CN115232120A - pH fluorescent probe and synthetic method and application thereof - Google Patents
pH fluorescent probe and synthetic method and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of organic small-molecule fluorescent probes, and provides a pH fluorescent probe which comprises a p-tertiary aminophenyl group and a counter ion, wherein the p-tertiary aminophenyl group is connected with a quinolinium ion skeleton structure through a carbon-carbon single bond; the fluorescence intensity of the pH fluorescent probe changes obviously in the change range of pH 4-7, and the fluorescence intensity of the pH fluorescent probe is gradually enhanced along with the reduction of pH; in addition, the molecular structure of the compound takes 410nm as an excitation wavelength, and the maximum emission wavelength of the compound is 478-509nm; the molecular structure of the compound has special penetrability, can penetrate through a cell membrane, but cannot penetrate through a nuclear membrane, so that the compound can be selectively marked at a special position of a subcellular level; meanwhile, the method has larger Stokes shift, can reduce the signal-to-noise ratio, and is suitable for being widely applied to the detection of the pH in the weak acid environment inside and outside cells.
Description
Technical Field
The application belongs to the technical field of organic small-molecule fluorescent probes, and particularly relates to a pH fluorescent probe and a synthetic 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 in cells and organelles, such as: ion transport, cell proliferation and apoptosis, endocytosis, multiple drug resistance, cell adhesion, muscle contraction, and the like, and therefore maintaining pH balance is critical to human health. Often, small changes in pH can lead to many dysfunctions such as membrane contractile breakdown, protein denaturation, enzyme dysfunction, and failure of free radical generation. For example, lysozyme may cause 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. For example, during the development of tumors, hypoxia occurs, which causes acidosis inside and outside cells, thus showing a decrease in pH, and ultimately leading to abnormal cell activities. Therefore, the real-time accurate and rapid detection of the pH inside and outside human cells is of great significance for the study of human physiological and pathological processes and the diagnosis of diseases.
Compared with methods for detecting the pH value of a biological sample, such as microelectrode, nuclear magnetic resonance spectroscopy (NMR) and absorption spectroscopy, the fluorescence spectroscopy has unique advantages: 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 generally consist of three parts: a fluorophore, a linker, and also a recognition group. Before the proton is not combined, the probe molecule does not emit fluorescence or the fluorescence is very weak, once the recognition group acts with the proton, the condition for inhibiting the fluorescence in the molecule disappears, and the fluorophore emits strong fluorescence, thereby realizing the aim of detecting the pH. Commonly used fluorescent groups in currently developed pH fluorescent probes include Fluorescein (Fluorescein), rhodamine (Rhodamine), coumarin (Coumarin), borofluorfen (BODIPY), and anthocyanin (Cyanine). Most of the fluorescent groups cannot select labeled organelles, a targeting group needs to be additionally connected, and a detection recognition group is used as an auxiliary, 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 emission wavelength, it becomes difficult to simultaneously detect different kinds of molecules or molecules with the same structure in different organelles. Moreover, the fluorescent groups on these fluorescent probes have some disadvantages: suboptimal excitation and emission wavelengths, photobleaching, weak spectral variation, etc. Therefore, the prior art has no targeting effect because the light-emitting wavelength of the pH fluorescent probe can not be adjusted, and the wide application of the pH fluorescent probe is influenced.
Disclosure of Invention
The application aims to provide a pH fluorescent probe and a synthesis method and application thereof, and aims to solve the problems that the luminous wavelength of the pH fluorescent probe cannot be adjusted and the pH fluorescent probe does not have a targeting effect in the prior art.
In order to achieve the purpose 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 a quinolinium ion as a skeleton, a p-tertiary aminophenyl group and a counter ion, wherein the p-tertiary aminophenyl group is connected with the quinolinium ion skeleton through a carbon-carbon single bond, the structural general formula of the pH fluorescent probe is shown as a formula I,
wherein R is 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Is selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate 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 with a general structural formula
Providing a second compound, wherein the structural general formula of the second compound is
Dissolving the first compound and the second compound in an organic solvent under inert atmosphere, carrying out catalytic addition reaction by using a monovalent gold complex as a catalyst, and carrying out first purification treatment to obtain a third compound, wherein the structural general formula of the third compound is
Carrying out reflux treatment on the third compound, then carrying out second purification treatment to obtain a pH fluorescent probe,
wherein the structural general formula of the pH fluorescent probe is
And, R 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Is selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate ions.
In a third aspect, the application provides an application of the pH fluorescent probe or the pH fluorescent probe prepared by the preparation method in fluorescence qualitative detection of pH.
