Carboxylesterase response type azulene two-photon fluorescent dye as well as preparation method and application thereof

Abstract

The invention discloses a carboxylesterase response type azulene two-photon fluorescent dye as well as a preparation method and application thereof, and the carboxylesterase response type azulene two-photon fluorescent dye is prepared by taking an azulene derivative as a fluorophore. The invention also specifically discloses a preparation method of the azulene dye molecule and application of the azulene dye molecule in preparation of CEs imaging detection reagents in different biological samples of a single organism. The azulene two-photon fluorescent dye prepared by the invention can be highly suitable for different biological membrane permeability, and can detect the activity of carboxylesterase in different environments such as serum, cells, bacteria and tissues; meanwhile, the phototoxicity and the photobleaching property are low, the biotoxicity is proper, the raw materials are easy to obtain, the structure is simple, and preparation and industrialization are easy.

Description

Technical Field

[0001] The present invention relates to the technical field of design, synthesis and application of azulene two-photon fluorescent dyes for specific identification of carboxylesterase in biological analysis, and in particular to a class of carboxylesterase-responsive azulene two-photon fluorescent dyes, a preparation method and application thereof. Background Art

[0002] Carboxylester esterases (CEs), key members of the serine hydrolase superfamily, are important phase I metabolic enzymes belonging to group B esterases. CEs are ubiquitously expressed in a wide variety of animal species and localized in the endoplasmic reticulum lumen of tissues such as the lung, liver, intestine, kidney, and skin. They participate in the metabolism of numerous exogenous and endogenous substances and drugs, including angiotensin-converting enzyme inhibitors, anticancer drugs, and immunomodulators. They play a key role in a range of pathophysiological processes, including lipid homeostasis, drug metabolism, lipid metabolism, and biological detoxification. Therefore, studying the activity of CEs in organisms is of paramount importance.

[0003] Currently, a variety of detection technologies have been reported for detecting CEs activity, including chromatography, protein reconstitution, protein immunoblotting, and fluorescence imaging. Among them, fluorescence imaging technology with fluorescent dyes as the core has been used to monitor changes in CEs activity in different pathological environments due to its high selectivity, high sensitivity, spatial and temporal resolution, and negligible biotoxicity. Researchers have focused on designing fluorescent dyes to monitor changes in CEs activity in different pathological environments. For example, dimethylcarbamoyl esters were introduced into the hemicyanine structure to detect fluctuations in CEs activity in diabetic patients under specific enzymatic hydrolysis of CEs; near-infrared CEs fluorescent dyes constructed with carbamate as the recognition group were successfully used to track the CEs activity of Dili in situ. Based on the "hydroxyl protection-deprotection" dye design strategy, high sensitivity and specificity of CEs were achieved, and it was successfully used as an intermediary method for clinical liver cancer diagnosis and surgical guidance. The above fluorescent dyes showed excellent performance in the detection of CEs in different pathological environments. They provide promising tools for the diagnosis and treatment of related diseases and also provide important guidance for further optimizing the performance of CEs dyes. However, there is still a lack of "integrated" universal dyes for detecting CEs in various environments, especially those containing bacteria, which greatly limits the progress of related pathological mechanisms and clinical treatment. Therefore, there is an urgent need to construct a new fluorescent dye for comprehensive detection of CEs in multiple biological environments from a single organism (including bacteria), thereby providing a potential universal and "integrated" tool for the diagnosis and treatment of CE-related diseases. Summary of the Invention

[0004] The technical problem solved by the present invention is to provide a class of carboxylesterase-responsive azulene two-photon fluorescent dyes and a preparation method thereof. The azulene two-photon fluorescent dyes prepared by this method can respond to carboxylesterase rapidly, specifically and with high sensitivity. By simultaneously detecting the activity of carboxylesterase under multiple environments, they can be used as a general tool for monitoring carboxylesterase activity and as a means of diagnosing and treating carboxylesterase-related diseases.

[0005] The present invention adopts the following technical solutions to solve the above technical problems:

[0006] A class of carboxylesterase-responsive azulene two-photon fluorescent dyes, the structure of which is shown in Formula I:

[0007]

[0008] In formula I, R1 and R2 are each independently -H, -CN, -NH2, -OH, -(CH2) x CH3, -(CH2) x CN, -(CH2) x COOH, -(CH2) x OH, -(CH2) x COCH3, -(CH2) x COO(CH2) x CH3, -COO(CH2) x CH3, -COO(CH2) x COOCH3, -CH[(CH2) x COOH]2 or -CH[(CH2) x OH]2, x is an integer from 1 to 8; R3 is -H, -CH3, -(CH2) x CH3, -[(CH2) x CH3]2CH, -CH[(CH2) x COOH]2、-CH[(CH2) x COOCH3]2, -(CH2) x COCH3, -(CH2) x COOH, -CH[(CH2) x COONH2]2, -CH[(CH2) x CONH2]2 or -CH[(CH2) x OH]2, x is an integer from 1 to 8.

