A fluorescent probe for labeling and bioimaging of adjacent thiol proteins and application
By designing a fluorescent probe based on the active dimethyl thioether site of cyclopentanedione, the problems of nonspecificity and poor stability of fluorescent probes in the prior art are solved, and high specificity and high stability labeling of adjacent thiol proteins are achieved, which is suitable for in vitro detection of adjacent thiol proteins and in situ fluorescent labeling in cells.
Patent Information
- Application Number
- CN202310094249.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-08
- Publication Date
- 2025-11-25
- Estimated Expiration
- 2043-02-08
AI Technical Summary
Existing fluorescent probes suffer from nonspecificity and poor stability when detecting adjacent thiol proteins, resulting in inaccurate recognition sites.
A fluorescent probe based on the active dimethyl thioether site on cyclopentanedione was designed. It achieves highly specific and stable labeling by undergoing a specific thiol substitution reaction with adjacent thiol proteins and reversibly decoupling after binding.
It achieves highly specific and stable labeling of adjacent thiol proteins, enabling in vitro detection and in situ intracellular fluorescent labeling. The chemical reaction selectivity and reversibility of the probe were verified by optical signal response and mass spectrometry analysis.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biomedical functional material preparation, and particularly relates to a fluorescent probe for adjacent sulfhydryl protein labeling and biological imaging and application. BACKGROUND
[0002] Cell redox homeostasis refers to the dynamic balance between oxidizing species and reducing species in cells, and is an intrinsic defense mechanism of cells, which plays an important role in maintaining the physiological activities of cells. All biological systems exist in redox balance, and balance oxidation and reduction reactions to achieve conditions suitable for life. Redox signal transduction regulation is mainly achieved by changing the redox state of target proteins. Compared with small molecule sulfhydryl compounds, the sulfhydryl groups in proteins are not only abundant but also more susceptible to various redox chemistry to produce various oxidative modifications.
[0003] There are two methods for adjacent sulfhydryl protein detection and imaging based on small molecule fluorescent probes. One is a method of specific binding of trivalent arsenic to adjacent sulfhydryl groups, but the cytotoxicity of arsenic atoms greatly limits the application of such probes. The other is a fluorescent probe with a bismaleimide as a recognition group, but such probes have non-specificity in the fluorescent labeling reaction due to the addition reaction of some small molecule thiols in cells, such as glutathione, and the addition product of maleimide and sulfhydryl has the defect of hydrolytic instability, so the reliable specific labeling of intracellular adjacent sulfhydryl proteins by these probes is still a challenge.
[0004] Based on the above scientific problems and the problems existing in the previous research, the exploration of adjacent sulfhydryl protein recognition and its physiological and pathological significance is limited by the types of recognition groups and the prominent defect of inaccurate recognition sites, therefore, developing new adjacent sulfhydryl protein recognition receptors and deriving more functional fluorescent probes has important significance for enriching the adjacent sulfhydryl protein detection toolkit and developing disease screening and diagnosis equipment. SUMMARY
[0005] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a fluorescent probe for adjacent sulfhydryl protein labeling and biological imaging and application, so as to solve the technical problem of inaccurate adjacent sulfhydryl protein recognition sites caused by non-specificity and poor stability of the fluorescent probes reported in the prior art.
[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is adopted:
[0007] The present application discloses a fluorescent probe (IDAs) for adjacent sulfhydryl protein labeling and biological imaging, and the structure general formula of the fluorescent probe is as follows:
[0008]
[0009] wherein R is selected from conjugated or non-conjugated groups with electron donating property; R1 and R2 are selected from any aliphatic groups.
[0010] Preferably, R is -NH2, p-dimethylaminobenzene or triphenylamine group.
[0011] Preferably, R1 and R2 are selected from methyl groups.
[0012] Further preferably, the fluorescent probe reversibly labels the adjacent thiol peptide segment, and has the reversible labeling property of recovering the initial peptide segment after decoupling by dithiothreitol.
[0013] Further preferably, the adjacent thiol protein is a class of proteins containing one or more pairs of spatially adjacent thiol groups, in which the adjacent thiol groups are spatially close enough to form disulfide bonds under oxidative conditions and exist in the form of adjacent thiol groups under reducing conditions.
[0014] Still further preferably, the fluorescent probe is one of the following compounds:
[0015]
[0016] The application also discloses application of the fluorescent probe for labeling and bioimaging of the adjacent thiol protein in detection of the adjacent thiol protein.
[0017] Further, the fluorescent probe for labeling and bioimaging of the adjacent thiol protein is administered to a detection object, and in-vitro detection of the adjacent thiol protein is realized through a spectroscopy signal response.