According to the pH fluorescent probe provided by the first aspect of the application, the structural general formula of the pH fluorescent probe is shown as formula I, the pH fluorescent probe comprises a p-tertiary amino phenyl group and a counter ion, wherein the p-tertiary amino phenyl group is connected with the quinoline onium ion skeleton structure through a carbon-carbon single bond, and the pH fluorescent probe also comprises R 1 、R 2 、R 3 Three substituents; by changing the types of the substituent groups on the structural general formula, different steric hindrance effects are formed between the dialkylamino group and the phenyl group, so that the electron donating capability of the substituent groups is changed, and the light-emitting wavelength and pK of the molecule can be selectively adjustedand a value, and further achieving the fluorescent qualitative detection of the pH. In the fluorescent qualitative detection process, when the pH is more than 7, the pH fluorescent probe is non-fluorescent, namely, a fluorescence radical quinolinium ion skeleton in the probe is quenched, along with the reduction of the pH, an amino group is used as a sensitive group of the pH fluorescent probe, nitrogen atoms of dialkyl amino groups in tertiary amino phenyl groups are protonated to form quaternary ammonium salt ions, and the fluorescence of the quinolinium ion skeleton is recovered, so that the fluorescent light-emitting effect is realized, the fluorescent intensity of the pH fluorescent probe is obviously changed in the change range of the pH between 4 and 7, and the fluorescent 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 of the compound is 478-509nm; the molecular structure of the compound has special penetrability, can penetrate through a cell membrane, but cannot penetrate through a nuclear membrane, so that the compound can be selectively marked at a special position of a subcellular level; meanwhile, the probe has larger Stokes shift and can reduce the signal-to-noise ratio, the luminous wavelength of the pH fluorescent probe can be adjusted according to different substituents, and the probe has a certain targeting effect based on the molecular structure, so that the probe is suitable for being widely applied to the detection of the pH in the weak acid environment inside and outside cells.
According to the preparation method of the pH fluorescent probe, two small molecular compounds are used as reactants, catalytic addition reaction is carried out under the action of a monovalent gold catalyst, and then backflow and purification treatment are carried out.
The application of the pH fluorescent probe provided by the third aspect of the application or the pH fluorescent probe prepared by the preparation method in fluorescence qualitative detection of pH. The pH fluorescent probe can adjust the light-emitting wavelength 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 acid environment inside and outside cells, so that the obtained pH fluorescent probe has high detection sensitivity and good effect in the fluorescence qualitative detection of the pH, and is beneficial to wide application.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph of the UV absorption spectrum of the pH fluorescent probe II obtained in example 1 of the present invention as a function of pH.
FIG. 2 is a graph showing the change of fluorescence intensity of the pH fluorescent probe II according to the present invention obtained in example 1.
FIG. 3 is a graph of the UV absorption spectrum of the pH fluorescent probe III according to the variation of pH value obtained in example 2 of the present invention.
FIG. 4 is a graph showing the fluorescence intensity of the pH fluorescent probe III according to the pH variation obtained in example 2 of the present invention.
FIG. 5 is a graph of the UV absorption spectrum of the pH fluorescent probe IV varying with pH according to the invention obtained in example 3.
FIG. 6 is a graph showing the fluorescence intensity of the pH fluorescent probe IV according to the present invention obtained in example 3.
FIG. 7 is a graph showing the UV absorption spectrum of the pH fluorescent probe V according to the variation of pH in example 4 of the present invention.
FIG. 8 is a graph showing the change of fluorescence intensity with pH of the pH fluorescent probe V obtained in example 4 of the present invention.
FIG. 9 is a graph of the UV absorption spectrum of the pH fluorescent probe VI according to the variation of pH value obtained in example 5 of the present invention.
FIG. 10 is a graph showing the fluorescence intensity of pH fluorescent probe VI according to the present invention obtained in example 5.
FIG. 11 is a graph showing fluorescence intensities of pH fluorescent probe II obtained in example 1 of the present invention at different pH values at a measurement wavelength of 478 nm.
FIG. 12 is a graph showing fluorescence intensities of pH fluorescent probe III according to example 2 of the present invention 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 of the present invention at different pH values at the test wavelength of 476 nm.
FIG. 14 is a graph showing fluorescence intensities of pH fluorescent probe V of example 4 of the present invention at different pH values at a test wavelength of 482 nm.
FIG. 15 is a graph showing fluorescence intensities of pH fluorescent probes VI of example 5 of the present invention at different pH values at a test wavelength of 509nm.
FIG. 16 is an image of cells obtained in example 1 of the present invention after incubation of HeLa cells with pH fluorescent probe II of 5. Mu.M to 25. Mu.M.
FIG. 17 shows IC of pH fluorescent probe II obtained in example 1 of the present invention 50 Drawing.