[0009] A method for preparing a carboxylesterase-responsive azulene two-photon fluorescent dye, the specific preparation steps of which are:

[0010] Step S1: uniformly mix the compound represented by Formula III and hydrogen peroxide, add them to a reaction solvent, and stir to react to obtain the compound represented by Formula II, wherein the corresponding structural formulas of the compound represented by Formula III and the compound represented by Formula II are:

[0011]

[0012] Step S2, dissolving the compound represented by Formula II obtained in Step S1 in acetic anhydride and heating under reflux to react to obtain the compound represented by Formula 1-1, i.e., a carboxylesterase-responsive azulene two-photon fluorescent dye. The reaction equation of the synthesis process is:

[0013]

[0014] Furthermore, in step S1, the molar ratio of the compound represented by formula III to hydrogen peroxide is 1:5 to 1:10.

[0015] Furthermore, the reaction solvent in step S1 is one or more of dichloromethane, ethanol, methanol, acetic acid, sulfuric acid, tetrahydrofuran, sodium hydroxide aqueous solution and N,N-dimethylformamide.

[0016] Furthermore, the reaction solvent in step S2 is one or more of acetic anhydride, anhydrous ethanol, methanol, acetic acid, sulfuric acid, tetrahydrofuran, sodium hydroxide aqueous solution and N,N-dimethylformamide.

[0017] Furthermore, the heating reflux reaction temperature in step S2 is 50-70°C.

[0018] The carboxylesterase-responsive azulene two-photon fluorescent dye of the present invention is used in the preparation of a reagent for imaging detection of carboxylesterase in different biological samples of a single organism.

[0019] The carboxylesterase-responsive azulene two-photon fluorescent dye of the present invention is used in the preparation of a reagent for imaging detection of carboxylesterase in serum, cells or bacteria.

[0020] The carboxylesterase-responsive azulene two-photon fluorescent dye of the present invention is used in the preparation of a reagent for imaging the fluctuation of carboxylesterase activity in different biological samples of a single organism under drug-induced liver injury.

[0021] The carboxylesterase-responsive azulene two-photon fluorescent dye of the present invention can respond to carboxylesterase rapidly, specifically, and with high sensitivity. By simultaneously detecting the potential of carboxylesterase activity under multiple environments, it can be used as a universal tool for monitoring carboxylesterase activity and as a means of diagnosing and treating carboxylesterase-related diseases.

[0022] The carboxylesterase-responsive azulene-based two-photon fluorescent dye of the present invention exhibits a specific, highly sensitive response to carboxylesterase. Simultaneously, the design of the amphiphilic molecular structure allows the fluorescent dye to exhibit good membrane permeability to both cell membranes and bacterial cell walls. Therefore, the fluorescent dye can not only independently detect carboxylesterase activity in serum, cells, or bacteria, but can also simultaneously monitor fluctuations in carboxylesterase activity in different biological samples from a single organism under conditions of drug-induced liver injury. These characteristics make it a promising comprehensive and versatile tool for studying and diagnosing the pathology of carboxylesterase-related diseases.

[0023] The present invention has the following advantages and beneficial effects: The dye molecules described in the present invention have two-photon properties and can image deeper tissues. The dye molecules described in the present invention can trigger excellent photochemical reactions under the action of intramolecular charge transfer, and respond specifically and sensitively to carboxylesterase. The dye molecules described in the present invention have low biological toxicity and can simultaneously monitor fluctuations in CE activity in different biological samples from a single organism under drug-induced liver injury. The raw materials are readily available, the structure is simple, and the dye molecules are easy to prepare and industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the H NMR spectrum of the fluorescent dye 1-1 prepared in Example 1.

[0025] Figure 2 This is the water solubility test result of the fluorescent dye 1-1 prepared in Example 1.

[0026] Figure 3 This is the pH stability test result of the fluorescent dye 1-1 prepared in Example 1.

[0027] Figure 4 This is a diagram showing the enzyme interference test results of the fluorescent dye 1-1 prepared in Example 1.

[0028] Figure 5 This is a graph showing the photostability test results of the fluorescent dye 1-1 prepared in Example 1.