[0018] The application also discloses application of the fluorescent probe for labeling and bioimaging of the adjacent thiol protein in cell localization.
[0019] Further, the fluorescent probe for labeling and bioimaging of the adjacent thiol protein is co-incubated with mitochondrial and lysosomal localization dyes, and subcellular localization of the adjacent thiol protein in living cells is realized through in-situ fluorescent labeling of the living cells.
[0020] Further, in-vitro detection of the adjacent thiol protein is realized through optical signal responses such as fluorescence spectroscopy and ultraviolet absorption spectroscopy; proteomic analysis through liquid chromatography-mass spectrometry; in-situ fluorescent labeling of the living cells through fluorescence confocal microscopy, and in-situ bioimaging and localization detection labeling of the adjacent thiol protein in organelles at the cell level.
[0021] Compared with the prior art, the application has the following beneficial effects:
[0022] The application discloses a fluorescent probe for labeling and bioimaging of adjacent thiol proteins, and is based on an active dimethyl sulfide site on a cyclopentanone, a series of compounds with high specificity, high stability and biocompatibility of adjacent thiol proteins are constructed and screened, the compound can specifically in-situ detect adjacent thiol proteins as a fluorescent probe, and realizes in-vitro fluorescent detection of adjacent thiol proteins and in-situ fluorescent labeling of adjacent thiol proteins in cells.
[0023] The application characterizes the structure of the series of probes by means of nuclear magnetic resonance, high-resolution mass spectrometry and the like, reveals the response relationship between optical signals (ultraviolet absorption, fluorescence intensity) and adjacent thiol protein concentration dependence and time dependence by means of ultraviolet absorption spectrum, fluorescence spectrum, confocal imaging and organelle co-localization imaging and the like, and discusses key problems such as in-situ fluorescent labeling of adjacent thiol proteins and intracellular distribution. Meanwhile, the chemical reaction selectivity of the probe and the adjacent thiol site and the reversible labeling characteristics of the probe-peptide segment coupling product after decoupling by dithiothreitol (DTT) are verified by high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the labeled coupling product of the peptide segment analog and the probe. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 A model schematic diagram of the fluorescent probe IDAs and adjacent thiol protein binding and decoupling effect described in the application.
[0025] Figure 2 Fluorescence spectra of the probe IDA-1 and IDA-2 with adjacent thiol proteins described in the application; wherein, A is an ultraviolet-visible absorption spectrum of the titration reaction of IDA-1 and reduced protein (rBSA); B is an ultraviolet-visible absorption spectrum of the titration reaction of IDA-2 and rBSA; C is a fluorescence emission spectrum of the titration reaction of IDA-1 and rBSA; D is a fluorescence emission spectrum of the titration reaction of IDA-2 and rBSA;
[0026] Figure 3 Linear relationship and detection limit of the probe IDA-2 for ex vivo fluorescence detection of adjacent thiol protein concentration described in the application; wherein, A is a linear relationship and detection limit diagram of IDA-1 for rBSA fluorescence detection; B is a linear relationship and detection limit diagram of IDA-2 for rBSA fluorescence detection;
[0027] Figure 4Mass spectrum analysis of the probe IDA-1 and IDA-2 coupled with adjacent thiol peptide segment analogs and liquid phase tracking of the uncoupling process; wherein, A is a schematic diagram of the coupling and uncoupling of IDAs with the peptide sample; B is a mass spectrum of the coupling product after the peptide segment Ac-YCGCT is combined with IDA-1; C is a mass spectrum of the coupling product after the peptide segment Ac-YCGCT is combined with IDA-2; D is an extracted ion chromatogram of the peptide segment Ac-YCGCT combined with IDA-1; E is an extracted ion chromatogram of the peptide segment Ac-YCGCT combined with IDA-2.
