CN112047876A - Red two-photon fluorescent AIE compound and synthesis and application thereof - Google Patents
Red two-photon fluorescent AIE compound and synthesis and application thereof Download PDFInfo
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Abstract
The invention discloses a red two-photon fluorescent AIE compound and synthesis and application thereof. The structure of the compound is shown as a formula (I), and the chemical name of the compound is 2, 6-bis [ (1E) -2- [4- (dibutylamino) phenyl group]Vinyl radical]-1- (2-hydroxyethyl) pyridinium bromide. The invention provides a synthesis method of a compound shown as a formula (I), which comprises the following steps: (1) quaternization reaction is carried out on 2, 6-lutidine and 2-bromoethanol to prepare 1- (2-hydroxyethyl) -2, 6-dimethylpyridinium bromide, namely the corresponding compound shown in the formula (II); (2) the compound shown in the formula (II) and 4- (dibutylamino) benzaldehyde are subjected to bilateral dehydration condensation reaction to prepare the corresponding compound shown in the formula (I). The invention provides application of a compound shown as a formula (I) in preparation of a two-photon fluorescence imaging reagent in living cells.The compound provided by the invention has red two-photon fluorescence emission, obvious AIE effect, larger two-photon fluorescence activity absorption cross section in water, good living cell penetrability and low cytotoxicity.
Description
(I) technical field
The invention relates to a compound, a synthetic method thereof and application thereof in preparing a two-photon fluorescence imaging reagent in living cells.
(II) background of the invention
Under the irradiation of common light, molecules can only generate linear absorption, namely, one photon is absorbed to reach an excited state, and the process is called single photon absorption; if the molecule subsequently excited in the singlet state undergoes a radiative transition, giving off a photon that falls back to the stable ground state, this radiation is called single photon fluorescence. If strong laser is used as excitation light source, the electric field intensity of laser light frequency is close to the electric field intensity in substance atoms, so that the nonlinear polarization response of the substance can be caused. Two-photon absorption is a three-order nonlinear optical effect, which refers to a transition process in which molecules absorb two photons simultaneously and reach a high-energy excited state through a virtual intermediate state; if it subsequently undergoes radiative transitions, the resulting frequency up-converted fluorescence is called two-photon fluorescence.
Compared with single photon absorption, two-photon absorption has the following characteristics: (1) the single photon absorption is a linear absorption process, and the two-photon absorption is a nonlinear absorption process; (2) the single photon absorption process is that matter molecules absorb a photon with high energy and short wavelength to reach an excited state, and the two-photon absorption process is that matter molecules absorb two photons with low energy and long wavelength to reach the excited state; (3) in the two-photon absorption process, the absorption intensity and the electron transition probability of substance molecules are in direct proportion to the square of the excitation light intensity; (4) for fluorescent molecules, the ability of the molecule to absorb a photon is generally expressed in terms of an absorption cross-section. The larger the absorption cross-section, the stronger the absorption capacity of the substance molecule for photons. Generally, the single photon absorption cross section is 1032-1033In the GM range, less optical density is required; the two-photon absorption section is generally 1-104In the GM range, the probability of two-photon absorption of common molecules is very small; (5) two-photon absorption occurs at the focal point λ3(λ is the excitation wavelength) and single photon absorption occurs over the entire focused optical path.
Based on the above characteristics, the two-photon fluorescence imaging technology based on two-photon absorption has many incomparable advantages compared with the single-photon technology: (1) the phototoxicity of light on the tested biological sample is greatly reduced; (2) the bleaching effect of light on fluorescence is greatly reduced, and the observation time of the fluorescence of the detected sample is effectively prolonged; (3) the penetration depth and the spatial resolution of the tested sample are greatly improved. Therefore, the two-photon fluorescence imaging technology is used in the host-guest molecule recognition process taking fluorescence as a conduction signal, such as: biological fluorescence recognition, medical fluorescence diagnosis and the like, and has immeasurable application potential and prospect.
