CN103992791B - A kind of rhodamine base pH value fluorescent probe in the linear response of faintly acid scope and preparation method thereof - Google Patents

Abstract

The invention discloses a rhodamine-based pH fluorescent probe with a linear response in a weakly acidic range and a preparation method thereof. The fluorescent probe has the following general structural formula: Wherein: R ₁ , R ₂ , R ₃ , R ₄ use hydrogen at the same time, or R ₁ , R ₂ , R ₃ or R ₄ are selected from hydrogen, 2-6 carbon atom alkyl or 4-8 carbon atom cycloalkane one of the bases. The fluorescence response of the pH fluorescent probe has good linearity in the range of pH=5.0-7.0, and this linear range can meet the requirements for pH detection of most biological cells and their organelles. The color of the probe solution changes from colorless to deep rose as the pH value decreases, and can be used for naked-eye detection. The probe selectively recognizes hydrogen ions without interference from other common cations. The probe has good sensitivity, and the fluorescence quantum yield can reach 0.599 at pH=5.13.

Description

A rhodamine-based pH fluorescent probe with linear response in weakly acidic range and preparation method thereof

technical field

The invention relates to a rhodamine-based pH fluorescent probe with linear response in weak acid range, in particular to a rhodamine-based pH fluorescent probe with linear response in weak acid range and a preparation method thereof.

Background technique

pH is an important parameter in the study of physiology, pharmacology, pathology, etc. The pH in human cells plays an important role in many physiological and pathological processes (Nature Rev. Mol. Cell. Biol., 2010, 11(1), 50-61). Excessive acidity or alkalinity can cause cell dysfunction, lead to heart, lung disease or neurological diseases, and even life-threatening in severe cases. Therefore, monitoring changes in intracellular pH can provide important information for studying physiological and pathological processes. Glass electrodes are generally used to measure pH, but they are not suitable for pH monitoring of living cells due to defects such as electrochemical interference and possible mechanical damage (Cell Mol. Biol., 2000, 46(8), 1361-1374). Fluorescent probe detection of cell pH is a non-invasive method that will not damage the sample. At the same time, it has the characteristics of high sensitivity, good selectivity, flexible instrument design, small sample volume, simple operation, and is suitable for high-throughput screening. Ideal method for cell pH (Trends in Analytical Chemistry, 2010, 29(9), 1004-1013).

In general, there are two main pH ranges in normal cells: the cytoplasm is mainly in Between 6.8-7.4, the pH of acidic organelles is 4.5-6.0. A large number of pH probes for these two ranges have been reported in the literature, and have been used for qualitative research of cytoplasmic pH and detection of acidic organelles (Chemical Journal of Chinese Universities, 2010, 13(6), 1148-1151). However, for cancer cells, due to abnormal anaerobic metabolism through the glycolytic pathway, the pH value of the cells is 0.55 units lower than that of normal cells on average, and the pH value is probably in the range of 5.8-7.7 (Bioorganic & Medicinal Chemistry Letters 22 (2012) 2440-2443). NMR and electrochemical cancer diagnosis methods targeting this abnormal pH value have been reported in the literature. Since there are few pH fluorescent probes that have a linear response in this range, cancer diagnosis methods based on pH fluorescent probes are rarely reported in the literature. Therefore, the development of a pH fluorescent probe with a linear response in the weakly acidic range (pH=5.0-7.0) is of great significance for cancer research and cancer diagnosis. In addition, most of the pH fluorescent probes that have been developed so far have disadvantages such as poor sensitivity and narrow linear range. Therefore, the development of a pH fluorescent probe with a linear response in the weakly acidic range (pH=5.0-7.0) is still a research hotspot in this field.

