阅读说明：本技术 可避免细胞内GSH干扰的Cys荧光探针及制备和应用 (Cys fluorescent probe capable of avoiding interference of intracellular GSH (glutathione) and preparation and application thereof ) 是由 刘景� 张洪星 于 2021-09-02 设计创作，主要内容包括：本发明涉及荧光探针领域,具体涉及一种可避免细胞内GSH干扰的Cys荧光探针及制备和应用。近年来一系列高选择性Cys荧光探针被相继开发,这些探针尽管在一定程度上排除了GSH引发的荧光信号的干扰,但GSH消耗探针所带来的敏感性降低的限制仍然没有有效地克服。为解决上述问题,本发明开发了一种不受GSH干扰的特异性Cys荧光探针及其制备和应用,该荧光探针不仅能直接与Cys反应产生大的荧光关-开响应,而且其与GSH的反应产物能进一步被Cys取代,产生同样的荧光产物和荧光响应,因此不仅能避免细胞内高浓度GSH对荧光信号的干扰,而且能避免GSH对探针的消耗。(The invention relates to the field of fluorescent probes, in particular to a Cys fluorescent probe capable of avoiding interference of intracellular GSH (glutathione) and preparation and application thereof. In recent years a series of highly selective Cys fluorescent probes have been developed in succession, which, although eliminating to some extent the interference of GSH-induced fluorescence signals, still do not effectively overcome the limitation of reduced sensitivity brought about by GSH-consuming probes. In order to solve the problems, the invention develops a specific Cys fluorescent probe which is not interfered by GSH, and preparation and application thereof, the fluorescent probe can directly react with Cys to generate large fluorescence off-on response, and the reaction product of the fluorescent probe and the GSH can be further replaced by Cys to generate the same fluorescent product and fluorescent response, thereby not only avoiding the interference of high-concentration GSH in cells on fluorescent signals, but also avoiding the consumption of the probe by the GSH.)

1. A Cys fluorescent probe capable of avoiding interference of intracellular GSH, which is characterized in that the structural formula of the probe is as follows:

2. a method of preparing a Cys fluorescent probe capable of avoiding intracellular GSH interference according to claim 1, comprising the steps of:

(1) under the protection of nitrogen, 4' -methylenebis (N, N-dimethylaniline) is dissolved in ultra-dry tetrahydrofuran, N-butyllithium is dropwise added into the reaction solution under the condition of controlling the temperature to be-78 ℃, and the temperature is continuously controlled for reaction for 2 hours; at the temperature, dropwise adding dichlorodimethylsilane into the reaction solution gradually, heating the reaction solution to room temperature, stirring for reacting for 2 hours, adding a hydrochloric acid aqueous solution to neutralize the reaction solution after the reaction is finished, evaporating tetrahydrofuran, extracting residual liquid by using diethyl ether, and respectively using saturated NaHCO for combined organic phases₃Washing the solution, water and a saturated sodium chloride aqueous solution, and drying to obtain dihydrosilicone red;

(2) dissolving dihydroprasugrel in acetone, controlling the temperature to be 15 ℃ below zero in an ice salt bath, adding potassium permanganate powder into the solution, returning the reaction solution to room temperature, continuing to react for 2 hours, and filtering, drying and separating by column chromatography to obtain the silatrane-erythrone;

(3) dissolving the silicone-prasuzurine in dry dichloromethane, dropwise adding oxalyl chloride into the solution, stirring the reaction solution at a room temperature for 10 minutes, after the reaction is finished, drying the solvent, and separating the crude product by column chromatography to obtain the probe.

3. The method for preparing Cys fluorescent probe capable of avoiding intracellular GSH interference according to claim 2, wherein the molar ratio of 4, 4' -methylenebis (N, N-dimethylaniline), N-butyllithium and dichlorodimethylsilane in the step (1) is 1: 4: 1.8.

4. the method for preparing Cys fluorescent probe capable of avoiding interference of intracellular GSH according to claim 2, wherein the concentration of the aqueous hydrochloric acid solution in the step (1) is 1 mol/L.

5. The method for preparing Cys fluorescent probe capable of avoiding interference of intracellular GSH (glutathione) according to claim 2, wherein the molar ratio of the silicone-prasugrel to the oxalyl chloride in the step (3) is 1: 1.2.

