CN116199675B - Rhodamine fluorescence quenching agent and synthesis and application thereof - Google Patents

Abstract

The invention provides a rhodamine fluorescent quenching agent, synthesis and application thereof, wherein the rhodamine fluorescent quenching agent further increases C-N bond torsion and non-radiative transition of excited molecules between a ligand and a parent of rhodamine through enhancing intramolecular charge transfer (TICT), achieves the effect of strong fluorescent quenching, has a structure shown in (1), has quantum yield of less than 0.01 in water, has absorption wavelength of 400-1000 nm, covers a visible light region and a near infrared light region, has half-peak width of 70-150 nm, and is an effective fluorescent quenching agent. The dye can keep stable quenching capacity under different microenvironments, and is insensitive to temperature, polarity and the like.

Description

A class of rhodamine fluorescence quenchers and their synthesis and applications

Technical Field

The invention belongs to the field of fluorescence imaging and labeling, and specifically relates to a rhodamine fluorescence quencher and synthesis and application thereof.

Background Art

In order to achieve a good signal-to-noise ratio before and after the fluorescent probe recognizes the substrate and further reduce the fluorescence background, fluorescent quenchers are widely used in the design of probes to achieve accurate recognition of the substrate. Before the probe recognizes the target, due to the fluorescence resonance energy transfer between the quencher and the fluorophore, the energy that the fluorophore should have released in the form of radiation transition is absorbed by the quencher, making the probe in a fluorescent dark state; after the probe recognizes the target, the fluorescent quencher leaves, the probe is in a fluorescent state, and the opening of fluorescence is used to reflect the information of the target. Therefore, the performance of the quencher, including quantum yield, quenching range, absorption intensity, etc., occupies an important position in the design of such probes.

At present, the commonly used fluorescence quenchers mainly include azo, BHQ series, ATTO series, MPQ series, QSY series, etc. However, these quenchers have narrow absorption wavelengths, limited quenching ability, and cannot quench fluorescence in the near-infrared region. In addition, although the QC-1 quencher has quenching ability in the near-infrared region, due to its parent cyanine dye, it has poor stability and is difficult to perform effective fluorescence quenching for a long time. Therefore, fluorescence quenchers still face many problems such as narrow quenching range, poor stability, and lack of full-spectrum quenching ability. The development of excellent fluorescence quenchers will also be of great significance to the fields of probe design and in vivo detection.

Summary of the invention

One of the purposes of the present invention is to provide a type of rhodamine fluorescence quencher with strong stability, good biocompatibility and a wide quenching range. The quantum yield of this type of fluorescence quencher is less than 0.01 and can effectively quench fluorescence within the absorption wavelength range.

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

The present invention provides a rhodamine fluorescence quencher, which quenches the fluorescence of rhodamine dye by enhancing the TICT effect of the rhodamine matrix and the C-N bond of the ligand, and has the advantages of a wide absorption spectrum and a wide quenching range, is insensitive to the microenvironment, has good stability, and is easy to be derived and connected to a fluorophore.

A type of rhodamine fluorescence quencher, this series of fluorescence quenchers has the following structure:

for One of them.

A method for synthesizing a type of rhodamine fluorescence quencher with strong stability, good biocompatibility and a wide quenching range. The synthesis route of this series of fluorescence quenchers 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 rhodamine fluorescence quencher

Dissolve aromatic amine compounds, 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;

The aromatic amine compounds are 1,2,3,4-tetrahydroquinoline, 2,3,4,5-tetrahydro-1H-benzo[b]azepine, and 1-methyl-1,2,3,4-tetrahydroquinoxaline.

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 fluorescein difluoromethanesulfonate, aromatic amine compound, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, and cesium carbonate is 1:0.5-3:0.1-1:0.1-1:0.5-2; the mass ratio of fluorescein difluoromethanesulfonate to the volume ratio of dioxane is 1:50-200 g/mL.

A class of rhodamine fluorescence quenchers is used 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, is insensitive to the microenvironment, has strong stability, is easy to derive and connect fluorophores, 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 TICT for enhancing the C-N bond between the rhodamine parent and the ligand, resulting in fluorescence quenching of the dye, with a quantum yield of less than 0.01 in water and an absorption spectrum covering the entire visible light region and the 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.