Rhodamine is a dye containing xanthene structure, which has the advantages of high absorption coefficient, long excitation and emission wavelength, high fluorescence quantum yield and good photostability, etc. It has been widely used as the parent of fluorescent probe for the construction of pH fluorescent probes (Chem. Rev., 2012, 112, 1910-1956). It is generally believed that when the lactam structure of the rhodamine-based pH value probe is in a five-membered spirocyclic state, the molar absorptivity and fluorescence quantum yield are very low, and there is almost no fluorescence. When the carbonyl of the rhodamine lactam is protonated, it can lead to The carbon-nitrogen bond in the five-membered helical ring of the needle is broken to form an open ring structure, and the fluorescence intensity is significantly enhanced, thereby realizing the selective response to pH value (Org. Biomol. Chem., 2014, 12, 526-533). Lin Weiying et al. studied the recognition mechanism and found that the response interval of the rhodamine probe can be adjusted by adjusting the ring tension of the rhodamine five-membered helix (Org. Biomol. Chem., 2011, 9, 1723-1726). According to this principle, the present invention adopts a large steric hindrance group to adjust the response range of the rhodamine pH value probe, and constructs a rhodamine-based pH value fluorescent probe with a linear response in the weakly acidic range (pH=5.0-7.0) .

Contents of the invention

The technical problem to be solved by the present invention is to provide a rhodamine-based pH value fluorescent probe with a linear response in the weakly acidic range and a preparation method thereof. The fluorescent probe prepared by the method induces rhodamine to undergo spiral The opening of the ring makes the probe molecule change in color (colorless to deep rose) and the fluorescence signal is enhanced, and the color change before and after recognition can be observed by the naked eye. The probe is highly selective to hydrogen ions, and has no obvious interference from other common ions. The fluorescence signal of the probe can generate a linear response in the range of pH=5.0-7.0, and has a promising application prospect in detecting pH value in biological cells.

One of the purposes of the present invention is to provide a rhodamine-based pH fluorescent probe with a linear response in the weakly acidic range, and its structural formula is as follows:

Wherein: R ₁ , R ₂ , R ₃ , R ₄ can be hydrogen at the same time, or R ₁ , R ₂ , R ₃ or R ₄ are selected from hydrogen, 2-6 carbon atom alkyl or 4-8 carbon atom cycloalkane one of the bases.

The second object of the present invention is to provide a method for preparing a rhodamine-based pH fluorescent probe with a linear response in the weakly acidic range. The method includes the following steps:

(1) Weigh the rhodamine dye and dissolve it in a dry organic solvent according to the molar ratio of rhodamine dye to phosphorus oxychloride as 1:2-5, then slowly add the proportioned amount of phosphorus oxychloride, and heat up to reflux Reaction, after the reaction is finished, it is down to room temperature to form a reaction solution;

(2) According to the molar ratio of 2,6-diisopropylaniline and acid-binding agent of 1:10-30, it is prepared by weighing 2,6-diisopropylaniline and acid-binding agent and dissolving them in a dry organic solvent solution, add this solution dropwise to the reaction solution formed in step (1), heat up to reflux reaction, wash with NaHCO ₃ solution after the reaction, collect the organic layer, dry over anhydrous magnesium sulfate, filter with suction, and evaporate under reduced pressure solvent, the target compound was separated by column chromatography.

In the step, the organic solvent can be conventional in the art, preferably at least one selected from 1,2-dichloroethane, acetonitrile, methylene chloride, dimethyl sulfoxide, chloroform, N,N-dimethylformaldehyde amides or tetrahydrofuran.

The acid-binding agent in the step (2) is conventional in the art, and the acid-binding agent is preferably pyridine or triethylamine.

The filler used in the column chromatography in the step (2) can be conventional in the field, preferably alumina, and the eluent can be conventional in the field, preferably dichloromethane and ethanol with a volume ratio of 20:1.

Specifically, the preparation method of the rhodamine-based pH fluorescent probe having a linear response in the weakly acidic range of the present invention comprises the following steps:

1. Dissolve 1 mole of rhodamine dyes in a dry organic solvent, then slowly add 2-5 moles of phosphorus oxychloride, and heat up to reflux. The reaction was stirred for 4-6h and then cooled to room temperature.

