CN109369565B

CN109369565B - Benzothiazole derivative and preparation method and application thereof

Info

Publication number: CN109369565B
Application number: CN201811347673.9A
Authority: CN
Inventors: 林博; 樊丽; 贾晶; 黄文成; 双少敏; 董川
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2018-11-13
Filing date: 2018-11-13
Publication date: 2022-03-18
Anticipated expiration: 2038-11-13
Also published as: CN109369565A

Abstract

The invention discloses a benzothiazole derivative and a preparation method and application thereof. The derivative is specifically named as 6- (2- (benzothiazol-2-yl) vinyl) naphthalene-2-ol (BTNO). The preparation method comprises the following steps: dissolving 6-hydroxy-2-naphthaldehyde and 2-methylbenzothiazole in a small amount of dichloromethane, adding trimethylchlorosilane, and heating to react to prepare a crude product; and removing the solvent from the crude product, and separating by a silica gel column to obtain a pure product. BTNO shows a blue shift in fluorescence spectrum with pH decrease from 11.50 to 4.00, showing a ratiometric fluorescence emission (F)_456nm/F_526nm) The characteristics, simultaneously have the advantages of high sensitivity to pH response, good selectivity, high quantum yield, large Stokes shift and the like. In addition, BTNO is used as a probe to permeate cell membranes to mark cells, and can be used for monitoring pH fluctuation in a cytoplasm range.

Description

Benzothiazole derivative and preparation method and application thereof

Technical Field

The invention relates to a heterocyclic compound and a fluorescent probe, in particular to a benzothiazole derivative (BTNO), a preparation method thereof and application of the derivative in monitoring cytoplasmic pH change.

Background

The intracellular pH is one of the important parameters of cell metabolism, and plays an important role in various physiological and pathological processes of cells, such as enzyme activity regulation, protein morphology maintenance, cell division and apoptosis, ion transport, endocytosis, cell energy generation and transformation, membrane potential balance, information transfer, and the like. Meanwhile, researches show that the growth and metastasis of cancer (tumor cells), multidrug resistance, Parkinson's disease, Alzheimer's disease and other diseases are closely related to intracellular pH abnormality. Therefore, it is of great importance to monitor the intracellular pH change accurately in real time.

In recent years, the literature reports that many small organic molecule fluorescent probes are used for the detection of intracellular pH changes, and most of these probes are fluorescent probes for acidic pH responses, such as lysosomes (pH 4.0-5.5). However, the pH of the cytoplasmic matrix and the subcellular compartments such as nucleus, mitochondria and the like which exist in a large amount in the cell are nearly neutral or weakly alkaline, and reports about such pH probes are not many, and especially probes based on the ratiometric fluorescence emission technology are rare. Ratiometric fluorescence emission can be effectively avoided by simultaneously recording the fluorescence intensities of two different emission wavelengths and calculating their ratio, for example: the polarity of the solvent, the negative uneven staining of the probe concentration in the cell, the interference of temperature, instruments and other environmental factors, thereby achieving the accurate quantitative analysis of the analyte. Therefore, it is very necessary to design and develop a novel proportional emission type pH fluorescent probe for monitoring the change of pH in the cytoplasm.

Disclosure of Invention

The invention aims to provide a benzothiazole derivative BTNO and a preparation method thereof; another purpose is to provide the application of the derivative as a ratio type pH fluorescent probe in monitoring the change of cytoplasmic pH.

The invention provides a benzothiazole derivative BTNO, which has a structural formula as follows:

the invention provides a preparation method of benzothiazole derivative BTNO, which comprises the following steps:

(1) dissolving 6-hydroxy-2-naphthaldehyde, 2-methylbenzothiazole and trimethylchlorosilane in a molar ratio of 2:3-10:10 in a small amount of dichloromethane, placing the dichloromethane in a high-pressure reaction kettle for sealing, and reacting at 105 ℃ for 24 hours; after the reaction was cooled to room temperature, concentrated under reduced pressure, the resulting solid was dissolved in water and Na was added₂CO₃Adjusting the pH of the solution to 6.0; then extracting with dichloromethane, removing the solvent to obtain a crude product;

(2) and concentrating the crude product, and separating by a silica gel column to obtain a pure product.

The molar ratio of the 6-hydroxy-2-naphthaldehyde, the 2-methylbenzothiazole and the trimethylchlorosilane in the step (1) is 2:3: 10.

The synthetic route of the derivative BTNO is as follows:

the derivative BTNO of the invention has good cell membrane permeability as a probe and can be used for monitoring the pH change in cytoplasm.

