Fluorescent compound with polycation structure and synthesis and application thereof

Abstract

The invention discloses a fluorescent compound with a polycation structure as well as synthesis and application of the fluorescent compound. The structure of the compound is shown as a formula (I). The invention provides a synthesis method of the compound, which comprises the following step: carrying out bilateral dehydration condensation reaction on a compound shown as a formula (II) and a compound shown as a formula (III) to prepare a corresponding compound shown as a formula (I). The invention provides application of the compound as a two-photon fluorescent probe or a single-photon fluorescent probe for quantitatively detecting DNA and / or viscosity. The invention provides a novel compound which has effective DNA recognition capability, sensitive viscosity responsiveness, large two-photon fluorescence activity absorption cross section and red fluorescence emission, and quantitative detection of DNA and viscosity can be realized. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a compound with a polycationic structure, a synthesis method thereof, and application of the compound as a fluorescent probe for quantitatively detecting DNA and / or viscosity. Background Art

[0002] Fluorescent probe is a kind of molecular device. When it recognizes the target substance, its own fluorescence changes, so that the relevant information of the target substance can be obtained by detecting its fluorescence signal. Compared with other detection technologies, fluorescent probe has the advantages of high sensitivity, good selectivity, simple operation, strong visibility, fast response speed, low damage and in-situ detection. Therefore, fluorescent probe has been widely used in biology, medicine, environment and other fields.

[0003] Two-photon fluorescent probes are a type of third-order nonlinear optical material based on two-photon absorption properties. Under strong light excitation, they can simultaneously absorb two photons of equal or different energy, then transition to a high-energy excited state through a virtual intermediate state, and then undergo a radiative transition to produce frequency up-conversion fluorescence induced by two-photon absorption, which is two-photon fluorescence. Compared with traditional single-photon fluorescent probes based on linear absorption, two-photon fluorescent probes have many incomparable advantages: (1) Single-photon fluorescence is short-wave excitation and long-wave emission, while two-photon fluorescence is long-wave excitation and short-wave emission. The wavelength used in the two-photon excitation process is red-shifted by nearly one-fold and is within the biological optical window (650-950nm), thus avoiding the UV-visible light damage that the living system cannot bear, enabling the imaging of living cells and the direct monitoring of life processes; (2) Since the linear absorption and Rayleigh scattering of this wavelength band of light in biological tissues are relatively small, the light penetration is good, which greatly increases the penetration of the excitation light in the organism, thus enabling deep three-dimensional imaging of the organism; (3) The probability of electron transition induced by two-photons is proportional to the square of the incident light intensity. Under the condition of tight laser beam focusing, the range of material excitation is limited to a micro-area volume equivalent to the cube of the incident wavelength, while the light intensity at other positions is insufficient to cause two-photon absorption, so that the excitation of the material has a high degree of spatial selectivity, which can greatly improve the axial resolution and contrast of imaging; (4) It can greatly reduce photobleaching and phototoxicity to the organism, and the background fluorescence interference is small, which is easy to observe. Therefore, two-photon fluorescent probes show revolutionary application value in the fields of medicine and life sciences.

[0004] Deoxyribonucleic acid (DNA) is the main carrier of biological genetic information and the material basis of gene expression. It is not only involved in the normal life processes of organisms such as growth, development, reproduction, and aging, but is also closely related to abnormal life phenomena such as cancer, mutation, and aberration. DNA is a biological macromolecule, and its purine and pyrimidine bases absorb in the ultraviolet region, but because its molar absorption coefficient is only 103 The order of magnitude and low sensitivity make it unsuitable for trace or trace detection and analysis. The fluorescence of DNA itself is also very weak, and its natural fluorescence cannot be directly used for research. As an important parameter of the intracellular microenvironment, viscosity plays a vital role in regulating physiological processes such as transport, diffusion and signal transmission between biomolecules. Abnormal viscosity often reflects the imbalance of the system in the organism, organelle damage and biomolecule damage, and can also cause diabetes, atherosclerosis, Alzheimer's disease, Parkinson's disease, cancer and many other diseases. There are many methods for measuring the viscosity of ordinary fluids, such as rotational viscometer, falling ball viscometer, vibration viscometer, capillary viscometer, etc., but none of them are suitable for the detection of biological samples such as cells and tissues. By labeling DNA or responding to viscosity with fluorescent probes, the detection of DNA and the viscosity of the intracellular microenvironment can be achieved quickly and effectively. Therefore, the development of a dual-functional high-performance two-photon fluorescent probe that can detect DNA and viscosity is of great significance for disease prevention and clinical diagnosis.