Most conventional fluorescent molecules exhibit strong fluorescence in dilute solutions, but fluorescence tends to decrease or even disappear completely in high concentration solutions or in the solid state, and the main reason for this concentration Quenching is related to the formation of aggregates, so this phenomenon is called "Aggregation-induced Quenching (ACQ)". Unlike conventional fluorescent molecules with ACQ effect, molecules with "Aggregation-Induced Emission (AIE)" effect emit very little or no light in solution, but fluorescence is sharply enhanced in the aggregated state and in the solid-state thin film, thus greatly broadening their application range in biological systems. The AIE molecule can be widely applied to the fields of biological detection, biochemical process tracing, organelle staining, microorganism and tissue organ imaging and the like.
During fluorescence imaging, the red fluorescence emission with long wavelength can not only effectively reduce light damage and enhance the transmittance and the transmittance depth of light, but also avoid the interference of autofluorescence in cells in a blue light/green light region, so that the background noise is reduced to the minimum, the signal-to-noise ratio of imaging is improved, and better tomography can be obtained.
Therefore, a novel compound with red two-photon fluorescence emission and obvious AIE effect is designed and synthesized, and the novel compound has a large two-photon fluorescence activity absorption cross section, good living cell penetrability and low cytotoxicity, so that the practical application of the novel compound in two-photon fluorescence imaging of living cells is realized, and the novel compound has both theoretical significance and practical significance.
Disclosure of the invention
The primary object of the present invention is to provide a compound that combines red two-photon fluorescence emission, a significant AIE effect, a large two-photon fluorescence active absorption cross section in water, good living cell penetration, and low cytotoxicity.
The second object of the present invention is to provide a process for the preparation of the compound.
The third purpose of the invention is to provide the application of the compound in preparing a two-photon fluorescence imaging reagent in living cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a compound having the structure shown in formula (I), wherein the chemical name of the compound is 2, 6-bis [ (1E) -2- [4- (dibutylamino) phenyl ] vinyl ] -1- (2-hydroxyethyl) pyridinium bromide:
in a second aspect, the present invention provides a method for synthesizing the compound represented by formula (i), comprising the following steps:
(1) quaternization reaction is carried out on 2, 6-lutidine and 2-bromoethanol to prepare 1- (2-hydroxyethyl) -2, 6-dimethylpyridinium bromide, namely the corresponding compound shown in the formula (II);
(2) the compound shown in the formula (II) and 4- (dibutylamino) benzaldehyde are subjected to bilateral dehydration condensation reaction to prepare the corresponding compound shown in the formula (I).
The general synthetic route for the compounds (I) of the present invention is as follows:
the solvent adopted in the quaternization reaction in the step (1) is acetonitrile or ethyl acetate, the molar amount of the solvent is 5-20 times of that of 2, 6-dimethylpyridine, and the solvent can be omitted. The molar use ratio of the 2, 6-dimethylpyridine to the 2-bromoethanol is 1: 1-2. The reaction temperature is 50-140 ℃ (preferably the reflux temperature), and the reaction time is 3-15 h. After the reaction is finished, the product can be purified by a washing method, and a washing reagent is preferably dichloromethane or tetrahydrofuran.
Preferably, the step (1) is carried out as follows:
adding 2-bromoethanol into 2, 6-dimethylpyridine, heating to 135-140 ℃, carrying out reflux reaction for 8h, cooling to room temperature, carrying out suction filtration, and washing the obtained solid with tetrahydrofuran to obtain a white compound shown as a formula (II).
