CN109369664B - Rhodamine acylhydrazone derivative, preparation method and application thereof, and fluorescent probe - Google Patents
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Abstract
The invention provides a rhodamine acylhydrazone derivative, a preparation method and application thereof, and a fluorescent probe, and belongs to the field of organic synthesis. The invention takes 4-morpholine benzaldehyde and rhodamine 6G hydrazide as reactants, and the rhodamine acylhydrazone derivative can be obtained through condensation reaction; the rhodamine acylhydrazone derivative has a structure shown in a formula I, is very sensitive to pH, does not emit obvious fluorescence when the pH is greater than 4.0, emits obvious green fluorescence when the pH is less than 4.0, and generates a jump in the range of pH 3.0-4.0; the rhodamine acylhydrazone derivative provided by the invention can target lysosomes of cells and can be used for distinguishing normal cells and tumor cells by fluorescence.
Description
Technical Field
The invention relates to the technical field of organic synthesis, in particular to a rhodamine acylhydrazone derivative, a preparation method and application thereof, and a fluorescent probe.
Background
The fluorescence sensor is a powerful tool for monitoring cations, anions and small molecules, and has the advantages of high sensitivity, simple and convenient operation, and real-time and on-line detection. The detection and marking of tumor cells have very important significance, and are helpful for explaining cancer-related processes and facilitating clinical diagnosis. A decrease in pH compared to normal cells is an important characteristic of tumor cells. The lysosome pH difference between tumor cells (pH <4.0) and normal cells (4.0< pH <6.0) is utilized to realize tumor cell labeling, which is one of the effective strategies for designing a pH fluorescent probe.
However, the conventional lysosome targeted tumor cell labeled fluorescent probe is complex to synthesize and high in cost, a large amount of organic solvent is required to be added for assisting dissolution when in use, and the volume ratio of the cosolvent is required to be more than 10% to effectively dissolve the compound serving as the fluorescent probe, so that the further application of the fluorescent probe is limited.
Disclosure of Invention
In view of the above, the present invention aims to provide a rhodamine acylhydrazone derivative, a preparation method and an application thereof, and a fluorescent probe. The rhodamine acylhydrazone derivative provided by the invention is very sensitive to the pH value, has different fluorescence emissions under different pH value conditions, can realize the fluorescence differentiation of normal cells and tumor cells, is convenient to use, and only needs a very small amount of organic solvent (the volume ratio is 0.5%) for assisting in dissolution.
In order to achieve the above object, the present invention provides the following technical solutions:
a rhodamine acylhydrazone derivative has a structure shown as a formula I:
the invention provides a preparation method of the rhodamine acylhydrazone derivative in the scheme, which comprises the following steps:
mixing rhodamine 6G hydrazide, 4-morpholine benzaldehyde and an alcohol solvent for condensation reaction to obtain the rhodamine acylhydrazone derivative with the structure shown in the formula I.
Preferably, the alcohol solvent is ethanol.
Preferably, the molar ratio of the rhodamine 6G hydrazide to the 4-morpholine benzaldehyde is 1: 1-1.1.
Preferably, the volume ratio of the rhodamine 6G hydrazide substance to the alcohol solvent is 0.01-0.02 mol: 0.3-0.6L.
Preferably, the condensation reaction is carried out under reflux stirring conditions; the condensation reaction is carried out at normal pressure for 3-5 h.
Preferably, the condensation reaction further comprises, after completion: post-treating the condensation reaction liquid, wherein the post-treating comprises the following steps:
and cooling the condensation reaction liquid to room temperature to separate out solids, and washing filter residues after filtering to obtain the rhodamine acylhydrazone derivative with the structure shown in the formula I.
The invention provides application of the rhodamine acylhydrazone derivative prepared by the preparation method in the scheme or the rhodamine acylhydrazone derivative prepared by the preparation method in the scheme as a pH fluorescent probe.
The invention provides a fluorescent probe for distinguishing tumor cells from normal cells, which comprises the rhodamine acylhydrazone derivative prepared by the preparation method in the scheme or the rhodamine acylhydrazone derivative prepared by the preparation method in the scheme.
