Water-soluble fluorescent probe with multiple responses to copper ions, pH value and serum protein as well as preparation method and application of water-soluble fluorescent probe

Abstract

The invention discloses a water-soluble fluorescent probe with copper ion, pH value and serum protein multi-response and a preparation method and application thereof.The structural formula of the fluorescent probe is # imgabs0 #, the fluorescent probe is based on a DO2A group and a DNS group, the copper ion, pH value and serum protein multi-response is effectively achieved, and the water-soluble fluorescent probe has the advantages that the water-soluble fluorescent probe can be applied to water-soluble fluorescent probes; and beneficial reference is provided for subsequent research and development of DNS fluorescent probes.

Description

Technical Field

[0001] The invention belongs to the technical field of functional fluorescent probe molecule development, and relates to a fluorescent probe with multiple responses to copper ions, pH values ​​and serum proteins, and a preparation method and application thereof. Background Art

[0002] Fluorescence detection technology has the characteristics of being fast, sensitive, efficient and rapid. The use of small molecule probes with fluorescent activity is an important means of current biomolecule analysis and detection. Fluorescent probes are a special type of fluorescent molecule that has characteristic fluorescence in the ultraviolet-visible-near infrared region, and their fluorescence properties (such as excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can change sensitively with the properties of the environment (such as polarity, refractive index, viscosity, etc.). This property makes fluorescent probes widely used in various detection and labeling applications. pH-responsive fluorescent probe molecules can be widely used in biomedical research, food safety testing and environmental monitoring; copper ion-responsive fluorescent probe molecules can be widely used in environmental monitoring, biomedical research and chemical analysis, while protein-responsive fluorescent probe molecules can be widely used in biomedical research, neuroscience research, immunology research, drug screening and new drug development. At present, most fluorescent probes have a single function, either responding to copper ions or responding to pH. There are few probe molecules that have good response effects on multiple detections and multiple detection objects. Therefore, the current scope of use of fluorescent probe molecules is limited. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention provides a fluorescent probe with multiple responses to copper ions, pH values ​​and serum proteins, and a preparation method and application thereof, thereby solving the technical problem of single response of fluorescent probe molecules in the prior art.

[0004] The present invention is achieved through the following technical solutions: A water-soluble fluorescent probe with multiple responses to copper ions, pH value and serum protein, the structural formula of which is shown in formula (I): Formula (I).

[0005] The method for preparing the water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein comprises the following steps: S1: The organic solution of benzyl chloroformate is added dropwise to the organic solution of 1,4,7,10-tetraazacyclododecane, and reacted at room temperature to obtain intermediate 1, whose structural formula is shown in formula (II): Formula (II); S2: adding an organic solution of N,N-diisopropylethylamine and an organic solution of tert-butyl bromoacetate to the organic solution of the intermediate 1 in sequence, and after reaction, obtaining an intermediate 2, the structural formula of which is shown in formula (III): Formula (III); S3: Add a catalyst to the organic solution of the intermediate 2, and stir the mixture under a hydrogen atmosphere to obtain an intermediate 3, the structural formula of which is shown in formula (IV): Formula (IV) S4: adding potassium carbonate to the organic solution of the intermediate 3 and adding an organic solution of dansyl chloride, and after the reaction is completed, an intermediate 4 is obtained, and its structural formula is shown in formula (V): Formula (V); S5: adding a dichloromethane solution of trifluoroacetic acid to the organic solution of the intermediate 4, stirring for reaction, and obtaining the fluorescent probe with multiple responses to copper ions, pH value and protein, the structural formula of which is shown in formula (I).

[0006] Preferably, in step S1, the reaction is carried out at room temperature for 24 to 30 h.

[0007] Preferably, in step S2, the reaction temperature is 60-80 °C.

[0008] Preferably, in step S3, the catalyst is any one of palladium-carbon, palladium hydroxide-carbon, palladium-polyethyleneimine, palladium-polyethyleneimine / formic acid and HBr / HOAc.

[0009] Preferably, in step S3, the reaction is carried out for 12 to 16 hours.

[0010] Preferably, in step S4, the reaction is carried out for 12 to 16 hours. After the reaction is completed, the mixture is washed with a saturated sodium bicarbonate solution and water in sequence, the organic phase is collected, anhydrous sodium sulfate is added, the mixture is allowed to stand, and the intermediate 4 is obtained after filtration, concentration and purification.