[0005] At present, fluorescent probes are widely used in biological imaging. The emission wavelength of most fluorescent probes is between 450-560nm. If the emission wavelength of the probe can be extended to the red light region above 600nm, it can not only effectively reduce the light damage of the organism, enhance the transmittance and penetration depth of light, but also avoid the interference of autofluorescence in cells in the blue / green / yellow light region, minimize the background noise, thereby improving the signal-to-noise ratio of imaging and obtaining better tomographic imaging.

[0006] In summary, the design and synthesis of a bifunctional two-photon fluorescent probe that has a large two-photon fluorescence active absorption cross section and red fluorescence emission and can quantitatively detect DNA and viscosity has both theoretical and practical significance. Summary of the invention

[0007] The primary purpose of the present invention is to provide a novel compound with a polycationic structure, which has effective DNA recognition ability, sensitive viscosity responsiveness, a large two-photon fluorescence active absorption cross section and red fluorescence emission.

[0008] The second object of the present invention is to provide a method for synthesizing the compound.

[0009] The third object of the present invention is to provide the use of the compound as a bifunctional two-photon fluorescent probe for quantitative detection of DNA and / or viscosity.

[0010] The fourth object of the present invention is to provide the use of the compound as a dual-functional single-photon fluorescent probe for quantitative detection of DNA and / or viscosity.

[0011] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0012] In the first aspect, the present invention provides a compound, the structure of which is shown in formula (I), and the chemical name of the compound of formula (I) is 4,4′-[(phenylimino)bis[4,1-phenylene-(1E)-2,1-vinylene]]bis[1-[3-(triethylammonium)propyl]pyridinium]tetrabromide:

[0013]

[0014] In a second aspect, the present invention provides a method for synthesizing the compound of formula (I) described in the first aspect, comprising the following steps:

[0015] The compound of formula (II) undergoes a bilateral dehydration condensation reaction with the compound of formula (III) to obtain the corresponding compound of formula (I);

[0016]

[0017] The bilateral dehydration condensation reaction of the present invention is specifically implemented as follows: add the compound of formula (II), the compound of formula (III) and the solvent to the reaction bottle, stir and dissolve, then add the base, and then react at 30-150°C (preferably reflux temperature) for 5-24h (preferably 10-15h). After the reaction is completed, the reaction mixture is separated and purified to obtain the target compound of formula (I). The base used is generally piperidine, triethylamine or potassium hydroxide, and the molar amount of the base is 1.2-4 times the molar amount of the compound of formula (II). The solvent is generally methanol, ethanol, chloroform, dichloromethane, acetonitrile, DMF or a mixture of several, and the molar amount of the solvent is 200-700 times the molar amount of the compound of formula (II). The molar ratio of the compound of formula (II) to the compound of formula (III) is 1:2-3. After the reaction is completed, the separation and purification method is preferably: the reaction mixture is cooled to room temperature, separated and purified by alumina column chromatography, and the eluting reagent is dichloromethane and ethanol (volume ratio is 8-30:1).

[0018] Preferably, the bilateral dehydration condensation reaction is carried out as follows:

[0019] Add the compound of formula (II), the compound of formula (III) and methanol into the reaction flask, stir to dissolve, then add piperidine, and then heat to reflux to react for 10 to 15 hours, then cool to room temperature, and separate and purify the reaction mixture by alumina column chromatography (eluting reagents are dichloromethane and ethanol) to obtain the target compound of formula (I).

