CN107090191A - One class rhodamine fluorescent dyes and preparation method thereof - Google Patents

Abstract

The invention discloses a class of rhodamine fluorescent dyes and a preparation method thereof. Based on a classic rhodamine synthesis method, through reasonable structural modification, chemical groups with different functions are introduced into the rhodamine matrix to increase the conjugation effect and change the color enhancement. Groups, such as: hydroxyl, diethylamino, azabicyclic and other structures. The novel rhodamine dyes prepared by the present invention have larger emission wavelength and larger light quantum yield, and also have more reaction sites, which makes the follow-up reaction more convenient; and has a moderate Stokes shift, which can In the subsequent fluorescence detection, the interference of the excitation wavelength is eliminated, so that the analysis results are more accurate.

Description

A kind of rhodamine fluorescent dye and preparation method thereof

technical field

The invention belongs to the technical field of organic synthesis, and in particular relates to a class of rhodamine fluorescent dyes and a preparation method thereof.

Background technique

Rhodamine-like compounds are basic xanthene dyes based on xanthene. Due to their special structure and corresponding fluorescent properties, rhodamine-like fluorescent dyes have become a relatively extensive research topic in the field of chemical and biological analysis. There have been many reports on the synthesis, ionization, structure, optical properties and analysis of rhodamine fluorescent dyes. Compared with other commonly used fluorescent dyes, rhodamine-based fluorescent dyes have the advantages of good photostability, insensitivity to pH, wide wavelength range and high fluorescence quantum yield, so they are widely used in pharmacology, physiology, etc. , Molecular biology, cell biology, molecular genetics, environmental chemistry, single molecule detection, information science, fluorescent labeling, laser dyes, etc., are the most commonly used fluorescent dyes in biotechnology fields such as analytical chemistry and biomedical science.

Rhodamine derivatives are the most widely used dyes in the field of organic small molecule probes. Rhodamine 6G and Rhodamine B have the advantages of large molar absorptivity, high fluorescence quantum yield, and good photostability. The specific reaction or non-covalent binding of bright fluorescent probes can induce the switch of the rhodamine lactam helix, causing the off-on change of the fluorescent signal, so as to realize the fluorescent detection of the target. This mechanism has been widely used in rhodamine Design of bright small molecule fluorescent probes. Using this principle to construct fluorescent probes has become a research hotspot in the field of sensing.

As the scope of application becomes wider and wider, the research on rhodamine-based fluorescent dyes develops rapidly and has received more attention. A large number of literatures have reported that fluorescent probes based on rhodamine spirocyclic derivatives are used for the detection of various targets. Such as metal ions (Cu ²⁺ , Hg ²⁺ , Fe ³⁺ , Zn ²⁺ , Cr ³⁺ , Ag ⁺ , Au ⁺ , Pb ²⁺ and Pd ²⁺ ), anions (OCl ^- , CN ^- and P ₂ O ₇ ^4- ), active oxygen species/active nitrogen species, thiol compounds, pH value and temperature, etc. Based on the broad research space and application value of rhodamine fluorescent dyes, it is necessary to design and synthesize a series of new rhodamine-based fluorescent dyes.

Contents of the invention

The object of the present invention is to provide a class of novel rhodamine fluorescent dyes with greater photon quantum yield and moderate Stokes shift, and the present invention also provides a preparation method of the rhodamine fluorescent dyes.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A class of rhodamine fluorescent dyes, such fluorescent dyes are any one of dye 1, dye 2 and dye 3, wherein the structural formulas of dye 1, dye 2 and dye 3 are respectively as follows:

The preparation method of above-mentioned rhodamine fluorescent dye comprises the following steps:

(1) Resorcinol reacts with phthalic anhydride to obtain compound 1; 3-hydroxyl-N, N-diethylaniline reacts with phthalic anhydride to obtain compound 2; m-aminophenol reacts with 1-bromo-3 -Chloropropane reacts to obtain compound 3, and compound 3 reacts with phthalic anhydride to obtain compound 4; the chemical formulas of compound 1, compound 2, compound 3 and compound 4 are as follows:

(2) Using methanesulfonic acid as a catalyst and toluene as a reaction solvent, compound 1, compound 2 and compound 4 were respectively reacted from acenaphthylphenol to obtain dye 1, dye 2 and dye 3.