[0011] Preferably, in step S5, the reaction is stirred for 12 to 20 hours. After the reaction is completed, dichloromethane and toluene are added in sequence to remove the solvent to obtain the fluorescent probe.

[0012] The above-mentioned water-soluble fluorescent probe with multiple responses to copper ions, pH values ​​and serum proteins is used in the preparation of products for detecting copper ions, pH values ​​and proteins.

[0013] Preferably, the product comprises a detection reagent or a kit.

[0014] Compared with the prior art, the present invention has the following beneficial technical effects: The present invention discloses a water-soluble fluorescent probe with multiple responses to copper ions, pH value and serum protein. The structural formula of the fluorescent probe is shown in formula (I): Formula (I) The derivative DO2A of Cyclen containing N-acetic acid side arm in this fluorescent probe has strong metal coordination ability for copper ions, so it can realize the specific detection of copper ions; in addition, the fluorescent probe contains DNS. The nitrogen atom in the dimethylamine group of the DNS part in this structure can be protonated in acidic medium, resulting in changes in fluorescence intensity within a certain pH range. Due to the protonation of the dimethylamine group, it can realize the specific detection of pH value in the environment; in addition, when the DNS group of this fluorescent probe enters the hydrophobic cavity of proteins such as HAS and BSA, the fluorescence intensity increases sharply due to the inhibition of TICT, so it can realize the specific detection of proteins in the environment. Based on the DO2A group and the DNS group, this fluorescent probe effectively realizes multiple responses of copper ions, pH value and serum proteins, which provides a useful reference for the subsequent research and development of DNS fluorescent probes.

[0015] Furthermore, in step S1, reacting at room temperature for 24 to 30 h can effectively improve the reaction conversion rate.

[0016] Furthermore, in step S2, the reaction temperature is 60-80°C, which can effectively increase the reaction rate and shorten the reaction time.

[0017] Furthermore, in step S3, the catalyst is any one of palladium-carbon, palladium hydroxide-carbon, palladium-polyethyleneimine, palladium-polyethyleneimine / formic acid and HBr / HOAc, which can effectively complete the adsorption-reaction-desorption cycle process.

[0018] Furthermore, in step S3, the reaction is carried out for 12 to 16 hours to complete the reaction.

[0019] Furthermore, in step S4, the reaction is carried out for 12 to 16 hours to complete the reaction. After the reaction is completed, the mixture is washed with a saturated sodium bicarbonate solution and water in sequence, the organic phase is collected, anhydrous sodium sulfate is added, and after standing, the intermediate 4 is obtained by filtering, concentrating and purifying, thereby effectively improving the purity of the product and ensuring the fluorescence properties of the material.

[0020] Furthermore, in step S5, the reaction is stirred for 12 to 20 hours, which can effectively cause the tert-butyl carboxyl protecting group to fall off. After the reaction is completed, dichloromethane and toluene are added in sequence to remove the solvent, so that trifluoroacetic acid and toluene can be azeotroped to remove trifluoroacetic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 A synthetic route for a method for preparing a fluorescent probe with multiple responses to copper ions, pH values ​​and serum proteins in the present invention; Figure 2 The mass spectrum of the fluorescent probe molecule prepared in Example 1 of the present invention; Figure 3 The nuclear magnetic resonance hydrogen spectrum of the fluorescent probe molecule prepared in Example 1 of the present invention; Figure 4 The fluorescence spectra of the fluorescent probe molecules prepared in Example 1 of the present invention at different pH values; Figure 5 Fluorescence spectra of blank group-pure probe DO2A-DNS (10 μM) and HSA, BSA, Ubi, T4lys (10 μM) in PBS buffer (pH=7.4, 10 μM); Figure 6 The fluorescence spectra of probe DO2A-DNS (10 μM) with different concentrations of HSA (0-20 μM) added in PBS buffer (pH=7.4, 10 μM); Figure 7 The fluorescence spectra of different concentrations of BSA (0-20 μM) added to the probe DO2A-DNS (10 μM) in PBS buffer (pH=7.4, 10 μM); Figure 8 The fluorescence spectra of the probe DO2A-DNS (10 μM) in aqueous solution (pH = 7.4, 10 μM) with different copper ions (40 μM) added; Fig. 9 is the fluorescence intensity of DO2A-DNS (10 μM) in the presence of blank DO2A-DNS and different metal ions (40 μM) at pH 7.6; Fig.10 The fluorescence spectra of blank DO2A-DNS (10 μM) and different concentrations of copper ions at pH 7.6. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0024] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0025] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0026] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0027] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0028] The present invention provides a water-soluble fluorescent probe with multiple responses to copper ions, pH value and serum protein, and its structural formula is shown in formula (I): Formula (I).

