CN116199662B - Visible light and near infrared light rhodamine fluorescent quenching agent and synthesis and application thereof - Google Patents

Abstract

The invention provides a rhodamine fluorescent quenching agent capable of quenching visible light and near infrared light, a synthesis method and application thereof, wherein the fluorescent quenching agent takes dimethylaniline as a ligand of rhodamine, the structural formula of the fluorescent quenching agent is shown as (1), and the fluorescent quenching agent enhances C-N bond torsion between the ligand and a parent body through enhancing intramolecular charge transfer (TICT). In addition, the torsion of benzene ring further increases the non-radiative transition of excited state molecules, achieves the effect of strong fluorescence quenching, and has quantum yield smaller than 0.001. The absorption wavelength is red shifted by the introduction of four aniline-like structures; the torsion of the material also increases molecular relaxation so that the absorption wavelength covers the whole visible light region and the near infrared region, and the half-peak width in water reaches 246nm. The quenching agent has stable quenching capability under different microenvironments and is insensitive to temperature, polarity and the like.

Description

A visible light and near-infrared light rhodamine fluorescence quencher and its synthesis and application

Technical Field

The invention belongs to the field of fluorescence imaging and labeling, and in particular relates to a visible light and near-infrared light rhodamine fluorescence quencher and synthesis and application thereof.

Background Art

The key point in the design of fluorescent probes is how to achieve a strong change in the fluorescence signal before and after the target is recognized. Among them, using a fluorescent quencher to connect with a fluorophore is one of the common methods: the fluorescent probe is in a fluorescence-off state before the target is recognized; after the fluorescent probe recognizes the target, the fluorophore is separated from the fluorescent quencher group, and the fluorescence of the probe is restored to achieve a high fluorescence enhancement multiple. Therefore, the quenching effect of the fluorescent quencher directly determines the quality of such probes.

In biological applications, especially in living organisms, near-infrared fluorescent probe imaging has become the current development direction and difficulty of in vivo biological imaging due to the low biological background, strong penetration and low phototoxicity of near-infrared light. However, at present, the quenching ability of the fluorescent quenchers used in fluorescent probes is limited, and the quenching range is generally within 200nm, and there are few quenchers that can quench fluorescence in the near-infrared region or even longer bands. Therefore, the development of fluorescent quenchers with the ability to quench near-infrared light will inevitably promote the design of fluorescent probes in the near-infrared region, which also requires the quenchers to have good biocompatibility and good biostability.

Summary of the invention

One of the purposes of the present invention is to provide a rhodamine fluorescence quencher with strong stability, good biocompatibility and a quenching range including visible light and near-infrared light. The quantum yield of the fluorescence quencher is less than 0.001 and can effectively quench fluorescence within the absorption wavelength range.

Another object of the present invention is to provide a method for synthesizing a rhodamine fluorescence quencher, which has high yield and good versatility.

The invention provides a rhodamine fluorescence quencher, which quenches the rhodamine fluorescence by utilizing the TICT effect of the rhodamine matrix and the C-N bond of the ligand, the steric hindrance caused by the benzene ring leading to the destruction of the molecular planarity, and the photoinduced charge transfer effect. The introduction of the benzene ring enables the absorption of the quencher to cover the near-infrared region, and the quencher has strong stability and good biocompatibility.

A rhodamine fluorescence quencher, the fluorescence quencher having the following structure:

A method for synthesizing a rhodamine fluorescence quencher with a quenching range covering the visible light region and the near-infrared region. The synthesis route of the fluorescence quencher is as follows:

The specific synthesis steps are as follows:

(1) Synthesis of intermediate fluorescein difluoromethanesulfonate:

Dissolve fluorescein in dichloromethane, add pyridine and trifluoromethanesulfonic anhydride dropwise while stirring in an ice bath; stir at room temperature for 1-4 hours, add deionized water; extract with dichloromethane, collect the organic phase, and dry the organic phase with anhydrous sodium sulfate; remove the solvent from the organic phase under reduced pressure, separate on a silica gel column, use dichloromethane and methanol in a volume ratio of 1 to 100:1 as eluent, remove the solvent, and obtain white solid fluorescein difluoromethanesulfonate.