The invention provides a rhodamine acylhydrazone derivative with a structure shown in a formula I. The rhodamine acylhydrazone derivative provided by the invention is sensitive to pH abnormality, exists in a spiro structure at low acidity, does not show fluorescence, and emits strong fluorescence when the spiro structure is opened at high acidity; in addition, lysosome positioning base morpholine exists in the structure of the rhodamine acylhydrazone derivative provided by the invention, so that the derivative can target lysosomes of cells when being used as a fluorescent probe, and the fluorescent differentiation of normal cells and tumor cells is realized.
The invention provides a preparation method of the rhodamine acylhydrazone derivative in the scheme, and the rhodamine acylhydrazone derivative can be obtained by taking 4-morpholine benzaldehyde and rhodamine 6G hydrazide as reactants through a condensation reaction. The preparation method provided by the invention has simple steps and easily obtained raw materials.
The invention provides application of the rhodamine acylhydrazone derivative in the scheme as a pH fluorescent probe. The example result shows that the rhodamine acylhydrazone derivative provided by the invention has no obvious fluorescence emission when the pH value is greater than 4.0 and emits obvious green fluorescence when the pH value is less than 4.0, and a jump is generated in the range of pH value of 3.0-4.0, which indicates that the rhodamine acylhydrazone derivative can be used as a high-acidity pH fluorescent probe; and the light-emitting effect of the fluorescent material is not interfered by cations, anions and amino acids.
The invention also provides a fluorescent probe for distinguishing tumor cells from normal cells, and the fluorescent probe comprises the rhodamine acylhydrazone derivative in the scheme. The rhodamine acylhydrazone derivative provided by the invention is used as a fluorescent probe to carry out a co-localization experiment with a commercial lysosome localization dye, and the result shows that the rhodamine acylhydrazone derivative provided by the invention can be specifically localized in the lysosome of living cells as the fluorescent probe, and can effectively distinguish normal cells from tumor cells.
Drawings
FIG. 1 is a crystal structure diagram of a rhodamine acylhydrazone derivative prepared in example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrogram of the rhodamine acylhydrazone derivative prepared in the embodiment 1 of the invention;
FIG. 3 is a mass spectrum of the rhodamine acylhydrazone derivative prepared in example 1 of the present invention;
FIG. 4 is a fluorescence spectrum of rhodamine acylhydrazone derivatives prepared in example 1 of the present invention in different disodium hydrogen phosphate-citric acid buffer solutions;
FIG. 5 is a fluorescence emission spectrogram obtained by dissolving the rhodamine acylhydrazone derivative prepared in example 1 in a disodium hydrogen phosphate-citric acid buffer solution with a pH value of 2.0 and then adding different metal ions, anions and amino acids;
FIG. 6 is a fluorescence emission spectrogram obtained by dissolving the rhodamine acylhydrazone derivative prepared in example 1 in a disodium hydrogen phosphate-citric acid buffer solution with a pH value of 4.0 and then adding different metal ions, anions and amino acids;
FIG. 7 is a fluorescence emission spectrogram obtained by dissolving the rhodamine acylhydrazone derivative prepared in example 1 in a disodium hydrogen phosphate-citric acid buffer solution with a pH value of 4.0 and then adding different metal ions, anions and amino acids;
FIG. 8 is a cell confocal fluorescence image of the rhodamine acylhydrazone derivative prepared in example 1 of the present invention as a fluorescence probe co-staining normal cells 239T, tumor cells Hpeg2 and Hela with a commercial lysosome positioning dye L ysoTracker Red.
Detailed Description
The invention provides a rhodamine acylhydrazone derivative which has a structure shown in a formula I:
the invention provides a preparation method of the rhodamine acylhydrazone derivative in the scheme, which comprises the following steps:
mixing rhodamine 6G hydrazide, 4-morpholine benzaldehyde and an alcohol solvent for condensation reaction to obtain the rhodamine acylhydrazone derivative with the structure shown in the formula I.
In the invention, the molar ratio of the rhodamine 6G hydrazide to the 4-morpholine benzaldehyde is preferably 1: 1-1.1, more preferably 1:1, the alcohol solvent is preferably ethanol, and the volume ratio of the amount of the rhodamine 6G hydrazide to the alcohol solvent is preferably 0.01-0.02 mol: 0.3-0.6L, more preferably 0.01 mol: 0.3-0.5L.