[0029] In addition, if Figure 1 As shown, the present invention also provides a method for preparing a water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein, comprising the following steps: S1: The organic solution of benzyl chloroformate is added dropwise to the organic solution of 1,4,7,10-tetraazacyclododecane, and reacted at room temperature to obtain intermediate 1, whose structural formula is shown in formula (II): Formula (II); S2: Adding an organic solution of N,N-diisopropylethylamine (DIPEA) and an organic solution of tert-butyl bromoacetate to the organic solution of the intermediate 1 in sequence, after reaction, an intermediate 2 is obtained, and its structural formula is shown in formula (III): Formula (III); S3: Add a catalyst to the organic solution of the intermediate 2, and stir the mixture under a hydrogen atmosphere to obtain an intermediate 3, the structural formula of which is shown in formula (IV): Formula (IV); The catalyst is any one of palladium-carbon, palladium hydroxide-carbon, palladium-polyethyleneimine, palladium-polyethyleneimine / formic acid and bromic acid / acetic acid (HBr / HOAc), and in the palladium-carbon catalyst, the mass fraction of palladium to carbon is 5%~10%, and in the palladium hydroxide-carbon catalyst, the mass fraction of palladium hydroxide to carbon is 10%~20%. S4: adding potassium carbonate to the organic solution of the intermediate 3 and adding an organic solution of dansyl chloride, and after the reaction is completed, an intermediate 4 is obtained, and its structural formula is shown in formula (V): Formula (V); S5: adding trifluoroacetic acid to the organic solution of the intermediate 4, stirring for reaction, and obtaining the fluorescent probe with multiple responses to copper ions, pH value and protein, the structural formula of which is shown in formula (I).

[0030] Specifically, the above process is: S1: Synthesis of intermediate 1, i.e. 1,7- N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane: 1,4,7,10-tetraazacyclododecane was dissolved in chloroform, and benzyl chloroformate was dissolved in chloroform and slowly added to the chloroform solution of 1,4,7,10-tetraazacyclododecane at low temperature, and then reacted at room temperature for 24-30 hours, and then filtered, the organic layer was evaporated and the solvent was concentrated, and it was recrystallized in ether to obtain intermediate 1. The slow addition method effectively controls the reaction rate and avoids the formation of by-products.

[0031] S2: Synthesis of intermediate 2, i.e. 1,7-N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: Dissolve 1,7-N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane in acetonitrile, add a solution of DIPEA mixed in acetonitrile, and add tert-butyl bromoacetate dissolved in acetonitrile. Heat the solution at 60-90°C, monitor the reaction by thin layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS), concentrate the solvent, and extract the residue with ether and water. Wash the organic layer further with water, sodium hydroxide solution, and water. 2 SO 4 The solvent was dried and evaporated. The crude product was purified by column chromatography to afford Intermediate 2.

[0032] S3: Synthesis of intermediate 3, i.e. 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: Dissolve 1,7-N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane in anhydrous methanol and add the catalyst. 2 The mixture was stirred for 12-16 h under the same conditions. The solvent and by-products were removed under reduced pressure to obtain intermediate 3.

[0033] S4: Synthesis of intermediate 4, i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: Dissolve 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane in chloroform, add potassium carbonate, dissolve dansyl chloride in chloroform, and stir at room temperature. Continue to react for 12-16 hours after the addition is complete. Cool to room temperature and filter. The solution is then diluted with saturated sodium bicarbonate solution and H 2 O, collect the organic phase, add anhydrous sodium sulfate, and let stand overnight. Filter and concentrate the solution. Purify the crude product with a silica gel column to obtain intermediate 4.

[0034] S5: Synthesis of fluorescent probe, 1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane: Intermediate 4 was dissolved in dichloromethane, trifluoroacetic acid was added and mixed at ambient temperature for 12-16 h. Dichloromethane was added and the solvent was removed, and then an additional amount of toluene was added and the solvent was removed a second time to obtain the final fluorescent probe. This fluorescent probe can be represented as DO2A-DNS.