The present invention provides a rhodamine fluorescence quencher and its synthesis and application. The fluorescence quencher further increases the C-N bond torsion between the ligand and the parent of rhodamine and the non-radiative transition of the excited state molecule by enhancing intramolecular charge transfer (TICT), thereby achieving a strong fluorescence quenching effect. The structure is shown in (1). The quantum yield of the quencher in water is less than 0.01, the absorption wavelength is 400nm-1000nm, covering the visible light region and the near-infrared light region, and the half-peak width is 70nm-150nm. The dye can maintain a stable quenching ability under different microenvironments and is insensitive to temperature, polarity, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the H NMR spectrum of Rho-6P prepared in Example 1.

Figure 2 shows the H NMR spectrum of Rho-7P prepared in Example 2.

Figure 3 shows the hydrogen spectrum of the NMR spectrum of Rho-n6P prepared in Example 3.

FIG4 is a normalized absorption spectrum of the fluorescence quencher Rho-6P 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 2 μM.

FIG5 is a fluorescence emission spectrum of the fluorescence quencher Rho-6P prepared in Example 1 in different solvents, wherein the abscissa is the wavelength, the ordinate is the fluorescence intensity, and the concentration of the fluorescent dye is 2 μM.

FIG6 is a normalized absorption spectrum of the fluorescence quencher Rho-7P prepared in Example 2 in different solvents, wherein the abscissa is the wavelength, the ordinate is the normalized intensity, and the concentration of the fluorescent dye is 2 μM.

FIG7 is a fluorescence emission spectrum of the fluorescence quencher Rho-7P prepared in Example 2 in different solvents, wherein the abscissa is the wavelength, the ordinate is the fluorescence intensity, and the concentration of the fluorescent dye is 2 μM.

Figure 8 is a normalized absorption spectrum of the fluorescence quencher Rho-n6P prepared in Example 3 in different solvents, where the horizontal axis is wavelength and the vertical axis is normalized intensity, and the concentration of the fluorescent dye is 2 μM.

FIG9 is a fluorescence emission spectrum of the fluorescence quencher Rho-n6P prepared in Example 3 in different solvents, wherein the abscissa is the wavelength, the ordinate is the fluorescence intensity, and the concentration of the fluorescent dye is 2 μM.

DETAILED DESCRIPTION

Example 1

Synthesis of Rho-6P

(1) Synthesis of intermediate fluorescein difluoromethanesulfonate:

Dissolve fluorescein (2.50g, 7.52mmol) in dichloromethane (30mL), add pyridine (4.66g, 60.2mmol) and trifluoromethanesulfonic anhydride (8.49g, 30.1mmol) dropwise under stirring in an ice bath; stir at room temperature for 4h, add 20mL of 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 5:1 as the eluent, remove the solvent, and obtain 3.72g 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 rhodamine fluorescence quencher Rho-6P

1,2,3,4-tetrahydroquinoline (175 mg, 1389 mmol), tris(dibenzylideneacetone)dipalladium (15.89 mg, 17.3 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (24.0 mg, 50.8 mmol), cesium carbonate (159 mg, 487 mmol), fluorescein difluoromethanesulfonate (100 mg, 173 mmol) were dissolved in dry dioxane (10 mL); the reaction solution was heated to 110 ° C under nitrogen protection and stirred for 16 h; the solvent was removed under reduced pressure, separated by silica gel column, and the solvent was removed with dichloromethane and methanol in a volume ratio of 10:1 as eluent to obtain 24 mg of white solid with a yield of 25%. The hydrogen NMR spectrum of the rhodamine fluorescence quencher Rho-6P prepared in Example 1 is shown in Figure 1, and the specific data are as follows:

¹ H NMR (400MHz, DMSO) δ8.02(d,J＝7.6Hz,1H),7.82(t,J＝7.5Hz,1H),7.74(t,J＝7.5Hz,1H),7.37(d, J＝7.4Hz,1H),7.09(d,J＝7.4Hz,2H),7.06(d,J＝2.2Hz,2H),6.97(m,6H),6.80(m,2H),6.65(d, J＝8.7Hz,2H),3.62(t,J＝5.8Hz,4H),2.72(t,J＝6.2Hz,4H),1.91(m,4H).

