CN111635388B - Pyrene and coumarin derivative-based bisulfite fluorescent probe, and preparation method and application thereof - Google Patents
Pyrene and coumarin derivative-based bisulfite fluorescent probe, and preparation method and application thereof Download PDFInfo
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention provides a pyrene and coumarin derivative-based bisulfite fluorescent probe, a preparation method and application thereof, wherein the structural formula of the fluorescent probe is as follows:
dissolving 1-acetylpyrene and 4- (diethylamino) salicylaldehyde in methanesulfonic acid, reacting for a period of time at 90 ℃, pouring the reaction solution into ice water after the reaction is completed, adding perchloric acid, generating a large amount of precipitates, performing suction filtration to obtain a solid crude product, and performing silica gel column chromatography separation on the solid crude product to obtain the fluorescent probe (B1). The probe is in CH3Reaction with SO in CN-HEPES (v/v: 6:4, v/v, pH 7.4) buffer solution2And carrying out specific nucleophilic reaction, thereby realizing specific recognition and detection of the bisulfite in the aqueous solution.
Description
Technical Field
The invention relates to the field of bisulfite detection agents, and in particular relates to a pyrene and coumarin derivative-based bisulfite fluorescent probe, a preparation method and application.
Background
Sulfur dioxide (SO)2) Can cause atmospheric pollution, sulfur dioxide is easy to dissolve in water, and the bisulphite (HSO) derivative thereof can be used in water3 -) Or Sulfite (SO)3 2-) Exist in the form of (1). Excessive contact with sulfur dioxide can be detrimental to the health of the organism. The long-term exposure in sulfur dioxide gas can bring harm to the respiratory system of people, and the risks of cardiovascular diseases and respiratory diseases are greatly increased. For example, excessive sulfur dioxide intake can cause gastrointestinal damage in humans. Therefore, it is very necessary to detect the residual amount of sulfur dioxide. Several conventional techniques, including electrochemical method, chromatography and capillary electrophoresis, have been developed for detecting sulfur dioxide derivatives, but most of these methods take a long time and damage the structure of the substance to be detected.
The fluorescent probe detection method has the outstanding advantages of high sensitivity, simplicity in operation, non-invasiveness, non-destructiveness, high selectivity, high time sequence, high spatial resolution, good biocompatibility and the like, and is particularly suitable for real-time monitoring. Fluorescent probes can detect analytes both in the environment and in vivo. As such, it has very general applications in chemistry, environment, medicine, and biology. As such, methods for detecting the residual amount of sulfur dioxide using fluorescent probes have been receiving increasing attention. The fluorescent probe can obtain results in a short time, has good timeliness, and cannot damage detected substances.
Disclosure of Invention
The invention provides a pyrene and coumarin derivative-based bisulfite fluorescent probe, a preparation method and application, and solves the technical problems that a conventional bisulfite detection agent in the prior art cannot be applied to actual medical treatment, the preparation method is complex and the cytotoxicity is high.
This application is based on SO2The nucleophilicity of the probe designs a fluorescent probe containing pyrene and coumarin structures, and the probe is on CH3Reaction with SO in CN-HEPES (v/v: 6:4, v/v, pH 7.4) buffer solution2And carrying out specific nucleophilic reaction, thereby realizing specific recognition and detection of the bisulfite in the aqueous solution.
The technical scheme for realizing the invention is as follows:
a bisulfite fluorescent probe based on pyrene and coumarin derivatives has the following structural formula:
the preparation method of the bisulfite fluorescent probe based on pyrene and coumarin derivatives comprises the following steps: dissolving 1-acetylpyrene and 4- (diethylamino) salicylaldehyde in methanesulfonic acid, reacting for a period of time at 90 ℃, pouring the reaction solution into ice water after the reaction is completed, adding perchloric acid, generating a large amount of precipitates, performing suction filtration to obtain a solid crude product, and performing silica gel column chromatography separation on the solid crude product to obtain the fluorescent probe (B1).
The synthetic route is as follows:
the molar ratio of the 1-acetylpyrene to the 4- (diethylamino) salicylaldehyde is 1: (1-6) the reaction time is 8-18 h.
The mol ratio of the 1-acetylpyrene to the methane sulfonic acid is 1: (25-50), wherein the molar ratio of the 1-acetylpyrene to the perchloric acid is 1: (6-16).
The silica gel column chromatography separation adopts the eluent of methanol and dichloromethane with the volume ratio of 1 (20-40), and the yield of the fluorescent probe is 45-85%.
