CN115232120B - PH fluorescent probe and synthesis method and application thereof - Google Patents
PH fluorescent probe and synthesis method and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of organic micromolecular fluorescent probes, and provides a pH fluorescent probe which comprises quinolinium ions serving as a framework, p-tertiary aminophenyl and counter ions, wherein the p-tertiary aminophenyl and the counter ions are connected with the quinolinium ion framework through carbon-carbon single bonds; the pH fluorescent probe has obvious fluorescence intensity change in the change range of pH of 4-7, and the fluorescence intensity is gradually enhanced along with the decrease of pH; in addition, the molecular structure of the compound takes 410nm as an excitation wavelength, and the maximum emission wavelength is 478-509nm; the molecular structure of the compound has special penetrability, can penetrate through cell membranes but cannot penetrate through cell nuclear membranes, so that the compound can be selectively marked at a special position of subcellular hierarchy; meanwhile, the sensor has larger Stokes displacement, can reduce the signal to noise ratio, and is suitable for being widely applied to the detection of pH in weak acidic environments inside and outside cells.
Description
Technical Field
The application belongs to the technical field of organic micromolecular fluorescent probes, and particularly relates to a pH fluorescent probe, and a synthesis method and application thereof.
Background
Hydrogen ions are one of the most important ions in the human body and play an important role in numerous physiological processes within cells and organelles, such as: ion transport, cell proliferation and apoptosis, endocytosis, multiple drug resistance, cell adhesion, muscle contraction, etc., so maintaining pH balance is critical to human health. Often small changes in pH can lead to many dysfunctions such as membrane contractility disruption, protein denaturation, enzyme dysfunction and radical generation failure. For example, lysozyme may lead to degradation of proteins in cellular metabolism at pH 4.5-5.5. At the same time, pH is also closely related to many serious degenerative diseases of the nervous system, cancer and cardiopulmonary diseases. If the tumor is in the development process, hypoxia occurs, so that acidosis is caused inside and outside the cell, thereby showing a decrease in pH and finally causing abnormal cell activities. Therefore, the real-time accurate and rapid detection of the pH value inside and outside the cells of the human body has important significance for researching the physiological and pathological processes of the human body and diagnosing diseases.
Fluorescence spectroscopy has unique advantages over microelectrodes, nuclear magnetic resonance spectroscopy (NMR), absorbance spectroscopy, and other methods of detecting biological sample pH values: simple operation, high selectivity, high sensitivity, real-time detection, low cost, no influence of external electromagnetic field, and no harm to cells in most cases. pH fluorescent molecular probes are typically composed of three parts: fluorescent groups, linking groups, and recognition groups. Before the proton is not combined, the probe molecule does not emit fluorescence, or the fluorescence is weak, once the recognition group acts with the proton, the condition of inhibiting fluorescence in the molecule disappears, and the fluorescent group emits strong fluorescence, so that the purpose of detecting the pH is realized. Examples of the fluorescent groups commonly used in the pH fluorescent probes currently developed include Fluorescein (Fluorescein), rhodamine (Rhodamine), coumarin (Coumarin), BODIPY (BODIPY), and anthocyanin (Cyanine). Most of such fluorescent groups cannot select labeled organelles per se, and a targeting group needs to be additionally connected, and then a detected recognition group is assisted, so that the synthesis steps of the molecular probe become more complicated, and the synthesis cost is higher. In addition, because it is difficult to simply change the luminescence wavelength thereof, it becomes difficult to achieve a process of simultaneously detecting different kinds of molecules or molecules of the same structure in different organelles. Furthermore, the fluorophores on these fluorescent probes have some disadvantages: sub-optimal excitation and emission wavelengths, photobleaching, weak spectral changes, etc. Therefore, the luminous wavelength of the pH fluorescent probe in the prior art is not adjustable, so that the pH fluorescent probe has no targeting effect, and the wide application of the pH fluorescent probe is affected.
Disclosure of Invention
The application aims to provide a pH fluorescent probe, a synthesis method and application thereof, and aims to solve the problems that the luminous wavelength of the pH fluorescent probe in the prior art is not adjustable and the pH fluorescent probe does not have a targeting effect.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the application provides a pH fluorescent probe, which comprises quinolinium ions as a framework, p-tertiary aminophenyl and counter ions connected with the quinolinium ion framework structure through carbon-carbon single bonds, wherein the structural general formula of the pH fluorescent probe is shown as formula I,
wherein R is 1 And R is 2 Each independently selected from any one of piperidine, cyclohexylimine, morpholine and methyl, R 3 Selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethane sulfonate ions.
In a second aspect, the present application provides a method for preparing a pH fluorescent probe, comprising the steps of:
providing a first compound having the structural formula
Providing a second compound having the structural formula
Dissolving the first compound and the second compound in an organic solvent under an inert atmosphere, carrying out catalytic addition reaction by taking monovalent gold complex as a catalyst, and then carrying out first purification treatment to obtain a third compound, wherein the structural general formula of the third compound is
Reflux treatment is carried out on the third compound, then second purification treatment is carried out, thus obtaining the pH fluorescent probe,
wherein the structural general formula of the pH fluorescent probe isAnd R is 1 And R is 2 Each independently selected from any one of piperidine, cyclohexylimine, morpholine and methyl, R 3 Selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethane sulfonate ions.
In a third aspect, the application provides an application of a pH fluorescent probe or the pH fluorescent probe prepared by the preparation method in fluorescence qualitative detection of pH.