6. The method of claim 2, wherein the Cys fluorescent probe is used for the column chromatography separation of the developing reagent CH in step (3)₂Cl₂：CH₃The volume ratio of CN is 5: 2.

7. Use of a Cys fluorescent probe according to claim 1 to avoid intracellular GSH interference, in the preparation of a reagent for detecting Cys in a cell.

Technical Field

The invention relates to the field of fluorescent probes, in particular to a Cys fluorescent probe capable of avoiding interference of intracellular GSH (glutathione) and preparation and application thereof.

Background

In addition to providing energy for cell survival and development, mitochondrial respiration also induces large amounts of Reactive Oxygen Species (ROS), mainly including superoxide radicals (O), due to electron leakage from the respiratory chain₂ ^·-) Hydrogen peroxide (H)₂O₂) Hypochlorous acid (HClO), hypobromous acid (HBrO), and a hydroxyl radical (HO.). In thatUnder the condition of unbalanced cellular redox balance, the excessively generated ROS can oxidize various biological macromolecules in cells, so that the normal functions of the cells are damaged, and various diseases are finally caused. This imbalance between oxidation and oxidation resistance, which tends to oxidize, is called Oxidative Stress (Oxidative Stress). In fact, in order to maintain redox balance, cells naturally express a variety of antioxidant systems, of which small molecule biological thiols constitute one of the important antioxidant systems in the cell, playing a key role in maintaining redox balance in the cell. Among them, cysteine (Cys) is an important biological thiol. Cys, homocysteine (Hcy) and Glutathione (GSH) maintain the oxidation-reduction balance in organisms together; secondly, due to the strong nucleophilic ability and coordination ability of sulfhydryl in Cys structure, Cys can be condensed with poisonous aromatic compound to form thioether ammonia acid, and can also be complexed with heavy metals such as copper, mercury and the like, thereby playing a role in detoxification. Cys is also involved in the structure of various proteins, and is involved in biological reactions such as biocatalysis and posttranslational modification of proteins. Due to these important physiological functions, an abnormal content of Cys in the body will lead to serious disease.

In view of this, the development of a biocompatible, highly selective and sensitive method for Cys detection is of great importance not only for the study of various known and unknown physiopathological functions of Cys, but also for the development of related therapeutic drugs.

Among various detection methods, the fluorescent probe technology is an indispensable tool for modern biomedical research due to the characteristics of short analysis time, simple operation, high sensitivity, visualization, sample nondestructiveness and the like, and plays a great role in revealing the positioning and functions of bioactive molecules and ions. In view of this, in recent decades, a large number of bio-thiol fluorescent probes have been reported successively by using a series of specific chemical reactions occurring with bio-thiols. However, since Cys has similar structure and reactivity to GSH and intracellular Cys concentration (200. mu.M) is much lower than that of GSH (1-10mM), the development of Cys fluorescent probes that avoid intracellular GSH interference is quite challenging. It is to be noted that, although of the same type, halfThe structure and reactivity of cystine (Hcy) with Cys are very similar (only one-CH difference)₂Groups) which, due to their very low intracellular concentration (-10 μ M), cause negligible interference. Fluorescent probes capable of selectively sensing intracellular Cys reported so far are mainly classified into three types, aldehyde cyclization type, addition-cyclization type and substitution-rearrangement type. Although fluorescent probes based on the above three strategies are capable of highly selective fluorescence sensing of Cys, there are some drawbacks in practical use. For example, most Cys fluorescent probes with the aldehyde group in a ring shape have weak reactivity and low water solubility, which results in poor sensitivity, long fluorescence response time and low biocompatibility, and the fluorescent regulation mechanism of the probes is not clear, so that the fluorescent response mode is difficult to grasp in design; although the Cys fluorescent probes of the addition-cyclization type and the substitution-rearrangement type can avoid the interference of a GSH-induced fluorescent signal, the Cys fluorescent probes can be consumed by GSH with high intracellular concentration, so that the intracellular probe concentration is reduced, and the detection sensitivity of Cys in cells is influenced. Therefore, how to avoid these disadvantages, especially the problem of GSH-depleted probes, is a significant challenge currently faced in designing Cys fluorescent probes. The silatrane red is a near-infrared fluorescent dye with high molar extinction coefficient and fluorescence quantum yield, strong pH tolerance, good water solubility and good light stability. Importantly, the carbon atom number 9 of the silatrane red and the derivatives thereof has strong electropositivity and can perform addition or aromatic nucleophilic substitution reaction with a nucleophilic reagent. By utilizing the property, aiming at the challenges, the invention synthesizes the Cys fluorescent probe SiPyCl which can avoid GSH interference by utilizing the reversibility of substitution reaction and the irreversibility of rearrangement reaction.