[0035] The present invention uses the N-acetic acid side arm DO2A contained in the cyclen derivative as a direct or indirect recognition group, and on this basis, simultaneously introduces the double-armed dansyl group as a connecting arm for other functional modifications. That is, the cyclen derivative contains the N-acetic acid side arm DO2A as a direct recognition group, which can coordinate copper ions. On this basis, simultaneously introduces the double-armed DNS as a connecting arm for other functional modifications. It has its unique advantages in constructing fluorescent probes. Among them, the DNS group is a typical fluorophore with environment-dependent fluorescence emission, which promotes its good potential candidate for the development of fluorescent probes. DNS acts as a linking arm and is substituted onto the nitrogen heterocyclic DO2A ring by nucleophilic substitution. Based on the design and coordination between the above molecular parts, a dansyl-based fluorescent probe is designed and tested for the detection of Cu 2+ , pH responsive and protein-detectable probes.

[0036] The fluorescent probe structure and experimental steps in the present invention enable the reaction to maintain high efficiency and low by-products in a dripping manner in the initial stage, successfully preparing a high-purity product, and the probe is sensitive to the microenvironment, the system preparation is simple, the chemical stability is good, and the selectivity is good. The fluorescent probe copper ion obtained by the present invention has good selectivity, and the good pH response gives DO2A-DNS the potential to detect pH changes in a physiological environment. In addition, the probe can achieve high-selective detection of proteins such as BSA and HSA in the aqueous phase, and can also be used in serum.

[0037] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0038] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio.

[0039] Example 1 A method for preparing a water-soluble fluorescent probe with multiple responses to copper ions, pH value and serum protein comprises the following steps: S1: Synthesis of intermediate 1, i.e. 1,7- N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane: 1,4,7,10-tetraazacyclododecane (1 g) was dissolved in chloroform (10 mL). Benzyl chloroformate (1.32 g, 9.56 mmol) in chloroform was slowly added dropwise to the above solution at 0°C and reacted for 7 h. Then, the reaction was continued at room temperature for 24 h. The organic layer was then filtered and concentrated by vacuum distillation. The crude product was recrystallized in ether to obtain intermediate 1 (2.1 g, yield 84%).

[0040] S2: Synthesis of intermediate 2, i.e. 1,7-N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: 1,7-N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (1 g, 1.95 mmol, 1 eq.) was dissolved in acetonitrile (20 mL), and DIPEA (1.259 g, 9.74 mmol, 5 eq.) was added and mixed in acetonitrile (8 mL) to clarify the solution. Tert-butyl bromoacetate (1.14 g, 5.84 mmol, 3 eq.) was dissolved in acetonitrile (4 mL) and mixed and added. The solution was heated at 60 °C and the reaction was monitored by TLC (DCM:MeOH, 20:1) and LC-MS to confirm that the starting material of intermediate 1 had been consumed. It was then allowed to cool to ambient temperature. The solvent was evaporated and the residue was extracted with ether (20 mL) and water (20 mL). The organic layer was further washed with water (10 mL), NaOH solution (0.5 g / 10 mL) and water (10 mL). The organic layer was washed with anhydrous Mg 2 SO 4 The solvent was dried and evaporated. 2 O 3 , CH 2 Cl 2 / CH 3 The crude product was separated and purified by 4% paraformaldehyde (2:1:4)-1:2:1 (OH = 50:1-20:1) to obtain the final intermediate 2 (2.17 g, 63%) as a colorless oil.

[0041] S3: Synthesis of intermediate 3, i.e. 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: 1,7- N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane (1.1 g, 1.64 mmol) was dissolved in anhydrous methanol (10 mL) and Pd-C catalyst (10% Pd, 50% weight, 0.55 g) was added. The mixture was stirred under hydrogen for 12 h. The solvent and by-products were removed under reduced pressure to obtain the final oily colorless intermediate 3 (quantitative yield).

[0042] S4: Synthesis of intermediate 4, i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: Dissolve 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane in 10 mL of chloroform, add potassium carbonate (1.509 g, 10.8 mmol), dissolve dansyl chloride (2.509 g, 9.6 mmol) in 10 mL of chloroform, add dropwise to the reaction flask, and stir at room temperature for 16 h. Cool to room temperature and filter. The solution is successively treated with saturated NaHCO 3 solution (20 mL × 3) and H 2 O (20 mL × 2), collect the organic phase, add Na 2 SO 4 , and left overnight. Filter and concentrate the solution to obtain a pale yellow crude product. 2 O 3 , CH 2 Cl 2 / CH 3 OH=100:1-70:1) to separate and purify the crude product to obtain a yellow powder with a yield of 54%.