The structural general formula of the pH fluorescent probe is shown as formula I, the pH fluorescent probe comprises quinolinium ions serving as a framework, p-tertiary aminophenyl and counter ions which are connected with the quinolinium ion framework structure through carbon-carbon single bonds, wherein the pH fluorescent probe further comprises R 1 、R 2 、R 3 Three substituents; different steric effects are formed between the dialkylamino and the phenyl by changing the types of the substituents on the general structural formula, so that the electron donating capacity of the substituents is changed, the luminous wavelength and the pKa value of the molecules can be selectively adjusted, and further the fluorescence qualitative detection of the pH value is achieved. In the fluorescence qualitative detection process, when the pH is greater than 7, the pH fluorescent probe is non-fluorescent, namely a fluorophore quinolinium ion skeleton in the probe is quenched, as the pH is reduced, amino is taken as a sensitive group of the pH fluorescent probe, nitrogen atoms of dialkyl amino in tertiary aminophenyl are protonated to form quaternary ammonium salt ions, the quinolinium ion skeleton is subjected to fluorescence recovery, so that the effect of fluorescence is realized, the change of fluorescence intensity of the pH fluorescent probe is very obvious in the change range of pH of 4-7, and the fluorescence intensity of the pH fluorescent probe is gradually enhanced along with the reduction of the pH; in addition, the molecular structure of the compound takes 410nm as an excitation wavelength, and the maximum emission wavelength is 478-509nm; and the molecular structure of the compound has special propertiesThe special penetrability can penetrate through cell membranes but cannot penetrate through nuclear membranes, so that the special positions of subcellular layers can be selectively marked; meanwhile, the pH fluorescent probe has larger Stokes displacement, can reduce the signal to noise ratio, can adjust the wavelength of the luminescence according to different substituents, has a certain targeting effect based on the molecular structure, and is suitable for being widely applied to the detection of the pH in the weak acidic environment inside and outside the cell.
According to the preparation method of the pH fluorescent probe, provided by the second aspect of the application, two small molecular compounds are used as reactants, catalytic addition reaction is carried out under the action of a monovalent gold catalyst, and then reflux and purification treatment are carried out, so that the pH fluorescent probe can be effectively and quickly obtained.
The application provides a pH fluorescent probe or application of the pH fluorescent probe prepared by the preparation method in qualitative detection of pH fluorescence. The pH fluorescent probe can adjust the wavelength of the luminescence according to different substituents, has a certain targeting effect based on the molecular structure, and is suitable for being widely applied to the detection of pH in weak acidic environments inside and outside cells, so that the obtained pH fluorescent probe has high detection sensitivity in the qualitative detection of pH fluorescence, good effect and is beneficial to wide application.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe II obtained in example 1 according to the present application.
FIG. 2 is a graph showing the change in fluorescence intensity with pH of pH fluorescent probe II obtained in example 1 of the present application.
FIG. 3 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe III according to the pH value obtained in example 2 of the present application.
FIG. 4 is a graph showing the change in fluorescence intensity of pH fluorescent probe III with pH value obtained in example 2 of the present application.
FIG. 5 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe IV obtained in example 3 according to the present application.
FIG. 6 is a graph showing the change in fluorescence intensity with pH of pH fluorescent probe IV obtained in example 3 of the present application.
FIG. 7 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe V according to the pH value obtained in example 4 of the present application.
FIG. 8 is a graph showing the change in fluorescence intensity with pH of pH fluorescent probe V according to example 4 of the present application.
FIG. 9 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe VI according to the pH value obtained in example 5 of the present application.
FIG. 10 is a graph showing the change in fluorescence intensity of pH fluorescent probe VI according to the present application in example 5.
FIG. 11 is a graph showing the fluorescence intensity of pH fluorescent probe II obtained in example 1 of the present application at different pH values at a test wavelength of 478 nm.
FIG. 12 is a graph showing the fluorescence intensity of pH fluorescent probe III of example 2 of the present application at different pH values at a test wavelength of 478 nm.
FIG. 13 is a graph showing the fluorescence intensity of pH fluorescent probe IV of example 3 according to the present application at various pH values at 476 nm.
FIG. 14 is a graph showing the fluorescence intensity of pH fluorescent probe V of example 4 of the present application at different pH values at a test wavelength of 482 nm.
FIG. 15 is a graph showing the fluorescence intensity of pH fluorescent probe VI of example 5 of the present application at different pH values at a test wavelength of 509nm.
FIG. 16 is a cell image of pH fluorescent probe II of 5. Mu.M to 25. Mu.M obtained in example 1 of the present application after incubation with HeLa cells.
FIG. 17 shows an IC of pH fluorescent probe II obtained in example 1 of the present application 50 A drawing.
FIG. 18 shows HeLa cells of the present application with pH fluorescent probe II or with pH fluorescent probe II, 50nMDeep red (Invitrogen) TM ) Cell imaging after incubation.
FIG. 19 is an image of cells treated with pH fluorescent probe II and concanavalin A according to the application.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The first aspect of the embodiment of the application provides a pH fluorescent probe, which comprises quinolinium ions as a framework, p-tertiary aminophenyl and counter ions connected with the quinolinium ion framework structure through carbon-carbon single bonds, wherein the structural general formula of the pH fluorescent probe is shown as formula I,
wherein R is 1 And R is 2 Each independently selected from any one of piperidine, cyclohexylimine, morpholine and methyl, R 3 Selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethane sulfonate ions.