The invention preferably dissolves rhodamine 6G hydrazide in an alcohol solvent, and then adds 4-morpholine benzaldehyde into the solution. In a specific embodiment of the present invention, it is preferable that 2 to 3 drops of acetic acid is dropped into a mixture of 6G hydrazide 4-morpholinobenzaldehyde and an alcohol solvent, and then condensation reaction is performed, and in the present invention, the acetic acid as a catalyst can catalyze the reaction to proceed rapidly.
In the present invention, the condensation reaction is preferably carried out under reflux stirring conditions; the pressure of the condensation reaction is preferably normal pressure, and the time is preferably 3-5 h, more preferably 3.5-4.5 h, and further preferably 4 h.
In the present invention, the reaction formula of the condensation reaction is shown in formula II:
in the present invention, the condensation reaction preferably further comprises: the condensation reaction liquid is subjected to post-treatment, which preferably comprises the following steps:
and cooling the condensation reaction liquid to room temperature to separate out solids, and washing filter residues after filtering to obtain the rhodamine acylhydrazone derivative with the structure shown in the formula I.
In the invention, the washing agent for washing filter residue is preferably ethanol; the filtration is preferably a reduced pressure filtration; the present invention does not require any particular operation method for the reduced pressure filtration and washing, and a method for reduced pressure filtration and washing known to those skilled in the art may be used.
In the present invention, the washing preferably further comprises recrystallizing the washed product, and the solvent for recrystallization is preferably acetonitrile; the present invention does not require any particular method for carrying out the recrystallization, and a recrystallization method known to those skilled in the art may be used.
The invention provides application of the rhodamine acylhydrazone derivative prepared by the preparation method in the scheme or the rhodamine acylhydrazone derivative prepared by the preparation method in the scheme as a pH fluorescent probe. The rhodamine acylhydrazone derivative provided by the invention has no obvious fluorescence emission when the pH value is less than 4.0 and less than 6.0, emits obvious green fluorescence when the pH value is less than 4.0, and generates a jump in the range of pH 3.0-4.0.
The invention also provides a fluorescent probe for distinguishing tumor cells from normal cells, and the fluorescent probe comprises the rhodamine acylhydrazone derivative in the scheme. In the invention, the rhodamine acylhydrazone derivative structure comprises lysosome positioning base morpholine, so that the derivative can be specifically positioned in cell lysosomes when being used as a fluorescent probe, the lysosomes of tumor cells (the pH is less than 4.0) and normal cells (the pH is less than 4.0 and less than 6.0) have pH difference, and the rhodamine acylhydrazone derivative can generate different fluorescence emission under different pH conditions, thereby realizing the differentiation of the tumor cells and the normal cells. The rhodamine acylhydrazone derivative provided by the invention can be directly dissolved in a solvent when being applied as a fluorescent probe, the solvent preferably comprises a main solvent and a cosolvent, the main solvent is preferably a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 6.0, the cosolvent is preferably DMSO, the volume ratio of the cosolvent in the solvent is preferably 0.5%, the rhodamine acylhydrazone derivative provided by the invention only needs a very small amount of organic solvent for dissolution assistance, the solubility of the rhodamine acylhydrazone derivative is obviously superior to that of the conventional lysosome targeted tumor cell labeled fluorescent probe in the field, and the rhodamine acylhydrazone derivative is more convenient to use.
The rhodamine acylhydrazone derivative, the preparation method and the application thereof and the fluorescent probe provided by the present invention are described in detail below with reference to the following examples, but the rhodamine acylhydrazone derivative, the preparation method and the application thereof and the fluorescent probe are not to be construed as limiting the scope of the present invention.
Example 1
428mg of rhodamine 6G hydrazide is dissolved in 30m L ethanol, 191mg of 4-morpholine benzaldehyde is added, 2 drops of acetic acid are dropwise added under normal pressure to perform catalytic reflux stirring for 3 hours, the mixture is cooled to room temperature, solid is separated out, the mixture is filtered under reduced pressure, filter residue is washed by ethanol, white solid is obtained, the target product is the target product, and the yield of the target product is 89%.
Recrystallizing the obtained product with acetonitrile, performing single crystal diffraction analysis on the obtained recrystallized product, and determining the crystal structure of the obtained product, wherein the obtained result is shown in figure 1; the crystal structure shows that the obtained rhodamine acylhydrazone derivative exists in the form of acetonitrile compound.