[0043] S5: Synthesis of fluorescent probe (DO2A-DNS), i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane: Intermediate 4 was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added and mixed at ambient temperature for 12 h. Dichloromethane (5 mL) was added and the solvent was removed, then an additional amount of toluene (5 mL) was added and the solvent was removed a second time to give the final product as a yellow powder.

[0044] Figure 2 This is the mass spectrum of the fluorescent probe molecule (DO2A-DNS) prepared in Example 1 of the present invention. As can be seen from the figure, the probe MS (ESI) m / z: C 36 H 46 N 6 O 8 S2 The measured value of [M+H]+ was 755.26, which was basically consistent with the predicted value of 755.28, indicating that the structure of the synthesized probe molecule was correct.

[0045] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe molecule (DO2A-DNS) prepared in Example 1 of the present invention, 1 H NMR (600 MHz, DMSO-d) δ 8.54 (d, J = 8.6 Hz, 2H), 8.30 (d, J = 8.7 Hz, 2H), 8.06 (dd, J = 7.4, 1.2 Hz, 2H), 7.68 (dd, J = 8.5, 7.3 Hz, 2H), 7.63 (dd, J = 8.7,7.5 Hz, 2H), 7.28 (d, J = 7.6 Hz, 2H), 3.87 (s, 4H), 3.55 (s, 8H), 3.32 (s, 7H), 2.83 (s, 12H).

[0046] In order to further test and evaluate the performance of the fluorescent probe molecules obtained by the present invention, the following tests are used to illustrate: The pH response selectivity of the probe DO2A-DNS prepared in Example 1 was tested.

[0047] Specifically: Use Tris-HCl buffer solution to adjust the pH value of the system to 10.5, 9.8, 8.9, 7.8, 6.7, 5.8, and 3.8, respectively. Dissolve the probe DO2A-DNS in dimethyl sulfoxide to prepare a 10 mM stock solution. Add 2 μL of PBS stock solution (10.0 μM) of DO2A-DNS to the centrifuge tube, and use solutions under different pH conditions to make the volume 2 mL. Scan with a fluorescence spectrophotometer and measure the fluorescence spectrum. Perform the experiment in parallel 3 times. Test as Figure 4 As shown in the figure, DO2A-DNS has sensitive fluorescence emission when the pH value in aqueous solution changes in the range of 3 to 10. These characteristics give DO2A-DNS the potential to detect pH changes in physiological environments.

[0048] In addition, the protein response selectivity of the probe DO2A-DNS prepared in Example 1 was tested.

[0049] Specifically: human serum albumin (HSA), bovine serum albumin (BSA), ubiquitin (UBi), and T4 lysozyme (T4lys) were prepared into 10 mM solutions, respectively, and stored in a refrigerator at -20°C. 2 μL of DO2A-DNS PBS stock solution (10.0 mM) was added to the PE tube, and each group was added with the prepared (10 mM) BSA, 2 μL, 4 μL, 6 μL, and 8 μL of HSA, respectively. Two groups of PBS solutions with different concentrations were prepared and the volume was fixed to 2 mL. After incubation at 37°C for 30 min, the solution was scanned with a fluorescence spectrophotometer, and then the fluorescence spectrum was measured. The experiment was repeated 3 times in parallel. The test results are as follows: Figure 5 As shown by Figure 5 It can be seen that in aqueous solution, the intrinsic fluorescence of the probe itself is weak. Due to the twisted intramolecular charge transfer (TICT) inside DO2A-DNS, when DO2A-DNS enters the hydrophobic cavity of proteins HAS and BSA, TICT is inhibited, causing a sharp increase in fluorescence intensity. In addition, in PBS buffer (pH=7.4, 10μM), the fluorescence spectra of probe DO2A-DNS (10 μM) with different concentrations of HSA (0-20 μM) added, and in PBS buffer (pH=7.4, 10μM), the fluorescence spectra of probe DO2A-DNS (10 μM) with different concentrations of BSA (0-20 μM) added, are shown as follows: Figure 6 and Figure 7 As shown by Figure 6 and Figure 7 It can be seen that the fluorescence intensity of HAS and BSA gradually increases with the increase of concentration, indicating that the probe is expected to monitor the production of HAS and BSA.

[0050] Furthermore, a copper ion selectivity test experiment of the probe DO2A-DNS prepared in Example 1 was implemented.