[0028] Figure 5 In-situ imaging and subcellular distribution of the probe IDA-2 on endogenous adjacent thiol proteins in living cells;
[0029] Figure 6 Analysis of the changes of adjacent thiol proteins in different oxidation states of living cells by flow cytometry and quantitative statistics of the fluorescence intensity of each group of experiments by the probe IDA-2; wherein, A is a graph of the influence of adjacent dithiol on redox regulation of endogenous proteins in HepG2 cells analyzed by flow cytometry; B is a fluorescence statistical column chart of flow cytometry analysis;
[0030] Figure 7 Hydrogen spectrum of the molecule NO2-IDA in the embodiment;
[0031] Figure 8 Carbon spectrum of the molecule NO2-IDA in the embodiment;
[0032] Figure 9 Mass spectrum of the molecule NO2-IDA in the embodiment;
[0033] Figure 10 Hydrogen spectrum of the molecule IDA-1 in the embodiment;
[0034] Figure 11 Carbon spectrum of the molecule IDA-1 in the embodiment;
[0035] Figure 12 Mass spectrum of the molecule IDA-1 in the embodiment;
[0036] Figure 13 Hydrogen spectrum of the molecule IDA-2 in the embodiment;
[0037] Figure 14 Carbon spectrum of the molecule IDA-2 in the embodiment;
[0038] Figure 15 Mass spectrum of the molecule IDA-2 in the embodiment;
[0039] Figure 16 Hydrogen spectrum of the molecule IDA-3 in the embodiment;
[0040] Figure 17 Carbon spectrum of the example molecule IDA-3;
[0041] Figure 18 Mass spectrum of the example molecule IDA-3. DETAILED DESCRIPTION
[0042] In order to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present application.
[0043] It should be noted that the terms "first", "second", and the like in the specification and claims of the present application and the above-described accompanying drawings are used to distinguish similar objects, and do not necessarily have to be used to describe a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device that includes a series of steps or units does not have to be limited to only those steps or units clearly listed, but can include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.
[0044] The present application will be described in further detail below in conjunction with the accompanying drawings:
[0045] The present application discloses a fluorescent probe for labeling and bioimaging of adjacent thiol proteins, and the structural general formula is as follows:
[0046]
[0047] In the formula, the molecule has a dimethyl sulfide ester vinyl active site connected with an alpha carbonyl carbon.
[0048] The R group is different from a substitution group, which is an amino group, a p-xylylenediamine group, a triphenylamine group or other electron-donating conjugated or non-conjugated groups; R1 and R2 are methyl groups or other aliphatic chain substitution groups.
[0049] The indenone moiety and the moiety connected in conjugation with the indenone are a fluorophore.
[0050] The term "proximal thiol protein" refers to a class of proteins that contain one or more pairs of proximal thiols, in which the proximal thiols are not necessarily in close sequence proximity to each other, but must be in close enough spatial proximity to enable them to form disulfide bonds under oxidizing conditions and exist as proximal thiols under reducing conditions.
[0051] Reference is made to Figure 1 A model of the binding and uncoupling of the fluorescent probe IDAs of the present application to proximal thiol proteins is shown in the figure. As can be seen, the fluorescent probe IDAs of the present application bind to proximal thiol proteins by specific thiol displacement reaction of the dimethyl sulfide vinyl active site with the proximal thiols on the protein. The probe-peptide segment conjugate recovers the reversible labeling properties of the initial peptide segment after decoupling by dithiothreitol (DTT).
[0052] Example 1. Synthesis of fluorescent probe IDA-1
[0053] The synthesis route is as follows:
[0054]
[0055] NO2-ID: Compound 4-nitrophenyl anhydride (1 g, 5.18 mmol) was dissolved in a solution of acetic anhydride (2 mL) and triethylamine (1.5 mL) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 12 hours. After this time, 1.7 g of ice water and concentrated hydrochloric acid (1.6 mL) were added to the mixture, which was stirred at room temperature for 30 min. Additional hydrochloric acid (5 mL, 5M) was added to the above solution, which was then refluxed for 2 h. After the reaction mixture was cooled to room temperature, water (100 mL) was added, and the mixture was extracted with dichloromethane (3 x 100 mL). The organic layers were combined, washed with brine, and then dried over anhydrous sodium sulfate. After the solvent was removed, it was used directly in the next step.
[0056] NO2-IDA: NO2-ID (0.5 g, 2.62 mmol, 1 eq) and sodium hydride (NaH (60 wt% in mineral oil), 0.21 g 2.01 mmol, 2 eq) were dissolved in 30 mL of dry DMF, and carbon disulfide (0.158 mL, 2.62 mmol, 1 eq) was added dropwise over 5 min. The reaction mixture was stirred for 1 h, and then iodomethane (0.407 mL, 6.54 mmol, 2.5 eq.) was added dropwise over 10 min in an ice bath at 0 °C. The solution was allowed to reach room temperature slowly, and then it was allowed to react for 18 h. The solution was then added dropwise to ice water, and it was stirred for an additional 30 min. The mixture was extracted with dichloromethane, and the organic phase was collected and purified by silica gel column chromatography (100:1 dichloromethane:methanol). The product was a dark yellow solid (150 mg, 19% yield).