Disclosure of Invention

Aiming at the problems, the invention provides a Cys fluorescent probe capable of avoiding GSH interference and preparation and application thereof. The probe can perform substitution-rearrangement reaction with Cys to generate a red fluorescent 'amino-silatrane red' dye, and only performs substitution reaction with GSH to generate a non-fluorescent 'thio-silatrane red' dye. Importantly, the 'sulfo-silicone-pyrrazone' dye generated after the reaction of SiPyCl and GSH can further perform 'substitution-rearrangement' reaction with Cys to generate red fluorescent 'amino-silicone-pyrrazone'; moreover, the efficiency of the reaction is high, even in the presence of millimolar levels of GSH, within minutes. Therefore, the probe SiPyCl can react with Cys to generate red fluorescent 'amino-silatrane' dye no matter whether GSH exists or not. In other words, SiPyCl is a Cys fluorescent probe that avoids GSH interference.

In order to achieve the purpose, the invention adopts the following technical scheme:

a Cys fluorescent probe that avoids intracellular GSH interference, the probe being SiPyCl having the formula:

a preparation method of Cys fluorescent probe capable of avoiding interference of intracellular GSH comprises the following steps:

(1) under the protection of nitrogen, dissolving 4, 4' -methylenebis (N, N-dimethylaniline) (compound 1) in ultra-dry tetrahydrofuran, dropwise adding N-butyllithium into the reaction solution under the condition of controlling the temperature to be-78 ℃, and continuing to control the temperature to react for 2 hours; at the temperature, dropwise adding dichlorodimethylsilane into the reaction solution gradually, heating the reaction solution to room temperature, stirring for reacting for 2 hours, adding a hydrochloric acid aqueous solution to neutralize the reaction solution after the reaction is finished, evaporating tetrahydrofuran, extracting residual liquid by using diethyl ether, and respectively using saturated NaHCO for combined organic phases₃Washing the solution, water and a saturated sodium chloride aqueous solution, drying and spin-drying to obtain dihydrogensenoxadine red (compound 2), wherein the compound 2 is directly subjected to the next reaction without purification;

(2) dissolving dihydropyranthrone (compound 2) in acetone, controlling the temperature to be-15 ℃ under the condition of an ice salt bath, adding potassium permanganate powder into the solution, recovering the reaction solution to the room temperature, continuing to react for 2 hours, and filtering, drying and separating by column chromatography to obtain the silatrane-redketone (compound 3) which is a yellow solid;

(3) dissolving the silicone-prasuzurine (compound 3) in dry dichloromethane, dropwise adding oxalyl chloride into the solution, reacting for 10 minutes while stirring at a room temperature in a reaction solution, after the reaction is finished, drying the solvent in a rotary manner, and separating the crude product by column chromatography to obtain the probe SiPyCl.

Further, in the step (1), the molar ratio of 4, 4' -methylenebis (N, N-dimethylaniline), N-butyllithium to dichlorodimethylsilane is 1: 4: 1.8.

further, the concentration of the hydrochloric acid aqueous solution in the step (1) is 1 mol/L.

Further, the molar ratio of the silicone prasuzurine to the oxalyl chloride in the step (3) is 1: 1.2.

Further, the column chromatography separation developing agent CH in the step (3)₂Cl₂：CH₃The volume ratio of CN is 5: 2.

An application of Cys fluorescent probe capable of avoiding interference of intracellular GSH in preparing a Cys reagent for detecting intracellular GSH.