The fluorescent probe is applied to the field of detecting the bisulfite.
The recognition mechanism of the fluorescent probe based on pyrene and coumarin derivatives on bisulfite is as follows:
the invention has the beneficial effects that:
(1) the fluorescent probe has the advantages of high fluorescence quantum yield, simple raw materials, simple synthetic method, high yield, easiness in obtaining and the like.
(2) The fluorescence emission wavelength (630 nm) of the fluorescent probe to which the present application relates is in the near infrared region. Some endogenous fluorophores in the environment and organisms and tissues generate 'autofluorescence', the emission wavelength of the autofluorescence is in a visible light region (400-600 nm), and meanwhile, the biological organism tissues have strong scattering on visible light, so that the fluorescence signals of the exogenous fluorescent probes are often seriously interfered. Background fluorescence in the near infrared region (600-900 nm) in the environment and organism tissues is obviously weakened, the absorption coefficient is minimum, scattering of near infrared light is less, and near infrared light energy can have strong tissue penetrability in organisms. The fluorescent probes of the present application greatly reduce the associated background interference and thus increase the sensitivity and penetration of the fluorescent technology.
(3) The fluorescent probe has high-efficiency and specific recognition performance on the bisulfite, and has the advantages of strong anti-interference performance, high response speed (the response time is 30 s) and high sensitivity (the minimum detection limit of the bisulfite is 39 nM).
(4) The fluorescent probe is mainly used for identifying the bisulfite, and the probe and the bisulfite generate specific nucleophilic addition. The recognition mechanism is confirmed by high-resolution mass spectrometry. The high resolution mass spectrometry data of the probe alone in the positive ion mode was 402.1850 (theoretical value of 402.1852), and the high resolution mass spectrometry data of the probe after the specific recognition reaction with bisulfite emission in the negative ion mode was 482.1434 (theoretical value of 482.1432) (FIG. 10). High-resolution mass spectrometry data verifies the identification mechanism of the bisulfite to be identified by the fluorescent probe, and lays a foundation for further developing more bisulfite fluorescent probes.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the NMR spectrum of fluorescent probe B1 (DMSO is the solvent) in example 1 of the present invention.
FIG. 2 shows the NMR spectrum of fluorescent probe B1 (DMSO is the solvent) in example 1 of the present invention.
FIG. 3 is a high resolution mass spectrum of fluorescent probe B1 (solvent is CH) in example 1 of the present invention3OH）。
FIG. 4 is a graph showing fluorescence selectivity of the fluorescent probe B1 of the present invention, with an excitation wavelength of 550 nm.
FIG. 5 is a graph showing the fluorescence interference resistance of bisulfite being recognized by the fluorescent probe B1 of the present invention, wherein the excitation wavelength is 550 nm and the emission wavelength is 630 nm.
FIG. 6 is a graph showing the fluorescence titration of bisulfite being recognized by the fluorescent probe B1 of the present invention, with an excitation wavelength of 550 nm.
FIG. 7 is a diagram showing the lowest detection limit of bisulfite being recognized by fluorescent probe B1 of the present invention, wherein the excitation wavelength is 550 nm and the emission wavelength is 630 nm.
FIG. 8 is a pH adaptation graph for bisulfite identification by fluorescent probe B1 of the present invention, with an excitation wavelength of 550 nm and an emission wavelength of 630 nm.
FIG. 9 is a fluorescence kinetic diagram of bisulfite identification by fluorescent probe B1 of the present invention, with an excitation wavelength of 550 nm and an emission wavelength of 630 nm.
FIG. 10 is a high-resolution mechanism verification diagram of bisulfite discrimination by the fluorescent probe B1 of the present invention (solvent CH)3OH, negative ion mode).
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art based on the embodiments of the present invention without inventive step, are within the scope of the present invention.
Example 1
1-acetylpyrene (244.3 mg, 1 mmol) and 4- (diethylamino) salicylaldehyde (193.3 mg, 1 mmol) were dissolved in methanesulfonic acid (2.4 g, 25 mmol), heated to 90 ℃ and stirred for reaction for 8 hours, after completion of the reaction, the reaction mixture was poured into ice water, perchloric acid (603 mg, 6 mmol) was added, a large amount of precipitate was generated, and the crude product was filtered with suction to obtain a solid. The crude product was chromatographed on a silica gel column (methanol: dichloromethane =1:25, vol.% eluent) to give 225 mg of a purple solid as product B1 in 45% yield.