Preferably, the preparation method of compound 1 in step (1) is as follows: dissolving resorcinol and phthalic anhydride in toluene, stirring and refluxing until a solid is formed, extracting the solid by suction filtration, and recrystallizing with methanol to obtain ; Among them, the dosage of resorcinol in toluene is 2~3mol/L, and the molar ratio of resorcinol to phthalic anhydride is 1:1.

Preferably, the preparation method of compound 2 in step (1) is as follows: dissolve 3-hydroxy-N,N-diethylaniline and phthalic anhydride in toluene, stir and reflux until a solid is formed, and extract the solid by suction filtration , recrystallized with methanol to get that; wherein, the dosage of 3-hydroxy-N, N-diethylaniline in toluene is 2～3mol/L, 3-hydroxy-N, N-diethylaniline and o- The molar ratio of phthalic anhydride is 1:1.

Preferably, the preparation method of compound 3 in step (1) is as follows: adding m-aminophenol, sodium bicarbonate and 1-bromo-3-chloropropane to dimethylformamide, stirring and mixing, and stirring at 65-75 Continue to stir at ℃, TLC detects that compound 3 has been generated in the reaction, and then further separates it by column chromatography to obtain it; The molar ratio of sodium hydrogen to 1-bromo-3-chloropropane is 1:3~3.5:2.5~3, and the eluent used for column chromatography is petroleum ether:ethyl acetate=20:1.

Preferably, the preparation method of compound 4 in step (1) is as follows: dissolving compound 3 and phthalic anhydride in toluene, stirring and refluxing until a solid is formed, extracting the solid by suction filtration, and recrystallizing with methanol to obtain; wherein , the dosage of compound 3 in toluene is 2-3mol/L, and the molar ratio of compound 3 to phthalic anhydride is 1:1.

Preferably, the preparation method of acenaphthylphenol in step (2) comprises the following steps:

(1) Add phosphorus oxychloride and N,N-dimethylformamide to acenaphthene in sequence, stir and mix well, then raise the temperature to 90-95°C, and continue to stir for 2.5-3.5 hours to obtain a reaction solution; add the reaction solution to ice In the water mixture, a viscous solid is formed, and after suction filtration and vacuum drying, the crude product of 5-acenaphthylaldehyde is obtained; wherein, the molar ratio of acenaphthylene, phosphorus oxychloride and N,N-dimethylformamide is 1:2～2.5 : 5~6;

(2) The crude product of 5-acenaphthylaldehyde obtained in step (1) is separated by column chromatography, and vacuum-dried to obtain the pure product of 5-acenaphthylaldehyde; wherein, the eluent during column chromatography separation is petroleum ether: ethyl acetate=38～42 :1;

(3) Add pure 5-acenaphthylenaldehyde, selenium dioxide, hydrogen peroxide and glacial acetic acid to dichloromethane, stir at 20-30°C for 35-40 hours, filter the filtrate, let it stand for stratification, and take the organic The solvent was evaporated from the layer, and dissolved in methanol solution of potassium hydroxide in an ice-bath environment, then hydrolyzed at room temperature for 2.5 to 3.5 hours, acidified by adding hydrochloric acid, extracted with ethyl acetate, and washed with ethyl acetate. Dry to obtain the crude product of acenaphthylphenol; wherein, the dosage of pure 5-acenaphthylaldehyde in dichloromethane is 80～100g/L, the dosage of selenium dioxide is 2～3g/L, the dosage of hydrogen peroxide The dosage is 1.6～2mol/L, the dosage of glacial acetic acid is 0.29～0.35mol/L, and the concentration of potassium hydroxide in the methanol solution of potassium hydroxide is 0.13～0.16g/mL;

(4) The crude product of acenaphthylphenol obtained in step (3) is separated by column chromatography and dried in vacuum to obtain the pure product of acenaphthylphenol; wherein, the eluent is petroleum ether: ethyl acetate = 28-32:1.