(2) Synthesis of intermediate N,N-bis(4-dimethylaminophenyl)amine:

Dissolve tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl and potassium tert-butoxide in toluene, heat the reaction solution to 50-100°C under nitrogen protection, and stir for 10-30 minutes; add 4-bromo-N,N-dimethylaniline and 4-(dimethylamino)aniline; heat the reaction solution to 60-110°C, and stir for 4-12 hours; remove the solvent by distillation under reduced pressure, separate by silica gel column, remove the solvent using petroleum ether and ethyl acetate in a volume ratio of 1 to 100:1 as eluent, and obtain light yellow solid N,N-bis(4-dimethylaminophenyl)amine;

(3) Synthesis of rhodamine fluorescence quencher Rho-Q3

Dissolve N,N-bis(4-dimethylaminophenyl)amine, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, cesium carbonate and fluorescein difluoromethanesulfonate in dry dioxane; heat the reaction solution to 60-120°C under nitrogen protection and stir for 12-24 hours; remove the solvent under reduced pressure, separate on a silica gel column, use dichloromethane and methanol in a volume ratio of 1 to 200:1 as an eluent, remove the solvent and obtain a rhodamine fluorescence quencher.

In step (1), the mass ratio of fluorescein, trifluoromethanesulfonic anhydride and pyridine is 1:1-5:1-5; the volume ratio of fluorescein to dichloromethane is 1:10-50 g/mL;

In step (2), the mass ratio of 4-bromo-N,N-dimethylaniline, 4-(dimethylamino)aniline, potassium tert-butoxide, tris(dibenzylideneacetone)dipalladium, and 2-dicyclohexylphosphine-2',4',6'-triisopropylamine is 1:0.5-3:0.5-2:0.05-0.1:0.05-0.1; the mass ratio of 4-bromo-N,N-dimethylaniline to toluene is 1:20-50 g/mL;

In step (3), the mass ratio of fluorescein difluoromethanesulfonate, N,N-bis(4-dimethylaminophenyl)amine, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, and cesium carbonate is 1:1-3:0.1-1:0.1-1:0.5-2; the mass ratio of fluorescein difluoromethanesulfonate to dioxane is 1:10-50 g/mL;

The invention discloses an application of a rhodamine fluorescence quencher in the field of fluorophore quenching.

The above-mentioned rhodamine fluorescence quencher can effectively quench fluorescence in the visible light region to the near-infrared region, has a wide quenching range, and has strong biological stability, and has great application potential in the field of fluorescent probe imaging and labeling. The present invention has the following characteristics:

The fluorescence quencher involved in the present invention has the advantages of high synthesis yield, simple and universal method, etc.

The invention relates to enhancing the TICT between a rhodamine matrix and a ligand, utilizing a photoinduced charge transfer effect to cause fluorescence quenching of rhodamine, with a quantum yield in water of less than 0.001; utilizing a benzene ring to expand the conjugated system of rhodamine, with an absorption spectrum covering the entire visible light region and near-infrared region.

The fluorescence quencher involved in the present invention is insensitive to microenvironments such as temperature, viscosity, polarity, etc., and can ensure the fluorescence quenching ability of the quencher in a biological body.

This fluorescence quencher enhances the torsion of the C-N bond between the ligand and the parent by enhancing intramolecular charge transfer (TICT). In addition, the torsion of the benzene ring further increases the non-radiative transition of the excited state molecule, achieving a strong fluorescence quenching effect with a quantum yield of less than 0.001. The introduction of four aniline-like structures red-shifts the absorption wavelength; its torsion also increases molecular relaxation so that the absorption wavelength covers the entire visible light region and near-infrared region, and the half-peak width in water reaches 246nm. The quencher has a stable quenching ability under different microenvironments and is insensitive to temperature, polarity, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the H NMR spectrum of Rho-Q3 prepared in Example 1;

FIG2 is a high-resolution mass spectrum of the compound Rho-Q3 prepared in Example 1;

FIG3 is a normalized absorption spectrum of the fluorescence quencher Rho-Q3 prepared in Example 1 in different solvents, wherein the abscissa is the wavelength, the ordinate is the normalized intensity, and the concentration of the fluorescent dye is 20 μM.

