Fluorescent compounds with polycationic structure, synthesis and application thereof

Abstract

The application discloses a fluorescent compound with a multi-cation structure and synthesis and application thereof. The structure of the compound is shown as formula (I). The application provides a synthesis method of the compound, which comprises the following steps: a double-side dehydration condensation reaction of a compound of formula (II) and a compound of formula (III) to prepare a corresponding compound of formula (I). The application provides application of the compound as a two-photon fluorescent probe or a one-photon fluorescent probe for quantitative detection of DNA and / or viscosity. The application provides a novel compound with effective DNA recognition ability, sensitive viscosity responsiveness, a large two-photon fluorescence activity absorption cross section and red fluorescence emission, which can realize quantitative detection of DNA and viscosity.

Description

Technical Field

[0001] The present 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 probes are molecular devices that, upon recognizing a target substance, produce a change in their own fluorescence. Detection of this fluorescence signal can provide information about the target substance. Compared to other detection technologies, fluorescent probes offer advantages such as high sensitivity, good selectivity, ease of operation, strong visibility, rapid response, minimal damage, and the ability to detect in situ. Consequently, fluorescent probes have found widespread application in biology, medicine, the environment, and other fields.

[0003] Two-photon fluorescence 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 fluorescence probes based on linear absorption, two-photon fluorescence 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 living systems cannot withstand, enabling the imaging of living cells and the direct monitoring of life processes. (2) Since the linear absorption and Rayleigh scattering of light in this wavelength band by 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 locations is insufficient to cause two-photon absorption, thus making the excitation of the material highly spatially selective, which can greatly improve the axial resolution and contrast of imaging. (4) It can greatly reduce photobleaching and phototoxicity to organisms, and the background fluorescence interference is small, making it easy to observe. Therefore, two-photon fluorescent probes have shown 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 distortion. DNA is a biological macromolecule. Its purine and pyrimidine bases absorb in the ultraviolet region, but because its molar absorption coefficient is only 103 The viscosity of ordinary fluids is measured in a variety of ways, with low sensitivity and unsuitable for micro or trace detection and analysis. The fluorescence of DNA itself is also very weak, and its natural fluorescence cannot be directly utilized for research. Viscosity, as an important parameter of the intracellular microenvironment, plays a vital role in regulating physiological processes such as transport, diffusion, and signal transmission between biomolecules. Abnormal viscosity often reflects imbalances in the biological system, organelle damage, and biomolecule damage, and can also cause a variety of diseases such as diabetes, atherosclerosis, Alzheimer's disease, Parkinson's disease, and cancer. There are many methods for measuring the viscosity of ordinary fluids, such as rotational viscometers, falling ball viscometers, vibration viscometers, and capillary viscometers, 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 viscosity of DNA and the intracellular microenvironment can be detected 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] Currently, fluorescent probes are widely used in biological imaging. Most fluorescent probes have emission wavelengths between 450 and 560 nm. Extending the probe's emission wavelength to the red region, above 600 nm, not only effectively reduces photodamage to organisms and enhances light transmittance and penetration depth, but also avoids interference from autofluorescence within cells in the blue, green, and yellow light regions, minimizing background noise and improving the signal-to-noise ratio (SNR) of imaging, enabling superior tomographic imaging.

[0006] In summary, the design and synthesis of a dual-functional two-photon fluorescent probe with a large two-photon fluorescence active absorption cross section and red fluorescence emission that 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 object of the invention, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a compound having a structure as 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:

[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 prepare the corresponding compound of formula (I);

[0016]

[0017] The bilateral dehydration condensation reaction described in the present invention is specifically carried out as follows: a compound of formula (II), a compound of formula (III), and a solvent are added to a reaction flask, stirred to dissolve, and then a base is added. The mixture is then reacted at 30-150°C (preferably reflux temperature) for 5-24 hours (preferably 10-15 hours). After the reaction, the resulting reaction mixture is separated and purified to obtain the target compound of formula (I). The base used is generally piperidine, triethylamine, or potassium hydroxide, with the molar amount of the base being 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 thereof, with the molar amount of the solvent being 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, the separation and purification method is preferably as follows: the reaction mixture is cooled to room temperature and purified by alumina column chromatography, with the eluting reagent being dichloromethane and ethanol (volume ratio of 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 to 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. The recommended synthetic routes are as follows:

[0021]