Preferably, the preparation method of dye 1 in step (2) is as follows: add compound 1, acenaphthylphenol and methanesulfonic acid to toluene, continue stirring at 60-70°C for 25-35 minutes, and extract the reaction solution with ethyl acetate , then wash the ethyl acetate extract phase with water, and dry to obtain the crude product of dye 1; the crude product of dye 1 is further separated by column chromatography, and dried in vacuum to obtain the pure product of dye 1; wherein, the dosage of compound 1 in toluene is 0.09～1.0mol /L, the molar ratio of compound 1, acenaphthylphenol and methanesulfonic acid is 1:1:26-27, and the eluent used in column chromatography separation is dichloromethane:methanol=48-52:1.

Preferably, the preparation method of dye 2 in step (2) is as follows: add compound 2, acenaphthylphenol and methanesulfonic acid to toluene, continue stirring at 60-70°C for 25-35 minutes, and extract the reaction solution with ethyl acetate , then wash the ethyl acetate extract phase, and dry to obtain the crude product of dye 2; the crude product of dye 2 is further separated by column chromatography, and dried in vacuum to obtain the pure product of dye 2; wherein, the dosage of compound 2 in toluene is 0.18～0.19mol /L, the molar ratio of compound 2, acenaphthylphenol and methanesulfonic acid is 1:1.0-1.1:8-9, and the eluent used in column chromatography separation is dichloromethane:methanol=38-42:1.

Preferably, the preparation method of dye 3 in step (2) is as follows: add compound 4, acenaphthylphenol and methanesulfonic acid to toluene, continue stirring at 60-70°C for 25-35 minutes, and extract the reaction solution with dichloromethane , and then wash the dichloromethane extract phase and dry to obtain the crude product of dye 3; the crude product of dye 3 is further separated by column chromatography and dried in vacuum to obtain the pure product of dye 3; wherein, the dosage of compound 4 in toluene is 0.035～0.045mol /L, the molar ratio of compound 4, acenaphthylphenol and methanesulfonic acid is 1:2~3:64~66, and the eluent used in column chromatography separation is dichloromethane:methanol=78~82:1.

Application of the above-mentioned rhodamine fluorescent dye in fluorescence detection and pH probe.

The synthetic route of above-mentioned acenaphthylphenol is as follows:

The synthetic route of above-mentioned rhodamine class fluorescent dye is as follows:

Various raw materials used in the present invention are common commercially available products, or are obtained by methods known to those skilled in the art or methods disclosed in the prior art.

Compared with the classic rhodamine dyes, the three novel rhodamine-based dyes of the present invention have better performance: (1) more reaction sites, making subsequent reactions more convenient; (2) larger emission wavelengths, dyes The fluorescence emission wavelength of the third is 606nm, which belongs to the near-infrared region, and can be excellently applied to the dyeing and analysis of living cells; (3) Greater light quantum yield, inheriting and developing rhodamine-based fluorescent dyes with high light quantum yield Features; (4) Moderate Stokes shift can eliminate the interference of excitation wavelength in subsequent fluorescence detection, making the analysis results more accurate.

Description of drawings

Fig. 1 is the nuclear magnetic hydrogen spectrogram of dye 1;

Fig. 2 is the carbon nuclear magnetic spectrogram of dye 1;

Fig. 3 is the mass spectrogram of dye 1;

Fig. 4 is the nuclear magnetic hydrogen spectrogram of dye 2;

Fig. 5 is the carbon nuclear magnetic spectrogram of dye 2;

Fig. 6 is the mass spectrogram of dye 2;

Fig. 7 is the nuclear magnetic hydrogen spectrogram of dye 3;

Fig. 8 is the mass spectrogram of dye 3;

Fig. 9 is the photograph of the ultraviolet-visible absorption spectrum of dyestuff 1, dyestuff 2 and dyestuff 3 and the color shown under sunlight thereof;

Fig. 10 is the photo of the fluorescent spectrum of dye 1, dye 2 and dye 3 and the color shown under the 365nm ultraviolet lamp;

Fig. 11 is the ultraviolet-visible spectrum and fluorescence spectrum of dye 1 at different pH;

Fig. 12 is the ultraviolet-visible spectrum and fluorescence spectrum of dye 2 at different pH;

Fig. 13 is the UV-visible spectrum and fluorescence spectrum of dye 3 at different pHs.

detailed description

The present invention will be described in further detail below through preferred embodiments, but the protection scope of the present invention is not limited thereto.