The structural general formula of the pH fluorescent probe is shown as formula I, the pH fluorescent probe takes quinolinium ions as a framework and comprises p-tertiary aminophenyl and counter ions which are connected with the quinolinium ion framework structure through carbon-carbon single bonds, wherein the pH fluorescent probe also comprises R 1 、R 2 、R 3 Three substituents; by changing the type of substituent groups on the general structural formula, different steric hindrance effects are formed between the dialkylamino group and the phenyl group, and then the electron donating ability of the substituent groups is changed, so that the compound can be selectively adjustedThe luminescence wavelength and pKa value of the whole molecule, and further achieve the fluorescence qualitative detection of pH. In the fluorescence qualitative detection process, when the pH is greater than 7, the pH fluorescent probe is non-fluorescent, namely a fluorophore quinolinium ion skeleton in the probe is quenched, as the pH is reduced, amino is taken as a sensitive group of the pH fluorescent probe, nitrogen atoms of dialkyl amino in tertiary aminophenyl are protonated to form quaternary ammonium salt ions, the quinolinium ion skeleton is subjected to fluorescence recovery, so that the effect of fluorescence is realized, the change of fluorescence intensity of the pH fluorescent probe is very obvious in the change range of pH of 4-7, and the fluorescence intensity of the pH fluorescent probe is gradually enhanced along with the reduction of the pH; in addition, the molecular structure of the compound takes 410nm as an excitation wavelength, and the maximum emission wavelength is 478-509nm; the molecular structure of the compound has special penetrability, can penetrate through cell membranes but cannot penetrate through cell nuclear membranes, so that the compound can be selectively marked at a special position of subcellular hierarchy; meanwhile, the pH fluorescent probe has larger Stokes displacement, can reduce the signal to noise ratio, can adjust the wavelength of the luminescence according to different substituents, has a certain targeting effect based on the molecular structure, and is suitable for being widely applied to the detection of the pH in the weak acidic environment inside and outside the cell.
The pH detection range of a pH fluorescent probe can be reflected in the pKa, which is the negative logarithm of the ionization constant (Ka) of an acid, and therefore it represents the pH at which the concentration of acid is the same as the conjugated base form of the molecule. Ka refers to the strength of the acid form in solution and is the equilibrium constant of the acid dissociation reaction. Therefore, if the acid form of the molecule is strong, ka is small and the acid is relatively difficult to dissociate, thus resulting in a larger pKa. The pKa value reveals the pH required for the molecule to donate a proton, i.e.: if the protonation of the pH fluorescent probe is more favourable, the acid form is more difficult to dissociate, the smaller Ka, the greater pKa, the more the pH detection range is biased towards the higher pH range, and vice versa.
Specifically, the provided structural general formula of the pH fluorescent probe takes quinolinium ions as a framework, and the structural general formula of the pH fluorescent probe comprises p-tertiary aminophenyl connected with a quinolinium ion framework structure through a carbon-carbon single bond. Wherein the significant increase in fluorescence intensity of the pH fluorescent probe is due to the protonation of the nitrogen atom of the dialkylamino group in the tertiary aminophenyl group. Dialkylamino groups are electron donating groups that cause fluorescence quenching upon photoexcitation by transferring electrons from their HOMO to the half-filled HOMO of the excited fluorescent probe. As a result, in the neutral form of the probe, fluorescence is quenched by the amine upon Photoinduced Electron Transfer (PET). When the proton concentration increases and the pH reaches the pKa value, the protonation of the amino group results in a decrease in its ability to donate electrons to the HOMO of the excited fluorescent dye, and thus increases in fluorescence intensity. Therefore, the responsiveness of the pH fluorescent probe is related to the ease of protonation of the nitrogen atom of the dialkylamino group, which is a pKa value, so the easier the protonation of the dialkylamino group, the higher the pKa value obtained.
Specifically, in the structural general formula I of the pH fluorescent probe, R 1 And R is 2 Each independently selected from any one of piperidine, cyclohexylimine, morpholine and methyl, R 3 Selected from hydrogen or methyl. The degree of protonation of the nitrogen atom of the dialkylamino group will have different effects depending on the different conformations of the tertiary aminophenyl group, the more planar the geometry, the more difficult it is to protonate. The conformation of the tertiary aminophenyl group will depend on R 1 、R 2 、R 3 The three substituents are different in type, so that different steric hindrance effects are formed between the dialkylamino group and the phenyl group, and the different steric hindrance effects enable the geometric structure of the nitrogen atom to trend towards a triangular cone (sp 3 Hybridization) or plane (sp 2 Hybridization). Therefore, in the general structural formula of the pH fluorescent probe, R needs to be controlled simultaneously 1 、R 2 、R 3 The three substituent groups can be used for determining whether the formed geometric structure tends to be triangular cone-shaped or plane-shaped, so as to judge the protonation degree of the dialkyl amino group and clearly obtain the pKa value.
Wherein when R is 1 And R is 2 Selected from cyclohexylimine, R 3 Selected from hydrogen; in the structural general formula of the obtained pH fluorescent probe, the geometry of the tertiary aminophenyl is closer to a plane (sp 2 Hybridization), the steric effect has smaller effect on resonance, so the resonance effect of the arc couple electron pair of the nitrogen atom and the aromatic ring is larger, the stronger resonance interaction between the nitrogen atom and the aromatic ring leads to better conjugation, and the stronger resonance interaction between the nitrogen atom and the aromatic ring leads toSo that the dialkylamino group has a stronger donor strength. The higher conjugation of the nitrogen atom to the aromatic ring results in a higher degree of difficulty in protonation of the dialkylamino group and therefore a lower pKa, at which point the pH detection range is biased towards a lower pH range.
Wherein when R is 1 And R is 2 Selected from piperidine, R 3 Selected from hydrogen; or when R is 1 Selecting methyl, R 2 Selected from methyl, R 3 Selected from methyl; in the structural general formula of the obtained pH fluorescent probe, the geometry of the tertiary aminophenyl is more similar to that of a triangular pyramid (sp 3 Hybridization), steric effects have a greater effect on resonance, so that the arc dipole pair of the nitrogen atom has less resonance with the aromatic ring, the weaker resonance interaction between the nitrogen atom and the aromatic ring results in poorer conjugation, and the poorer resonance interaction between the nitrogen atom and the aromatic ring results in a dialkylamino group having poorer donor strength. The lower conjugation of the nitrogen atom to the aromatic ring results in easier protonation of the dialkylamino group and therefore a higher pKa, in which case the pH detection range is biased towards a higher pH range.
