CN109369566B

CN109369566B - Benzothiazole derivative NTNO, and preparation method and application thereof

Info

Publication number: CN109369566B
Application number: CN201811349638.0A
Authority: CN
Inventors: 樊丽; 林博; 贾晶; 王晓东; 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: CN109369566A

Abstract

The invention discloses a benzothiazole derivative NTNO, a preparation method and application thereof. The derivative is specifically named as 6- (2- (6-nitrobenzothiazol-2-yl) vinyl) naphthalene-2-ol (NTNO). The preparation method comprises the following steps: dissolving 6-hydroxy-2-naphthaldehyde and 2-methyl-6-nitrobenzothiazole in a small amount of ethanol, adding piperidine, 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. NTNO as a probe does not fluoresce under acidic conditions, and as the pH is increased, a new fluorescence emission appears at 526nm and gradually increases. pK_aThe value is 7.85 +/-0.022, the pH response linear range is 6.90-8.20, and the monitoring of the change of the pH (6.8-7.4) in the neutral cytoplasm range is facilitated. Meanwhile, NTNO has the function of coupling OH^‑Has the advantages of good selectivity, large Stokes displacement and the like. In addition, NTNO has excellent cell membrane penetrating and cytoplasm staining capacity, and is suitable for monitoring pH change in cytoplasm matrix range.

Description

Benzothiazole derivative NTNO, and preparation method and application thereof

Technical Field

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

Background

Intracellular pH plays an irreplaceable role in many physiological and pathological processes of the cell, such as cell division and apoptosis, regulation of enzymatic activity, maintenance of protein structure and morphology, ion transport, cellular energy generation and transformation, membrane potential balance and information transfer, etc. Meanwhile, studies have shown that the growth, metastasis and drug resistance of cancerous cells (tumor cells), Down syndrome, Alzheimer's disease and other diseases are closely related to intracellular pH abnormality. Therefore, it is very important to monitor the intracellular pH.

In recent years, many fluorescent probes for measuring changes in intracellular pH have been reported one after another, but most of these probes are based on a weakly acidic pH-responsive fluorescent probe, such as lysosomes (pH 4.0-5.5). However, the intracellular cytoplasmic matrix and nucleus, subcellular compartments such as mitochondria, etc. existing in large quantities in the cell have nearly neutral or weakly alkaline pH, so that it is necessary to develop a novel fluorescent probe that can be used for monitoring the nearly neutral or weakly alkaline pH.

Disclosure of Invention

One of the purposes of the invention is to provide a benzothiazole derivative NTNO and a preparation method thereof; the other purpose is to provide the application of NTNO as a probe in monitoring the cytoplasmic pH change.

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

the synthetic route is as follows:

the preparation method of benzothiazole derivative NTNO provided by the invention comprises the following steps:

(1) dropwise adding 2-methylbenzothiazole into a mixed solution of concentrated nitric acid and concentrated sulfuric acid in an ice bath overnight; adding crushed ice into the solution after reaction, adjusting the pH value to 10.0 by using NaOH solution, and filtering; recrystallizing the obtained solid in ethanol to obtain 2-methyl-6-nitrobenzothiazole;

(2) dissolving 2-methyl-6-nitrobenzothiazole and 6-hydroxy-2-naphthaldehyde in a small amount of ethanol according to the molar ratio of 2:3-5, adding a small amount of piperidine, refluxing the obtained mixed solution for 24 hours, and removing the solvent to obtain a crude product of NTNO;

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

The NTNO of the invention has good cell membrane permeability as a probe, and can be used for monitoring pH change, cell marking and the like in a cytoplasmic matrix range.