Example 1

(1) Preparation of acenaphthylphenol

Add 5.0g (32mmol) of acenaphthene to a 100mL three-necked flask, then slowly add 6.5mL of phosphorus oxychloride (POCl ₃ ) into the three-necked flask with a constant pressure funnel (dropping is completed within 15 minutes), then add 13.5mL of N, N-dimethylformamide (DMF), after stirring for 5 minutes, the temperature was slowly raised to 90-95 ° C, and the stirring was continued for 3 hours to obtain a reaction solution; the reaction solution was added to 270 mL of ice-water mixture, and a large amount of viscous solid appeared after stirring, and the Suction filtration under reduced pressure and vacuum drying to obtain the crude product of acenaphthylaldehyde; then, it was separated by column chromatography, the eluent was petroleum ether: ethyl acetate = 40:1, and vacuum dried to obtain 2.759 g of light yellow needle-like solid, namely 5-acenaphthyl Pure aldehyde, the yield is 46.74%.

Add 11 mL of dichloromethane and 1.0 g of 5-acenaphthylenal to a 50 mL round bottom flask, then add 30 mg of selenium dioxide (SeO ₂ ) and 2 mL of 30% hydrogen peroxide (H ₂ O ₂ ), then add 4 drops of ice Acetic acid (about 0.2mL), stirred in a water bath at 25°C for 38 hours, filtered to take the filtrate, stood to separate layers, took the organic layer and rotary evaporated the solvent, and dissolved it in methanol solution of potassium hydroxide (from 0.85g Potassium hydroxide was dissolved in 6 mL of methanol), and then hydrolyzed at room temperature for 3 hours, then acidified by adding 5 mL of dilute hydrochloric acid with a mass fraction of 10%, extracted with ethyl acetate, washed with deionized water and extracted with ethyl acetate phase twice, and then dried with an appropriate amount of anhydrous sodium sulfate for 30 minutes to obtain crude acenaphthylphenol; the crude product of acenaphthylphenol was separated by column chromatography, and the eluent was petroleum ether: ethyl acetate = 30:1, and vacuum-dried to obtain 573mg shallow Yellow needle-like solid, that is, pure acenaphthylphenol, the yield is 61.3%. ¹ HNMR (400MHz, CDCl ₃ ) δ7.72(d, J=8.3Hz, 1H), 7.46–7.40(m, 1H), 7.28(d, J=6.8Hz, 1H), 7.08(d, J=7.3 Hz, 1H), 6.79 (d, J = 7.3Hz, 1H), 5.08 (s, 1H), 3.43–3.38 (m, 2H), 3.31 (dd, J = 8.2, 5.0Hz, 2H).

The synthetic route is as follows:

(2) Preparation of compound 1

Add 40mL of toluene (PhMe) to a 150mL round bottom flask, then add 11.0g (0.1mol) of resorcinol and 14.8g (0.1mol) of phthalic anhydride, stir to dissolve, stir and reflux for 5 hours, and gradually generate a solid, The solid was removed by suction filtration, and recrystallized from methanol to obtain an orange solid.

The synthetic route is as follows:

(3) Preparation of Dye 1

Add 3mL of toluene to a 50mL round bottom flask, then add 75mg (0.29mol) of compound 1, 50mg (0.29mol) of pure acenaphthylphenol and 0.5mL of methanesulfonic acid (MeSO ₃ H), heat in an oil bath at 65°C, and stir For 30 minutes, the reaction solution was extracted with ethyl acetate, and the ethyl acetate extract phase was washed twice with water and dried with anhydrous Na ₂ SO ₄ to obtain the crude product of dye 1; the crude product of dye 1 was further separated by column chromatography, and the eluent was Dichloromethane:methanol (DCM:MeOH)=50:1, the solvent was evaporated by rotary evaporation, and dried in vacuo to obtain 85 mg of a dark red solid (yield, 74%), namely the pure dye 1.