According to the bilateral dehydration condensation reaction in the step (2), alkali is used as a catalyst, the adopted alkali is piperidine, tetrahydropyrrole, sodium hydroxide or potassium carbonate generally, and the molar amount of the alkali is 2-8 times of that of the compound in the formula (II). The solvent adopted by the invention is generally methanol, ethanol, butanol, chloroform, water or a mixture of the methanol, the ethanol, the butanol, the chloroform and the water, and the molar amount of the solvent is 100-800 times of that of the compound shown in the formula (II). The molar using ratio of the compound shown in the formula (II) to the 4- (dibutylamino) benzaldehyde is 1: 2-3. The reaction temperature is 20-120 ℃, and the reaction time is 5-24 h. After the reaction is finished, purifying the product by recrystallization or silica gel column chromatography, wherein in the recrystallization operation, a recrystallization solvent is methanol or ethanol, and in the column chromatography operation, an elution reagent is ethyl acetate and methanol (the volume ratio is 10-20: 1).
Preferably, the step (2) is performed as follows:
adding the compound shown in the formula (II) into methanol, stirring for dissolving, adding 4- (dibutylamino) benzaldehyde and piperidine, heating to 65 ℃, refluxing for reaction for 12 hours, cooling to room temperature, performing suction filtration, and separating and purifying the obtained solid by silica gel column chromatography to obtain the compound shown in the red formula (I).
The compound (I) provided by the invention takes pyridine cations with strong electron-withdrawing property as a pi-center, styrene with excellent electron transmission capacity as a pi-conjugate bridge and dibutylamino with strong electron-donating property as an end capping group, and under the excitation of light, the push-pull-push configuration is favorable for symmetric charge transfer of electrons from two ends to the middle. The density functional theory calculation proves that: on the HOMO molecular orbit, electron clouds are mainly distributed on a styrene conjugate bridge and a dibutylamino electron donor, while on the LUMO molecular orbit, the electron cloud density on the dibutylamino electron donors at two ends of the molecule is reduced, the electron cloud density on the central pyridine cation electron acceptor is obviously increased, and the strong electron cloud delocalization in the pi conjugated chain is favorable for improving the two-photon absorption performance of the compound (I). The electron donor (D) and the electron acceptor (A) are introduced into the conjugated chain of the compound (I) to form a symmetrical D-pi-A-pi-D configuration. The introduced dibutylamino electron donor can effectively raise the HOMO energy level of the whole molecule, and the introduced pyridine cation electron acceptor can effectively lower the LUMO energy level, so that the energy gap between the emission excited state and the ground state of the compound (I) is narrowed, and red two-photon fluorescence emission above 620nm is favorably generated. Compound (i) also exhibited a significant AIE effect: the fluorescence is very weak in dimethyl sulfoxide (DMSO), but with the addition of poor solvent water, the red fluorescence is increased sharply after the aggregates are generated, and the fluorescence can be identified by naked eyes. It is known that cells are in water environment, and the compound (I) has a large two-photon fluorescence activity absorption cross section (fluorescence quantum yield phi multiplied by two-photon absorption cross section) in water, so that enough brightness can be obtained when living cells are subjected to two-photon fluorescence imaging. In addition, the compound (I) contains four lipophilic butyl chains and N-hydroxyethyl bonded hydrophilic pyridine cations, so that the oil-water distribution coefficient of the compound (I) is well modulated, the compound (I) has good cell membrane permeability, and cytotoxicity experiments show that the compound (I) has low cytotoxicity.
In a third aspect, the invention provides the use of a compound of formula (i) in the preparation of a two-photon fluorescence imaging agent in living cells.
Compared with the prior art, the invention has the beneficial effects that: the compound provided by the invention has red two-photon fluorescence emission, a remarkable AIE effect, a larger two-photon fluorescence activity absorption cross section in water, good living cell penetrability and low cytotoxicity, and can be applied to two-photon fluorescence imaging in living cells.
(IV) description of the drawings
FIG. 1 shows the DMSO-H ratio of compound (I) at various ratios2Fluorescence emission spectrum in O mixed solvent. The ordinate represents the fluorescence intensity and the abscissa represents the wavelength.