Nuclear magnetic resonance measurement:1H NMR (DMSO-d 6 , 400 MHz) δ 1.23 (t, J = 7.2 Hz, 6 H), 3.66 (d, J = 5.2 Hz, 4 H), 7.06 (d, J = 1.6 Hz, 1 H), 7.38 (dd, J = 2.0 Hz, 1 H), 7.89 (q, J = 7.6 Hz, 2 H), 8.20 (m,2 H), 8.43 (m,6 H), 8.58 (d, J = 9.2 Hz, 1 H), 8.68 (d, J = 8.0 Hz, 1 H); 13C NMR (DMSO- d 6 100 MHz) delta 12.5, 46.2, 55.4, 96.0, 114.9, 119.4, 119.7, 123.6, 123.8, 124.3, 124.9, 125.6, 127.2, 127.6, 127.7, 128.9, 129.3, 130.4, 130.9, 131.1, 132.9, 134.4, 148.5, 156.8, 160.3, 167.4. The hydrogen spectrum and carbon spectrum of nuclear magnetic resonance are shown in FIG. 1 and 2, respectively.
High-resolution mass spectrometry: HR-ESI-MS calcd for C29H24NO+: 402.1852, found 402.1850 [M+H+]. The high resolution mass spectrum is shown in figure 3.
Example 2
Dissolving 1-acetylpyrene (244.3 mg, 1 mmol) and 4- (diethylamino) salicylaldehyde (386.6 mg, 2 mmol) in methanesulfonic acid (3.8 g, 40 mmol), heating to 90 ℃, stirring, reacting for 12 hours, pouring the reaction solution into ice water after the reaction is completed, adding perchloric acid (804 mg, 6 mmol), generating a large amount of precipitate, and filtering to obtain a solid crude product. The crude product was chromatographed on a silica gel column (methanol: dichloromethane =1:30 as eluent, vol.%) to give 275 mg of a purple solid as product B1 in 55% yield.
Nuclear magnetic resonance measurement:1H NMR (DMSO-d 6 , 400 MHz) δ 1.23 (t, J = 7.2 Hz, 6 H), 3.66 (d, J = 5.2 Hz, 4 H), 7.06 (d, J = 1.6 Hz, 1 H), 7.38 (dd, J = 2.0 Hz, 1 H), 7.89 (q, J = 7.6 Hz, 2 H), 8.20 (m,2 H), 8.43 (m,6 H), 8.58 (d, J = 9.2 Hz, 1 H), 8.68 (d, J = 8.0 Hz, 1 H); 13C NMR (DMSO- d 6 100 MHz) delta 12.5, 46.2, 55.4, 96.0, 114.9, 119.4, 119.7, 123.6, 123.8, 124.3, 124.9, 125.6, 127.2, 127.6, 127.7, 128.9, 129.3, 130.4, 130.9, 131.1, 132.9, 134.4, 148.5, 156.8, 160.3, 167.4. The hydrogen spectrum and carbon spectrum of nuclear magnetic resonance are shown in FIG. 1 and 2, respectively.
High-resolution mass spectrometry: HR-ESI-MS calcd for C29H24NO+: 402.1852, found 402.1850 [M+H+]. The high resolution mass spectrum is shown in figure 3.
Example 3
Dissolving 1-acetylpyrene (244.3 mg, 1 mmol) and 4- (diethylamino) salicylaldehyde (773.2 mg, 4 mmol) in methanesulfonic acid (4.3 g, 45 mmol), heating to 90 ℃, stirring for reaction for 13 hours, pouring the reaction solution into ice water after the reaction is completed, adding perchloric acid (1.2 g, 12 mmol), generating a large amount of precipitate, and filtering to obtain a solid crude product. The crude product was chromatographed on a silica gel column (methanol: dichloromethane =1:35 as eluent, vol.%) to give 350 mg of a purple solid as product B1 in 70% yield.
Nuclear magnetic resonance measurement:1H NMR (DMSO-d 6 , 400 MHz) δ 1.23 (t, J = 7.2 Hz, 6 H), 3.66 (d, J = 5.2 Hz, 4 H), 7.06 (d, J = 1.6 Hz, 1 H), 7.38 (dd, J = 2.0 Hz, 1 H), 7.89 (q, J = 7.6 Hz, 2 H), 8.20 (m,2 H), 8.43 (m,6 H), 8.58 (d, J = 9.2 Hz, 1 H), 8.68 (d, J = 8.0 Hz, 1 H); 13C NMR (DMSO- d 6 100 MHz) delta 12.5, 46.2, 55.4, 96.0, 114.9, 119.4, 119.7, 123.6, 123.8, 124.3, 124.9, 125.6, 127.2, 127.6, 127.7, 128.9, 129.3, 130.4, 130.9, 131.1, 132.9, 134.4, 148.5, 156.8, 160.3, 167.4. The hydrogen spectrum and carbon spectrum of nuclear magnetic resonance are shown in FIG. 1 and 2, respectively.