Compared with the existing pH fluorescent probe, the probe BTNO synthesized by the invention has the following advantages: (1) the probe belongs to a ratio emission type pH fluorescent probe (F)_456nm/F_526nm) The interference caused by factors such as the concentration of the probe and uneven dyeing can be effectively reduced; (2) pK of probe BTNO_aThe pH linear response range is 7.91 +/-0.034, and the pH linear response range is 7.00-9.50, so that the pH linear response device is suitable for monitoring pH change in neutral cytoplasm and weak alkaline range; (3) the probe is designed based on an Intramolecular Charge Transfer (ICT) effect, namely hydroxyl on a naphthalene ring is used as an electron donor, benzothiazole is used as an electron acceptor, and hydroxyl of BTNO loses electrons and is changed into phenoxidyl anion under an alkaline condition, so that the ICT effect of the whole molecule is enhanced, the absorption spectrum and the emission spectrum of the probe are red-shifted, and obvious color change can be observed by naked eyes under the irradiation of natural light and ultraviolet light; (4) the fluorescence quantum yield is high (the fluorescence quantum yield of the probe is 0.88 measured by taking quinine sulfate as a reference substance), and the interference of background fluorescence can be effectively reduced during biological imaging; (5) the probe has excellent light stability, and is beneficial to keeping higher signal-to-noise ratio and reducing errors when the pH change of a biological sample is monitored in real time; (6) the large Stokes displacement (about 132nm) can effectively reduce the interference of exciting light; (7) the probe has excellent anti-interference capability on pH response and is free from common anions, cations, amino acid Reactive Oxygen Species (ROS) and reactive nitrogen species (reactive nitrogen species) ((ROS))RNS), etc.; (8) the probe has excellent cell membrane permeability, and can realize real-time monitoring on the change of pH in cytoplasm by using a laser confocal imaging technology; (9) the probe has simple synthesis steps and low cost.

Drawings

FIG. 1 is a graph showing an ultraviolet absorption spectrum of BTNO as a probe according to the present invention as a function of pH.

FIG. 2 shows that the color of BTNO as a probe changes from yellow to light green before and after binding with protons under natural light.

FIG. 3 shows fluorescence emission spectrum of BTNO as probe according to the present invention as a function of pH.

FIG. 4 shows the color change from green to blue before and after the BTNO probe of the present invention binds protons under an ultraviolet lamp.

FIG. 5 BTNO of the present invention as a probe, F_456nm/F_526nmBoltzmann function relationship, pK, as a function of pH_a＝7.91±0.034。

FIG. 6 shows BTNO of the present invention as a probe, F_456nm/F_526nmThe linear range is pH 7.00-9.50 as a function of pH.

FIG. 7 shows that BTNO of the present invention can be used as a probe for OH in the presence of common anions and cations and some common amino acids, active oxygen, active nitrogen, etc. in vivo^-Selectivity of (2).

FIG. 8 is a laser confocal image of the incubation of the probe BTNO of the present invention with HeLa cells at pH 5.50, pH 6.50, pH 7.00, pH 7.40, pH 8.00, pH 8.50 and pH 9.50 for 30 min.

FIG. 9 shows that the BTNO of the present invention is used as a probe to incubate with HeLa cells for 30min, and NH is added₄Cl treatment, and laser confocal imaging at 0min, 10min, 20min, 30min and 40min respectively.

FIG. 10 shows that after BTNO of the present invention is used as a probe to incubate with HeLa cells for 30min, H is added to each of the BTNO and HeLa cells₂O₂Confocal laser imaging after 1h incubation with NAC (N-acetylcysteine).

Detailed Description

Example 1

Preparation of 1, 6- (2- (benzothiazol-2-yl) vinyl) naphthalen-2-ol (BTNO):

(1) a mixed solution of 6-hydroxy-2-naphthaldehyde (10mmol, 1.7218g), 2-methylbenzothiazole (15mmol, 1.89mL), chlorotrimethylsilane (50mmol, 6.40mL), and 10mL CH2Cl2 was reacted at 105 ℃ in a high-pressure autoclave for 24 hours. Removing the solvent under reduced pressure, dissolving the obtained solid in water, and adding Na₂CO₃Adjusting the pH of the solution to 6.0 and then adding CH₂Cl₂And (4) extracting. Removing the solvent to obtain a crude product;

(2) separating and purifying the crude product by a silica gel column, v_{Ethyl acetate}:v_N-hexane1:6 as eluent, to give BTNO as a reddish brown solid product (1.58g, 52%).¹H NMR(400MHz,DMSO)δ10.62(s,1H),10.34(s,1H),10.04(s,1H),9.97(s,1H),9.65(s,1H),8.43(s,1H),8.02(d,J＝8.7Hz,2H),7.80(dd,J＝18.1,8.5Hz,2H),7.21(d,J＝8.4Hz,2H),7.02(s,1H).¹³C NMR(101MHz,d⁶-DMSO)δ197.80(s),197.53(s),163.67(s),143.29(s),140.03(s),136.82(s),136.54(s),136.50(s),132.27(s),131.85(s),127.94(s),127.87(s),125.01(s),123.31(s),114.40(s),100.00(s),84.53(s),84.20(s),83.86(s).MS(ESI)m/z:Calcd 304.0791；found 304.0787[M+H]⁺.