[0029] Figure 6 This is a test result of the response of the fluorescent dye 1-1 prepared in Example 1 to carboxylesterase.

[0030] Figure 7 This is a graph showing the cytotoxicity test results of the fluorescent dye 1-1 prepared in Example 1. DETAILED DESCRIPTION

[0031] The specific technical contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0032] Example 1

[0033] The fluorescent dye 1-1 was prepared using the following synthetic route:

[0034]

[0035] Synthesis of fluorescent dye 1-1:

[0036] The compound represented by Formula II (1.00 g, 3.30 mmol) was dissolved in acetic anhydride and heated to 55°C under reflux for 2 h. After the reaction was complete, dichloromethane (2 × 20 mL) was added to the reaction system for extraction. The combined organic extracts were dried over anhydrous sodium sulfate and filtered. The crude product was purified by silica gel column chromatography with dichloromethane to obtain an orange powder, fluorescent dye 1-1, in a 40% yield. 1 HNMR (600MHz,CDCl3)δ9.11(d,J=11.6Hz,2H),7.76(s,2H),7.30(d,J=11.6Hz,2H),4.46(q,J=7.1Hz,4H),2.35(s,3H),1.47(t,J=7.1Hz,6H). The H NMR spectrum of the obtained product is shown in Figure 1 shown.

[0037] Water solubility test of fluorescent dye 1-1:

[0038] Weigh a certain amount of the above-synthesized fluorescent dye 1-1 to prepare a 6mM DMSO solution. Use a pipette to transfer the above mother solution to 3mL of water and add it in a certain gradient concentration. Stir and fully dissolve it. At the same time, ensure that the final volume of DMSO added is less than one thousandth of the total volume. Measure its absorbance value. The test results are as follows: Figure 2 The results are shown in Figure 2 As shown: when the concentration of fluorescent dye 1-1 is greater than 6 μM, the absorbance value deviates from the linear relationship, that is, the maximum solubility of fluorescent dye 1-1 in water is 6 μM.

[0039] pH stability test of fluorescent dye 1-1:

[0040] The fluorescent dye 1-1 (6 μM) synthesized in Example 1 was added to solutions with different pH values ​​and its fluorescence intensity was measured. The test results are as follows: Figure 3 The results showed that the fluorescent dye 1-1 has good pH stability, which is beneficial for analyzing the activity of CEs in different biological samples under different pathological conditions.

[0041] Interference test of fluorescent dye 1-1:

[0042] The fluorescent dye 1-1 (6 μM) synthesized in Example 1 was added to a PBS buffer solution (pH = 7.4). Then, enzyme interferors (interferors 1 to 19 are lysozyme; β-amylase; trypsin; pepsin; proteinase K; SnCl2; CdCl2; MnCl2; CoCl2; CuSO4; HgCl2; Zn(NO3)2·6H2O; FeCl2·7H2O; K2HPO4·H2O; Na2CO3; MgSO4; LiCO3; Al(NO3)3; CEs) were added to the test system in sequence, and their fluorescence emission spectra were measured. The test results are shown in Figure 2. Figure 4 The results showed that fluorescent dye 1-1 is not interfered with by other substances in the body and has a highly sensitive and specific detection analysis for CEs, which is expected to be used for the detection of CEs activity in different biological samples of a single organism.

[0043] Photostability test of fluorescent dye 1-1:

[0044] Fluorescent dye 1-1 was prepared into a DMSO solution with a concentration of 6 μM and placed in a cuvette. The cuvette was then placed 30 cm away from the light source, and cold hydrazine containing a saturated sodium nitrite solution was placed between the cuvette and the light source. The fluorescence emission spectra of the cuvette were then tested in sequence under different illumination time intervals. The results are shown in Figure 2. Figure 5 As shown, the fluorescence signal of fluorescent dye 1-1 remained almost unchanged under 500W iodine tungsten lamp for 5.0h. Results The surface fluorescent dye 1-1 has good photostability.

[0045] Fluorescent dye 1-1 response test to carboxylesterase:

[0046] The fluorescent dye 1-1 (6 μM) synthesized in Example 1 was added to a PBS buffer solution (pH = 7.4), and then the carboxylesterase activity equivalent was increased in sequence (0.2 U / mL, 0.4 U / mL, 0.6 U / mL, 0.8 U / mL, 1 U / mL), and the fluorescence spectrum change was measured by a fluorescence spectrophotometer. The results are shown in FIG. Figure 6 As shown, the fluorescence signal intensity of fluorescent dye 1-1 at 490 nm increases significantly in sequence, indicating that fluorescent dye 1-1 can perform highly sensitive and quantitative detection of carboxylesterase activity in solution.