FIG. 18 shows HeLa cells of the present invention with pH fluorescent probe II or with pH fluorescent probe II, 50nM
Deep red (Invitrogen) TM ) Imaging of the cells after incubation.
FIG. 19 is an image of cells treated with pH fluorescent probe II and concanavalin A according to the present invention.
Detailed Description
In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of and not restrictive on the broad application.
The first aspect of the embodiments of the present application provides a pH fluorescent probe, which includes a quinolinium ion as a skeleton, a p-tertiary aminophenyl group and a counter ion, wherein the p-tertiary aminophenyl group and the counter ion are connected to the quinolinium ion skeleton through a carbon-carbon single bond, the structural general formula of the pH fluorescent probe is as shown in formula I,
wherein R is 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Is selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate ions.
According to the pH fluorescent probe provided by the first aspect of the application, 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 a carbon-carbon single bond, and R 1 、R 2 、R 3 Three substituents; by changing the types of the substituent groups on the general structural formula, different steric hindrance effects are formed between the dialkylamino group and the phenyl group, so that the electron donating capability of the substituent groups is changed, the light-emitting wavelength and the pKa value of the molecule can be selectively adjusted, and the fluorescent qualitative detection of pH is further achieved. In the fluorescent qualitative detection process, when the pH is more than 7, the pH fluorescent probe is non-fluorescent, namely, a fluorescence radical quinolinium ion skeleton in the probe is quenched, along with the reduction of the pH, an amino group is used as a sensitive group of the pH fluorescent probe, nitrogen atoms of dialkyl amino groups in tertiary amino phenyl groups are protonated to form quaternary ammonium salt ions, and the fluorescence of the quinolinium ion skeleton is recovered, so that the fluorescent light-emitting effect is realized, the fluorescent intensity of the pH fluorescent probe is obviously changed in the change range of the pH between 4 and 7, and the fluorescent 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 of the compound is 478-509nm; the molecular structure of the compound has special penetrability, can penetrate through a cell membrane, but cannot penetrate through a nuclear membrane, so that the compound can be selectively marked at a special position of a subcellular level; meanwhile, the probe has larger Stokes shift and can reduce the signal-to-noise ratio, the luminous wavelength of the pH fluorescent probe can be adjusted according to different substituents, and the probe has a certain targeting effect based on the molecular structure, so that the probe is suitable for being widely applied to the detection of the pH in the weak acid environment inside and outside cells.
The pH detection range of pH fluorescent probes can be reflected in the pKa, which is the negative logarithm of the ionization constant (Ka) of an acid, and thus represents the pH at which the concentration of the acid is the same as the conjugate base form of the molecule. Ka refers to the strength of the acid form in solution and is the equilibrium constant for 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, resulting in a large pKa. The pKa value reveals the pH value required for the molecule to donate a proton, i.e.: if protonation of the pH fluorescent probe is more favorable and the acid form is difficult to dissociate, the Ka is smaller and the pKa is larger, the pH detection range is more biased toward the higher pH range, and vice versa.
Specifically, the structural general formula of the provided pH fluorescent probe takes quinolinium ions as a skeleton, and the structural general formula of the provided pH fluorescent probe comprises p-tertiary aminophenyl connected with the quinolinium ion skeleton structure through a carbon-carbon single bond. Among them, the remarkable increase in fluorescence intensity of the pH fluorescent probe is due to protonation of the nitrogen atom of the dialkylamino group in the tertiary aminophenyl group. Dialkylamino is an electron donating group that upon photoexcitation causes fluorescence quenching by transferring an electron from its 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 amines upon photo-induced electron transfer (PET). As the concentration of protons 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 the fluorescence intensity increases. 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, and thus the easier the protonation of the dialkylamino group is, the higher the pKa value obtained.
Specifically, in the structural general formula I of the pH fluorescent probe, R 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Selected from hydrogen or methyl. The degree of protonation of the nitrogen atom of the dialkylamino group will be affected differently depending on the conformation of the tertiary aminophenyl group, and the more planar the geometry is, the more difficult it is to protonate. The tertiary aminophenyl moiety will be conformationally modified according to R 1 、R 2 、R 3 The three substituents are different in type, so that different steric hindrance effects are formed between the dialkylamino and the phenyl, and the different steric hindrance effects enable the geometrical structure of the nitrogen atom to tend to be triangular pyramid (sp) 3 Hybridization) or planar (sp) 2 Hybridization). Therefore, in the structural general formula of the pH fluorescent probe, R needs to be controlled simultaneously 1 、R 2 、R 3 The three substituent groups can determine whether the formed geometric structure tends to be triangular pyramid or planar, so as to judge the protonation degree of the dialkyl amido and to clearly obtain the pKa value.