Example 2

The BTNO concentration of example 1 was maintained at 150 μ M, the pH was adjusted in a DMSO/water (2: 1 by volume) system with a small volume of HCl and NaOH at high concentration, and the absorption spectrum was recorded (fig. 1). As the pH value is reduced, the absorption peak at 385nm is gradually reduced, the absorption peak at 321nm is correspondingly enhanced, and an isoabsorption point exists at 349 nm. The color of the solution also changed from yellow to pale green (fig. 2).

Example 3

The BTNO concentration of example 1 was maintained at 10. mu.M, the pH was adjusted in a DMSO/water (volume ratio 2:1) system with a small volume of HCl and NaOH at a high concentration, the excitation wavelength was 349nm, andthe fluorescence emission spectrum was recorded (FIG. 3). As the pH value is lowered, the fluorescence peak at 526nm gradually decreases, and the fluorescence peak at 456nm gradually increases. The color of the solution changed from green to blue under the uv lamp (figure 4). Fitting F by Boltzmann function_456nm/F_526nmThe curve of the change of the pH value is calculated to calculate the pK_aThe value was 7.91. + -. 0.034 (FIG. 5), and the linear range of pH response was 7.00-9.50. Linear regression equation of F_456nm/F_526nm2.13641-0.21119 × pH, correlation coefficient R²0.9995 (fig. 6).

Example 4

The BTNO concentration in example 1 was kept at 10. mu. mol/L, and the response of the probe to common anions, cations, and some amino acids, ROS, RNS, etc. in the living body was examined. As shown in FIG. 7, the probe showed almost no response to the above-mentioned substances, demonstrating that the probe responded to OH^-Has excellent selectivity. The order and concentration of the substances in fig. 7 are, in order: 1, Blank; 2, K⁺(140mM)；3,Cd²⁺(1mM),4,Mg²⁺(1mM)；5,Li⁺(0.1mM)；6,Co²⁺(0.1mM)；7,Hg²⁺(0.1mM)；8,Ba²⁺(0.1mM)；9,Ni²⁺(0.1mM)；10,Ca²⁺(0.1mM)；11,Fe²⁺(0.1mM)；12,Zn²⁺(0.1mM)；13,Cu²⁺(0.1mM)；14,Mn²⁺(0.1mM)；15,F^-(1mM)；16,Br^-(1mM)；17,I^-(1mM)；18,SO₄ ^2-(1mM)；19,S₂SO₃ ^2-(1mM)；20,SO₃ ^2-(HSO₃ ^-,0.1mM)；21,S^-(HS^-,1mM)；22,NO₃ ^-(1mM)；23,NO₂ ^-(1mM)；24,AC^-(1mM)；25,CO₃ ^2-(HCO₃ ^-,1mM)；26,ClO^-(1mM)；27,ClO₄ ^-(1mM)；28,O₂ ^2-(1mM)；29,ONNO^-(0.1mM)；30,O^2-(1mM)；31,L-GSH(1mM)；32,Hcy(1mM)；33,Cys(1mM)。

Example 5

Adherent HeLa cells were incubated with BTNO of example 1 at pH 7.40, 37 ℃ and 5% CO₂Then gently washed 3 times with phosphate buffer (pH 7.40) to remove excess BTNO, and then used with high K at pH 5.50, 6.50, 7.00, 7.40, 8.00, 8.50 and 9.50, respectively⁺Buffer (30mM NaCl, 120mM KCl, 1mM CaCl)₂、0.5mM MgSO₄、1mM NaH₂PO₄5mM glucose, 20mM HEPES and 20mM NaOAC) and nigericin for 10min, observed under a confocal laser microscope. The fixed excitation wavelength was 405nm for collecting the blue channel 430-480nm and the green channel 500-550 nm. As shown in fig. 8, bright luminescence was observed in both blue and green channels when pH 5.50 cells; when the pH is increased to 9.50, the blue fluorescence of the cells is obviously weakened, the green fluorescence is obviously enhanced, and the change trend of the pH can be obviously seen through the ratio imaging of green light/blue light.

Example 7

To demonstrate the rapid response capability of BTNO of example 1 to cytoplasmic range pH changes, we used NH separately₄Cl(5mM)、H₂O₂HeLa cells stained with BTNO (0.1mM) and NAC (0.5mM) were treated and the change in intracellular fluorescence at each channel was recorded over time. By NH₄The HeLa cell treated by Cl has gradually weakened blue channel fluorescence and gradually strengthened green channel fluorescence, which means NH₄The pH of HeLa cells after Cl treatment was gradually increased (fig. 9). As shown in FIG. 10, using H₂O₂The pH of treated HeLa cells was significantly reduced compared to untreated cells, while NAC treated cells were significantly increased. These results indicate that BTNO can respond rapidly to fluctuations in intracellular pH.

Claims

1. The application of a benzothiazole derivative in preparing a probe for monitoring cytoplasmic pH, wherein the structural formula of the benzothiazole derivative is as follows:

。