High-resolution mass spectrometry: HR-ESI-MS calcd for C29H24NO+: 402.1852, found 402.1850 [M+H+]. The high resolution mass spectrum is shown in FIG. 3.
Example 4
Dissolving 1-acetylpyrene (244.3 mg, 1 mmol) and 4- (diethylamino) salicylaldehyde (1159.8 mg, 6 mmol) in methanesulfonic acid (4.8 g, 50 mmol), heating to 90 ℃, stirring for reaction for 18 hours, pouring the reaction solution into ice water after the reaction is completed, adding perchloric acid (1.6 g, 16 mmol), generating a large amount of precipitate, and filtering to obtain a solid crude product. The crude product was chromatographed on a silica gel column (methanol: dichloromethane =1:40 as eluent, vol.) to give 425 mg of a purple solid as product B1 in 85% yield.
Nuclear magnetic resonance measurement:1H NMR (DMSO-d 6 , 400 MHz) δ 1.23 (t, J = 7.2 Hz, 6 H), 3.66 (d, J = 5.2 Hz, 4 H), 7.06 (d, J = 1.6 Hz, 1 H), 7.38 (dd, J = 2.0 Hz, 1 H), 7.89 (q, J = 7.6 Hz, 2 H), 8.20 (m,2 H), 8.43 (m,6 H), 8.58 (d, J = 9.2 Hz, 1 H), 8.68 (d, J = 8.0 Hz, 1 H); 13C NMR (DMSO-d 6 , 100 MHz) δ 12.5, 46.2, 55.4, 96.0, 114.9, 119.4, 119.7, 123.6, 123.8, 124.3, 124.9, 125.6, 127.2, 127.6, 127.7, 128.9, 129.3, 130.4, 130.9,131.1, 132.9, 134.4, 148.5, 156.8, 160.3, 167.4. The hydrogen spectrum and carbon spectrum of nuclear magnetic resonance are shown in FIG. 1 and 2, respectively.
High-resolution mass spectrometry: HR-ESI-MS calcd for C29H24NO+: 402.1852, found 402.1850 [M+H+]. The high resolution mass spectrum is shown in FIG. 3.
Examples of the effects of the invention
1 mM probe solution preparation: the probe (B1) prepared in example 1 was accurately weighed, and B1 was dissolved in acetonitrile (CH)3CN) solution to prepare a 1 mM solution for later use.
Fluorescence selectivity experiments:
the specific selectivity is the first condition to examine the superiority of fluorescent probes. A HEPES buffer solution at a pH of 7.4 and a concentration of 10 mM was prepared, and a probe B1 acetonitrile solution at a concentration of 1 mM was prepared using acetonitrile. The selectivity of probe B1 for bisulfite was examined using fluorescence spectroscopy. As shown in FIG. 4, the individual probe probes B1 (10 μ M) were in CH under fluorescence excitation at 550 nm3CN-HEPES (v/v: 6:4, v/v, pH 7.4) buffer solution has a strong fluorescence emission intensity at 630 nm, and when bisulfite (10 eq.) is added, the fluorescence emission at 630 nm is almost completely quenched, but when other active small molecule substances (10 eq.) are added, the fluorescence emission intensity of the solution system is not significantly changed from that of the probe system alone. The experimental results show that the probe has good specific selectivity on bisulfite.
And (3) interference resistance experiment:
in order to test the anti-interference capability of the probe molecules on the detection of the bisulfite, the anti-interference capability of the probe molecules on identifying the bisulfite on common amino acid micromolecule substances is respectively tested in a fluorescence emission spectrum. A HEPES buffer solution at a pH of 7.4 and a concentration of 10 mM was prepared, and a probe B1 acetonitrile solution at a concentration of 1 mM was prepared using acetonitrile. As can be seen from FIG. 5, when bisulfite is added in the presence of other common amino acid small molecule substances, the fluorescence emission intensity (630 nm) obtained by adding bisulfite alone is substantially the same, and the result shows that probe B1 has strong anti-interference capability on bisulfite detection.