In some embodiments, the pH fluorescent probe comprises a compound of the formula:
in the formula II, the substituent of the pH fluorescent probe is a six-membered piperidine ring which is in a chair conformation, and the torsion angle of the piperidine ring relative to the aromatic ring is large, so that the degree of conjugation of nitrogen atoms to the aromatic ring is small, and the resonance interaction between the nitrogen atoms and the aromatic ring is reduced. Thus, the six-membered piperidine ring favors protonation of the dialkylamino group and results in a higher pKa value.
In formula III, the substituent of the pH fluorescent probe is a seven-membered piperidine ring, which exhibits a smaller twist angle, resulting in a greater degree of conjugation of the nitrogen atom to the aromatic ring and increased resonance interaction between the nitrogen atom and the aromatic ring. Thus, protonation of the dialkylamino group is difficult and results in a lower pKa value.
In formula IV, the substituent of the pH fluorescent probe is morpholine, the oxygen atom in morpholine is an electron withdrawing group, tending to attract electrons to itself and thus reducing the basicity of the nitrogen atom. Thus, protonation of the dialkylamino group is difficult, and the lowest pKa value is obtained.
In the formula V, the substituent of the pH fluorescent probe is methyl, and the ortho carbon in the obtained N, N,2, 6-tetramethylaniline has the substituent. The strong steric hindrance between the o-dimethylamino group and the dimethylamino group in N,2, 6-tetramethylaniline results in a twisting out of plane of the dimethylamino group relative to the benzene ring, and thus, a greater twisting of the dimethylamino group reduces the resonant interaction between the nitrogen atom and the aromatic ring and promotes protonation of the dialkylamino group, resulting in the highest pKa value.
In the formula VI, the quinolinium ion skeleton of the pH fluorescent probe is polycyclic aromatic hydrocarbon consisting of 5 condensed heterocyclic benzene rings, and the substituent is a six-membered piperidine ring. The difference between them compared to the structure of formula II is that the quinolinium of formula VI has an additional benzene ring. Since the substituents of formula II and formula VI are both six-membered piperidine rings, it is shown that the resonance interactions between the piperidine and the benzene ring are identical, resulting in similar pKa values. The presence of an additional benzene ring on the quinolinium does not significantly affect the resonance interaction between the piperidine and the benzene ring. Instead, the presence of an additional benzene ring on the quinolinium of formula V expands the conjugation system, causing a long wavelength shift in which the emission wavelength increases.
The second aspect of the embodiment of the application provides a preparation method of a pH fluorescent probe, which comprises the following steps:
s01, providing a first compound with a structural general formula of
S02, providing a second compound with a structural general formula of
S03, dissolving a first compound and a second compound in an organic solvent under an inert atmosphere, carrying out catalytic addition reaction by taking a monovalent gold complex as a catalyst, and carrying out first purification treatment to obtain a third compound with a structural general formula of
S04, carrying out reflux treatment on the third compound, then carrying out second purification treatment to obtain the pH fluorescent probe,
wherein the structural general formula of the pH fluorescent probe isAnd R is 1 And R is 2 Each independently selected from any one of piperidine, cyclohexylimine, morpholine and methyl, R 3 Selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethane sulfonate ions.
According to the preparation method of the pH fluorescent probe, provided by the second aspect of the application, two small molecular compounds are used as reactants, catalytic addition reaction is carried out under the action of a monovalent gold catalyst, and then reflux and purification treatment are carried out, so that the pH fluorescent probe can be effectively and quickly obtained.
Wherein, the whole flow of the preparation method is shown in the following reaction equation,
in the step S01, a first compound is provided, the structural general formula of the first compound is shown as a formula VII, and the formula VII isWherein R is 1 And R is 2 Each independently selected from any one of piperidine, cyclohexylimine, morpholine and methyl, R 3 Selected from hydrogen or methyl, which can be prepared by prior art literature.
In step S02, a second compound is provided, the structural general formula of the second compound is shown as formula VIII, and the formula VIII isWherein, Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethane sulfonate ion, and can be prepared by the prior art literature.
In step S03, the first compound and the second compound are dissolved in an organic solvent under inert atmosphere, a monovalent gold complex is used as a catalyst to perform catalytic addition reaction, and then the first purification treatment is performed to obtain a third compound with the structural general formula of
In some embodiments, the reaction is performed under an inert atmosphere, ensuring that the purity of the resulting product is high and that no side effects such as oxidation occur during the reaction. Wherein the inert atmosphere is selected from any one of nitrogen, helium, neon, argon, krypton, xenon and radon.
In some embodiments, the first compound and the second compound are dissolved in an organic solvent, wherein the organic solvent is selected from acetonitrile, or a mixed solution of acetonitrile and at least one of methanol, ethanol, propanol, butanol, toluene, chloroform. Based on the principle of similar compatibility, the first compound and the second compound have high solubility in acetonitrile solution, are favorable for reaction, and the obtained organic solvent cannot react with the compounds.
In some embodiments, monovalent gold complexes selected from the group consisting of chloro [ tris (p-trifluoromethylphenyl) phosphine ] gold (I) are used as catalysts, with which the reaction can be efficiently catalyzed, increasing the efficiency of the reaction.