Wherein when R is 1 And R 2 Selected from cyclohexylimine, R 3 Selected from hydrogen; in the structural general formula of the obtained pH fluorescent probe, the geometric structure of tertiary aminophenyl is closer to plane (sp) 2 Hybridization), steric effect has less effect on resonance, so that the resonance effect of the arc couple electron pair of the nitrogen atom and the aromatic ring is larger, the conjugation is better due to stronger resonance interaction between the nitrogen atom and the aromatic ring, and the stronger resonance interaction between the nitrogen atom and the aromatic ring causes that the dialkyl amino group has stronger donor strength. The higher conjugation of the nitrogen atom to the aromatic ring results in a higher degree of difficulty in protonating the dialkylamino group and therefore a lower pKa value, with a shift in pH detection to lower pH ranges.
Wherein when R is 1 And R 2 Selected from piperidine, R 3 Selected from hydrogen; or when R is 1 Selecting methyl, R 2 Selected from methyl, R 3 Is selected from methyl; in the structural general formula of the obtained pH fluorescent probe, the geometric structure of tertiary aminophenyl is closer to triangular pyramid (sp) 3 Hybridization), steric effects have a greater effect on resonance, and therefore the resonance of the electron pair coupled with the nitrogen atom and the aromatic ring is less, and 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 poorer donor strength for the dialkylamino group. The lower conjugation of the nitrogen atom to the aromatic ring results in easier protonation of the dialkylamino group and therefore higher pKa values, where the pH range of detection is more biased toward higher pH ranges.
In some embodiments, the pH fluorescent probe comprises a compound of the formula:
in the formula II, the substituent group of the pH fluorescent probe is a six-membered piperidine ring which is in a chair conformation, and the twist 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, a six-membered piperidine ring favors the protonation of the dialkylamino group and leads to higher pKa values.
In the formula III, the substituent of the pH fluorescent probe is a seven-membered piperidine ring, and the seven-membered ring shows a smaller twist angle, so that the degree of conjugation of nitrogen atoms to aromatic rings is larger, and the resonance interaction between the nitrogen atoms and the aromatic rings is increased. Thus, the protonation of the dialkylamino group is less difficult and results in a lower pKa value.
In formula IV, the substituent of the pH fluorescent probe is morpholine, and 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. Therefore, the dialkylamino group is less difficult to protonate, and the lowest pKa value is obtained.
In the formula V, the substituent of the pH fluorescent probe is methyl, and the ortho-position carbon in the obtained N, N,2, 6-tetramethylaniline has a substituent. The strong steric hindrance between the ortho-dimethyl group and the dimethylamino group in N,2, 6-tetramethylaniline causes the dimethylamino group to distort out of plane with respect to the benzene ring, so that a greater distortion of the dimethylamino group reduces the resonance interaction between the nitrogen atom and the aromatic ring and promotes protonation of the dialkylamino group, thereby achieving 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 suggested that the resonance interaction between their piperidines and the benzene ring is the same, resulting in similar pKa values. The presence of the additional phenyl ring on the quinolinium does not significantly affect the resonance interaction between the piperidine and phenyl rings. Instead, an extra benzene ring exists on the quinolinium in the formula V, which expands a conjugation system and causes an long wavelength displacement, wherein the emission wavelength is increased.
In a second aspect, the embodiments of the present application provide a method for preparing a pH fluorescent probe, including the following steps:
s01, providing a first compound, wherein the structural general formula of the first compound is
S02, providing a second compound, wherein the structural general formula of the second compound is
S03, dissolving the first compound and the second compound in an organic solvent under an inert atmosphere, carrying out catalytic addition reaction by using a monovalent gold complex as a catalyst, and carrying out first purification treatment to obtain a third compound, wherein the structural general formula of the third compound is
S04, carrying out reflux treatment on the third compound, then carrying out second purification treatment to obtain a pH fluorescent probe,
wherein, the general structural formula of the pH fluorescent probe is
And R is 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Is selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate ions.
According to the preparation method of the pH fluorescent probe, two small molecular compounds are used as reactants, catalytic addition reaction is carried out under the action of a monovalent gold catalyst, and then backflow and purification treatment are carried out.
Wherein, the whole flow of the preparation method is shown as the following reaction equation,
in step S01, a first compound is provided, wherein the structural general formula of the first compound is as formula VII, and the formula VII is
Wherein R is 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Selected from hydrogen and methyl, which can be prepared by the prior art literature.
In step S02, a second compound is provided, wherein the structural general formula of the second compound is as shown in formula VIII, and the formula VIII is
Wherein Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate ions, which can be prepared by the prior art documents.