[0020] In the present invention, the compounds represented by formula (II) and formula (III) can be synthesized by methods reported in the literature, and the recommended synthetic routes are as follows:

[0021]

[0022] The compound of formula (I) provided by the present invention uses triphenylamine at the center as an electron donor, and then symmetrically introduces pyridinium and quaternary ammonium electron acceptors at the 4,4′-position thereof through a trans-ethylene conjugated bridge, thereby forming an electron pull-push-pull configuration with a tetracationic structure. The lone pair of electrons on the nitrogen of triphenylamine forms p-π conjugation with the benzene ring, resulting in an increase in the electron cloud density on the benzene ring, thereby making triphenylamine have good electron-donating ability, and the pyridinium group and quaternary ammonium group at both ends have strong electron-withdrawing properties due to their positive charge, so that under the action of the electron donor-acceptor, the entire molecule can undergo significant intramolecular charge transfer, which is conducive to improving the two-photon absorption performance of the compound of formula (I). The compound of formula (I) presents an A-π-D-π-A quadrupole configuration. During the radiation transition, the triphenylamine electron donor introduced therein can effectively increase the HOMO energy level of the entire molecule, while the pyridinium and quaternary ammonium electron acceptors introduced therein can effectively reduce the LUMO energy level, thereby narrowing the energy gap between the emission excited state and the ground state of the compound of formula (I), thereby facilitating the red shift of the emission wavelength to the red light region. The compound of formula (I) contains a tetracationic structure connected by a flexible propyl chain, which not only improves the electrostatic adsorption between the negatively charged phosphate backbone in DNA and makes the action site more flexible, thereby enabling targeted recognition and high bonding of DNA, but also significantly increases the water solubility of the entire molecule, making it easier to disperse in the water environment where living cells survive. The connection between the electron donor and the electron acceptor of the compound of formula (I) uses non-rigid trans-ethylene, which helps it become a fluorescent molecular rotor. In an environment with low viscosity, the intramolecular rotation of the fluorescent molecular rotor causes it to form a non-radiative "twisted intramolecular charge transfer (TICT)" state and quench the fluorescence; in an environment with high viscosity, the intramolecular rotation is inhibited, and the fluorescence can be restored. This TICT effect can achieve a viscosity-sensitive fluorescence response of the compound of formula (I). In addition, whether the compound of formula (I) is used to label DNA or respond to viscosity, its single-photon and two-photon fluorescence intensities show a good linear relationship with DNA concentration or viscosity, so it can be used as a dual-function probe to complete the quantitative detection of DNA and viscosity.

[0023] Therefore, in a third aspect, the present invention provides the use of the compound of formula (I) described in the first aspect as a two-photon fluorescent probe for quantitative detection of DNA and / or viscosity.

[0024] In a fourth aspect, the present invention provides the use of the compound of formula (I) described in the first aspect as a single-photon fluorescent probe for quantitative detection of DNA and / or viscosity.

[0025] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a novel compound having effective DNA recognition ability, sensitive viscosity responsiveness, a large two-photon fluorescence active absorption cross section and red fluorescence emission, which can realize the practical application of quantitative detection of DNA and viscosity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The single-photon fluorescence emission spectra of the compound of formula (I) acting on calf thymus DNA at different molar concentrations are shown in FIG. The ordinate represents the single-photon fluorescence intensity, and the abscissa represents the wavelength.

[0027] Figure 2 The linear relationship between the logarithm of the single-photon fluorescence intensity of the compound of formula (I) and the logarithm of the calf thymus DNA molar concentration is fitted. The ordinate represents the logarithm of the single-photon fluorescence intensity, and the abscissa represents the logarithm of the DNA molar concentration.

[0028] Figure 3 The two-photon fluorescence emission spectra of the compound of formula (I) acting on calf thymus DNA at different molar concentrations are shown in FIG. The ordinate represents the two-photon fluorescence intensity, and the abscissa represents the wavelength.