2. Dissolve 1 mole of 2,6-diisopropylaniline and 10-30 moles of acid-binding agent in a dry organic solvent, add this solution dropwise to the above reaction system, raise the temperature to reflux, and react for 3-7 hours. After the reaction was completed, it was washed with 0.1M NaHCO ₃ solution, and the organic layer was collected and dried over anhydrous magnesium sulfate. After suction filtration, the solvent was evaporated under reduced pressure, and the target compound was separated by column chromatography.

The organic solvent in the step is 1,2-dichloroethane, acetonitrile, dichloromethane, dimethyl sulfoxide, chloroform, N,N-dimethylformamide, tetrahydrofuran, or a mixture thereof.

The acid-binding agent in the step 2 is pyridine or triethylamine.

The filler used in the column chromatography in the step 2 is alumina, and the eluent is dichloromethane and ethanol with a volume ratio of 20:1.

The synthesis reaction formula of the rhodamine-based pH fluorescent probe with linear response in the weakly acidic range of the present invention is:

The pH fluorescent probe of the present invention has no emission peak at 540-660nm in a neutral or basic system, indicating that the probe is in a five-membered spirocyclic state within this pH range. As the pH value decreased, the probe had an emission peak at 595nm and the fluorescence intensity gradually increased. The change trend of the ultraviolet absorption spectrum was consistent with that of the fluorescence spectrum, and the solution changed from colorless to deep rose, indicating that the response of the probe to the pH value could be observed under naked eye conditions. The probe has good linearity in the pH range of 5.0-7.0. The fluorescence quantum yield of the probe can reach 0.599 at pH=5.13. According to the Henderson-Hasselbach type equation: (log[(I _max - I)/(II _min )]= pKa-pH, the calculated pKa of the probe is 5.826. This probe is expected to be used in the detection of pH value of biological cells, especially It is the study and diagnosis of cancer.

The beneficial effects of the present invention are: the fluorescent response of the pH value fluorescent probe described in the present invention has good linearity in the interval of pH=5.0-7.0, and this linear range can meet the requirements for pH value detection of most biological cells and their organelles . The color of the probe solution changes from colorless to deep rose as the pH value decreases, and can be used for naked-eye detection. The probe selectively recognizes hydrogen ions without interference from other common cations. The probe has good sensitivity, and the fluorescence quantum yield can reach 0.599 at pH=5.13. The probe is expected to be used in the pH value detection of biological cells, especially in the research and diagnosis of cancer.

Description of drawings

Fig. 1 is a graph of the fluorescence emission spectra of the fluorescent probe in Example 1 of the present invention under different pH conditions. The probe concentration is 50 μM, the abscissa is the wavelength (nm), the ordinate is the fluorescence intensity, and the excitation wavelength is 520 nm.

Fig. 2 is a curve diagram of the fluorescence intensity at 595 nm of the fluorescent probe in Example 1 of the present invention as a function of the pH value. (Small picture in the upper right corner: the linear relationship between the fluorescence intensity at 595nm and the pH value). The probe concentration is 50 μM, the abscissa is the wavelength (nm), the ordinate is the fluorescence intensity, and the excitation wavelength is 520 nm.

Fig. 3 is a color change diagram of the fluorescent probe in Example 1 of the present invention in different pH buffer solutions. The pH value from left to right is 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.2, 5.5, 5.8, 6.0, 6.5, 7.0, 8.0, 9.0.

4 is the fluorescence emission spectrum of the fluorescent probe in Example 1 of the present invention at pH=2.95 and the fluorescence emission spectrum when it coexists with different metal ions (10 mM) in a buffer solution of pH=7.4. The probe concentration is 50 μM, the ion concentration is 10 mM, the abscissa is the wavelength (nm), the ordinate is the fluorescence intensity, and the excitation wavelength is 520 nm.