In step S03, dissolving the first compound and the second compound in an organic solvent under an inert atmosphere, performing a catalytic addition reaction with a monovalent gold complex as a catalyst, and performing a first purification treatment to obtain a third compound, wherein the third compound has a general structural formula
In some embodiments, the reaction is carried out under an inert atmosphere to ensure that the purity of the obtained product is high and that side effects such as oxidation and the like do not occur in the reaction process. 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 and chloroform. Based on the principle of similar compatibility, the first compound and the second compound have high solubility in acetonitrile solution, so that the reaction is facilitated, and the obtained organic solvent can not react with the compounds.
In some embodiments, a monovalent gold complex is used as a catalyst, and the monovalent gold complex is selected from chloro [ tri (p-trifluoromethylphenyl) phosphine ] gold (I), and the monovalent gold complex can be used for effectively catalyzing the reaction and improving the reaction efficiency.
Further, in order to improve 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 (1.0-1.5): (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, the product obtained by adopting the proportion for reaction 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 under irradiation of visible light for 16 to 18 hours, wherein the visible light is selected from a blue light source, and the power of the blue light source 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 a corresponding product is obtained. Further, the reaction is carried out under the condition of room temperature, namely, the room temperature is controlled to be 10-30 ℃.
In some embodiments, a first purification treatment is performed, and the first purification treatment is performed by silica gel column chromatography. In a specific embodiment of the present invention, the specific method of the first purification treatment is as follows: performing rotary evaporation on a product obtained by the catalytic addition reaction to remove the organic solvent to obtain a residue; providing a silica gel column, performing gradient elution by using a mixed solution of dichloromethane and methanol as an eluent, combining rotary evaporation to remove a solvent, adding dichloromethane, performing multiple extraction by using water, combining water phases, and finally, removing water by rotary evaporation to obtain a third compound, wherein the structural general formula of the third compound is as shown in formula IX, formula IX
Is composed of
R 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Is selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate ions.
In step S04, the third compound is subjected to reflux treatment and then to second purification treatment to obtain the pH fluorescent probe.
In some implementations, the third compound is subjected to a reflow process, including the steps of: dissolving the third compound in water, heating to 180-185 deg.C, and reflux-treating for 16-18 hr.
In some embodiments, a second purification treatment is performed, and the second purification treatment is performed by using a silica gel column chromatography method. In a specific embodiment of the present invention, the specific method of the second purification treatment is as follows: removing water by rotary evaporation 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, combining, and removing a solvent by rotary evaporation to obtain a pH fluorescent probe, wherein the structural general formula of the pH fluorescent probe is shown in the specification
And R is 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Is selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate ions.
The preparation method utilizes the synergy of visible light and a catalyst univalent gold complex for catalysis to obtain the pH fluorescent probe shown as the structural formula I, and the reaction method is efficient and easy to control; the obtained reaction product has high purity and no other impurities are generated.
In a third aspect of the embodiments, the present application provides a use 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 of the pH fluorescent probe or the pH fluorescent probe prepared by the preparation method provided by the third aspect of the application in fluorescence qualitative detection of pH. The pH fluorescent probe can adjust the light-emitting wavelength 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 acid environment inside and outside cells, so that the obtained pH fluorescent probe has high detection sensitivity and good effect in the fluorescence qualitative detection of the pH, and is beneficial to wide application.
The following description is given with reference to specific examples.
Example 1
pH fluorescent probe II and preparation method thereof
pH fluorescent Probe IIHas the structural formula
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;
trimethylsilane and a diazonium salt having a quinoline structure were dissolved in 5mL of acetonitrile solvent under an inert nitrogen atmosphere to obtain (CF) 3 Ph) 3 Performing catalytic addition reaction with PAuCl (0.05 mmol) as catalyst under irradiation of blue LED light source at room temperature for 16 hr, performing rotary evaporation to remove organic solvent, providing silica gel column, performing gradient elution with mixed solution of dichloromethane and methanol, combining rotary evaporation to remove organic solventRemoving the solvent, adding dichloromethane, extracting with water for multiple times, mixing the water phases, and finally removing water by rotary evaporation to obtain a third compound;
dissolving the obtained third compound in water, heating to 180 ℃, carrying out 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, carrying out rotary evaporation to remove the solvent, and finally obtaining a dark red solid product, namely a pH fluorescent probe type II with the yield of two steps being 22%.
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＝9.0 Hz,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 IIIHas the structural formula
preparation method of pH fluorescent probe III
The preparation method of the pH fluorescent probe III is shown in the following reaction formula,
specific procedures for the preparation of pH fluorescent Probe III are shown in example 1, and the product is obtained as a dark red solid with a 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＝9.1 Hz,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 IVHas the structural formula
preparation method of pH fluorescent probe IV
The preparation method of the pH fluorescent probe IV is shown in the following reaction formula,
specific procedures for the preparation of pH fluorescent Probe IV as shown in example 1, the product was obtained as a dark red solid in 38% yield.
