroxybenzaldehyde to obtain 3- (3- (4-hydroxyphenyl) acryloyl) nopinone;
2) carrying out complex reaction on 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and boron trifluoride-diethyl ether to obtain a 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex;
3) carrying out esterification reaction on the 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex and acryloyl chloride to obtain a 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex;
the specific reaction formula is as follows:
3. the method for preparing a nopinone-based ratiometric fluorescent probe for detecting cysteine 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 boron trioxide, 10-20 mmol of p-hydroxybenzaldehyde and 10-20 mmol of tributyl borate into 100-200 mL of ethyl acetate, reacting at 70-90 ℃ for 30-60 min, dropwise adding 5-30 mu L of n-butylamine, and continuing to react for 15-20 h; cooling the reaction liquid to 60 ℃, adding 0.4M hydrochloric acid to adjust the pH value of the solution to be neutral, and stirring for 30-60 min;
(2) after the reaction is finished, adding 50-100 mL of distilled water and stirring for 5-10 min, washing the separated organic layer with distilled water and saturated salt solution to be neutral, drying with anhydrous sodium sulfate, filtering, and distilling to obtain a crude product of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone;
(3) and purifying the crude product of the 3- (3- (4-hydroxyphenyl) acryloyl) nopinone by a silica gel chromatographic column to obtain the light yellow solid 3- (3- (4-hydroxyphenyl) acryloyl) nopinone.
4. The method for preparing a nopinone-based ratiometric fluorescent probe for detecting cysteine according to claim 2, wherein the specific steps of step 2) are as follows:
(1) adding 2-4 mmol of 3- (3- (4-hydroxyphenyl) acryloyl) nopinone and 3-6 mmol of boron trifluoride-diethyl ether into 30-60 mL of dichloromethane, and reacting for 12h at 40 ℃;
(2) and after the reaction is finished, cooling to room temperature, distilling the reaction liquid under reduced pressure to remove dichloromethane, recrystallizing in normal hexane, and drying to obtain a light yellow solid 3- (3- (4-hydroxyphenyl) acryloyl) nopinone boron difluoride complex.
5. The method for preparing a nopinone-based ratiometric fluorescent probe for detecting cysteine 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 liquid to remove the solvent to obtain a crude product of the 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex;
(3)3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex the crude product was isolated by silica gel column separation (petroleum ether/ethyl acetate 10/1, v/v) to give 3- (3- ((4-acryloyloxy) phenyl) acryloyl) nopinone boron difluoride complex as a white solid.
6. The use of the nopinone-based rate fluorescent probe according to claim 1 for detecting cysteine to detect cysteine.
7. The use of claim 6, wherein the THF-PBS solution of nopinone-based ratiometric fluorescent probe changes its fluorescence from blue to yellow-green under 365nm UV light.
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| CN108409765A (en) * | 2018-03-27 | 2018-08-17 | 南京林业大学 | Nopinane base beta-diketon boron difluoride complex compound and its preparation method and application |
| CN109734738A (en) * | 2019-02-21 | 2019-05-10 | 南京林业大学 | A kind of fluorescence probe and the preparation method and application thereof that can quickly detect sulfurous acid hydrogen radical ion |
| CN110172336A (en) * | 2019-05-09 | 2019-08-27 | 南京林业大学 | A kind of detection HSO3-And difunctional fluorescence probe of hydrazine hydrate and the preparation method and application thereof |
| CN110357913A (en) * | 2019-08-16 | 2019-10-22 | 南京林业大学 | A kind of fluorescence probe and its preparation method and application for detecting hypochlorite ion |
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| CN108409765A (en) * | 2018-03-27 | 2018-08-17 | 南京林业大学 | Nopinane base beta-diketon boron difluoride complex compound and its preparation method and application |
| CN109734738A (en) * | 2019-02-21 | 2019-05-10 | 南京林业大学 | A kind of fluorescence probe and the preparation method and application thereof that can quickly detect sulfurous acid hydrogen radical ion |
| CN110172336A (en) * | 2019-05-09 | 2019-08-27 | 南京林业大学 | A kind of detection HSO3-And difunctional fluorescence probe of hydrazine hydrate and the preparation method and application thereof |
| CN110357913A (en) * | 2019-08-16 | 2019-10-22 | 南京林业大学 | A kind of fluorescence probe and its preparation method and application for detecting hypochlorite ion |
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