[0047] Evaluation experiment of cytotoxicity of fluorescent dye 1-1:

[0048] HepG2 cells were selected as the research object. The cell survival rate was used to characterize the cytotoxicity of the dye. The cells were seeded in a 96-well plate at a density of 5*104 cells / mL, with a volume of 100μL in each well, and cultured at 37°C and a volume fraction of 5% CO2 for 24 hours. Then, fluorescent dye 1-1 was added to the culture medium in gradient concentrations, and 5 replicate wells were set for each gradient concentration. A blank control was set, and the cell survival rate was detected after culture. During the test, 20μL of 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) solution was first added to each well, and cultured at 37°C and a volume fraction of 5% CO2 for 4 hours. Then the original culture medium was removed and DMSO (150μL / well) was added. The OD value was then measured with an enzyme reader. This was repeated three times. The results are as follows. Figure 7 The results showed that when the concentration of the fluorescent dye was 20 μM, the cell survival rate was still above 90%, indicating that the synthesized fluorescent dye 1-1 has existing biological safety.

[0049] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A carboxylesterase-responsive azulene two-photon fluorescent dye, characterized in that The structure is shown in Formula I: In formula I, R1 and R2 are each independently -H, -CN, -NH2, -OH, -(CH2) x CH3, -(CH2) x CN, -(CH2) x COOH, -(CH2) x OH, -(CH2) x COCH3, -(CH2) x COO(CH2) x CH3, -COO(CH2) x CH3, -COO(CH2) x COOCH3, -CH[(CH2) x COOH]2 or -CH[(CH2) x OH]2, x is an integer from 1 to 8; R3 is -H, -CH3, -(CH2) x CH3, -[(CH2) x CH3]2CH, -CH[(CH2) x COOH]2、-CH[(CH2) x COOCH3]2, -(CH2) x COCH3, -(CH2) x COOH, -CH[(CH2) x COONH2]2, -CH[(CH2) x CONH2]2 or -CH[(CH2) x OH]2, x is an integer from 1 to 8.

2. A method for preparing the carboxylesterase-responsive azulene two-photon fluorescent dye according to claim 1, characterized in that The specific preparation steps are: Step S1: uniformly mix the compound represented by Formula III and hydrogen peroxide, add them to a reaction solvent, and stir to react to obtain the compound represented by Formula II, wherein the corresponding structural formulas of the compound represented by Formula III and the compound represented by Formula II are: Step S2, dissolving the compound represented by Formula II obtained in Step S1 in acetic anhydride and heating under reflux to react to obtain the compound represented by Formula 1-1, i.e., a carboxylesterase-responsive azulene two-photon fluorescent dye. The reaction equation of the synthesis process is:

3. The method for preparing the carboxylesterase-responsive azulene two-photon fluorescent dye according to claim 1, wherein: The molar ratio of the compound represented by formula III to hydrogen peroxide in step S1 is 1:5 to 1:

10.

4. The method for preparing the carboxylesterase-responsive azulene two-photon fluorescent dye according to claim 1, wherein: The reaction solvent in step S1 is one or more of dichloromethane, ethanol, methanol, acetic acid, sulfuric acid, tetrahydrofuran, sodium hydroxide aqueous solution and N,N-dimethylformamide.

5. The method for preparing the carboxylesterase-responsive azulene two-photon fluorescent dye according to claim 1, wherein: The reaction solvent in step S2 is one or more of acetic anhydride, anhydrous ethanol, methanol, acetic acid, sulfuric acid, tetrahydrofuran, sodium hydroxide aqueous solution and N,N-dimethylformamide.

6. The method for preparing the carboxylesterase-responsive azulene two-photon fluorescent dye according to claim 1, wherein: The heating reflux reaction temperature in step S2 is 50-70°C.

7. Use of the carboxylesterase-responsive azulene two-photon fluorescent dye according to claim 1 in preparing a reagent for imaging and detecting carboxylesterase in different biological samples of a single organism.

8. Use of the carboxylesterase-responsive azulene two-photon fluorescent dye according to claim 1 in the preparation of a reagent for imaging detection of carboxylesterase in serum, cells or bacteria.

9. Use of the carboxylesterase-responsive azulene two-photon fluorescent dye according to claim 1 in the preparation of a reagent for imaging the fluctuation of carboxylesterase activity in different biological samples of a single organism under drug-induced liver injury.

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