Compared with the prior art, the invention has the following advantages:

GSH concentration in cells is millimolar (1-10mM), and most of Cys fluorescent probes reported at present have the limit of being easily consumed by intracellular GSH. The probe provided by the invention can directly perform substitution-rearrangement reaction with Cys to generate highly-fluorescent 'amino-pyrrazone' dye, and the non-fluorescent 'thio-pyrrazone' dye generated by the reaction of the probe and GSH can further perform substitution-rearrangement reaction with Cys to generate highly-fluorescent 'amino-pyrrazone' dye. Therefore, the probe provided by the invention is a high-selectivity Cys fluorescent probe capable of avoiding the consumption of the probe by high-concentration GSH in cells.

Drawings

FIG. 1 is a drawing of Compound 3¹H NMR chart (CDCl)₃,600MHz)；

FIG. 2 is a drawing of Compound 3¹³C NMR chart (CDCl)₃,150MHz)；

FIG. 3 is a HRMS profile of Compound 3;

FIG. 4 shows SiPyCl compounds¹H NMR chart (CDCl)₃,600MHz)；

FIG. 5 shows SiPyCl compounds¹³C NMR chart (CDCl)₃,150MHz)；

FIG. 6 is a HRMS map of SiPyCl compound;

FIG. 7 is a graph showing the change of fluorescence spectrum of the probe in PBS with time (0-30 min).

FIG. 8 shows the change in absorption and fluorescence spectra resulting from the reaction of the probe SiPyCl with Cys/GSH, (FIG. 8A) the change in absorption spectra resulting from Cys/GSH; (FIG. 8B) Cys/GSH induced change in fluorescence spectrum of SiPyCl; (FIG. 8C) fluorescence titration spectra of SiPyCl against Cys in GSH deficiency; (FIG. 8D) fluorescence titration spectra of SiPyCl against Cys in the presence of GSH (1 mM); (FIG. 8E) results of selectivity of probe SiPyCl for Cys;

FIG. 9 is a schematic diagram of the mechanism of Cys sensing by the probe SiPyC1 of the present invention.

Detailed Description

Example 1

A Cys fluorescent probe capable of avoiding interference of intracellular GSH, which has a structural formula as follows:

a preparation method of the Cys fluorescent probe capable of avoiding interference of intracellular GSH comprises the following steps:

(1) under the protection of nitrogen, dissolving the compound 1(6.00g, 14.6mmol) in ultra-dry tetrahydrofuran (200mL), slowly dropping n-butyllithium (n-hexane solution with the concentration of 2.4M, 24.3mL, 58.4mmol, 4 equivalents) into the reaction solution at the temperature of-78 ℃, and continuing to react for 2 hours at the temperature; at this temperature, dichlorodimethylsilane (3.2mL, 26.28mmol, 1.8 equivalents) was slowly added dropwise to the reaction mixture, and the reaction mixture was slowly warmed to room temperature and stirred for reaction for 2 hours; after the reaction was complete, aqueous hydrochloric acid (1M, 50mL) was carefully added to neutralize the reaction; evaporating tetrahydrofuran, extracting residual liquid with diethyl ether, mixing organic phases, and respectively adding saturated NaHCO₃Washing the solution, water and saturated sodium chloride aqueous solution, drying and spin-drying to obtain a compound 2, combiningThe product 2 was directly subjected to the next reaction without purification.

(2) Compound 2 was dissolved in acetone (30mL), potassium permanganate powder (5.75g) was slowly added to the solution at-15 ℃ in an ice salt bath, and the reaction solution was allowed to return to room temperature for 2 hours. The reaction solution was filtered, dried and subjected to column Chromatography (CH)₂Cl₂) Compound 3 was obtained as a yellow solid after isolation (1.65g, 34.7% yield).

¹H NMR(600Hz,CDCl₃)δ8.10(d,J＝9.0Hz,2H),6.87(d,J＝9.0Hz,2H),6.83(s,2H),3.11(s,12H),0.49(s,6H).¹³C NMR(150MHz,CDCl₃)δ185.3,151.4,140.5,131.6,129.7,114.3,113.2,40.1,0.97.ESI-MS:[M+H]⁺calcd for 325.1736,Found 325.1734.