The synthetic route is as follows:

The H NMR spectrum of dye 1 is shown in Figure 1, ¹ H NMRδ _H (400MH _z , CDCl ₃ )8.11(1H,d,J 8.2),8.07(1H,dd,J 6.3,1.8),7.68–7.62 (2H,m),7.62–7.56(1H,m),7.41(1H,d,J 6.8),7.14(1H,dd,J 6.2,1.5),6.92(1H,d,J 2.5),6.73(1H ,d,J 8.6),6.58(1H,dd,J 8.6,2.5),6.51(1H,s),3.38(2H,dd,J 11.7,5.3),3.24–3.17(2H,m); The carbon NMR spectrum is shown in Figure 2, δ _C (101MH _z , CDCl ₃ ) 169.79, 157.27, 154.22, 152.10, 145.66, 140.92, 135.10, 129.65, 128.46, 126.48, 125.04, 124.02, 121.60, 1116.849 113.98, 112.31, 111.36, 103.14, 30.84, 29.62. The pure dye 1 was analyzed by mass spectrometry, and the results are shown in Figure 3, indicating that dye 1 was synthesized.

(4) Preparation of Compound 2

Add 40mL of toluene to a 150mL round bottom flask, then add 16.5g (0.1mol) of 3-hydroxy-N,N-diethylaniline and 14.8g (0.1mol) of phthalic anhydride, stir to dissolve, stir and reflux for 5 hours , gradually forming a solid, which was removed by suction filtration and recrystallized with methanol to obtain an orange solid.

The synthetic route is as follows:

(5) Preparation of Dye 2

Add 10 mL of toluene to a 50 mL round bottom flask, then add 580 mg (1.85 mol) of compound 2, 341 mg (2.0 mol) of pure acenaphthylphenol and 1.0 mL of methanesulfonic acid, heat in an oil bath at 65 ° C, stir for 30 minutes, and react The solution was extracted with ethyl acetate, and the ethyl acetate extract phase was washed twice with water and dried with anhydrous Na ₂ SO ₄ to obtain the crude product of dye 2; the crude product of dye 2 was further separated by column chromatography, eluent: DCM: MeOH=40 : 1, the solvent was evaporated by rotary evaporation, and dried in vacuo to obtain 305.7 mg (yield, 37%) of a dark red solid, namely the pure dye 2.

The synthetic route is as follows:

The H NMR spectrum of dye 2 is shown in Figure 4, ¹ H NMR (400MHz, CDCl ₃ ) δ8.17(d, J=8.2Hz, 1H), 8.05(dd, J=6.5, 1.4Hz, 1H) ,7.62(tdd,J=19.1,10.1,4.8Hz,3H),7.39(d,J=6.8Hz,1H),7.19–7.15(m,1H),6.63(dd,J=8.1,5.7Hz,2H ),6.51(s,1H),6.41(dd,J＝8.9,2.6Hz,1H),5.30(s,2H),3.47–3.28(m,6H),3.24–3.11(m,2H),1.21( t, J=7.1Hz, 6H); the carbon nuclear magnetic resonance spectrum of dye 2 is shown in Figure 5, ¹³ C NMR (101MHz, CDCl ₃ ) δ169.55, 164.04, 154.11, 151.75, 151.45, 145.67, 144.42, 141.24, 140.38 ,135.20,133.26,129.81,129.21,128.59,127.59,126.16,125.12,124.11,121.85,121.70,117.84,117.47,116.39,116.31,113.84,110.38,83.42,77.34,77.23,77.02,76.70,30.84,29.64,22.70 , 14.85, 14.21. The pure dye 2 was analyzed by mass spectrometry, and the results are shown in Figure 6, indicating that dye 2 was synthesized.

(6) Preparation of Compound 3

Add 6mL of dimethylformamide to a 100mL round bottom flask, then add 1.3g of m-aminophenol, 3.2g of sodium bicarbonate and 3.4mL of 1-bromo-3-chloropropane, stir and mix, and stir at 70°C, thin Compound 3 was detected by layer chromatography, and further separated by column chromatography, using petroleum ether: ethyl acetate = 20:1 as the eluent, to obtain compound 3.