[0029] Figure 4 The linear relationship between the logarithm of the two-photon fluorescence intensity of the compound of formula (I) and the logarithm of the calf thymus DNA molar concentration is fitted. The ordinate represents the logarithm of the two-photon fluorescence intensity, and the abscissa represents the logarithm of the DNA molar concentration.

[0030] Figure 5 The single-photon fluorescence emission spectra of the compound of formula (I) in glycerol-water systems with different mass fractions are shown in Table 1. The ordinate represents the single-photon fluorescence intensity, and the abscissa represents the wavelength.

[0031] Figure 6 The linear relationship between the logarithm of the single-photon fluorescence intensity and the logarithm of the viscosity of the compound of formula (I) in the glycerol-water system is fitted. The ordinate represents the logarithm of the single-photon fluorescence intensity, and the abscissa represents the logarithm of the viscosity.

[0032] Figure 7 The two-photon fluorescence emission spectra of the compound of formula (I) in glycerol-water systems with different mass fractions are shown in Table 1. The ordinate represents the two-photon fluorescence intensity, and the abscissa represents the wavelength.

[0033] Figure 8 The linear relationship between the logarithm of the two-photon fluorescence intensity and the logarithm of the viscosity of the compound of formula (I) in the glycerol-water system is fitted. The ordinate represents the logarithm of the two-photon fluorescence intensity, and the abscissa represents the logarithm of the viscosity. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme will be further described clearly and completely by examples below. The materials, reagents or instruments used in the examples without indicating the manufacturer are all conventional products that can be purchased commercially. If the specific conditions are not indicated in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0035] Example 1 Compound of formula (I)

[0036] 0.30 g (1 mmol) of the compound of formula (II), 0.83 g (2.1 mmol) of the compound of formula (III) and 20 mL of methanol were added to the reaction flask, stirred to dissolve, and then 0.21 g (2.5 mmol) of piperidine was added, and then heated under reflux for 10 h, and then cooled to room temperature. The reaction mixture was separated and purified by alumina column chromatography (eluting reagent was V 二氯甲烷 :V 乙醇 =10:1), to obtain 0.44 g of the red target compound of formula (I). 1 H NMR (DMSO-d6, 500MHz) δ: 9.11 (d, J = 6.6 Hz, 4H), 8.29 (d, J = 6.6 Hz, 4H), 8.10 (d, J = 16.2 Hz, 2H), 7.74(d,J=8.6Hz,4H), 7.46(d,J=16.2Hz,2H), 7.45(t,J=7.8Hz,2H), 7.25(t,J=7.4Hz ,1H), 7.18(d,J=7.8Hz,2H), 7.11(d,J=8.6Hz,4H), 4.65(t,J=7.1Hz,4H), 3.29(q,J=7.1Hz ,12H), 3.26-3.32(m,4H), 2.33-2.39(m,4H), 1.21(t,J=7.1Hz,18H); HRMS(ESI): m / zcalcd for C 50 H 67 N5[M-4Br] 4+ :184.3844; found:184.3848.

[0037] Example 2 Compound of formula (I)

[0038] 0.30 g (1 mmol) of the compound of formula (II), 0.95 g (2.4 mmol) of the compound of formula (III) and 20 mL of ethanol were added to the reaction flask, stirred to dissolve, and then 0.17 g (2 mmol) of piperidine was added, and then heated under reflux for 14 h, and then cooled to room temperature. The reaction mixture was separated and purified by alumina column chromatography (eluting reagent was V 二氯甲烷 :V 乙醇 =10:1), to obtain 0.42 g of the red target compound of formula (I).