(3) Compound 3(0.324g, 1.0mmol) was dissolved in dry dichloromethane (10mL), oxalyl chloride (1.2mmol, 1.2 equiv.) was slowly added dropwise to the above solution, and the reaction was stirred at room temperature for 10 minutes. After the reaction is finished, the solvent is dried by spinning, and the crude product is subjected to column Chromatography (CH)₂Cl₂/CH₃CN 5/2, v/v) was isolated as probe SiPyC1(0.313g, 82.8% yield).

¹H NMR(600Hz,CDCl₃)δ8.43(d,J＝9.6Hz,2H),7.14(s,2H),6.97(d,J＝9.6Hz,2H),3.48(s,12H),0.58(s,6H).¹³C NMR(150MHz,CDCl₃)δ164.0,155.6,148.7,140.9,127.4,121.4,119.4,42.7,0.41.ESI-MS:[M]⁺calcd for 343.1392,Found 343.1390.

Example 2

1. Test solution preparation

The probe was made up in 2mM stock with acetonitrile and subsequently diluted to the test concentration with 20mM PBS (pH 7.4).

2. Stability test of Probe

The stability of the probe in aqueous solution was first studied on a fluorescence spectrometer, prepared in PBS (2. mu.M) and placed in a cuvette, as shown in FIG. 7, with the emission wavelength of the probe in PBS being 688nm, and the fluorescence spectrum remained almost unchanged after continuous scanning for 30 minutes. The above results indicate that the cationic property, which is important for biological applications, imparts excellent water solubility to the probe and can be stably present in PBS.

3. Study of the reactivity of the Probe with Cys/GSH

In PBS buffer, SiPyCl itself has a main absorption peak at 663 nm; a blue-shifted absorption peak appeared at 470nm after Cys addition, and a red-shifted absorption peak appeared at 688nm after GSH addition. This result suggests that SiPyCl generates "amino-silazane" and "thiopyrrazoxane" dyes with Cys and GSH, respectively (fig. 8A). SiPyCl itself is almost non-fluorescent under excitation of 470nm excitation light; after addition of Cys, a strong fluorescence peak appears at 618 nm; after addition of GSH, hardly any fluorescence peaks were observed (fig. 8A). This result suggests that SiPyCl can avoid interfering fluorescence signals induced by GSH. Importantly, when GSH was added first and then Cys was added to the SiPyCl solution, the red-shifted absorption peak by GSH disappeared, the blue-shifted absorption peak by Cys appeared, and the absorption intensity of this blue-shifted absorption peak was nearly identical to that in the absence of GSH (fig. 8A). Furthermore, a strong fluorescence peak was observed when GSH was added first and then Cys was added to the SiPyCl solution, and the fluorescence intensity of this peak was almost identical to that in the absence of GSH (fig. 8B). Thus, SiPyCl is also a Cys fluorescent probe that avoids GSH depletion of the probe. Further fluorescence titration assays showed that SiPyC1 exhibited nearly consistent Cys sensing performance in the absence (fig. 8C) and presence (fig. 8D) of 1mM GSH, and that the latter exhibited a better fluorescence response to low concentrations of Cys. The selectivity experiments showed that no biologically relevant cation, anion, active oxide, amino acid induced fluorescence enhancement of SiPyCl (fig. 8E), and therefore the probe had very good selectivity for Cys.

4. Research on Cys sensing mechanism by probe

In pure PBS, SiPyCl and Cys can generate rapid 'substitution-rearrangement' reaction to generate high-fluorescence amido-silatrane red (lambda ex:470 nm); however, due to the unstable macrocyclic transition state, SiPyCl and GSH can only undergo a "substitution" reaction to generate non-fluorescent thiosilazole red under 470nm excitation; importantly, the sulpho-silatrane red can further perform a rapid 'substitution-rearrangement' reaction with Cys to generate high-fluorescence amino-pyrromone red; SiR is therefore a highly sensitive Cys fluorescent probe that avoids GSH interference (including signal interference and depletion probes). The inventors speculate that the driving force of Cys substitution for a GSH unit in SiR-GSH comes from two aspects: the first is the large electropositive force of Si-pyrrosia No. 9 carbon atoms, and the second is the irreversible rearrangement which occurs immediately after Cys replaces a GSH unit, wherein the former establishes a balanced reaction between Cys/GSH and a probe, and the latter promotes the irreversible movement of the total reaction to the right.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.