FIG. 2 shows two-photon fluorescence active absorption cross sections of compound (I) under different wavelength excitation. The ordinate represents a two-photon fluorescence active absorption cross section, and the abscissa represents a wavelength.
FIG. 3 shows two-photon fluorescence imaging of HepG2 live cells with Compound (I). (a) The measurement is cell bright field, (b) two-photon fluorescence imaging, and (c) superposition of the cell bright field and the two-photon fluorescence imaging, and the scale is 20 mu m.
(V) specific embodiment:
the technical solution of the present invention is illustrated by the following specific examples, but the scope of the present invention is not limited thereto:
EXAMPLE 1 Compound (II)
4.28g (40mmol) of 2, 6-lutidine and 7.50g (60mmol) of 2-bromoethanol are added into a reaction flask, and then the mixture is heated and refluxed for 8 hours. The reaction mixture was then cooled to room temperature, the precipitated solid was filtered with suction, and the filter cake was washed with tetrahydrofuran several times to give 7.63g of a white compound (II).1HNMR(DMSO-d6,500MHz)：8.28(t,J＝7.9Hz,1H)，7.83(d,J＝7.9Hz,2H)，4.59(t,J＝5.4Hz,2H)，3.82(t,J＝5.4Hz,2H)，2.81(s,6H)。
EXAMPLE 2 Compound (II)
A reaction flask was charged with 4.28g (40mmol) of 2, 6-lutidine, 5.50g (44mmol) of 2-bromoethanol and 20mL of ethyl acetate, and then reacted at 70 ℃ for 13 h. The reaction mixture was then cooled to room temperature, the precipitated solid was filtered with suction, and the filter cake was washed with methylene chloride several times to give 6.59g of a white compound (II).
EXAMPLE 3 Compound (II)
A reaction flask was charged with 4.28g (40mmol) of 2, 6-lutidine, 6.50g (52mmol) of 2-bromoethanol and 15mL of acetonitrile, and then reacted at 80 ℃ for 12 hours. The reaction mixture was then cooled to room temperature, the precipitated solid was filtered with suction, and the filter cake was washed with methylene chloride several times to give 7.21g of a white compound (II).
EXAMPLE 4 Compound (I)
2.32g (10mmol) of the compound (II) and 20mL of methanol were charged into a reaction flask, and dissolved by stirring, and then 5.59g (24mmol) of 4- (dibutylamino) benzaldehyde and 3.40g (40mmol) of piperidine were added, and the mixture was refluxed at 65 ℃ for 12 hours. The reaction mixture was then cooled to room temperature, the precipitated solid was filtered with suction, and the filter cake was separated by column chromatography over silica gel [ eluent: v (ethyl acetate): V (methanol) ═ 15:1]5.08g of the red compound (I) was obtained. m.p.245-247 deg.C;1H NMR(DMSO-d6,500MHz)：8.20(t,J＝8.1Hz,1H)，8.09(d,J＝8.1Hz,2H)，7.64(d,J＝15.7Hz,2H)，7.62(d,J＝8.9Hz,4H)，7.34(d,J＝15.7Hz,2H)，6.72(d,J＝8.9Hz,4H)，5.36(t,J＝5.6Hz,1H)，4.85(t,J＝5.0Hz,2H)，3.95(q,J＝5.4Hz,2H)，3.36(t,J＝7.6Hz,8H)，1.50-1.56(m,8H)，1.31-1.38(m,8H)，0.94(t,J＝7.4Hz,12H)；13C NMR(DMSO-d6,125MHz)：153.76，149.62，142.80，141.51，130.47，121.92，121.44，112.31，111.27，59.12，53.24，49.82，29.02，19.60，13.83；HRMS(ESI)：m/zcalcd for C39H56N3O[M-Br]+：582.8890；found：582.8879。
EXAMPLE 5 Compound (I)
2.32g (10mmol) of the compound (II) and 20mL of ethanol were added to a reaction flask, and stirred to dissolve, 4.66g (20mmol) of 4- (dibutylamino) benzaldehyde and 2.55g (30mmol) of piperidine were added, and the mixture was heated to 78 ℃ and refluxed for 8 hours. The reaction mixture was then cooled to room temperature, the precipitated solid was filtered with suction, and the filter cake was recrystallized from ethanol to give 4.54g of red compound (I).