[0039] Example 3 Single-photon fluorescence spectrum test of DNA interaction

[0040] The molar concentration of the compound of formula (I) in the Tris-HCl buffer solution is 5×10 -6 mol L -1 The concentration of calf thymus DNA (ctDNA) was gradually increased, and the single-photon fluorescence emission spectra of the compound of formula (I) acting on ctDNA of different molar concentrations were measured using an RF-5301PC fluorescence spectrophotometer. The specific results are shown in Figure 1 The compound of formula (I) hardly fluoresces before reacting with ctDNA. After reacting with ctDNA, the fluorescence intensity gradually increases with the increase of ctDNA concentration. When saturation is reached, the fluorescence intensity increases by more than 60 times, and the fluorescence emission wavelength is about 625nm, which is in the red light region. Figure 2 It is a linear relationship fitting between the logarithm of the single photon fluorescence intensity of the compound of formula (I) and the logarithm of the molar concentration of ctDNA. It can be seen from the figure that the single photon fluorescence intensity (lgI) of the compound of formula (I) and the molar concentration of ctDNA (lgc DNA ) showed a good linear relationship, with a linear correlation coefficient of 0.9916, which can realize the quantitative detection of DNA.

[0041] Example 4 Two-photon fluorescence spectrum test of DNA interaction

[0042] The molar concentration of the compound of formula (I) in the Tris-HCl buffer solution is 1×10 -5 mol L -1 The concentration of ctDNA was gradually increased, a mode-locked titanium sapphire femtosecond laser (Chameleon Ultra II, 680-1080nm, 80MHz, 140fs) was used as the pump light source, and a full-spectrum spectrometer (USB4000-FLG) was used to record the fluorescence spectrum. The two-photon fluorescence emission spectra of the compound of formula (I) acting on ctDNA of different molar concentrations were measured. The specific results are shown in Figure 3 The compound of formula (I) hardly fluoresces before reacting with ctDNA. After reacting with ctDNA, the fluorescence intensity gradually increases with the increase of ctDNA concentration. When saturation is reached, the fluorescence intensity increases by more than 30 times, and the fluorescence emission wavelength is about 605nm, which is in the red light region. Figure 4 It is a linear relationship fitting between the logarithm of the two-photon fluorescence intensity of the compound of formula (I) and the logarithm of the molar concentration of ctDNA. It can be seen from the figure that the two-photon fluorescence intensity (lgI) of the compound of formula (I) and the molar concentration of ctDNA (lgc DNA) showed a good linear relationship, with a linear correlation coefficient of 0.9919, which can realize the quantitative detection of DNA.

[0043] Example 5 Two-photon fluorescence active absorption cross section test with DNA

[0044] According to Example 4, a molar concentration of 1×10 -5 mol L -1 The two-photon fluorescence emission spectra of the compound of formula (I) acting on ctDNA of different molar concentrations, the same concentration of fluorescein as the compound of formula (I) was selected at 0.1 molL -1 The solution in sodium hydroxide was used as a reference, and the two-photon fluorescence active absorption cross section of the compound of formula (I) interacting with ctDNA was calculated using formula (1).

[0045]

[0046] Where: subscripts s and r represent the physical quantities corresponding to the sample and reference, respectively. δ is the two-photon absorption cross section, F is the two-photon fluorescence integrated intensity, Φ is the fluorescence quantum yield, n is the solution refractive index, and c is the solution concentration.

[0047] According to formula (1), the two-photon fluorescence active absorption cross section of the compound of formula (I) when the interaction with ctDNA reaches saturation is 47.5GM, which is bright enough to realize two-photon fluorescence imaging of DNA.

[0048] Example 6 Single-photon fluorescence response test of viscosity

[0049] In the single-photon fluorescence response test of the compound of formula (I) on viscosity, a glycerol-water system was used to simulate the viscosity environment. Glycerol-water was prepared into 11 gradient solutions with different viscosities (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%) according to different mass fractions. The constant sample concentration was 1×10 -5 mol L -1 , the test temperature was 20°C. The single-photon fluorescence emission spectrum was measured using a RF-5301PC fluorescence spectrophotometer. Figure 5 The single-photon fluorescence emission spectra of the compound of formula (I) in different mass fractions of glycerol-water systems. It can be seen from the figure that as the proportion of glycerol increases, that is, the viscosity increases, the fluorescence emission intensity of the compound of formula (I) continues to increase. From the pure water system to the 99% glycerol system, the fluorescence intensity increases by more than 80 times, and the fluorescence emission wavelength is about 610nm, which is in the red light region.