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＝9.1 Hz,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 VHas the structural formula
preparation method of pH fluorescent probe V
The preparation method of the pH fluorescent probe V is shown in the following reaction formula,
specific procedures for the preparation of pH fluorescent Probe V as shown in example 1, the product was obtained as an orange solid in 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＝9.1 Hz,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 VIHas the structural formula
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 the pH fluorescent probe VI is shown in example 1, and the obtained product is a black solid with a 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＝8.1 Hz,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 measured.
The test method comprises the following steps: 2mol/L aqueous HCl and 2mol/L aqueous NaOH solutions were mixed and 2mol/L HCl/NaOH buffers having various pH values were prepared under monitoring of a pH meter. By diluting the pH fluorescent probe to 5X10 in acetonitrile - 4 mol/L to prepare stock solutions. Mixing 2mol/L HCl/NaOH buffer solution with various pH values and acetonitrile stock solution in a volume ratio of 19 to 1 to obtain 2.5X 10 -5 mol/L of diluted solution.
pH fluorescent probe absorption and emission spectra measured at different pH values.
(2) pKa analysis of the pH fluorescent probes obtained in examples 1 to 5.
As can be seen from the absorption spectrum and the fluorescence spectrum in (1) (FIGS. 1 to 10), the excitation wavelength was 410nm, and the maximum emission wavelengths of the pH fluorescent probe II, the pH fluorescent probe III, the pH fluorescent probe IV and the pH fluorescent probe V and the pH fluorescent probe VI were 478nm,478nm, 476nm,482nm and 509nm, respectively. The fluorescence intensities obtained from the maximum emission wavelengths of the pH fluorescent probes obtained in examples 1 to 5 were summed with the pH values to obtain a normalized fluorescence intensity curve with respect to the pH values, wherein the pH value half of the normalized intensity was estimated as the pKa value.
(3) Cytotoxicity assay of the pH fluorescent Probe II obtained in example 1.
Providing a HeLa cell line, culturing Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) and 100ug/mL streptomycin (Gibco) supplemented with 44mmol/L sodium bicarbonate (Sigma-Aldrich), 10% v/v fetal bovine serum (Gibco) and 100U/mL penicillin (Gibco) at 37 ℃ with 5% CO 2 . The day before the cytotoxicity determination experiment, 5X10 3 Inoculating the cells into a 96-well plate, incubating the cells with a pH fluorescent probe II of 5-25. Mu. Mol/L at 37 ℃,5% 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/mLMTS, promega,1:20 Add to each well. Mixing the plate with 5% CO at 37 deg.C 2 Incubated for 1 hour, the absorbance at 490nm was measured and the IC was calculated 50 Numerical values.
(4) Cellular imaging of the pH fluorescent probe II obtained in example 1.
Seeding HeLa cells on 35mm glass-bottom dishes (SPL) and at 37 ℃ and 5% CO 2 Overnight adhesion in cell culture incubator. Cells were probed with either pH alone or pH fluorescent probes, 50nmol/L
Deep red (Invitrogen) TM ) Incubate together for 2 hours. The images were examined under a confocal microscope (Leica TCS SP8 MP).
(5) Intracellular pH change sensing of the pH fluorescent probe II obtained in example 1.
To further confirm whether pH fluorescent probe II fluoresced due to the acidity of lysosomes, cells were treated with concanavalin a, a V-ATPase inhibitor that causes an increase in lysosomal pH, and further analyzed for the presence of a fluorescent signal.
Analysis of results
(1) The optical properties of the pH fluorescent probes obtained in examples 1 to 5 were measured, and the results are shown in FIGS. 1 to 10.
FIG. 1 is a graph of an ultraviolet absorption spectrum of a pH fluorescent probe II obtained in example 1 of the present invention with a change in pH, and FIG. 2 is a graph of a fluorescence intensity of the pH fluorescent probe II obtained in example 1 of the present invention with a change in pH; as can be seen from FIGS. 1 and 2, the pH fluorescent probe II obtained in example 1 has a maximum absorption wavelength of 410nm in the visible light region when the pH is in the range of 1.49 to 3.57; when excited by the maximum absorption wavelength, a strong emission peak is observed at 478nm at a pH of 1.49, the luminosity of which becomes gradually weaker at a pH range of 1.49 to 5.52.
FIG. 3 is a graph of an ultraviolet absorption spectrum of a pH fluorescent probe III according to a change in pH value obtained in example 2 of the present invention, and FIG. 4 is a graph of a fluorescence intensity of the pH fluorescent probe III according to a change in pH value obtained in example 2 of the present invention; as can be seen from FIGS. 3 and 4, in the pH fluorescent probe III obtained in example 2, when the pH was in the range of 0.33 to 3.56, the maximum absorption wavelength was observed to be 410nm, which is in the visible light region; when excited by the maximum absorption wavelength, a strong emission peak is observed at 478nm at a pH of 0.33, the luminosity of which becomes gradually weaker at a pH range of 0.33 to 3.56.