[0051] Specifically, the probe DO2A-DNS was prepared into a 10 mM stock solution using PBS buffer, 2 μL of DO2A-DNS stock solution (10.0 μM) was added to the centrifuge tube, and 2 μL of different copper ion solutions (Cd 2+ , Cu 2+ 、Te 2+ 、Zn 2+ 、Ag + , Pd 2+ , Mn 2+ , Fe 3+ 、Co 2+ 、Ni 2+), so that the final concentration of copper ions is 40 μM, and finally PBS is used to make the volume 2 mL. After mixing evenly, the fluorescence spectrophotometer is scanned and the fluorescence spectrum is measured. The experiment is repeated 3 times in parallel. The test results are shown in Figure 8 as well as Fig. 9 ,Depend on Figures 8 and 9 It can be seen that the probe is sensitive to copper ions (Cu 2+ ) has the most obvious response, that is, the probe can be used for the response detection of copper ions. All the copper ion solutions above were prepared with ultrapure water.

[0052] In addition, further, the probe DO2A-DNS prepared in Example 1 was subjected to a test experiment on its selectivity to copper ions.

[0053] The fluorescence probe DO2A-DNS was used to measure the effects of different concentrations of Cu 2+ The fluorescence response of 2 ·2H 2 O) was prepared into a 0.1 M stock solution with ultrapure water for use as the Cu 2+ Source. The spectral properties were studied by mixing 2 μL of the probe stock solution (10 μM) and 5 μL of different concentrations of Cu in PBS. 2+ The solution was mixed to 2 mL and the fluorescence test results were shown in Fig.10 ,Depend on Fig.10 It can be seen that with Cu 2+ With the increase of Cu concentration, the fluorescence intensity of the fluorescent probe DO2A-DNS gradually weakened, which indicates that 2+ There is an interaction between the Cu and the fluorescent probe, and the fluorescence is quenched. 2+ Before and after, the absorption peak position of the fluorescent probe DO2A-DNS did not change much, but the absorption intensity decreased significantly, which is also related to the Cu 2+ The quenching process caused by

[0054] Example 2 A method for preparing a water-soluble fluorescent probe with multiple responses to copper ions, pH value and serum protein comprises the following steps: S1: Synthesis of intermediate 1, i.e. 1,7- N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane: 1,4,7,10-tetraazacyclododecane (1 g) was dissolved in chloroform (10 mL), and a chloroform solution of benzyl chloroformate (1.32 g, 9.56 mmol) was slowly added dropwise to the above solution at 0°C, and reacted for 7 hours, and then reacted for 25 hours at room temperature, and then filtered, and the organic layer was concentrated by vacuum distillation. The obtained crude product was recrystallized in diethyl ether to obtain intermediate 1 (2.1 g, yield 84%).

[0055] S2: Synthesis of intermediate 2, i.e. 1,7-N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: 1,7-N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (1 g, 1.95 mmol, 1 eq.) was dissolved in acetonitrile (20 mL), and DIPEA (1.259 g, 9.74 mmol, 5 eq.) was added and mixed in acetonitrile (8 mL) to clarify the solution. Tert-butyl bromoacetate (1.14 g, 5.84 mmol, 3 eq.) was dissolved in acetonitrile (4 mL) and mixed and added. The solution was heated at 80 °C and the reaction was monitored by TLC (DCM:MeOH, 20:1) and LC-MS to confirm that the starting material of intermediate 1 had been consumed. It was then allowed to cool to ambient temperature. The solvent was evaporated and the residue was extracted with ether (20 mL) and water (20 mL). The organic layer was further washed with water (10 mL), NaOH solution (0.5 g / 10 mL) and water (10 mL). The organic layer was washed with anhydrous Mg 2 SO 4 The solvent was dried and evaporated. 2 O 3 , CH 2 Cl 2 / CH 3 The crude product was separated and purified by 4% paraformaldehyde (2:1:4)-1:2:1 (OH = 50:1-20:1) to obtain the final intermediate 2 (2.17 g, 63%) as a colorless oil.

[0056] S3: Synthesis of intermediate 3, i.e. 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: 1,7- N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane (1.1 g, 1.64 mmol) was dissolved in anhydrous methanol (10 mL) and Pd-C catalyst (10% Pd, 50% weight, 0.55 g) was added. The mixture was stirred under hydrogen for 14 h. The solvent and by-products were removed under reduced pressure to obtain the final oily colorless intermediate 3 (quantitative yield).