The synthetic route is as follows:

(7) Preparation of Compound 4

Add 40 mL of toluene to a 150 mL round bottom flask, then add 18.9 g (0.1 mol) of compound 3, 14.8 g (0.1 mol) of phthalic anhydride, stir to dissolve, stir and reflux for 5 hours, gradually form a solid, remove the solid by suction filtration, Recrystallized from methanol to obtain a dark red solid.

The synthetic route is as follows:

(8) Preparation of Dye 3

Add 3mL of toluene to a 50mL round bottom flask, then add 40mg (0.119mol) of compound 4, 50mg (0.29mol) of pure acenaphthylphenol and 0.5mL of methanesulfonic acid, heat in an oil bath at 65°C, stir for 30 minutes, and the reaction The solution was extracted with ethyl acetate, and the ethyl acetate extract phase was washed twice with water, and dried with anhydrous Na ₂ SO ₄ to obtain the crude product of dye 3; the crude product of dye 3 was further separated by column chromatography, eluent: DCM: MeOH=80 : 1, the solvent was evaporated by rotary evaporation, and vacuum-dried to obtain 50 mg (yield, 89.3%) of a dark red solid, namely the pure product of dye 3.

The synthetic route is as follows:

The carbon nuclear magnetic resonance spectrum of dye 3 is shown in Figure 7, ¹ H NMR (400MHz, CDCl ₃ ) δ8.23–8.19 (m, 1H), 8.15 (d, J=8.2Hz, 1H), 7.71–7.63 ( m,2H),7.63–7.58(m,1H),7.47(d,J＝6.9Hz,1H),7.21–7.15(m,1H),6.64(s,1H),6.49(s,1H),3.46 –3.30(m,6H),3.27–3.18(m,2H),3.14(t,J＝6.4Hz,2H),2.69–2.56(m,3H),2.17–2.06(m,2H),1.93(dd , J=11.9, 5.9Hz, 2H). The pure dye 3 was analyzed by mass spectrometry, and the results are shown in Figure 10, indicating that dye 3 was synthesized.

Exploration of physical properties

(9) Application test

1) Configuration of stock solution for detection

a. Preparation of dye 1 sample solution (1.00×10 ^-3 mol·L ^-1 ): Dissolve 0.0039 g (M=392) dye 1 in 10 mL of ethanol to make a concentration of 1.00×10 ^-3 mol·L ^{- 1} solution.

b. Preparation of dye 2 sample solution (1.00×10 ^-3 mol·L ^-1 ): Dissolve 0.0045 g (M=448) of dye 2 in 10 mL of ethanol to make a concentration ^of 1.00×10 ^-3 mol·L -1 ¹ solution.

c. Preparation of Dye 3 sample solution (1.00×10 ^-3 mol·L ^-1 ): Dissolve 0.0047g (M=472) Dye 3 in 10mL ethanol to make a concentration of 1.00×10 ^-3 mol·L ^-1 The solution.

d. prepare the solution of pH=1 with 37% hydrochloric acid, determine pH with pH test paper; Be made into the solution of pH=14 with sodium hydroxide solution, determine pH with pH test paper; 13 solutions with different pH gradients from 1 to 14, use pH test paper to determine the pH.

2) Detection and analysis

Take 30 μL of dye 1 sample solution, dye 2 sample solution and dye 3 sample solution respectively, add them into cuvettes containing 3 mL of deionized water, and then conduct UV-visible spectrophotometry and fluorescence spectrophotometry tests on them, the results were respectively As shown in Figure 9 and Figure 10.

It can be seen from Figure 9 and Figure 10 that the physical properties of dye 1, dye 2 and dye 3 are shown in Table 1 (wherein, the Stokes shift is the difference between the maximum emission wavelength and the maximum absorption wavelength).

Table 1 The physical properties of dye 1, dye 2 and dye 3

Take 3mL of the solution at pH=1, add 30μL of dye 1 sample solution, put it into a UV-visible spectrometer to detect the change of absorbance of dye 1 at this pH condition, and use the same detection method to obtain the change of absorbance of dye 1 at pH=2-14.

Take 3mL of the solution at pH=1, add 30 μL of dye 1 sample solution, put it into a fluorescence spectrometer to detect the change of absorbance of dye 1 at this pH condition, and obtain the fluorescence intensity change of dye 1 at pH=2-14 by the same detection method.