EXAMPLE 6 Compound (I)
2.32g (10mmol) of the compound (II), 10mL of methanol and 10mL of chloroform were charged into a reaction flask, and dissolved with stirring, and then 6.06g (26mmol) of 4- (dibutylamino) benzaldehyde and 3.55g (50mmol) of tetrahydropyrrole were added, and the mixture was heated to 60 ℃ and reacted with reflux for 16 hours. The reaction mixture was then cooled to room temperature, the precipitated solid was filtered with suction, and the filter cake was separated by column chromatography over silica gel [ eluent: v (ethyl acetate): V (methanol) ═ 15:1], yielded 4.87g of red compound (i).
Example 7AIE Effect test
Test of Compound (I) in DMSO-H2Fluorescence spectra in O mixed solvent systems, where DMSO and H2O is its good solvent and poor solvent, respectively. This is a commonly used method for verifying the behavior of the fluorescent chromophore AIE, because when the poor solvent in the mixed solvent system reaches a certain proper ratio, the solubility of the molecule in the mixed solution changes, thereby promoting the generation of aggregates.
FIG. 1 shows the DMSO-H ratio of compound (I) at various ratios2Fluorescence emission spectrum in O mixed solvent is very weak in good solvent DMSO, and fluorescence peak intensity is only 0.72, along with poor solvent H2The fluorescence intensity gradually changes obviously when O is added: when H is present2When the volume fraction of O is less than 50%, the fluorescence intensity is not improved (fluorescence peak intensity)<1) Weak enough to be hardly visible; but when H2When the volume fraction of O is equal to 50%, the fluorescence intensity is obviously enhanced due to the generation of aggregates, and the fluorescence peak intensity reaches 414; when H is present2When the volume fraction of O is further increased to 80%, the fluorescence peak intensity is up to 648, which is 900 times of the fluorescence intensity in pure DMSO, and the fluorescence becomes very bright and is easy to see by naked eyes; but when H2When the volume fraction of O is further increased to 90%, the fluorescence intensity becomes rather weak due to the particle size of the aggregates. At H2And under different volume fractions of O, the maximum fluorescence emission wavelength of the compound (I) is always kept around 633nm, and red fluorescence is shown. Therefore, the compound (I) has remarkable AIE effect and red fluorescence emission performance.
Example 8 two-photon fluorescence Activity absorption Cross section test
Cells in organisms all live in water environment, so that the characterization of the two-photon fluorescence activity absorption cross section (fluorescence quantum yield phi multiplied by the two-photon absorption cross section) of the compound (I) in water is important, and the small value of phi multiplied means that the excitation light intensity must be increased in order to obtain good imaging during two-photon fluorescence imaging, thereby causing damage to biological samples. The two-photon fluorescence active absorption cross section of the compound (I) is tested by a two-photon induced fluorescence method.During testing, a mode-locked titanium gem femtosecond laser (Chameleon Ultra II, 680-1080nm, 80MHz, 140fs) is used as a pumping light source, and a full spectrum spectrometer (USB4000-FLG) is adopted to record fluorescence spectrum. The solvent of the sample is H2O (10% volume fraction of DMSO was added) was placed in a four-side-illuminated quartz cuvette with an excitation wavelength of 690 and 930nm at 20nm intervals. Selecting 0.1mol L of fluorescein-1The calculation formula of the two-photon fluorescence active absorption cross section is shown as the formula (1) by taking the sodium hydroxide aqueous solution as a reference:
in the formula, subscripts s and r represent physical quantities of the sample and the reference, respectively. Is a two-photon absorption cross section, F is the two-photon fluorescence integral intensity, phi is the fluorescence quantum yield, n is the solution refractive index, and c is the solution concentration.