[0057] S4: Synthesis of intermediate 4, i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: Dissolve 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane in 10 mL of chloroform, add anhydrous potassium carbonate (1.509 g, 10.8 mmol), dissolve dansyl chloride (2.509 g, 9.6 mmol) in 10 mL of chloroform, add dropwise to the reaction flask, and stir at room temperature for 12 h. Cool to room temperature and filter. The solution was successively treated with saturated NaHCO 3 solution (20 mL×3) and H 2 O (20 mL × 2), collect the organic phase, add Na 2 SO 4 , and left overnight. Filter and concentrate the solution to obtain a pale yellow crude product. 2 O 3 , CH 2 Cl 2 / CH 3 OH=100:1-70:1) to separate and purify the crude product to obtain a yellow powder with a yield of 54%.

[0058] S5: Synthesis of fluorescent probe (DO2A-DNS), i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane: Intermediate 4 was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added and mixed at ambient temperature for 15 h. Dichloromethane (5 mL) was added and the solvent was removed, then an additional amount of toluene (5 mL) was added and the solvent was removed a second time to give the final product as a yellow powder.

[0059] Example 3 A method for preparing a water-soluble fluorescent probe with multiple responses to copper ions, pH value and serum protein comprises the following steps: S1: Synthesis of intermediate 1, i.e. 1,7- N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane: 1,4,7,10-tetraazacyclododecane (1 g) was dissolved in chloroform (10 mL), and a chloroform solution of benzyl chloroformate (1.32 g, 9.56 mmol) was slowly added dropwise to the above solution at 0°C, and reacted for 7 hours, and then reacted for 30 hours at room temperature, and then filtered, and the organic layer was concentrated by vacuum distillation. The obtained crude product was recrystallized in diethyl ether to obtain intermediate 1 (2.1 g, yield 84%).

[0060] S2: Synthesis of intermediate 2, i.e. 1,7-N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: 1,7-N,N-di(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (1 g, 1.95 mmol, 1 eq.) was dissolved in acetonitrile (20 mL), and DIPEA (1.259 g, 9.74 mmol, 5 eq.) was added and mixed in acetonitrile (8 mL) to clarify the solution. Tert-butyl bromoacetate (1.14 g, 5.84 mmol, 3 eq.) was dissolved in acetonitrile (4 mL) and mixed and added. The solution was heated at 90 °C and the reaction was monitored by TLC (DCM:MeOH, 20:1) and LC-MS to confirm that the starting material of intermediate 1 had been consumed. It was then allowed to cool to ambient temperature. The solvent was evaporated and the residue was extracted with ether (20 mL) and water (20 mL). The organic layer was further washed with water (10 mL), NaOH solution (0.5 g / 10 mL) and water (10 mL). The organic layer was washed with anhydrous Mg 2 SO 4 The solvent was dried and evaporated. 2 O 3 , CH 2 Cl 2 / CH 3 The crude product was separated and purified by 4% paraformaldehyde (2:1:4)-1:2:1 (OH = 50:1-20:1) to obtain the final intermediate 2 (2.17 g, 63%) as a colorless oil.

[0061] S3: Synthesis of intermediate 3, i.e. 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: 1,7- N,N-di(benzyloxycarbonyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane (1.1 g, 1.64 mmol) was dissolved in anhydrous methanol (10 mL) and Pd-C catalyst (10% Pd, 50% weight, 0.55 g) was added. The mixture was stirred under hydrogen for 16 h. The solvent and by-products were removed under reduced pressure to obtain the final oily colorless intermediate 3 (quantitative yield).

[0062] S4: Synthesis of intermediate 4, i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane: Dissolve 1,7-N,N-di(tert-butyloxycarbonylmethylene)-1,4,7,10-tetraazacyclododecane in 10 mL of chloroform, add potassium carbonate (1.509 g, 10.8 mmol), dissolve dansyl chloride (2.509 g, 9.6 mmol) in 10 mL of chloroform, add dropwise to the reaction flask, and stir at room temperature for 15 h. Cool to room temperature and filter. The solution is successively treated with saturated NaHCO3 solution (20 mL×3) and H 2 O (20 mL × 2), collect the organic phase, add Na 2 SO 4 , and left overnight. Filter and concentrate the solution to obtain a pale yellow crude product. 2 O 3 , CH 2 Cl 2 / CH 3 OH=100:1-70:1) to separate and purify the crude product to obtain a yellow powder with a yield of 54%.