Fig. 5 is a graph of the fluorescence emission spectrum when the fluorescent probe in Example 1 of the present invention coexists with different metal ions (10 mM) in a buffer solution of pH = 3.0. The probe concentration is 50 μM, the ion concentration is 10 mM, the abscissa is the wavelength (nm), the ordinate is the fluorescence intensity, and the excitation wavelength is 520 nm.

detailed description

Example 1

(1) Dissolve 0.2395g (0.5mmol) Rhodamine B in 15mL dry 1,2-dichloroethane, slowly add 0.15mL (1.6mmol) POCl ₃ , raise the temperature to 83°C, reflux for 5h, and cool down to room temperature .

(2) Dissolve 0.1314g (0.6mmol) of 2,6-diisopropylaniline and 2mL (14.3mmol) of triethylamine in 10mL of 1,2-dichloroethane, and add this solution dropwise to the above step (1 ) in the reaction system, the temperature was raised to 83°C, and the reaction was refluxed for 5h. After the reaction was completed, it was washed with 0.1M NaHCO ₃ solution (3×20 mL), and the organic layer was collected and dried over anhydrous magnesium sulfate. After suction filtration, the solvent was evaporated under reduced pressure, and column chromatography was performed with neutral alumina as a filler (V _{dichloromethane} :V _methanol =20:1) to obtain 0.25 g of the target compound with a yield of 83.17%. mp: 305°C. ¹ HNMR (400 MHz, CDCl ₃ ) δ (ppm)=8.08 (dd, J=6.8, 1.5 Hz, 1H), 7.70-7.60 (m, 2H), 7.36-7.27 (m, 2H), 7.00 (d, J=7.7 Hz, 2H), 6.57 (d, J=8.8 Hz, 2H), 6.31 (dd, J=8.9, 2.6 Hz, 2H), 6.22 (d, J=2.6 Hz, 2H), 3.31 (q, J=7.0 Hz, 8H), 2.43 (dt, J=13.4, 6.7 Hz, 2H), 1.13 (t, J=7.0 Hz, 12H), 0.92 (d, J=6.7 Hz, 6H), 0.47 (t, J=8.4 Hz, 6H); ¹³ CNMR (100 MHz, CDCl ₃ ) δ (ppm)=167.35 (s), 156.57 (s), 149.71 (s), 149.48 (s), 148.72 (s), 133.18 (s ), 131.99 (s), 130.36 (s), 129.56 (s), 128.85 (s), 128.49 (s), 125.07 (s), 123.71 (s), 123.15 (s), 108.94 (s), 107.60 (s ), 98.48 (s), 77.34 (s), 76.91 (d, J=23.1 Hz), 76.71 (s), 69.94 (s), 44.44 (s), 29.67 (s), 26.71 (s), 21.88 (s ), 12.62 (s); HRMS: anal. calcd for C ₄₀ H ₄₇ N ₃ O ₂ : 601.82; found: 602.3762 (M+H ⁺ ); IR(KBr, cm ^-1 ): 2961.46, 2926.15, 2862.00, 1687.00 , 1615.05, 1515.56, 1465.77, 1353.88, 1266.48, 1220.38, 1118.04, 1014.60, 785.91, 761.03, 702.14, 543.67.

Example 2

(1) Dissolve 0.1684g (0.5mmol) Rhodamine 110 in 15mL dry 1,2-dichloroethane, slowly add 0.15mL (1.6mmol) POCl ₃ , raise the temperature to 83°C, reflux for 5h, and cool down to room temperature .