The nuclear magnetic resonance analysis is carried out on the prepared rhodamine acylhydrazone derivative, and the result is as follows:
1H NMR(400MHz,DMSO-d6),(ppm):8.59(s,1H,CH＝N),7.86-7.88(dd,1H,Ar-H),7.52–7.59(m,2H,Ar-H),7.24-7.26(d,2H,Ar-H),7.00-7.02(s,2H,Ar-H),6.87-6.89(d,2H,Ar-H),6.32(s,2H,Ar-H),6.16(s,2H,Ar-H),5.04-5.07(t,2H,2NH),3.68-3.70(t,4H,2CH2),3.09-3.16(m,8H,4CH2),1.84(s,6H,2CH3),1.18-1.22(t,6H,2CH3) The specific nuclear magnetic spectrum is shown in figure 2;
the prepared rhodamine acylhydrazone derivatives are subjected to mass spectrometry: ESI-MS: M/z 301.6587([ M + 2H)]2+)；602.3149([M+H]+)；1225.6054([2M+Na]+) (calc.1225.60), the specific mass spectrum is shown in FIG. 3.
Example 2
The rhodamine acylhydrazone derivative prepared in the example 1 is subjected to fluorescence property determination in buffer solutions with different pH values, and the steps are as follows:
(1) the rhodamine acylhydrazone derivative prepared in the example 1 is prepared into disodium hydrogen phosphate-citric acid buffer solutions (containing DMSO with the volume ratio of 0.5%) with the molar concentration of 1 × 10 in different pH values (1.0-6.0)-5The solution of mol/L is respectively subjected to fluorescence spectrum analysis (excitation wavelength is 480nm) by a fluorescence spectrometer, the obtained fluorescence spectrogram is shown in figure 4, and as can be seen from figure 4, the rhodamine acylhydrazone derivative prepared in the embodiment 1 of the invention is used as a fluorescent probe at 4.0<pH<No significant fluorescence emission at 6.0, whereas the pH<4.0 emits obvious green fluorescence and generates a jump in the pH range of 3.0 to 4.0,indicating that it can be used as a high acidity pH fluorescent probe.
The rhodamine acylhydrazone derivatives prepared in example 1 were respectively prepared in disodium hydrogen phosphate-citric acid buffer solutions (containing 0.5 vol% DMSO) with pH values of 2.0,4.0 and 6.0 to have a molar concentration of 1 × 10-5A solution of mol/L, to which different metal ions (Ag) are added+,Al3+,Ca2+,Cd2+,Co2+,Cr3+,Cu2+,Fe3+,Hg2+,K+,Mg2+,Na+,Ni2+,Pb2+And Zn2+Controlling the concentration of metal ions in the solution to be 5 × 10-5mol/L), anions (AcO)-,Br-,Cl-,ClO4 -,CN-,CO3 2-,F-,HCO3 -,HPO4 2-,H2PO4 -,HSO3 -,HSO4 -,I-,N3 -,PO4 3-,S2-,SCN-,SO3 2-And SO4 2-The concentration of the anion in the solution is controlled to be 5 × 10-4mol/L) and partial amino acids (arginine, cysteine, glutamic acid, glycine, histidine, homocysteine and lysine), wherein the concentration of the amino acids in the solution is controlled to be 5 × 10-4mol/L), and performing fluorescence spectrum analysis by using a fluorescence spectrometer (excitation wavelength is 480 nm).
The obtained fluorescence spectrogram is shown in fig. 5-7, wherein fig. 5 is a fluorescence emission spectrogram obtained by dissolving the rhodamine acylhydrazone derivative in a disodium hydrogen phosphate-citric acid buffer solution (containing 0.5% by volume of DMSO) with the pH value of 2.0 and then adding different metal ions, anions and amino acids, fig. 6 is a fluorescence emission spectrogram obtained by dissolving the rhodamine acylhydrazone derivative in a disodium hydrogen phosphate-citric acid buffer solution (containing 0.5% by volume of DMSO) with the pH value of 4.0 and then adding different metal ions, anions and amino acids, and fig. 7 is a fluorescence emission spectrogram obtained by dissolving the rhodamine acylhydrazone derivative in a disodium hydrogen phosphate-citric acid buffer solution (containing 0.5% by volume of DMSO) with the pH value of 4.0 and then adding different metal ions, anions and amino acids. As can be seen from fig. 5 to 7, the fluorescence emission of the rhodamine acylhydrazone derivative prepared in example 1 as a fluorescent probe at pH 2.0,4.0, and 6.0 is not substantially interfered by the coexisting cations, anions, and amino acids.