Minimum detection limit experiment:
good detection limits are one of the criteria for verifying whether a probe molecule has an application value. A HEPES buffer solution at a pH of 7.4 and a concentration of 10 mM was prepared, and a probe B1 acetonitrile solution at a concentration of 1 mM was prepared using acetonitrile. The concentration of the immobilized probe B1 is 10 mu M, the fluorescence emission response intensity of the immobilized probe B1 to bisulfite with different concentrations is measured, the fluorescence emission intensity of the system is continuously reduced at 630 nm along with the increase of the bisulfite concentration (figure 6), and the research shows that the fluorescence emission intensity of the solution is linear (R is between 0 and 0.8 mu M) in the bisulfite concentration2= 0.98, fig. 7), the lowest detection limit of bisulfite by the probe molecule was found to be 39 nM by calculation (3 σ/k).
Effect of pH on Probe discrimination ability
In order to examine whether the probe can recognize bisulfite under different pH values (pH), the application of the probe in an actual sample is discussed, and the influence of the probe B1 on the bisulfite recognition under different pH values (pH) is examined by using a fluorescence spectrometer. HEPES buffer solutions at pH 4, 5, 6, 7, 8, and 9, concentrations of 10 mM, and probe B1 solution at a concentration of 1 mM were prepared, respectively, with acetonitrile. In HEPES-CH3In CN (6:4, v/v) solution system, the fluorescence emission intensity (630 nm) of the single probe (10. mu.L) is not greatly changed by changing the pH value of the buffer system (4-9), but the fluorescence emission intensity (630 nm) of the system has more obvious quenching in the pH range of 4-9 when 10 equivalents of bisulfite are added (figure 8), and the result shows that the probe B1 can be applied to the identification and detection of bisulfite under physiological conditions.
Experiment of dynamics
Good recognition speed is one of the important indicators for inspecting probes. A fluorescence spectrometer is adopted to investigate the kinetic experiment of the probe on the bisulfite. In CH3CN-HEPES (v/v: 6:4, v/v, pH 7.4) buffer solution, the concentration of the immobilized probe B1 was 10. mu.M, and the fluorescence emission (630 nm) of the probe alone and the probe plus bisulfite was measured at 550 nm with time under excitationTo change in time. As shown in FIG. 9, the fluorescence intensity of the individual probes did not fluctuate much with time; when bisulfite was added, the fluorescence emission intensity of the solution system decreased instantaneously and reached a response plateau at 30 s. The results show that the probe is quick and sensitive in recognition response to the bisulfite, and the effect of real-time detection is achieved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. A pyrene and coumarin derivative-based bisulfite fluorescent probe is characterized in that the structural formula of the fluorescent probe is as follows:
the preparation method of the bisulfite fluorescent probe based on pyrene and coumarin derivatives comprises the following steps: dissolving 1-acetylpyrene and 4- (diethylamino) salicylaldehyde in methanesulfonic acid, reacting for a period of time at 90 ℃, pouring the reaction liquid into ice water after complete reaction, adding perchloric acid, generating a large amount of precipitates, performing suction filtration to obtain a solid crude product, and performing silica gel column chromatography separation on the solid crude product to obtain the fluorescent probe.
2. The method for preparing a bisulfite fluorescent probe based on pyrene and coumarin derivatives according to claim 1, wherein the method comprises the following steps: dissolving 1-acetylpyrene and 4- (diethylamino) salicylaldehyde in methanesulfonic acid, reacting for a period of time at 90 ℃, pouring reaction liquid into ice water after complete reaction, adding perchloric acid, generating a large amount of precipitates, performing suction filtration to obtain a solid crude product, and performing silica gel column chromatography separation on the solid crude product to obtain a fluorescent probe, wherein the molar ratio of the 1-acetylpyrene to the 4- (diethylamino) salicylaldehyde is 1: (1-6) the reaction time is 8-18 h.
3. The method for preparing a bisulfite fluorescent probe based on pyrene and coumarin derivatives according to claim 2, wherein the method comprises the following steps: the mol ratio of the 1-acetylpyrene to the methane sulfonic acid is 1: (25-50), wherein the molar ratio of the 1-acetylpyrene to the perchloric acid is 1: (6-16).
4. The method for preparing a bisulfite fluorescent probe based on pyrene and coumarin derivatives according to claim 2, wherein the method comprises the following steps: the silica gel column chromatography separation adopts the eluent of methanol and dichloromethane with the volume ratio of 1 (20-40), and the yield is 45-85%.
5. Use of the fluorescent probe according to claim 1 in the field of detection of bisulfite.
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