The results are shown in Figure 11 ((a) is the UV-Vis spectrum, (b) is the fluorescence spectrum), in the range of pH=4-14, the absorbance and fluorescence intensity of dye 1 gradually decrease with the increase of pH.

Take 3mL of the solution at pH=1, add 30μL of the dye 2 sample solution, put it into a UV-visible spectrometer to detect the change of the absorbance of the dye 2 at this pH condition, and obtain the change of the absorbance of the dye 2 at pH=2-14 by the same detection method.

Take 3mL of the solution at pH=1, add 30 μL of dye 2 sample solution, put it into a fluorescence spectrometer to detect the change of absorbance of dye 2 at this pH condition, and use the same detection method to obtain the change of fluorescence intensity of dye 2 at pH=2-14.

The results are shown in Figure 12 ((a) is the UV-Vis spectrum, (b) is the fluorescence spectrum), in the range of pH=2-14, the absorbance and fluorescence intensity of dye 2 gradually decrease with the increase of pH.

Take 3 mL of the solution at pH=1, add 30 μL of dye 3 sample solution, and put it into an ultraviolet-visible spectrometer to detect the change of absorbance of dye 3 at this pH condition.

Take 3 mL of the solution at pH=1, add 30 μL of dye 3 sample solution, put it into a fluorescence spectrometer to detect the change of absorbance of dye 3 at this pH condition, and obtain the fluorescence intensity change of dye 3 at pH=2-14 by the same detection method.

The results are shown in Figure 13 ((a) is the UV-Vis spectrum, (b) is the fluorescence spectrum), as can be seen from the UV spectrum, in the range of pH=1 to 14, the absorbance of the dye 3 increases with the pH It can be seen from the fluorescence spectrum that in the pH=1～7 range, the fluorescence intensity of the dye 3 gradually increases with the increase of pH, and in the pH=8～14 range, the fluorescence intensity of the dye Decreases gradually with increasing pH.

Claims (10)

1. a class rhodamine fluorescent dyes, it is characterised in that the fluorochrome is any in dyestuff 1, dyestuff 2 and dyestuff 3 One kind, wherein dyestuff 1, dyestuff 2, the structural formula of dyestuff 3 distinguish as follows：

2. the preparation method of rhodamine fluorescent dyes described in claim 1, it is characterised in that comprise the following steps：

(1) resorcinol obtains compound 1 with phthalic anhydride；3- hydroxy-ns, N- diethylanilines and O-phthalic Anhydride reaction obtains compound 2；Meta-aminophenol reacts to obtain compound 3, compound 3 and phthalic acid with the bromo- 3- chloropropanes of 1- Anhydride reactant obtains compound 4；Compound 1, compound 2, compound 3 and compound 4 chemical formula it is as follows：

(2) by catalyst of methanesulfonic acid, toluene be reaction dissolvent, by acenaphthene phenol distinguish compound 1, compound 2 and compound 4 it is anti- Should, obtain dyestuff 1, dyestuff 2 and dyestuff 3.

3. the preparation method of rhodamine fluorescent dyes according to claim 2, it is characterised in that compound 1 in step (1) Preparation method is specially：Resorcinol and phthalic anhydride are dissolved in toluene, are stirred at reflux to generation solid, suction filtration takes Solid, with recrystallizing methanol, is produced；Wherein, the mol ratio of resorcinol and phthalic anhydride is 1：1.

4. the preparation method of rhodamine fluorescent dyes according to claim 2, it is characterised in that compound 2 in step (1) Preparation method is specially：By 3- hydroxy-ns, N- diethylanilines and phthalic anhydride are dissolved in toluene, are stirred at reflux to life Into solid, suction filtration takes solid, with recrystallizing methanol, produces；Wherein, 3- hydroxy-ns, N- diethylanilines and phthalic anhydride Mol ratio be 1：1.

5. the preparation method of rhodamine fluorescent dyes according to claim 2, it is characterised in that compound 3 in step (1) Preparation method is specially：Meta-aminophenol, sodium acid carbonate and the bromo- 3- chloropropanes of 1- are added into dimethylformamide, is stirred and evenly mixed, And continue to stir in 65~75 DEG C, thin-layer chromatography detection reaction has generated compound 3, then is further separated using column chromatography, i.e., ；Wherein, the mol ratio of meta-aminophenol, sodium acid carbonate and the bromo- 3- chloropropanes of 1- is 1：3~3.5：2.5~3.