[0057] The NMR data for NO2-IDA are as follows:Figure 7 The carbon spectrum data is shown in Figure 2. Figure 8 The mass spectrum data is shown in Figure 3. Figure 9 The mass spectrum data is shown in Figure 4.
[0058] IDA-1: To a solution of NO2-ID A (300 mg, 1.02 mmol, 1 eq.) in absolute ethanol (10 mL), a solution of SnCl2(963 mg, 5.08 mmol, 5 eq.) and concentrated HCl (424 μL, 5.08 mmol, 5 eq.) in absolute ethanol (1 mL) was added dropwise and refluxed for 20 min. After the reaction was completed, 100 mL of water was added and the mixture was extracted with 100 mL of dichloromethane in three portions. The organic layers were combined and dried over anhydrous sodium sulfate. After the solvent was removed under vacuum, the residue was purified by flash column chromatography to give an orange solid (228.00 mg, 85%).
[0059] The hydrogen spectrum data of IDA-1 is shown in Figure 5. Figure 10 The carbon spectrum data is shown in Figure 6. Figure 11 The mass spectrum data is shown in Figure 7. Figure 12 The mass spectrum data is shown in Figure 8.
[0060] Synthesis of fluorescent probe IDA-2
[0061] The synthesis route is as follows:
[0062]
[0063] Br-ID: Compound 4-bromophthalic anhydride (1 g, 4.41 mmol) was dissolved in a solution of acetic anhydride (2.4 mL) and triethylamine (1.3 mL). The reaction mixture was stirred at room temperature for 12 hours. Then, 1.7 g of ice water and concentrated hydrochloric acid (1.6 mL) were added to the mixture, which was stirred at room temperature for 30 min. Hydrochloric acid (7 mL) was added to the above solution, and after the reaction system was cooled to room temperature, water was added and extracted with dichloromethane. The organic layers were combined, washed with brine, and then dried over anhydrous sodium sulfate. After the solvent was removed, a gray-white product was obtained, which was directly used as the raw material for the next step.
[0064] ID-DMA: Compound Br-ID (1 g, 4.44 mmol) was dissolved in tetrahydrofuran, a catalytic equivalent of Pd(PPh3)4was added and the mixture was stirred at room temperature for 30 minutes. 4-(dimethylamino) phenylboronic acid pinacol ester (1.32 g, 5.33 mmol) was added to the reaction flask stirred for 10 minutes, then a solution of Na2C03was added to the reaction, heated and left for 12 h. The reaction was followed by TLC. After the reaction was complete, the reaction mixture was cooled to room temperature. The solvent was removed in vacuo. Extraction was performed by adding 3 x 120 mL of chloroform, the organic phases were combined. Then washed with saturated brine, dried over anhydrous magnesium sulfate, the solvent was removed in vacuo, the residue was purified by flash column chromatography using dichloromethane / petroleum ether to give the product as an orange yellow solid (720 mg, yield 61 %).
[0065] IDA-2: ID-DMA (117 mg, 444 pmol) was dissolved in 2 ml of anhydrous DMSO, then triethylamine (2 eq.) and carbon disulfide (1 eq.) were added sequentially. After stirring at room temperature for 1 h, iodomethane (56 pL, 888 pmol, 2 eq.) was slowly added under an ice bath, then stirred at room temperature for 12 h. The reaction solution was poured into ice water, the mixture was extracted with dichloromethane, the organic phase was collected, and purified by silica gel column chromatography (dichloromethane:methanol) to give an orange solid (69 mg, yield 17 %).
[0066] The hydrogen spectrum data of IDA-2 is shown in Table 1, the carbon spectrum data is shown in Table 2, and the mass spectrum data is shown in Table 3. Figure 13 Figure 14 Figure 15
[0067] Example 3 Synthesis of IDA-3 probe
[0068] The synthesis route is as follows:
[0069]
[0070] ID-TPA: Compound Br-ID (1 g, 4.44 mmol) was dissolved in 100 mL of tetrahydrofuran, a catalytic equivalent of Pd(PPh3)4was added and the mixture was stirred at room temperature for 30 minutes. 4-triphenylamine boronic acid (1.98 g, 5.33 mmol) was added to the reaction flask stirred for 10 minutes, then 60 mL of Na2C03solution was added to the reaction, heated and left for 12 h. The reaction was followed by TLC. After the reaction was complete, the reaction mixture was cooled to room temperature. The solvent was removed in vacuo. Extraction was performed by adding 3 x 100 mL of chloroform, the organic phases were combined. Then washed with saturated brine, dried over anhydrous magnesium sulfate, the solvent was removed in vacuo, the residue was purified by flash column chromatography using dichloromethane / petroleum ether to give the product as a red-brown solid (800 mg, yield 43 %).