Example 3
The application of the rhodamine acylhydrazone derivative as a fluorescent probe in distinguishing tumor cells from normal cells is tested, and the method comprises the following steps:
293T cells, HpeG2 and He L a tumor cells are selected as experimental materials, and the experimental materials are mixed with the rhodamine acylhydrazone derivative (the rhodamine acylhydrazone derivative is prepared into a probe solution with the concentration of 1 × 10) prepared in the embodiment 1 of the invention-5mol/L, solvent disodium phosphate-citric acid buffer solution with pH6.0, containing 0.5% DMSO by volume) and lysosome commercial dye L ysoTracker Red at 37 ℃ for 30min, and fluorescence imaging was performed using an Olympus FV500-IX70 confocal laser microscope, the results of which are shown in FIG. 8.
In FIG. 8, a-e are fluorescence images obtained by co-staining a normal cell 293T, wherein a is a probe green channel fluorescence image, b is an L ysoTracker Red channel fluorescence image, c is a bright field image, d is a superposition of the bright field image and the fluorescence image, and e is a correlation image of the Red channel and the Green channel.
f-j are fluorescence imaging images of co-staining tumor cells Hep G2, f is a probe green channel fluorescence imaging image, G is a L ysoTracker Red Red channel fluorescence imaging image, h is a bright field image, i is a superposition image of the bright field image and the fluorescence image, and j is a Red channel and green channel correlation image.
k-o are fluorescence imaging images of co-staining tumor cells He L a, k is a probe green channel fluorescence imaging image, l is a L ysoTracker Red channel fluorescence imaging image, m is a bright field image, n is a superposition image of the bright field image and the fluorescence image, and o is a red channel and green channel correlation image.
As can be seen from FIG. 8, Hpeg2 and He L a tumor cells emit strong green light, while 293T cells only have slight background fluorescence, which indicates that the rhodamine acylhydrazone derivative prepared in the invention example 1 can effectively distinguish normal cells from tumor cells, the red channel and green channel correlation diagram shows that the co-infection indexes of the rhodamine acylhydrazone derivative prepared in the invention example 1 and L ysoTracker Red in 293T normal cells, Hpeg2 and He L a tumor cells are respectively 98.21%, 83.25% and 99.18%, which indicates that the rhodamine acylhydrazone derivative prepared in the invention example 1 can specifically locate in live cell lysosomes.
The embodiments show that the rhodamine acylhydrazone derivative provided by the invention is very sensitive to pH, can be used as a pH fluorescent probe, can target lysosomes of cells, realizes fluorescent differentiation of normal cells and tumor cells, and does not need an organic solvent for dissolution assistance when being applied; the preparation method provided by the invention has the advantages of simple steps and low cost.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The rhodamine acylhydrazone derivative is characterized by having a structure shown as a formula I:
2. the method for preparing the rhodamine acylhydrazone derivative according to claim 1, which comprises the steps of:
mixing rhodamine 6G hydrazide, 4-morpholine benzaldehyde and an alcohol solvent for condensation reaction to obtain the rhodamine acylhydrazone derivative with the structure shown in the formula I.
3. The method according to claim 2, wherein the alcoholic solvent is ethanol.
4. The preparation method according to claim 2, wherein the molar ratio of rhodamine 6G hydrazide to 4-morpholinylbenzaldehyde is 1:1 to 1.1.
5. The method according to claim 2, wherein the ratio of the amount of rhodamine 6G hydrazide to the volume of the alcohol solvent is 0.01 to 0.02 mol: 0.3 to 0.6L.
6. The production method according to claim 2, wherein the condensation reaction is carried out under reflux stirring conditions; the condensation reaction is carried out at normal pressure for 3-5 h.
7. The method of claim 2, wherein the condensation reaction is further performed after: post-treating the condensation reaction liquid, wherein the post-treating comprises the following steps:
and cooling the condensation reaction liquid to room temperature to separate out solids, and washing filter residues after filtering to obtain the rhodamine acylhydrazone derivative with the structure shown in the formula I.
8. A fluorescent probe for distinguishing tumor cells from normal cells, which is characterized by comprising the rhodamine acylhydrazone derivative as defined in claim 1 or the rhodamine acylhydrazone derivative prepared by the preparation method as defined in any one of claims 2 to 7.
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