[0022] The compound of formula (I) provided by the present invention uses triphenylamine as a central electron donor, and then symmetrically introduces pyridinium and quaternary ammonium electron acceptors at the 4,4'-position via a trans-ethylene conjugated bridge, forming an electron pull-push-pull configuration with a tetracationic structure. The lone electron pair 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 endowing the triphenylamine with good electron-donating ability. The pyridinium and quaternary ammonium groups at both ends have strong electron-withdrawing properties due to their positive charge. Therefore, under the action of the electron donor-acceptor, the entire molecule can undergo significant intramolecular charge transfer, which is beneficial for improving the two-photon absorption performance of the compound of formula (I). The compound of formula (I) exhibits an A-π-D-π-A quadrupole configuration. During radiative transitions, the introduced triphenylamine electron donor can effectively increase the HOMO energy level of the entire molecule, while the introduced pyridinium and quaternary ammonium electron acceptors can effectively lower the LUMO energy level, thereby narrowing the energy gap between the excited and ground states of the compound of formula (I), thereby facilitating a red-shift of the emission wavelength to the red region. The compound of formula (I) contains a tetracationic structure connected by flexible propyl chains. This not only enhances the electrostatic adsorption interaction with the negatively charged phosphate backbone of DNA and makes the interaction sites more flexible, thereby enabling targeted recognition and high bonding to DNA, but also significantly increases the water solubility of the entire molecule, making it easier to disperse in the aqueous environment of living cells. The connection between the electron donor and the electron acceptor of the compound of formula (I) uses a non-rigid trans-ethylene, which helps it become a fluorescent molecular rotor. In low-viscosity environments, the intramolecular rotation of the fluorescent molecular rotor leads to a non-radiative "twisted intramolecular charge transfer (TICT)" state, quenching fluorescence. In high-viscosity environments, however, intramolecular rotation is suppressed, allowing fluorescence to recover. This TICT effect enables the viscosity-sensitive fluorescence response of the compound of formula (I). Furthermore, whether labeling DNA or responding to viscosity, the single-photon and two-photon fluorescence intensities of the compound of formula (I) exhibit a good linear relationship with DNA concentration or viscosity, making it suitable as a dual-function probe for 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 present application has the beneficial effect that the present application provides a novel compound with effective DNA recognition ability, sensitive viscosity responsiveness, large two-photon fluorescence activity absorption cross section and red fluorescence emission, which can realize the practical application of quantitative detection of DNA and viscosity. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 6 is a one-photon fluorescence emission spectrum of the compound of formula (I) in the glycerol-water system with different mass fractions. The vertical coordinate represents the one-photon fluorescence intensity, and the horizontal coordinate represents the wavelength.

[0027] Figure 2 Figure 7 is a linear relationship fitting between the logarithm of the one-photon fluorescence intensity of the compound of formula (I) and the logarithm of the viscosity in the glycerol-water system. The vertical coordinate represents the logarithm of the one-photon fluorescence intensity, and the horizontal coordinate represents the logarithm of the viscosity.

[0028] Figure 3 Figure 8 is a two-photon fluorescence emission spectrum of the compound of formula (I) in the glycerol-water system with different mass fractions. The vertical coordinate represents the two-photon fluorescence intensity, and the horizontal coordinate represents the wavelength.

[0029] Figure 4 Figure 9 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 viscosity in the glycerol-water system. The vertical coordinate represents the logarithm of the two-photon fluorescence intensity, and the horizontal coordinate represents the logarithm of the viscosity.

[0030] Figure 5 Figure 6 is a one-photon fluorescence emission spectrum of the compound of formula (I) in the glycerol-water system with different mass fractions. The vertical coordinate represents the one-photon fluorescence intensity, and the horizontal coordinate represents the wavelength.

[0031] Figure 6 Figure 7 is a linear relationship fitting between the logarithm of the one-photon fluorescence intensity of the compound of formula (I) and the logarithm of the viscosity in the glycerol-water system. The vertical coordinate represents the logarithm of the one-photon fluorescence intensity, and the horizontal coordinate represents the logarithm of the viscosity.

[0032] Figure 7 Figure 8 is a two-photon fluorescence emission spectrum of the compound of formula (I) in the glycerol-water system with different mass fractions. The vertical coordinate represents the two-photon fluorescence intensity, and the horizontal coordinate represents the wavelength.

[0033] Figure 8 Figure 9 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 viscosity in the glycerol-water system. The vertical coordinate represents the logarithm of the two-photon fluorescence intensity, and the horizontal coordinate represents the logarithm of the viscosity. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions are further described in detail below through the use of examples. Materials, reagents, or instruments used in the examples, unless their manufacturers are indicated, are commercially available conventional products. For examples in which specific conditions are not indicated, conventional conditions or those recommended by the manufacturer were followed.