Further, in order to increase the yield and obtain a product with higher purity, the molar ratio of the first compound, the second compound and the monovalent gold complex is (1.0 to 1.5): (1.5-2.0): (0.05-0.1). Further, the molar ratio of the first compound, the second compound and the monovalent gold complex is 1.0:1.5:0.05, and the product obtained by the reaction with the ratio has high purity and high yield.
In some embodiments, in the step of performing the catalytic addition reaction, the catalytic addition reaction is performed under room temperature conditions and irradiation of visible light for 16 to 18 hours, wherein the visible light is selected from blue light sources, and the power of the blue light sources is more than or equal to 3 watts. The light source of the reaction is controlled to meet the conditions, so that the addition reaction can be catalyzed, and the corresponding product is obtained. Further, the reaction is carried out under the room temperature condition, namely, the room temperature is controlled to be 10-30 ℃.
In some embodiments, the first purification treatment is performed using a silica gel column chromatography method. In a specific embodiment of the present application, the specific method of the first purification treatment is as follows: carrying out rotary evaporation on a product obtained by the catalytic addition reaction to remove an organic solvent, and obtaining a residue; providing silica gel column, gradient eluting with mixed solution of dichloromethane and methanol as eluent, mixing rotary evaporation to remove solvent, adding dichloromethane, extracting with water for multiple times, mixing water phase, and rotary evaporation to remove water to obtain third compound with structural formula as shown in formula IX
Is thatR 1 And R is 2 Each independently selected from any one of piperidine, cyclohexylimine, morpholine and methyl, R 3 Selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethane sulfonate ions.
In step S04, the third compound is subjected to a reflux treatment and then to a second purification treatment to obtain a pH fluorescent probe.
In some implementations, the subjecting the third compound to the reflow process includes the steps of: and dissolving the third compound in water, heating to 180-185 ℃ and carrying out reflux treatment for 16-18 hours.
In some embodiments, a second purification treatment is performed, the second purification treatment being a purification treatment using a silica gel column chromatography method. In a specific embodiment of the present application, the specific method of the second purification treatment is as follows: spin-evaporating to remove water after the reaction is finished to obtain a residue; providing a silica gel column, performing gradient elution by using a mixed solution of dichloromethane and methanol as an eluent, and combining and spin-evaporating to remove the solvent to obtain a pH fluorescent probe, wherein the structural general formula of the pH fluorescent probe is as followsAnd R is 1 And R is 2 Each independently selected from any one of piperidine, cyclohexylimine, morpholine and methyl, R 3 Selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethane sulfonate ions.
The preparation method utilizes the cooperation of visible light and a catalyst monovalent gold complex to catalyze, and a pH fluorescent probe as shown in a structural formula I is obtained; the obtained reaction product has high purity and no other impurities.
In a third aspect, the embodiment of the application provides an application of a pH fluorescent probe or a pH fluorescent probe prepared by the preparation method of any one of claims 3 to 9 in fluorescence qualitative detection of pH.
The application provides a pH fluorescent probe or application of the pH fluorescent probe prepared by the preparation method in qualitative detection of pH fluorescence. The pH fluorescent probe can adjust the wavelength of the luminescence according to different substituents, has a certain targeting effect based on the molecular structure, and is suitable for being widely applied to the detection of pH in weak acidic environments inside and outside cells, so that the obtained pH fluorescent probe has high detection sensitivity in the qualitative detection of pH fluorescence, good effect and is beneficial to wide application.
The following description is made with reference to specific embodiments.
Example 1
PH fluorescent probe II and preparation method thereof
pH fluorescent probe IIIs of the structure of
preparation method of pH fluorescent probe II
The preparation method of the pH fluorescent probe II is shown in the following reaction formula,
the preparation method of the pH fluorescent probe II comprises the following steps:
providing 0.5mmol of trimethylsilane, 0.6mmol of diazonium salt having quinoline structure;
trimethylsilyl and diazonium salt having quinoline structure were dissolved in 5mL acetonitrile solvent under inert nitrogen atmosphere to give (CF) 3 Ph) 3 PAuCl (0.05 mmol) is used as a catalyst, catalytic addition reaction is carried out for 16 hours under the conditions of blue LED light source irradiation and room temperature, after the reaction is completed, organic solvent is removed by rotary evaporation, a silica gel column is provided for the obtained residue, the mixed solution of dichloromethane and methanol is used for gradient elution, the solvents are removed by rotary evaporation in a combined mode, the dichloromethane is added, water is used for multiple extraction, the water phase is combined, and finally water is removed by rotary evaporation, so that a third compound is obtained;
and dissolving the obtained third compound in water, heating to 180 ℃ for reflux reaction for 16 hours, removing the solvent under reduced pressure, providing a silica gel column, carrying out gradient elution by using a mixed solution of dichloromethane and methanol, combining and rotary evaporation to remove the solvent, and finally obtaining a dark red solid product, namely the pH fluorescent probe formula II, with the yield of 22 percent in two steps.
NMR results of pH fluorescent Probe II
1 H NMR(400MHz,DMSO-d 6 )δ9.41(d,J＝9.2Hz,1H),9.22(d,J＝8.6Hz,1H),9.08(d,J＝9.0Hz,1H),8.52(s,1H),8.38(d,J＝7.7Hz,1H),8.34(d,J＝8.1Hz,1H),8.22(t,J＝7.5Hz,1H),8.08(t,J＝7.8Hz,1H),7.99(d,J＝9.0Hz,1H),7.82(t,J＝7.5Hz,1H),7.60(t,J＝8.0Hz,1H),7.34(d,J＝8.6Hz,2H),6.99(d,J＝8.7Hz,2H),3.29–3.25(m,4H),1.59(s,6H).