FIG. 2 shows two-photon fluorescence active absorption cross sections of compound (I) under different wavelength excitation. The compound (I) emits red two-photon fluorescence under the excitation of long-wavelength strong laser, and emits red two-photon fluorescence under the excitation of H2The maximum two-photon fluorescence activity absorption cross section in O was 49GM, and this brightness enabled two-photon fluorescence imaging.
Example 9 cytotoxicity assays
The cytotoxicity test adopts a tetrazolium salt (MTT) colorimetric method, selects human liver cancer cells HepG2 as a research object, and represents the toxicity of the compound (I) to the cells by the cell survival rate. DMEM medium supplemented with 10% fetal bovine serum was used for cell culture. HepG2 cells were seeded at 10000 cell concentration per well in 96-well plates (100. mu.L per well medium) at 37 ℃ with 5% CO2Culturing for 24h under the condition. Then compound (i) was added in a gradient and HepG2 cells were incubated for 24 h. mu.L of MTT solution was added to each well and 5% CO was added at 37 ℃2Incubation was continued for 4h under conditions. And finally removing the culture medium, adding DMSO (each well is 100 mu L) to dissolve the blue-violet formazan crystallisate, detecting the photon density OD value of each well by using a microplate reader, and calculating the cell survival rate according to the formula (2).
According to the above cytotoxicity test, the cell survival rates of compound (I) at concentrations of 5, 10, 20, 40 and 80. mu.M were 94%, 90%, 83%, 78% and 75%, respectively, indicating that compound (I) had low cytotoxicity.
Example 10 two-photon fluorescence imaging in living cells
mu.M of Compound (I) was added to imaging dishes inoculated with HepG2 cells at 37 ℃ with 5% CO2Incubating HepG2 cells for 0.5h under the condition, then removing the culture medium, washing the cells for 2-3 times by using PBS buffer solution, and carrying out two-photon fluorescence imaging by adopting an Olympus BX61W1-FV1000 two-photon confocal microscope, wherein the two-photon excitation wavelength is 800nm, and the two-photon fluorescence emission signal collection channel is 575-630 nm.
FIG. 3 shows two-photon fluorescence imaging of HepG2 live cells with Compound (I). The results show that: the compound (I) has good living cell penetrability, can successfully enter the inside of a living cell, and realizes red two-photon fluorescence imaging on the living cell.
Claims (3)
1. A compound having the structure shown in formula (I), wherein the chemical name of the compound is 2, 6-bis [ (1E) -2- [4- (dibutylamino) phenyl ] vinyl ] -1- (2-hydroxyethyl) pyridinium bromide:
2. a process for the synthesis of a compound of formula (i) as claimed in claim 1, comprising the steps of:
(1) quaternization reaction is carried out on 2, 6-lutidine and 2-bromoethanol to prepare 1- (2-hydroxyethyl) -2, 6-dimethylpyridinium bromide, namely the corresponding compound shown in the formula (II);
(2) the compound shown in the formula (II) and 4- (dibutylamino) benzaldehyde are subjected to bilateral dehydration condensation reaction to prepare the corresponding compound shown in the formula (I).
3. Use of a compound according to claim 1 for the preparation of a reagent for two-photon fluorescence imaging in living cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115215849A (en) * | 2022-07-15 | 2022-10-21 | 浙江工业大学 | Red two-photon fluorescent compound with large Stokes displacement and synthesis and application thereof |
| CN115215849B (en) * | 2022-07-15 | 2024-01-12 | 浙江工业大学 | Red two-photon fluorescent compound with large Stokes displacement and synthesis and application thereof |
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| CN112047876B (en) | 2021-07-27 |
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