[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 and stirred to dissolve. 0.21 g (2.5 mmol) of piperidine was then added and heated under reflux for 10 h. The reaction mixture was then cooled to room temperature and purified by alumina column chromatography (elution reagent was V 二氯甲烷 :V 乙醇 =10:1), to give 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 and stirred to dissolve. 0.17 g (2 mmol) of piperidine was then added and heated under reflux for 14 h. The reaction mixture was then cooled to room temperature and purified by alumina column chromatography (elution reagent was V 二氯甲烷 :V 乙醇 =10:1), to give 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 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) emits almost no fluorescence before interacting with ctDNA. After interacting 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 The linear relationship between the logarithm of the single-photon fluorescence intensity of the compound of formula (I) and the logarithm of the molar concentration of ctDNA is fitted. As can be seen from the figure: the single-photon fluorescence intensity (logI) of the compound of formula (I) and the molar concentration of ctDNA (logc 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 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, 680-1080 nm, 80 MHz, 140 fs) 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) emits almost no fluorescence before interacting with ctDNA. After interacting 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 The linear relationship between the logarithm of the two-photon fluorescence intensity of the compound of formula (I) and the logarithm of the molar concentration of ctDNA is fitted. As can be seen from the figure: the two-photon fluorescence intensity (logI) of the compound of formula (I) and the molar concentration of ctDNA (logc 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 activity 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 were obtained by selecting 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) 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 spectra were measured using an RF-5301PC fluorescence spectrophotometer. Figure 5 The single-photon fluorescence emission spectra of compound (I) in glycerol-water systems with varying mass fractions are shown in Figure 1. As can be seen, the fluorescence intensity of compound (I) increases with increasing glycerol content, i.e., viscosity. From pure water to 99% glycerol, the fluorescence intensity increases by over 80-fold. The emission wavelength is approximately 610 nm, well within the red region.

[0050] The relationship between the single-photon fluorescence intensity of the compound of formula (I) and the ambient viscosity is shown 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 This figure shows a linear relationship between the logarithm of the single-photon fluorescence intensity of the compound of formula (I) and the logarithm of its viscosity in a glycerol-water system. The figure shows a good linear relationship between the single-photon fluorescence intensity (logI) and viscosity (logη), with a linear correlation coefficient of 0.9959, enabling quantitative viscosity measurement.

[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%, and 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, 680-1080 nm, 80 MHz, 140 fs) was used as the pump light source, and a full-spectrum spectrometer (USB4000-FLG) was used to record the two-photon fluorescence spectra. Figure 7 The two-photon fluorescence emission spectra of compound (I) in glycerol-water systems with varying mass fractions are shown in Figure 1. As can be seen, the fluorescence intensity of compound (I) increases with increasing glycerol content, i.e., viscosity. From pure water to 99% glycerol, the fluorescence intensity increases by over 150-fold. The emission wavelength is approximately 608 nm, well within the red 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 This figure shows a linear relationship between the logarithm of the two-photon fluorescence intensity of the compound of formula (I) and the logarithm of its viscosity in a glycerol-water system. The figure shows a good linear relationship between the two-photon fluorescence intensity (logI) and viscosity (logη), with a linear correlation coefficient of 0.9975, enabling quantitative viscosity measurement.

[0057] Example 8 Two-photon fluorescence absorption cross section test of 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 were obtained by selecting the same concentration of fluorescein as the compound of formula (I) at 0.1 mol L -1 Using a solution in sodium hydroxide as a reference, the two-photon fluorescence absorption cross section of the compound of formula (I) in response to different viscosities was calculated according to formula (1). From pure water to 99% glycerol, the two-photon fluorescence absorption cross section of the compound of formula (I) increased from less than 1GM to 41GM, a brightness that enables two-photon fluorescence imaging of viscosity.

[0059] The above description is merely an embodiment of the present application and is not intended to limit the scope of protection of the present application. For those skilled in the art, various modifications and variations of the present application are possible. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection 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-(1 E )-2,1-vinylidene]]bis[1-[3-(triethylammonium)propyl]pyridinium]tetrabromide: (Ⅰ)。 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 prepare the corresponding compound of formula (I); (Ⅱ) (Ⅲ)。 3. Use of the compound according to claim 1 in the preparation of a two-photon fluorescent probe for quantitative detection of DNA and / or viscosity.

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

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