DETAILED DESCRIPTION

Example 1

(1) Synthesis of intermediate fluorescein difluoromethanesulfonate:

Fluorescein (2.50 g, 7.52 mmol) was dissolved in dichloromethane (30 mL), and pyridine (4.66 g, 60.2 mmol) and trifluoromethanesulfonic anhydride (8.49 g, 30.1 mmol) were added dropwise under stirring in an ice bath; the mixture was stirred at room temperature for 4 h, and 20 mL of deionized water was added; the mixture was extracted with dichloromethane, and the organic phase was collected and dried over anhydrous sodium sulfate; the organic phase was decompressed to remove the solvent, and the mixture was separated by a silica gel column, and the solvent was removed using dichloromethane and methanol in a volume ratio of 5:1 as the eluent, to obtain 3.72 g of white solid fluorescein difluoromethanesulfonate, with a yield of 83%. Its NMR spectrum data are as follows:

¹ H NMR (400MHz, CDCl ₃ ) δ8.08 (d, J＝7.1Hz, 1H), 7.73 (dt, J＝15.0, 7.0Hz, 2H), 7.31 (s, 2H), 7.20 (d, J＝ 7.2Hz, 1H), 7.00 (dd, J＝27.7, 8.2Hz, 4H).

(2) Synthesis of intermediate N,N-bis(4-dimethylaminophenyl)amine

Dissolve tris(dibenzylideneacetone)dipalladium (21 mg, 0.023 mmol) and 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (22 mg, 0.046 mmol) in 5 mL of toluene, heat the reaction solution to 50°C under nitrogen protection, and stir for 10 min; add 4-bromo-N,N-dimethylaniline (231 mg, 1.15 mmol), 4-(dimethylamino)aniline (188 mg, 1.38 mmol), and potassium tert-butoxide (180 mg, 1.61 mmol); heat the reaction solution to 110°C and stir for 4 h; remove the solvent by distillation under reduced pressure, separate by silica gel column, use petroleum ether and ethyl acetate in a volume ratio of 2:1 as eluent, remove the solvent, and obtain 150 mg of light yellow solid N,N-bis(4-dimethylaminophenyl)amine with a yield of 51%. Its nuclear magnetic spectrum data are as follows: ¹ H NMR (400 MHz, DMSO) δ7.16 (s, 1H), 6.84 (d, J＝8.6 Hz, 4H), 6.67 (d, J＝8.8 Hz, 4H), 2.78 (s, 12H).

(3) Synthesis of rhodamine fluorescence quencher Rho-Q3

N,N-bis(4-dimethylaminophenyl)amine (256 mg, 1 mmol), tris(dibenzylideneacetone)dipalladium (30 mg, 0.033 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (50 mg, 0.1 mmol), cesium carbonate (300 mg, 0.94 mmol), and fluorescein difluoromethanesulfonate (200 mg, 0.33 mmol) were dissolved in dry dioxane (10 mL); the reaction solution was heated to 100° C. under nitrogen protection and stirred for 16 h; the solvent was removed under reduced pressure, and the mixture was separated by silica gel column. The solvent was removed using dichloromethane and methanol in a volume ratio of 4:1 as the eluent to obtain 80 mg of a blue solid with a yield of 30%. The H NMR spectrum of the rhodamine fluorescence quencher Rho-Q3 prepared in Example 1 is shown in FIG1 , and the specific data are as follows: ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.05 (d, J = 7.2 Hz, 1H), 7.60 (d, J = 7.2 Hz, 1H), 7.53 (t, J = 7.4 Hz, 1H), 6.92 (m, 23H), 3.00 (s, 24H).

Its high-resolution mass spectrum is shown in Figure 2, and the data are as follows: high-resolution mass spectrum theoretical value C ₅₂ H ₅₁ N ₆ O ₃ [M] ⁺ 807.4017, measured value 807.4012.

After testing, its structure is shown in the above formula Rho-Q3, and its spectral properties are as follows:

Rho-Q3 was dissolved in DMSO solution to prepare a 2 mM stock solution, and test solutions of different concentrations were prepared as needed to detect its fluorescence spectrum and ultraviolet absorption spectrum.

UV absorption and fluorescence emission spectra of Rho-Q3 in different solvents. Each time, 40 μL of Rho-Q3 mother solution was added to 3.96 mL of solvent, and then 4 μL of trifluoroacetic acid was added to prepare a 20 μM fluorescent dye test solution, which was placed in a quartz cuvette and placed in a UV-visible spectrophotometer for absorption spectrum testing and in a fluorescence spectrometer for fluorescence emission spectrum testing.

The normalized UV absorption spectra of Rho-Q3 in different solvents are shown in FIG3 : the maximum value of the maximum absorption wavelength of Rho-Q3 in different solvents is 672 nm, and the maximum value of the absorption half-peak width reaches 228 nm.