The high-resolution mass spectrum data are as follows: high-resolution mass spectrum theoretical value C ₃₈ H ₃₁ N ₂ O ₃ [M] ⁺ 563.2329, measured value 563.2333.

After testing, its structure is shown in the above formula Rho-6P, and its spectral properties are as follows:

Rho-6P 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-6P in different solvents. Take 4 μL of Rho-6P mother solution and add it to 4 mL of solvent each time, then add 4 μL of trifluoroacetic acid to prepare a 2 μM fluorescent dye test solution, place it in a quartz cuvette, put it in a UV-visible spectrophotometer for absorption spectrum test, and put it in a fluorescence spectrometer for fluorescence emission spectrum test.

The normalized UV absorption spectra of Rho-6P in different solvents are shown in FIG4 : the maximum value of the maximum absorption wavelength of Rho-6P in different solvents is 619 nm, and the maximum value of the absorption half-peak width reaches 92 nm.

The fluorescence emission spectra of Rho-6P in different solvents are shown in Figure 5: Rho-6P has no fluorescence emission in different solvents.

The fluorescence quantum yield of Rho-6P in various solvents is less than 0.001, which can effectively quench fluorescence.

Example 2

Synthesis of Rho-7P

(1) Synthesis of intermediate fluorescein difluoromethanesulfonate:

Dissolve fluorescein (2.50 g, 7.52 mmol) in dichloromethane (50 mL), add pyridine (7.5 g, 94.9 mmol) and trifluoromethanesulfonic anhydride (10 g, 35.5 mmol) dropwise while stirring in an ice bath; stir at room temperature for 1 h, add 30 mL of deionized water; extract with dichloromethane, collect the organic phase, and dry the organic phase over anhydrous sodium sulfate; remove the solvent from the organic phase under reduced pressure, separate on a silica gel column, and use dichloromethane and methanol in a volume ratio of 5:1 as the eluent to remove the solvent, and obtain 1.45 g of white solid fluorescein difluoromethanesulfonate with a yield of 32%. (2) Synthesis of rhodamine fluorescence quencher Rho-7P

2,3,4,5-tetrahydro-1H-benzo[b]azepine (58mg, 0.402mmol), tris(dibenzylideneacetone)dipalladium (12mg, 0.0134mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (19mg, 0.040mmol), cesium carbonate (122mg, 0.375mmol), fluorescein difluoromethanesulfonate (80mg, 0.134mmol) were dissolved in dry dioxane (10mL); the reaction solution was heated to 100°C under nitrogen protection and stirred for 18h; the solvent was removed under reduced pressure, separated by silica gel column, and the solvent was removed with dichloromethane and methanol in a volume ratio of 10:1 as eluent to obtain 46mg of white solid with a yield of 58%. The hydrogen NMR spectrum of the rhodamine fluorescence quencher Rho-7P prepared in Example 2 is shown in Figure 2, and the specific data are as follows:

¹ H NMR (400MHz, MeOD) δ8.04(m,1H),7.61(m,2H),7.39(dd,J＝6.1,3.0Hz,2H),7.32(m,4H),7.18(m,3H ),7.06(d,J＝9.5Hz,2H),6.69(m,4H),3.84(s,4H),2.65(m,4H),1.93(dd,J＝10.3,5.1Hz,4H),1.75 (s,4H).

The high-resolution mass spectrum data are as follows: high-resolution mass spectrum theoretical value C ₄₀ H ₃₅ N ₂ O ₃ [M] ⁺ 591.2642, measured value 591.2654.

After testing, its structure is shown in the above formula Rho-7P, and its spectral properties are as follows:

Rho-7P 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-7P in different solutions. Each time, 4 μL of Rho-7P mother solution was added to 4 mL of solvent, and then 4 μL of trifluoroacetic acid was added to prepare a 2 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-7P in different solutions are shown in FIG6 : the maximum value of the maximum absorption wavelength of Rho-7P in different solvents is 560 nm, and the maximum value of the absorption half-peak width reaches 62 nm.

The fluorescence emission spectra of Rho-7P in different solvents are shown in Figure 7: Rho-7P has no fluorescence emission in different solvents.

The fluorescence quantum yield of Rho-7P in various solvents is less than 0.01, which can effectively quench fluorescence.