(2) Dissolve 0.1314g (0.6mmol) of 2,6-diisopropylaniline and 2mL (14.3mmol) of triethylamine in 10mL of 1,2-dichloroethane, and add this solution dropwise to the above step (1 ) in the reaction system, the temperature was raised to 83°C, and the reaction was refluxed for 5h. After the reaction was completed, it was washed with 0.1M NaHCO ₃ solution (3×20 mL), and the organic layer was collected and dried over anhydrous magnesium sulfate. After suction filtration, the solvent was evaporated under reduced pressure, and column chromatography was performed with neutral alumina as a filler (V _{dichloromethane} :V _methanol =20:1) to obtain 0.17 g of the target compound.

Example 3

(1) Dissolve 0.2395g (0.5mmol) Rhodamine B in 15mL dry 1,2-dichloroethane, slowly add 0.15mL (1.6mmol) POCl ₃ , raise the temperature to 83°C, reflux for 5h, and cool down to room temperature .

(2) Dissolve 0.1314g (0.6mmol) 2,6-diisopropylaniline and 1.15mL (14.3mmol) pyridine in 10mL 1,2-dichloroethane, and add this solution dropwise to the above step (1) In the reaction system, the temperature was raised to 83° C., and the reaction was refluxed for 5 hours. After the reaction was completed, it was washed with 0.1M NaHCO ₃ solution (3×20 mL), and the organic layer was collected and dried over anhydrous magnesium sulfate. After suction filtration, the solvent was evaporated under reduced pressure, and column chromatography was performed with neutral alumina as a filler (V _{dichloromethane} :V _methanol =20:1) to obtain 0.23 g of the target compound.

Example 4

(1) Dissolve 0.1684g (0.5mmol) Rhodamine 110 in 15mL dry acetonitrile, slowly add 0.15mL (1.6mmol) POCl ₃ , raise the temperature to 80°C, reflux for 5h, and cool down to room temperature.

(2) Dissolve 0.1314g (0.6mmol) of 2,6-diisopropylaniline and 2mL (14.3mmol) of triethylamine in 10mL of acetonitrile, add this solution dropwise to the reaction system of the above step (1), and raise the temperature To 80 ° C, reflux reaction for 5h. After the reaction was completed, it was washed with 0.1M NaHCO ₃ solution (3×20 mL), and the organic layer was collected and dried over anhydrous magnesium sulfate. Suction filtration, evaporation of the solvent under reduced pressure, and column chromatography with neutral alumina as a filler (V _{dichloromethane} :V _methanol = 20:1) yielded 0.16 g of the target compound.

Example 5

The fluorescent probe in Example 1 of the present invention was used to test the change of the fluorescence emission spectrum with the pH value under different pH conditions. Fig. 1 is the ethanol/water (v/v, 1:1, pH=7.0) solution fluorescence spectrum. The fluorescence excitation wavelength is 520nm. It can be seen from the figure that the probe has no emission peak at 540-700 nm in the range of neutral and alkaline solutions, indicating that the probe is in a helical ring structure in this pH range. As the pH value decreased, the emission peak of the probe appeared at 595nm and the fluorescence intensity gradually increased, but when the pH value was less than 5.13, the fluorescence intensity decreased again. Taking rhodamine B as the benchmark (Φ=0.89), and calculated according to the data in the figure, the fluorescence quantum yield of the probe is 0.599 at pH=5.13.

Example 6

Using the fluorescent probe in Example 1 of the present invention to test the linear relationship between the fluorescence emission intensity and the pH value under different pH conditions. Figure 2 is the fluorescence intensity curve at 595nm of the ethanol/water (v/v, 1:1, pH=7.0) solution of the fluorescent probe prepared in Example 1 with a concentration of 50 μM at different pH values as a function of pH value picture. The fluorescence excitation wavelength is 520nm. It can be seen from the figure that there is a good linear relationship in the pH range of 5.0-7.0. According to the data in the figure, according to the Henderson-Hasselbach type equation (log[(I _max - I)/(II _min )]= pKa-pH, the pKa of the probe was calculated to be 5.826.