The fluorescence quantum yield of Rho-Q3 in various solvents is less than 0.001, which can effectively quench fluorescence.

Example 2

(1) Synthesis of intermediate fluorescein difluoromethanesulfonate:

Fluorescein (2.50 g, 7.52 mmol) was dissolved in dichloromethane (50 mL), and pyridine (7.5 g, 94.9 mmol) and trifluoromethanesulfonic anhydride (10 g, 35.5 mmol) were added dropwise under stirring in an ice bath; the mixture was stirred at room temperature for 1 h, and 30 mL of deionized water was added; the mixture was extracted with dichloromethane, and the organic phase was collected and dried over anhydrous sodium sulfate; the organic phase was decompressed to remove the solvent, and the mixture was separated by a silica gel column, and the solvent was removed using dichloromethane and methanol in a volume ratio of 5:1 as the eluent to obtain 1.45 g of white solid fluorescein difluoromethanesulfonate with a yield of 32%.

(2) Synthesis of intermediate N,N-bis(4-dimethylaminophenyl)amine

Dissolve tris(dibenzylideneacetone)dipalladium (12 mg, 0.013 mmol) and 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (12 mg, 0.025 mmol) in 8 mL of toluene, heat the reaction solution to 60°C under nitrogen protection, and stir for 20 min; add 4-bromo-N,N-dimethylaniline (231 mg, 1.15 mmol), 4-(dimethylamino)aniline (272 mg, 2 mmol), and potassium tert-butoxide (120 mg, 1.07 mmol); heat the reaction solution to 100°C and stir for 6 h; remove the solvent by distillation under reduced pressure, separate on a silica gel column, use petroleum ether and ethyl acetate in a volume ratio of 2:1 as eluent, remove the solvent, and obtain 120 mg of light yellow solid N,N-bis(4-dimethylaminophenyl)amine with a yield of 41%.

(3) Synthesis of rhodamine fluorescence quencher Rho-Q3

N,N-bis(4-dimethylaminophenyl)amine (200 mg, 0.78 mmol), tris(dibenzylideneacetone)dipalladium (20 mg, 0.022 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (20 mg, 0.04 mmol), cesium carbonate (100 mg, 0.31 mmol), and fluorescein difluoromethanesulfonate (200 mg, 0.33 mmol) were dissolved in dry dioxane (5 mL); the reaction solution was heated to 90° C. under nitrogen protection and stirred for 16 h; the solvent was removed under reduced pressure, and the mixture was separated by silica gel column. The solvent was removed using dichloromethane and methanol in a volume ratio of 4:1 as the eluent to obtain 72 mg of a blue solid with a yield of 27%.

Example 3

(1) Synthesis of intermediate fluorescein difluoromethanesulfonate:

Fluorescein (2.50 g, 7.52 mmol) was dissolved in dichloromethane (25 mL), and pyridine (2.5 g, 31.6 mmol) and trifluoromethanesulfonic anhydride (4.23 g, 15.0 mmol) were added dropwise under stirring in an ice bath; the mixture was stirred at room temperature for 3 h, and 15 mL of deionized water was added; the mixture was extracted with dichloromethane, and the organic phase was collected and dried over anhydrous sodium sulfate; the organic phase was decompressed to remove the solvent, and the mixture was separated on a silica gel column, and the solvent was removed using dichloromethane and methanol in a volume ratio of 5:1 as the eluent to obtain 3.5 g of white solid fluorescein difluoromethanesulfonate with a yield of 78%.

(2) Synthesis of intermediate N,N-bis(4-dimethylaminophenyl)amine

Dissolve tris(dibenzylideneacetone)dipalladium (23 mg, 0.025 mmol) and 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (23 mg, 0.048 mmol) in 10 mL of toluene, heat the reaction solution to 70°C under nitrogen protection, and stir for 30 min; add 4-bromo-N,N-dimethylaniline (231 mg, 1.15 mmol), 4-(dimethylamino)aniline (400 mg, 2.93 mmol), and potassium tert-butoxide (450 mg, 4.03 mmol); heat the reaction solution to 90°C and stir for 12 h; remove the solvent by distillation under reduced pressure, separate on a silica gel column, use petroleum ether and ethyl acetate in a volume ratio of 2:1 as eluent, remove the solvent, and obtain 120 mg of light yellow solid N,N-bis(4-dimethylaminophenyl)amine with a yield of 41%.