[0050] The relationship between the single photon fluorescence intensity of the compound of formula (I) and the ambient viscosity is given by -Hoffmann formula, that is, formula (2) is used for fitting:

[0051] lgI=C+xlgη Formula (2)

[0052] Where: I is the fluorescence intensity, C is a constant related to concentration and temperature, x is a constant related to the fluorescent dye, and η is the viscosity of the solvent system.

[0053] Figure 6 The linear relationship between the logarithm of the single photon fluorescence intensity of the compound of formula (I) and the logarithm of the viscosity in the glycerol-water system is fitted. As can be seen from the figure: the single photon fluorescence intensity (lgI) of the compound of formula (I) and the viscosity (lgη) show a good linear relationship, and the linear correlation coefficient reaches 0.9959, which can realize the quantitative detection of viscosity.

[0054] Example 7 Two-photon fluorescence response test of viscosity

[0055] In the two-photon fluorescence response test of the compound of formula (I) on viscosity, a glycerol-water system was also used to simulate the viscosity environment. Glycerol-water was prepared into six gradient solutions with different viscosities (0%, 20%, 40%, 60%, 80%, 99%) according to different mass fractions. The constant sample concentration was 2×10 -4 mol L -1 , the test temperature was 20°C. A mode-locked Ti:Sapphire femtosecond laser (Chameleon Ultra II, 680-1080nm, 80MHz, 140fs) was used as the pump light source, and a full-spectrum spectrometer (USB4000-FLG) was used to record the two-photon fluorescence spectrum. Figure 7 The two-photon fluorescence emission spectra of the compound of formula (I) in glycerol-water systems with different mass fractions are shown in the figure. As can be seen from the figure: with the increase of the glycerol ratio, that is, the increase of viscosity, the fluorescence emission intensity of the compound of formula (I) continues to increase. From the pure water system to the 99% glycerol system, the fluorescence intensity increases by more than 150 times, and the fluorescence emission wavelength is about 608nm, which is in the red light region.

[0056] The relationship between the two-photon fluorescence intensity of the compound of formula (I) and the ambient viscosity is fitted by formula (2). Figure 8 The linear relationship between the logarithm of the two-photon fluorescence intensity of the compound of formula (I) and the logarithm of the viscosity in the glycerol-water system is fitted. As can be seen from the figure: the two-photon fluorescence intensity (lgI) of the compound of formula (I) and the viscosity (lgη) show a good linear relationship, and the linear correlation coefficient reaches 0.9975, which can realize the quantitative detection of viscosity.

[0057] Example 8 Two-photon fluorescence active absorption cross section test for viscosity response

[0058] According to Example 7, a molar concentration of 2×10 -4 mol L -1 The two-photon fluorescence emission spectra of the compound of formula (I) in response to different viscosities are selected to select the same concentration of fluorescein as the compound of formula (I) at 0.1 mol L -1 The solution in sodium hydroxide was used as a reference, and the two-photon fluorescence active absorption cross section of the compound of formula (I) in response to different viscosities was calculated according to formula (1). From the pure water system to the 99% glycerol system, the two-photon fluorescence active absorption cross section of the compound of formula (I) increased from less than 1GM to 41GM, and this brightness can realize two-photon fluorescence imaging of viscosity.

[0059] The above description is only an embodiment of the present application and is not intended to limit the protection scope of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A compound, the structure of which is shown in formula (I), the chemical name of the compound of formula (I) is 4,4′-[(phenylimino)bis[4,1-phenylene-(1E)-2,1-vinylene]]bis[1-[3-(triethylammonium)propyl]pyridinium]tetrabromide:

2. A method for synthesizing the compound according to claim 1, characterized in that: The synthesis method comprises the following steps: The compound of formula (II) undergoes a bilateral dehydration condensation reaction with the compound of formula (III) to obtain the corresponding compound of formula (I); 3. Use of the compound as claimed in claim 1 as a two-photon fluorescent probe for quantitative detection of DNA and / or viscosity.

4. Use of the compound as claimed in claim 1 as a single-photon fluorescent probe for quantitative detection of DNA and / or viscosity.

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