Fig. 5 is an ultraviolet absorption spectrum of the pH fluorescent probe IV obtained in example 3 according to the present invention, and fig. 6 is a graph of the fluorescence intensity of the pH fluorescent probe IV obtained in example 3 according to the present invention, as a function of pH; as can be seen from FIGS. 5 and 6, the pH fluorescent probe IV obtained in example 3 has a maximum absorption wavelength of 410nm in the visible light region when the pH is in the range of 0.36 to 3.00; when excited by the maximum absorption wavelength, a strong emission peak is observed at 476nm at a pH of 0.36, with a luminosity that gradually weakens at a pH range of 0.36 to 3.00.
FIG. 7 is an ultraviolet absorption spectrum of the pH fluorescent probe V obtained in example 4 of the present invention as a function of pH, and FIG. 8 is a graph of the fluorescence intensity of the pH fluorescent probe V obtained in example 4 of the present invention as a function of pH; as can be seen from FIGS. 7 and 8, in the pH fluorescent probe V obtained in example 5, when the pH was in the range of 0.57 to 6.72, the maximum absorption wavelength of 410nm, which is in the visible light region, was observed; when excited by the maximum absorption wavelength, a strong emission peak is observed at 482nm at pH values of 0.57 to 5.26, and the luminosity thereof sharply decreases at a pH range of 6.72 or more.
FIG. 9 is an ultraviolet absorption spectrum of the pH fluorescent probe VI obtained in example 5 of the present invention varying with pH, FIG. 10 is a graph of fluorescence intensity of the pH fluorescent probe VI obtained in example 5 of the present invention varying with pH, and it can be seen from FIGS. 9 and 10 that the pH fluorescent probe VI obtained in example 5 has a maximum absorption wavelength of 410nm in the visible light region when the pH ranges from 0.56 to 4.61; when excited by the maximum absorption wavelength, a strong emission peak is observed at 509nm at a pH of 0.56, the luminosity of which becomes gradually weaker at a pH range of 0.56 to 4.61.
(2) pKa analysis results 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 was 410nm, while the maximum emission wavelengths of the pH fluorescence probe II, the pH fluorescence probe III, the pH fluorescence probe IV and the pH fluorescence probe V and the pH fluorescence probe VI were 478nm,476 nm,482nm and 509nm, respectively, and FIGS. 11 to 15 are fluorescence intensities obtained from the maximum emission wavelengths, and normalized fluorescence intensity curves with respect to pH values were obtained. The pH value, which is half of the normalized intensity, was estimated as the pKa value. When the normalized intensity reaches half of it, it means that the concentrations of the protonated form and the unprotonated form of the quinolinium compound are the same.
FIG. 11 is a graph showing fluorescence intensities of pH fluorescent probe II according to example 1 of the present invention at different pH values at a test wavelength of 478nm, and its pKa value is 4.6.
FIG. 12 is a graph showing fluorescence intensities of pH fluorescent probe III of example 2 of the present invention at different pH values at a test wavelength of 478nm, and the pKa value is 2.8.
FIG. 13 is a graph showing fluorescence intensities of pH fluorescent probe IV of example 3 of the present invention at different pH values at a test wavelength of 476nm, and the pKa value is found to be <2.2.
FIG. 14 is a graph showing fluorescence intensities of pH fluorescent probe V of example 4 of the present invention at different pH values at a test wavelength of 482nm, and the pKa value is 6.1.
FIG. 15 is a graph showing fluorescence intensities of pH fluorescent probe VI of example 5 of the present invention at different pH values at 509nm, and the pKa value is 4.3.
Wherein the pH value 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 the pH fluorescent Probe II obtained in example 1.
After incubation of the pH fluorescent probe II with HeLa cells at 490nm, measured with an EnSpire multimode plate reader (Perkinelmer) at 5. Mu. Mol/L to 25. Mu. Mol/L, the cell image was taken, and as shown in FIG. 16, the fluorescence intensity was increased with the increase in the concentration of the pH fluorescent probe, indicating that the pH fluorescent probe has a concentration-dependent signal-responsive ability.IC 50 Values were calculated by GraphPad Prism, as shown in FIG. 17, IC 50 The concentration was 28.08. Mu. Mol/L.
(4) Cellular imaging of the pH fluorescent probe II obtained in example 1.