(3) Synthesis of rhodamine fluorescence quencher Rho-Q3

N,N-bis(4-dimethylaminophenyl)amine (600 mg, 2.34 mmol), tris(dibenzylideneacetone)dipalladium (60 mg, 0.066 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (100 mg, 0.2 mmol), cesium carbonate (400 mg, 1.25 mmol), and fluorescein difluoromethanesulfonate (200 mg, 0.33 mmol) were dissolved in dry dioxane (8 mL); the reaction solution was heated to 80° C. under nitrogen protection and stirred for 24 h; the solvent was removed under reduced pressure, and the product was separated by silica gel column. The solvent was removed using dichloromethane and methanol in a volume ratio of 4:1 as the eluent to obtain 15 mg of a blue solid with a yield of 5.6%.

Claims (7)

1. A visible light and near-infrared light rhodamine fluorescence quencher, characterized in that the quencher structure is as follows: . 2. A method for synthesizing the rhodamine fluorescence quencher according to claim 1, characterized in that it comprises the following steps: (1) Synthesis of intermediate fluorescein difluoromethanesulfonate: Dissolve fluorescein in dichloromethane, add pyridine and trifluoromethanesulfonic anhydride dropwise under stirring in an ice bath; stir at room temperature for 1-4 hours, add deionized water; extract with dichloromethane, collect the organic phase, and dry the organic phase with anhydrous sodium sulfate; remove the solvent from the organic phase under reduced pressure, separate on a silica gel column, use dichloromethane and methanol in a volume ratio of 1-100:1 as eluent, remove the solvent, and obtain white solid fluorescein difluoromethanesulfonate; (2) Synthesis of intermediate N,N-bis(4-dimethylaminophenyl)amine: Dissolve tri(dibenzylideneacetone)dipalladium and 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl in toluene, heat the reaction solution to 50-100°C under nitrogen protection, and stir for 10-30 min; add 4-bromo-N,N-dimethylaniline, 4-(dimethylamino)aniline, and potassium tert-butoxide; heat the reaction solution to 60-110°C and stir for 4-12 h; remove the solvent by vacuum distillation, separate by silica gel column, use petroleum ether and ethyl acetate in a volume ratio of 1-100:1 as eluent, remove the solvent, and obtain N,N-bis(4-dimethylaminophenyl)amine; (3) Synthesis of rhodamine fluorescence quencher Rho-Q3: Dissolve N,N-bis(4-dimethylaminophenyl)amine, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, cesium carbonate, and fluorescein difluoromethanesulfonate in dry dioxane; heat the reaction solution to 60-120°C under nitrogen protection, and stir for 12-24 hours; remove the solvent under reduced pressure, separate on a silica gel column, use dichloromethane and methanol in a volume ratio of 1-200:1 as an eluent, remove the solvent, and obtain a rhodamine fluorescence quencher. 3. The synthesis method according to claim 2, characterized in that: in step (1), the mass ratio of fluorescein, trifluoromethanesulfonic anhydride and pyridine is 1:1-5:1-5; the volume ratio of fluorescein to dichloromethane is 1:10-50 g/mL. 4. The method for synthesizing a rhodamine fluorescence quencher according to claim 2, characterized in that: in step (2), the mass ratio of 4-bromo-N,N-dimethylaniline, 4-(dimethylamino)aniline, potassium tert-butoxide, tris(dibenzylideneacetone)dipalladium, and 2-dicyclohexylphosphine-2',4',6'-triisopropylamine is 1:0.5-3:0.5-2:0.05-0.1:0.05-0.1; the mass ratio of 4-bromo-N,N-dimethylaniline to toluene is 1:20-50 g/mL. 5. The method for synthesizing a rhodamine fluorescence quencher according to claim 2, characterized in that: in step (3), the mass ratio of fluorescein difluoromethanesulfonate, N,N-bis(4-dimethylaminophenyl)amine, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, and cesium carbonate is 1:1-3:0.1-1:0.1-1:0.5-2; the mass ratio of fluorescein difluoromethanesulfonate to dioxane is 1:10-50 g/mL. 6. Use of the rhodamine fluorescence quencher according to claim 1 for preparing a preparation for quenching a fluorophore. 7. The use according to claim 6, characterized in that the quenching range of the quencher includes the visible light region and the near infrared region.