6. the preparation method of rhodamine fluorescent dyes according to claim 2, it is characterised in that compound 4 in step (1) Preparation method is specially：Compound 3 and phthalic anhydride are dissolved in toluene, are stirred at reflux to generation solid, suction filtration takes Solid, with recrystallizing methanol, is produced；Wherein, compound 3 and the mol ratio of phthalic anhydride are 1：1.

7. the preparation method of rhodamine fluorescent dyes according to claim 2, it is characterised in that the system of acenaphthene phenol in step (2) Preparation Method comprises the following steps：

(1) POCl3 and DMF are sequentially added into acenaphthene, 90~95 DEG C, continuation are warming up to after stirring and evenly mixing Stirring 2.5~3.5 hours, obtains reaction solution；Reaction solution is added in mixture of ice and water, sticky solid is generated, through suction filtration and very Sky is dried, and obtains 5- acenaphthene aldehyde crude products；Wherein, the mol ratio of acenaphthene, POCl3 and DMF is 1：2~2.5：5 ~6；

(2) 5- acenaphthene aldehyde crude products obtained by step (1) are used into pillar layer separation, is dried in vacuo, obtains 5- acenaphthene aldehyde sterlings；

(3) 5- acenaphthene aldehyde sterling, selenium dioxide, hydrogen peroxide and glacial acetic acid are added into dichloromethane, 35 are stirred in 20~30 DEG C ~40 hours, filter to take after filtrate, stratification, take organic layer to evaporate solvent, and potassium hydroxide is dissolved in ice bath environment In methanol solution, then after being placed in and hydrolyzing 2.5~3.5 hours at room temperature, hydrochloric acid acidifying is added, then is extracted with ethyl acetate, is washed Ethyl acetate extraction phase, drying, obtain acenaphthene phenol crude product；Wherein, the dosage of 5- acenaphthenes aldehyde sterling is 80~100g/ in dichloromethane L, the dosage of selenium dioxide are that 2~3g/L, the dosage of hydrogen peroxide are that 1.6~2mol/L, the dosage of glacial acetic acid are 0.29~0.35mol/L；

(4) acenaphthene phenol crude product obtained by step (3) is used into pillar layer separation, is dried in vacuo, produces acenaphthene phenol sterling.

8. the preparation method of rhodamine fluorescent dyes according to claim 2, it is characterised in that dyestuff 1, dye in step (2) Material 2 preparation method be specially：Compound 1 or compound 2 are added into toluene, acenaphthene phenol and methanesulfonic acid are added, in 60~70 DEG C stirring 25~35 minutes, reaction solution is extracted with ethyl acetate, then wash ethyl acetate extraction phase, drying, respectively obtains dye Expect 1 crude product or the crude product of dyestuff 2；The crude product of dyestuff 1 or the crude product of dyestuff 2 are further used into pillar layer separation, vacuum drying obtains dyestuff 1 sterling or the sterling of dyestuff 2；Wherein, the mol ratio of compound 1, acenaphthene phenol and methanesulfonic acid is 1：1：26~27, compound 2, acenaphthene phenol and The mol ratio of methanesulfonic acid is 1：1.0~1.1：8~9.

9. the preparation method of rhodamine fluorescent dyes according to claim 2, it is characterised in that the system of dyestuff 3 in step (2) Preparation Method is specially：Compound 4, acenaphthene phenol and methanesulfonic acid are added into toluene, stirs 25~35 minutes, will react in 60~70 DEG C Liquid is extracted with dichloromethane, then washes dichloromethane extraction phase, drying, obtains the crude product of dyestuff 3；The crude product of dyestuff 3 is further used Pillar layer separation, vacuum drying, obtains the sterling of dyestuff 3；Wherein, the mol ratio of compound 4, acenaphthene phenol and methanesulfonic acid is 1：2~3： 64~66.

10. application of the rhodamine fluorescent dyes in fluoroscopic examination and pH probes described in claim 1.