[0063] S5: Synthesis of fluorescent probe (DO2A-DNS), i.e. 1,7-N,N-di(dansyl)-4,10-N,N-di-(carboxymethyl)-1,4,7,10-tetraazacyclododecane: Intermediate 4 was dissolved in dichloromethane (5 mL), trifluoroacetic acid (5 mL) was added and mixed at ambient temperature for 16 h. Dichloromethane (5 mL) was added and the solvent was removed, then an additional amount of toluene (5 mL) was added and the solvent was removed a second time to give the final product as a yellow powder.

[0064] Example 4 The difference from Example 1 is that in step S3, the catalyst is a palladium-carbon catalyst, wherein the mass fraction of palladium to carbon is 5%.

[0065] Example 5 The difference from Example 1 is that in step S3, the catalyst is a palladium-carbon catalyst, wherein the mass fraction of palladium to carbon is 10%.

[0066] Example 6 The difference from Example 1 is that in step S3, the catalyst is a palladium hydroxide-carbon catalyst, wherein the mass fraction of palladium hydroxide to carbon is 10%.

[0067] Example 7 The difference from Example 1 is that in step S3, the solvent is ethanol, wherein the mass fraction of palladium hydroxide to carbon is 20%.

[0068] Example 8 The difference from Example 1 is that in step S3, the catalyst is palladium-polyethyleneimine.

[0069] Example 9 The difference from Example 1 is that in step S3, the catalyst is palladium-polyethyleneimine / formic acid.

[0070] Example 10 The difference from Example 1 is that in step S3, the catalyst is HBr / HOAc.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A water-soluble fluorescent probe with multiple responses to copper ions, pH value and serum protein, characterized in that: Its structural formula is shown in formula (I): Formula (I).

2. A method for preparing a water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein as described in claim 1, characterized in that: The following steps are involved: S1: The organic solution of benzyl chloroformate is added dropwise to the organic solution of 1,4,7,10-tetraazacyclododecane, and reacted at room temperature to obtain intermediate 1, whose structural formula is shown in formula (II): Formula (II); S2: adding an organic solution of N,N-diisopropylethylamine and an organic solution of tert-butyl bromoacetate to the organic solution of the intermediate 1 in sequence, and after reaction, obtaining an intermediate 2, the structural formula of which is shown in formula (III): Formula (III); S3: Add a catalyst to the organic solution of the intermediate 2, and stir the mixture under a hydrogen atmosphere to obtain an intermediate 3, the structural formula of which is shown in formula (IV): Formula (IV) S4: adding potassium carbonate to the organic solution of the intermediate 3 and adding an organic solution of dansyl chloride, and after the reaction is completed, an intermediate 4 is obtained, and its structural formula is shown in formula (V): Formula (V); S5: adding a dichloromethane solution of trifluoroacetic acid to the organic solution of the intermediate 4, stirring for reaction, and obtaining the fluorescent probe with multiple responses to copper ions, pH value and protein, the structural formula of which is shown in formula (I).

3. The method for preparing a water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein according to claim 2, characterized in that: In step S1, the reaction is carried out at room temperature for 24 to 30 h.

4. The method for preparing a water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein according to claim 2, characterized in that: In step S2, the reaction temperature is 60-80°C.

5. The method for preparing a water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein according to claim 2, characterized in that: In step S3, the catalyst is any one of palladium-carbon, palladium hydroxide-carbon, palladium-polyethyleneimine, palladium-polyethyleneimine / formic acid and HBr / HOAc.

6. The method for preparing a water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein according to claim 2, characterized in that: In step S3, the reaction is carried out for 12 to 16 hours.

7. The method for preparing a water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein according to claim 2, characterized in that: In step S4, the reaction is carried out for 12 to 16 hours. After the reaction is completed, the mixture is washed with a saturated sodium bicarbonate solution and water in sequence. The organic phase is collected, anhydrous sodium sulfate is added, and the mixture is allowed to stand for 12 to 16 hours. The intermediate 4 is obtained after filtration, concentration and purification.

8. The method for preparing a water-soluble fluorescent probe having multiple responses to copper ions, pH value and serum protein according to claim 2, characterized in that: In step S5, the reaction is stirred for 12 to 20 hours. After the reaction is completed, dichloromethane and toluene are added in sequence to remove the solvent to obtain the fluorescent probe.

9. Use of the water-soluble fluorescent probe with multiple responses to copper ions, pH values ​​and serum proteins as claimed in claim 1 in the preparation of products for detecting copper ions, pH values ​​and proteins.

10. The use according to claim 9, characterized in that: The products include detection reagents or kits.

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