Example 2
PH fluorescent probe III and preparation method thereof
pH fluorescent probe IIIIs of the structure of
preparation method of pH fluorescent probe III
The preparation method of the pH fluorescent probe III is shown in the following reaction formula,
specific operation of the preparation method of pH fluorescent probe III is shown in example 1, and the obtained product is dark red solid with the yield of 14%.
NMR results of pH fluorescent Probe III
1 H NMR(400MHz,DMSO-d 6 )δ9.38(d,J＝9.2Hz,1H),9.20(d,J＝8.6Hz,1H),9.05(d,J＝9.1Hz,1H),8.51(s,1H),8.36(d,J＝7.9Hz,1H),8.32(d,J＝8.0Hz,1H),8.20(t,J＝7.5Hz,1H),8.07(d,J＝7.9Hz,1H),8.03(d,J＝8.8Hz,1H),7.82(t,J＝7.5Hz,1H),7.57(t,J＝8.1Hz,1H),7.30(d,J＝8.5Hz,2H),6.75(d,J＝8.6Hz,2H),3.52(t,J＝6.0Hz,4H),1.72(s,4H),1.47(s,4H).
Example 3
PH fluorescent probe IV and preparation method thereof
pH fluorescent probe IVIs of the structure of
preparation of pH fluorescent Probe IVPreparation method
The preparation method of the pH fluorescent probe IV is shown in the following reaction formula,
specific operation of the preparation method of pH fluorescent probe IV is shown in example 1, and the obtained product is dark red solid with a yield of 38%.
NMR results of pH fluorescent Probe IV
1 H NMR(400MHz,DMSO-d 6 )δ9.43(d,J＝9.2Hz,1H),9.24(d,J＝8.6Hz,1H),9.09(d,J＝9.1Hz,1H),8.54(s,1H),8.39(d,J＝7.9Hz,1H),8.35(d,J＝7.9Hz,1H),8.23(t,J＝7.5Hz,1H),8.10(t,J＝7.8Hz,1H),7.97(d,J＝9.0Hz,1H),7.83(t,J＝7.5Hz,1H),7.61(t,J＝8.0Hz,1H),7.39(d,J＝8.5Hz,2H),7.03(d,J＝8.6Hz,2H),3.73–3.78(m,4H),3.21–3.25(m,4H).
Example 4
PH fluorescent probe V and preparation method thereof
PH fluorescent probe VIs of the structure of
preparation method of pH fluorescent probe V
The preparation method of the pH fluorescent probe V is shown in the following reaction formula,
specific operation of the preparation method of pH fluorescent probe V is shown in example 1, and the obtained product is orange solid with 3% yield.
NMR results of pH fluorescent Probe V
1 H NMR(400MHz,DMSO-d 6 )δ.9.45(d,J＝9.2Hz,1H),9.26(d,J＝8.6Hz,1H),9.12(d,J＝9.1Hz,1H),8.58(s,1H),8.41(d,J＝8.1Hz,1H),8.36(d,J＝8.0Hz,1H),8.24(t,J＝7.5Hz,1H),8.12(t,J＝7.8Hz,1H),7.91(d,J＝8.9Hz,1H),7.85(t,J＝7.6Hz,1H),7.61(t,J＝8.0Hz,1H),7.17(s,2H),2.80(s,6H),2.21(s,6H).
Example 5
PH fluorescent probe VI and preparation method thereof
pH fluorescent probe VIIs of the structure of
preparation method of pH fluorescent probe VI
The preparation method of the pH fluorescent probe VI is shown in the following reaction formula,
specific operation of the preparation method of pH fluorescent probe VI is shown in example 1, and the obtained product is black solid with the yield of 23%.
NMR results of pH fluorescent Probe VI
1 H NMR(400MHz,DMSO-d 6 )δ.9.00(d,J＝8.2Hz,1H),8.89(d,J＝8.6Hz,1H),8.85(d,J＝8.1Hz,1H),8.81(d,J＝8.3Hz,1H),8.56(s,1H),8.40(d,J＝8.1Hz,1H),8.28–8.26(m,1H),8.26–8.23(m,1H),8.07(d,J＝7.3Hz,1H),8.03(d,J＝7.4Hz,1H),7.77(s,1H),7.76–7.73(m,1H),7.47(d,J＝7.9Hz,1H),7.45–7.39(m,2H),6.97(d,J＝8.7Hz,2H),3.21(s,4H),1.58(s,6H).
Property testing
(1) Optical properties of the pH fluorescent probes obtained in examples 1 to 5 were tested.
The testing method comprises the following steps: 2mol/L aqueous HCl and 2mol/L aqueous NaOH are mixed and 2mol/L HCl/NaOH buffers having various pH values are prepared under monitoring by a pH meter. By diluting the pH fluorescent probe to 5X10 in acetonitrile - 4 mol/L to prepare a stock solution. Respectively dissolving 2mol/L HCl/NaOH buffer solution with various pH values with acetonitrile stockMixing the solutions in a volume ratio of 19 to 1 to obtain a mixture of 2.5X10 -5 A diluted solution of mol/L.
pH fluorescent probes measured at different pH values absorb and emit spectra.
(2) pKa analysis of pH fluorescent probes obtained in examples 1 to 5.
As can be seen from the absorption spectrum and the fluorescence spectrum (FIGS. 1 to 10) in (1), the excitation wavelength is 410nm, and the maximum emission wavelengths of pH fluorescent probes II, III, IV, V, VI are respectively 178 nm, 470 nm,482nm and 509nm. The fluorescence intensities obtained at the maximum emission wavelengths of the pH fluorescent probes obtained in examples 1 to 5 were summarized with pH values to obtain normalized fluorescence intensity curves for pH values, wherein the pH value of half of the normalized intensity was estimated as the pKa value.
(3) Cytotoxicity assay of pH fluorescent Probe II obtained in example 1.
Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with 44mmol/L sodium bicarbonate (Sigma-Aldrich), 10% v/v fetal bovine serum (Gibco) and 100U/mL penicillin (Gibco) and 100ug/mL streptomycin (Gibco) at 37℃with 5% CO 2 . One day before the cytotoxicity assay experiments, 5x10 3 Cells were seeded in 96-well plates and incubated with pH fluorescent probe II at 37℃and 5% CO at 5. Mu. Mol/L to 25. Mu. Mol/L 2 Incubate for 72 hours. After incubation, the medium was removed and replaced with fresh medium. mu.L of MTS mixture (0.92 mg/mL PMS, sigma-Aldrich:2mg/mL MTS, promega, 1:20) was added to each well. The plates were incubated at 37℃and 5% CO 2 During 1 hour incubation, absorbance at 490nm was measured and IC was calculated 50 Numerical values.
(4) Cell imaging of pH fluorescent probe II obtained in example 1.
HeLa cells were seeded on 35mm glass bottom dishes (SPL) and incubated at 37℃and 5% CO 2 Cell culture chambers were allowed to adhere overnight. The cells were incubated with pH probe alone or with pH fluorescent probe, 50nmol/LDeep red (Invitrogen) TM ) Incubate for 2 hours. The images were examined under a confocal microscope (Leica TCS SP8 MP).
(5) Intracellular pH change sensing of pH fluorescent Probe II obtained in example 1.
To further confirm whether pH fluorescent probe II fluoresced due to lysosomal acidity, cells were treated with concanavalin A, a V-ATPase inhibitor that caused the lysosomal pH to rise, and further analyzed for fluorescent signal.
Analysis of results
(1) The pH fluorescent probes obtained in examples 1 to 5 were tested for optical properties, and the test results are shown in FIGS. 1 to 10.
FIG. 1 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe II according to the change of pH value obtained in example 1 of the present application, and FIG. 2 is a graph showing the change of fluorescence intensity of pH fluorescent probe II according to the change of pH value obtained in example 1 of the present application; from FIGS. 1 and 2, it can be seen that the pH fluorescent probe II obtained in example 1, when the pH is in the range of 1.49 to 3.57, can observe a maximum absorption wavelength of 410nm, which lies in the visible light region; when excited by the maximum absorption wavelength, a strong emission peak was observed at 478nm at a pH of 1.49, with the luminosity gradually decreasing at a pH ranging from 1.49 to 5.52.
FIG. 3 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe III according to the pH change obtained in example 2 of the present application, and FIG. 4 is a graph showing the fluorescence intensity of pH fluorescent probe III according to the pH change obtained in example 2 of the present application; from FIGS. 3 and 4, it can be seen that pH fluorescent probe III obtained in example 2, when the pH is in the range of 0.33 to 3.56, the maximum absorption wavelength is 410nm, which is located in the visible light region; when excited by the maximum absorption wavelength, a strong emission peak was observed at 478nm at a pH of 0.33, with the luminosity gradually decreasing in the pH range of 0.33 to 3.56.
FIG. 5 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe IV according to the change of pH value obtained in example 3 of the present application, and FIG. 6 is a graph showing the change of fluorescence intensity of pH fluorescent probe IV according to the change of pH value obtained in example 3 of the present application; from FIGS. 5 and 6, it can be seen that the pH fluorescent probe IV obtained in example 3, when the pH is in the range of 0.36 to 3.00, can observe a maximum absorption wavelength of 410nm, which lies in the visible light region; when excited by the maximum absorption wavelength, a strong emission peak was observed at 476nm at a pH of 0.36, with the luminosity gradually decreasing at a pH range of 0.36 to 3.00.
FIG. 7 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe V according to the pH change obtained in example 4 of the present application, and FIG. 8 is a graph showing the fluorescence intensity of pH fluorescent probe V according to the pH change obtained in example 4 of the present application; from FIGS. 7 and 8, it can be seen that pH fluorescent probe V obtained in example 5, when the pH is in the range of 0.57 to 6.72, the maximum absorption wavelength is 410nm, which is located in the visible light region; when excited by the maximum absorption wavelength, a strong emission peak was observed at 482nm at a pH of 0.57 to 5.26, and its luminosity was drastically reduced at a pH range of 6.72 or more.
FIG. 9 is a graph showing the ultraviolet absorption spectrum of pH fluorescent probe VI according to the present application obtained in example 5, and FIG. 10 is a graph showing the change of fluorescence intensity of pH fluorescent probe VI according to the present application obtained in example 5, and it can be seen from FIGS. 9 and 10 that pH fluorescent probe VI according to example 5 has a maximum absorption wavelength of 410nm in the visible region when the pH range is 0.56 to 4.61; when excited by the maximum absorption wavelength, a strong emission peak was observed at 509nm at a pH of 0.56, with the luminosity gradually decreasing at a pH range of 0.56 to 4.61.
(2) The results of pKa analysis of the pH fluorescent probes obtained in examples 1 to 5 are shown in FIGS. 11 to 15.
As can be seen from the absorption spectrum and the fluorescence spectrum (FIGS. 1 to 10), the excitation wavelength is 410nm, and the pH fluorescent probe II, the pH fluorescent probe III, the pH fluorescent probe IV, the pH fluorescent probe V, the maximum emission wavelengths of the pH fluorescent probe VI are respectively 178 nm, 470 nm, 4812 nm and 509nm, and FIGS. 11 to 15 are fluorescence intensities obtained from the maximum emission wavelengths, and normalized fluorescence intensity curves with respect to pH values are obtained. The pH of half the normalized intensity was estimated as pKa. When the normalized intensity reaches half of it, it means that the concentrations of the protonated and non-protonated forms of the quinolinium compound are the same.