Example 7

The color development experiment was carried out in different pH buffer solutions with the fluorescent probe in Example 1 of the present invention. Figure 3 is a buffer solution with different pH values of the fluorescent probe prepared in Example 1 with a concentration of 50 μM, and the pH values are 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.2, 5.5, 5.8, 6.0 from left to right , 6.5, 7.0, 8.0, 9.0, the photos after 20 minutes of placement. It can be seen from the figure that the probe solution gradually changes from colorless to dark rose as the pH value decreases. It shows that the probe can indicate the acidity change of the tested system under the naked eye condition.

Example 8

The fluorescent probe prepared in Example 1 of the present invention was used to conduct a selectivity experiment for hydrogen ions. Fig. 4 is a fluorescence emission spectrum diagram of the fluorescent probe in Example 1 of the present invention at a concentration of 50 μM at pH = 2.95, and when it coexists with different metal ions (10 mM) in a buffer solution of pH = 7.4. The fluorescence excitation wavelength is 520nm. As can be seen from the figure, 10 mM K ⁺ , Na ⁺ , Ca ²⁺ , Mg ²⁺ , Al ³⁺ , Fe ³⁺ , Cu ²⁺ , Ni ² were added to the fluorescent probe solution prepared in Example 1. ⁺ , Co ²⁺ , Mn ²⁺ , Sn ²⁺ , Zn ²⁺ and other metal ions, the fluorescence spectrum has no emission peak between 550-750nm. But at pH=2.95, the fluorescence intensity of the probe's fluorescence spectrum was significantly enhanced at 595nm, and a new emission peak appeared. This shows that the probe has good selectivity for hydrogen ions.

Example 9

The anti-interference experiment was carried out with the fluorescent probe prepared in Example 1 of the present invention. Figure 5 shows the interaction of the fluorescent probe with K ⁺ , Na ⁺ , Ca ²⁺ , Mg ²⁺ , Al ³⁺ , Fe ³⁺ , Cu ²⁺ , Ni ²⁺ in a buffer solution with pH=3.0 in Example 1 of the present invention. , Co ²⁺ , Mn ²⁺ , Sn ²⁺ , Zn ²⁺ different metal ions (10mM) coexistence fluorescence emission spectrum. The probe concentration in the experiment was 50 μM, and the excitation wavelength was 520 nm. As can be seen from the figure, various common metal ions K ⁺ , Na ⁺ , Ca ²⁺ , Mg ²⁺ , Al ³⁺ , Fe ³⁺ , Cu ²⁺ , Ni ²⁺ , Co ²⁺ , Mn ²⁺ , The presence of Sn ²⁺ , Zn ²⁺ had little or no interference on the pH response of the probe. It shows that the probe has good anti-interference ability to common metal ions.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Therefore, the invention is not limited to the specific details and examples shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims (4)

1. a kind of rhodamine base pH value fluorescent probe that has linear response in weakly acidic range, it is characterized in that this kind fluorescent probe has following general structural formula: Wherein: R ₁ , R ₂ , R ₃ or R ₄ are selected from hydrogen, an alkyl group with 2-6 carbon atoms or a cycloalkyl group with 4-8 carbon atoms. 2. claim 1 has the rhodamine-based pH value fluorescent probe of linear response in weakly acidic range, it is characterized in that described pH value fluorescent probe solution changes color from colorless to dark along with the reduction of pH value Rose color, can be used for naked eye detection. 3. the rhodamine-based pH fluorescent probe with linear response in the weakly acidic range as claimed in claim 1, is characterized in that the pH fluorescent probe can selectively recognize hydrogen ions, and is not interfered by other common cations . 4. the rhodamine-based pH fluorescent probe with linear response in the weakly acidic range of claim 1, characterized in that the fluorescent response of the pH fluorescent probe is at pH=5.0-7.0 interval, at pH=5.13 When the fluorescence quantum yield reaches 0.599, the probe can be used to detect the pH value of biological cells.