Images were examined under a confocal microscope (Leica TCS SP8 MP), as shown in FIG. 18, FIGS. 18A and 18B are fluorescence microscope images of pH fluorescent probes in different selected cells, and FIGS. 18C and 18D are fluorescence microscope images of pH fluorescent probes in different selected cells
Deep red (Invitrogen) TM ) Fluorescence microscopy images in different selected cells, bright field microscopy images of different selected cells in FIGS. 18E and 18F, pH fluorescence probes and pH fluorescence probes in FIGS. 18G and 18H
Deep red (Invitrogen) TM ) Combined fluorescence microscope images in different selected cells.
For pH fluorescent probe II and the lysosomal tracer, co-localization of the fluorescent signal was observed, indicating that the compound localized to the lysosome. The molecular structure can penetrate cell membrane and cell nucleus membrane, and can be selectively marked at special position of subcellular level.
(5) Intracellular pH change sensing of the pH fluorescent probe II obtained in example 1.
As shown in fig. 19, fig. 19A and 19B are fluorescence microscope images (without concanavalin a treatment) of the pH fluorescent probe of the control group on different selected cells, fig. 19C and 19D are bright field microscope images of different selected cells of the control group, fig. 19E and 19F are fluorescence microscope images of the pH fluorescent probe of different selected cells after concanavalin a treatment, and fig. 19G and 19H are bright field microscope images of different selected cells after concanavalin a treatment.
Cells treated with pH fluorescent probe II and concanavalin a showed complete loss of fluorescent signal, indicating that pH fluorescent probe II functions as a pH sensor in vivo.
Therefore, the pH fluorescent probe provided by the application has obvious change of the fluorescence intensity in the change range of pH 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 of the compound is 478-509nm; the molecular structure of the compound has special penetrability, can penetrate through a cell membrane, but cannot penetrate through a nuclear membrane, so that the compound can be selectively marked at a special position of a subcellular level; meanwhile, the pH fluorescent probe has larger Stokes shift, can reduce the signal-to-noise ratio, can adjust the luminescence wavelength according to different substituents, has certain targeting effect based on the molecular structure, and is suitable for being widely applied to the detection of the pH in the weak acid environment inside and outside cells.
The above description is only a preferred embodiment of the present application and should not be taken as limiting the present application, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A pH fluorescent probe is characterized by comprising a quinolinium ion as a skeleton, a p-tertiary aminophenyl group and a counter ion which are connected with the quinolinium ion skeleton structure through a carbon-carbon single bond, wherein the structural general formula of the pH fluorescent probe is shown as a formula I,
wherein R is 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Is selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate ions.
2. The pH fluorescent probe according to claim 1, characterized in that the pH fluorescent probe comprises a compound of the following structural formula:
3. the preparation method of the pH fluorescent probe is characterized by comprising the following steps of:
providing a first compound with a general structural formula
Providing a second compound with a structural general formula
Dissolving the first compound and the second compound in an organic solvent under inert atmosphere, carrying out catalytic addition reaction by using a monovalent gold complex as a catalyst, and carrying out first purification treatment to obtain a third compound, wherein the structural general formula of the third compound is
Carrying out reflux treatment on the third compound, then carrying out second purification treatment to obtain a pH fluorescent probe,
wherein the structural general formula of the pH fluorescent probe is
And, R 1 And R 2 Each independently selected from any one of piperidine, cycloheximide, morpholine and methyl, R 3 Is selected from hydrogen or methyl, and Q is selected from any one of tetrafluoroborate, hexafluorophosphate, perchlorate, hexafluoroantimony and trifluoromethanesulfonate ions.
4. The method according to claim 3, wherein the monovalent gold complex is selected from chloro [ tris (p-trifluoromethylphenyl) phosphine ] gold (I).
5. The production method according to claim 3, wherein 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).
6. The method according to claim 3, wherein the organic solvent is acetonitrile, or a mixed solution of acetonitrile and at least one of methanol, ethanol, propanol, butanol, toluene, and chloroform.
7. The method according to claim 3, wherein the step of performing the catalytic addition reaction comprises performing the catalytic addition reaction under irradiation of visible light at room temperature for 16 to 18 hours, wherein the visible light is selected from a blue light source, and the power of the blue light source is not less than 3W.
8. The method according to any one of claims 3 to 7, wherein the step of subjecting the third compound to a reflux treatment comprises the steps of: and dissolving the third compound in water, and heating to 180-185 ℃ for reflux treatment for 16-18 hours.
9. The production method according to any one of claims 3 to 7, wherein the first purification treatment and the second purification treatment are performed by silica gel column chromatography to isolate the target compound.
10. Use of the pH fluorescent probe according to claim 1 or 2 or prepared by the preparation method according to any one of claims 3 to 9 in the qualitative fluorescence detection of pH.
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