FIG. 11 is a graph showing the fluorescence intensity of pH fluorescent probe II of example 1 of the present application at different pH values at 478nm, resulting in pKa value of 4.6.
FIG. 12 is a graph showing the fluorescence intensity of pH fluorescent probe III of example 2 of the present application at different pH values at 478nm, resulting in pKa value of 2.8.
FIG. 13 is a graph showing the fluorescence intensity of pH fluorescent probe IV of example 3 of the present application at different pH values at 476nm, resulting in pKa value of <2.2.
FIG. 14 is a graph showing the fluorescence intensity of pH fluorescent probe V of example 4 of the present application at different pH values at a test wavelength of 482nm, resulting in a pKa value of 6.1.
FIG. 15 is a graph showing the fluorescence intensity of pH fluorescent probe VI of example 5 of the present application at different pH values at 509nm, resulting in a pKa value of 4.3.
Wherein the pH detection range of the pH fluorescent probe II of example 1, the pH fluorescent probe V of example 4 and the pH fluorescent probe VI of example 5 is between 4 and 7.
(3) Cytotoxicity assay of pH fluorescent Probe II obtained in example 1.
After incubation of HeLa cells with pH fluorescent probe II at 5. Mu. Mol/L to 25. Mu. Mol/L measured with EnSpire Multi-mode plate reader (Perkinelmer), the fluorescence intensity became stronger as the concentration of pH fluorescent probe increased as shown in FIG. 16, indicating that pH fluorescent probe has a concentration-dependent signal-reacting ability. IC (integrated circuit) 50 The values were calculated by GraphPad Prism as shown in FIG. 17, IC 50 28.08. Mu. Mol/L.
(4) Cell imaging of pH fluorescent probe II obtained in example 1.
The images were examined under confocal microscopy (Leica TCS SP8 MP) as shown in fig. 18, fig. 18A and 18B are fluorescence microscopy images of pH fluorescent probes in different selected cells, fig. 18C and 18D areDeep red (Invitrogen) TM ) In different selected cellsFluorescence microscopy images, FIGS. 18E and 18F are bright field microscopy images of different selected cells, FIGS. 18G and 18H are pH fluorescence probe and +.>Deep red (Invitrogen) TM ) Pooled fluorescence microscopy images in different selected cells.
For pH fluorescent probe II and lysosome tracer, co-localization of the fluorescent signal was observed, indicating that the compound was localized to the lysosome. The molecular structure can penetrate through cell membrane, but not through nuclear membrane, and can be selectively marked at specific positions of subcellular hierarchy.
(5) Intracellular pH change sensing of pH fluorescent Probe II obtained in example 1.
As shown in fig. 19, fig. 19A and 19B are fluorescence microscopy images of pH fluorescence probe of the control group at different selected cells (without treatment with concanavalin a), fig. 19C and 19D are bright field microscopy images of different selected cells of the control group, fig. 19E and 19F are fluorescence microscopy images of pH fluorescence probe of the control group at different selected cells after treatment with concanavalin a, and fig. 19G and 19H are bright field microscopy images of different selected cells after treatment with concanavalin a.
Cells treated with pH fluorescent probe II and concanavalin A showed complete loss of fluorescent signal, indicating that pH fluorescent probe II acts as pH sensing in vivo.
Therefore, the pH fluorescent probe provided by the application has obvious change of fluorescence intensity in the change range of pH 4-7, and the fluorescence intensity is gradually enhanced along with the decrease of pH; in addition, the molecular structure of the compound takes 410nm as an excitation wavelength, and the maximum emission wavelength is 478-509nm; the molecular structure of the compound has special penetrability, can penetrate through cell membranes but cannot penetrate through cell nuclear membranes, so that the compound can be selectively marked at a special position of subcellular hierarchy; meanwhile, the pH fluorescent probe has larger Stokes displacement, can reduce the signal to noise ratio, can adjust the wavelength of the luminescence according to different substituents, has a certain targeting effect based on the molecular structure, and is suitable for being widely applied to the detection of the pH in the weak acidic environment inside and outside the cell.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Claims (6)
1. A pH fluorescent probe, characterized in that the pH fluorescent probe is a compound of the following structural formula:
a formula II; />Formula III;
a formula IV; />Formula VI.
2. A method for preparing a pH fluorescent probe of the formula II according to claim 1,
。
3. a method for preparing a pH fluorescent probe of the formula III as claimed in claim 1, as shown in the following reaction scheme,
。
4. a method for preparing a pH fluorescent probe of the formula IV as set forth in claim 1, as shown in the following reaction scheme,
。
5. a method for preparing a pH fluorescent probe of the formula VI as set forth in claim 1, as shown in the following reaction scheme,
。
6. use of the pH fluorescent probe according to claim 1 or prepared by the preparation method according to any one of claims 2 to 5 in fluorescence qualitative detection of pH for non-diagnostic purposes.
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| CN104177357A (en) * | 2013-05-28 | 2014-12-03 | 中国科学院兰州化学物理研究所 | Isoquinoline salt synthesis method |
| CN107964257A (en) * | 2016-10-20 | 2018-04-27 | 香港理工大学深圳研究院 | Quinoline cation matrix structure organic dyestuff compound and its preparation method and application |
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| CN107964257A (en) * | 2016-10-20 | 2018-04-27 | 香港理工大学深圳研究院 | Quinoline cation matrix structure organic dyestuff compound and its preparation method and application |
| WO2021015544A1 (en) * | 2019-07-23 | 2021-01-28 | 재단법인 대구경북첨단의료산업진흥재단 | Benzoquinolizinium-based compound, preparation method therefor, and contrast medium composition comprising same |
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