Compared with the existing pH fluorescent probe, the NTNO synthesized by the invention has the following advantages: (1) pK of NTNO_aThe value is 7.85 +/-0.022, the pH response linear range is 6.90-8.20, and the monitoring of the change of the pH range (6.8-7.4) of the neutral cytoplasmic matrix is very facilitated. (2) NTNO presents an 'off/on' response type to pH response, namely fluorescence does not occur under an acidic condition, fluorescence is gradually enhanced under a near-neutral and weakly alkaline condition, and the interference of background fluorescence, especially the interference of an acidic organelle, is favorably reduced. (3) NTNO has large Stokes displacement (113 nm), and can effectively reduce the interference from exciting light; (4) the NTNO has excellent selectivity on the pH response of the solution and is not interfered by common negative and positive ions, amino acid Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS) and other substances; (5) NTNO has good cell membrane permeability, and pH change in cytoplasm matrix range can be monitored by laser confocal microscopy imaging technology. (6) The NTNO synthesis steps are simple, the cost is low, and the potential commercial application value is realized.

Drawings

FIG. 1 is a graph of the UV absorption spectrum of NTNO according to the present invention, which varies with pH.

FIG. 2 shows that the color of NTNO of the present invention changes from yellow to dark purple before and after the NTNO is combined under natural light to lose proton.

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

FIG. 4 shows the color change of NTNO under UV lamp before and after proton loss, from colorless to green.

FIG. 5 shows Boltzmann function relationship of fluorescence intensity of NTNO at 526nm with pH value, pK_a＝7.85±0.022。

FIG. 6 shows the linear pH response range of 6.90-8.20 of NTNO according to the present invention, as a function of pH at 526 nm.

FIG. 7 shows the selectivity of the NTNO of the present invention in response to pH (pH 7.40) in the presence of common anions and cations and some common amino acids, active oxygen, active nitrogen, etc. in the organism.

FIG. 8 is a laser confocal image of the incubation of the inventive NTNO with HeLa cells for 30min at pH 6.50, pH 7.00, pH 7.40, pH 8.00, pH 8.50 and pH 9.00, respectively.

FIG. 9, after the NTNO of the present invention and HeLa cells were incubated for 30min, H was added thereto respectively₂O₂Confocal laser imaging after 1h incubation with NAC (N-acetylcysteine).

Detailed Description

Example 1

Preparation of 1, 6- (2- (6-nitrobenzothiazol-2-yl) vinyl) naphthalen-2-ol (NTNO):

(1) 19.9mL (0.157mol) of 2-methylbenzothiazole was dropped in a mixed solution of 19.0mL of concentrated nitric acid and 36.0mL of concentrated sulfuric acid in an ice-water bath overnight. 500g of ice was added to the reacted solution, and the pH was adjusted to 10.0 with NaOH solution. Filtering, washing with water for 3 times, and filtering₂H₅OH recrystallization purification gave the product as a yellow-green solid, 10.2g of 2-methyl-6-nitrobenzothiazole, 33%).¹H NMR(400MHz,CDCl₃)：δ8.78(s,1H),8.33(d,J＝8.9Hz,1H),8.04(d,J＝8.8Hz,1H),2.92(d,J＝0.8Hz,3H).¹³C NMR(101MHz,CDCl₃)：δ173.31(s),157.17(s),144.81(s),136.06(s),122.67(s),121.58(s),118.02(s),20.71(s).

(2) 2-methyl-6-nitrobenzothiazole (10mmol, 1.94g), 6-hydroxy-2-naphthaldehyde (15mmol, 2.58g) and 500. mu.L piperidine were placed in 100mL C₂H₅The OH solution is stirred and refluxed for 24 h. Removing solvent to obtain NTNO primary product. With v_{Ethyl acetate}:v_N-hexanePurification by silica gel column separation with 3:1 as eluent gave the product NTNO as a yellow solid (0.52g, 15%).¹H NMR(400MHz,DMSO₆):：δ10.06(s,1H),9.18(s,1H),8.34(d,J＝8.9Hz,1H),8.20–8.09(m,2H),7.93(t,J＝12.3Hz,2H),7.82(d,J＝8.8Hz,1H),7.79–7.71(m,2H),7.19–7.10(m,2H).¹³C NMR(101MHz,DMSO₆)：δ173.24(s),157.51(s),156.83(s),144.11(s),140.35(s),135.55(s),134.76(s),130.30(s),129.90(s),129.40(s),127.47(s),126.89(s),124.01(s),122.50(s),121.92(s),119.93(s),119.31(d,²J(C,N)＝5.9Hz),109.09(s).MS(ESI)m/z:Calcd 349.0641；found 349.0635[M+H]⁺.