Example 3

Synthesis of Rho-n6P

(1) Synthesis of intermediate fluorescein difluoromethanesulfonate:

Dissolve fluorescein (2.50 g, 7.52 mmol) in dichloromethane (25 mL), add pyridine (2.5 g, 31.6 mmol) and trifluoromethanesulfonic anhydride (4.23 g, 15.0 mmol) dropwise while stirring in an ice bath; stir at room temperature for 3 h, add 15 mL of deionized water; extract with dichloromethane, collect the organic phase, and dry the organic phase over anhydrous sodium sulfate; remove the solvent from the organic phase under reduced pressure, separate on a silica gel column, and use dichloromethane and methanol in a volume ratio of 5:1 as the eluent to remove the solvent, and obtain 3.5 g of white solid fluorescein difluoromethanesulfonate with a yield of 78%. (2) Synthesis of rhodamine fluorescence quencher Rho-n6P

1,2,3,4-tetrahydroquinoline (182 mg, 1.22 mmol), tris(dibenzylideneacetone)dipalladium (28.4 mg, 0.031 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (44.3 mg, 0.093 mmol), cesium carbonate (283 mg, 0.87 mmol), fluorescein difluoromethanesulfonate (182 mg, 0.31 mmol) were dissolved in dry dioxane (15 mL); the reaction solution was heated to 110°C under nitrogen protection and stirred for 24 h; the solvent was removed under reduced pressure, separated by silica gel column, and the solvent was removed with dichloromethane and methanol in a volume ratio of 4:1 as eluent to obtain 44 mg of white solid with a yield of 24%. The hydrogen NMR spectrum of the rhodamine fluorescence quencher Rho-n6P prepared in Example 3 is shown in Figure 3, and the specific data are as follows:

¹ H NMR (400MHz, DMSO) δ8.01(d,J＝7.6Hz,1H),7.81(td,J＝7.5,0.9Hz,1H),7.73(t,J＝7.4Hz,1H),7.35( d,J＝7.6Hz,1H),6.89(m,8H),6.72(d,J＝8.2Hz,2H),6.61(d,J＝8.8Hz,2H),6.53(t,J＝7.6Hz, 2H), 3.73 (m, 4H), 3.25 (t, J = 4.9Hz, 4H), 2.87 (s, 6H).

The high-resolution mass spectrum data are as follows: high-resolution mass spectrum theoretical value C ₃₈ H ₃₃ N ₄ O ₃ [M+H] ⁺ 593.2547, measured value 593.2547.

After testing, its structure is shown in the above formula Rho-n6P, and its spectral properties are as follows:

Rho-n6P 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-n6P in different solutions. Each time, 4 μL of Rho-n6P mother solution was added to 4 mL of solvent, and then 4 μL of trifluoroacetic acid was added to prepare a 2 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-n6P in different solutions are shown in Figure 8: the maximum value of the maximum absorption wavelength of Rho-n6P in different solvents is 666 nm, and the maximum absorption half-peak width reaches 222 nm.

The fluorescence emission spectra of Rho-n6P in different solvents are shown in Figure 9: Rho-n6P has no fluorescence emission in different solvents.

The fluorescence quantum yield of Rho-n6P in various solvents is less than 0.01, which can effectively quench fluorescence.

Claims (5)

1. A rhodamine fluorescence quencher, characterized in that: the rhodamine fluorescence quencher has the following structure: , for , , One or two or more of the following. 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 fluorescein difluoromethanesulfonate; (2) Synthesis of rhodamine fluorescence quencher Dissolve aromatic amine compounds, 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 h; 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 eluent, remove the solvent, and obtain a rhodamine fluorescence quencher; The aromatic amine compound is one or more of 1,2,3,4-tetrahydroquinoline, 2,3,4,5-tetrahydro-1H-benzo[b]azepine, and 1-methyl-1,2,3,4-tetrahydroquinoxaline. 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 synthesis method according to claim 2, characterized in that: in step (2), the mass ratio of fluorescein difluoromethanesulfonate, aromatic amine compound, tris(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl, and cesium carbonate is 1:0.5-3:0.1-1:0.1-1:0.5-2; the mass ratio of fluorescein difluoromethanesulfonate to dioxane is 1:50-200 g/mL. 5. Use of the rhodamine fluorescence quencher according to claim 1 in a rhodamine fluorophore quenching process.