Example 2

The NTNO concentration of example 1 was maintained at 100 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 increased, the absorption peak at 421nm is gradually reduced, the absorption peak at 487nm is correspondingly enhanced, and an equal absorption point exists at 443 nm. While the color of the solution changed from yellow to dark purple (fig. 2).

Example 3

The NTNO concentration of example 1 was maintained at 10 μ M, the pH was adjusted in a DMSO/water (2: 1 by volume) system with a small volume of HCl and NaOH solution at a high concentration, the fluorescence emission spectrum was recorded at 413nm as the excitation wavelength (fig. 3). Under weakly acidic or neutral conditions, NTNO hardly emits light, and a new fluorescence emission peak appears at 526nm and gradually increases with the increase of pH value. The color of the solution changed from none to green under the uv lamp (figure 4). Calculation of pK for NTNO by Boltzmann function fitting curves_aThe value was 7.85. + -. 0.022 (FIG. 5), and the pH response linearity ranged from 6.90 to 8.20. The linear regression equation is F ═ 7460000+1121740 × pH, correlation coefficient R²0.99667 (fig. 6).

Example 4

The NTNO concentration of example 1 was maintained at 10 μ M, and the probe was examined for responses to common anions, cations, and some amino acids, ROS, RNS, and the like in a living body at pH 7.40, respectively. As shown in FIG. 7, NTNO hardly responded to the above substances, that is, it was said thatIt is clear that NTNO has excellent selectivity for pH response. The order and concentration of the substances in fig. 7 are, in order: 1, pH 6.75; 2, pH 9.10; 3, K⁺(140mM)；4,Cd²⁺(1mM),5,Mg²⁺(1mM)；6,Li⁺(0.1mM)；7,Co²⁺(0.1mM)；8,Hg²⁺(0.1mM)；9,Ba²⁺(0.1mM)；10,Ni²⁺(0.1mM)；11,Ca²⁺(0.1mM)；12,Fe²⁺(0.1mM)；13,Zn²⁺(0.1mM)；14,Cu²⁺(0.1mM)；15,Mn²⁺(0.1mM)；16,F^-(1mM)；17,Br^-(1mM)；18,I^-(1mM)；19,SO₄ ^2-(1mM)；20,S₂SO₃ ^2-(1mM)；21,SO₃ ^2-(HSO₃ ^-,0.1mM)；22,S^-(HS^-,1mM)；23,NO₃ ^-(1mM)；24,NO₂ ^-(1mM)；25,AC^-(1mM)；26,CO₃ ^2-(HCO₃ ^-,1mM)；27,ClO^-(1mM)；28,ClO₄ ^-(1mM)；29,O₂ ^2-(1mM)；30,ONNO^-(0.1mM)；31,O₂ ^-(1mM)；32,L-GSH(1mM)；33,Hcy(1mM)；34,Cys(1mM)。

Example 5

Adherent HeLa cells were incubated with NTNO of example 1 at pH 7.40, 37 ℃ and 5% CO₂Then gently washed 3 times with phosphate buffer (pH 7.40) to remove excess NTNO, and then treated with high K at pH 6.50, 7.00, 7.40, 8.00, 8.50 and 9.00, 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 is 405nm, and the green fluorescence emission channel 475-. As shown in fig. 8, little fluorescence was observed when pH 6.50, and bright green fluorescence was observed and gradually increased as pH increased to 9.00, indicating that NTNO had a better ability to respond to pH changes in the cytoplasmic matrix.

Example 7

To further demonstrate that the NTNO of example 1 can effectively monitor the change in cytoplasmic matrix pH, H was used₂O₂HeLa cells stained with NTNO (0.1mM) and NAC (0.5mM) were treated separately and the cell status after 1h was recorded. As shown in FIG. 9, via H₂O₂The pH of treated HeLa cells was significantly reduced compared to untreated cells, while NAC-treated cells were significantly increased. These results indicate that NTNO is able to monitor changes in pH within the cytoplasmic matrix.

Claims

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

。