[0071] ID-TPA (200 mg, 0.514 mmol) was dissolved in anhydrous DMSO, then triethylamine (2 eq.) and carbon disulfide (1 eq.) were added successively. After stirring at room temperature for 1 h, iodomethane (56 μL, 1.3 mmol) was added slowly under ice-bath, then stirred at room temperature for 12 h. The reaction solution was poured into ice-water, the mixture was extracted with dichloromethane, the organic phase was collected and purified by silica gel column chromatography (dichloromethane: petroleum ether) to obtain a yellow solid (77 mg, yield 30%).
[0072] The hydrogen spectrum data of IDA-3 is shown in Table 1, the carbon spectrum data is shown in Table 2, and the mass spectrum data is shown in Table 3. Figure 16 Figure 17 The hydrogen spectrum data of IDA-3 is shown in Table 1, the carbon spectrum data is shown in Table 2, and the mass spectrum data is shown in Table 3. Figure 18
[0073] Spectral performance test of the fluorescent probe according to the present application
[0074] In a set of test tubes, 890 μL of PBS buffer (10 mM, pH 7.4) and 100 μL of target protein solution (0.1 mM) were added, and then 10 μL of IDAs (0.5 mM) was added. The resulting solution was mixed uniformly, and the absorption spectrum or fluorescence spectrum was recorded after being kept at room temperature for 60 min.
[0075] The results are shown in Figures 1 and 2, and it can be seen from the figures that the IDAs react rapidly with the target protein solution, and strong fluorescence turn-on occurs at the same time. IDA-1 has a good linear relationship of fluorescence response in the range of 0-14 μM, and the detection limit is 19.8 nM; IDA-1 has a good linear relationship of fluorescence response in the range of 0-8 μM, and the detection limit is 2.2 nM. Figure 2 Figure 3 The results are shown in Figures 1 and 2, and it can be seen from the figures that the IDAs react rapidly with the target protein solution, and strong fluorescence turn-on occurs at the same time. IDA-1 has a good linear relationship of fluorescence response in the range of 0-14 μM, and the detection limit is 19.8 nM; IDA-1 has a good linear relationship of fluorescence response in the range of 0-8 μM, and the detection limit is 2.2 nM.
[0076] Coupling and decoupling analysis of the fluorescent probe according to the present application and the peptide segment mimetic
[0077] The peptide mimetic ACHN-YCGCT was designed and purchased to simulate the lysine residue and the adjacent cysteine residue on the peptide chain of the protein. The IDAs probe (2.5 eq.) was added to the peptide segment in the PBS buffer (pH 7.4). IDA-1 was reacted in a 1% DMSO / PBS buffer solution system, and IDA-2 was reacted in a 30% DMSO / PBS buffer solution system. The reaction was placed in an open container at room temperature for 3 h, and the reaction was analyzed by HPLC-MS. The high performance liquid chromatography used 1% formic acid: water (solvent A): acetonitrile (solvent B); the detection wavelength was 220 nm. 5 mM dithiothreitol (DTT) was added to the labeled peptide-segment probe conjugate, and the mixture was left to stand for 3 h, and the decoupling process was tracked by HPLC.
[0078] The results are shown in Figures 3 and 4. Figure 4 As shown, it is confirmed that the product of IDA-1 and the peptide mimic ACHN-YCGCT is mainly the cyclization product obtained by thiol displacement reaction of sulfhydryl and dimethyl sulfide ethylene, and it is also confirmed that DTT decouples the cyclization product, which makes the cyclization product open ring and restores to the structure of the original peptide segment.
[0079] Example 6 In situ labeling and subcellular localization of adjacent thiol proteins in HepG2 cells using probe IDA-2
[0080] (1) In situ labeling of adjacent thiol proteins in living cells
[0081] HepG2 cells were cultured in a fluorescence confocal dish. The cells were pretreated with different redox stimuli to construct cell models with different redox levels, and then incubated with IDA-2 in a 37°C cell incubator. After washing, fresh medium was replaced. The cells were imaged under a 60x oil lens using a Leica TCS SP8 STED 3X confocal imaging system, and the fluorescence intensity was statistically analyzed using the imaging system software.
[0082] (2) Organelle co-localization study of adjacent thiol proteins in cells
[0083] After washing the HepG2 cells twice with PBS, they were first labeled with 50 nM Mito-tracker Deep Red (commercialized mitochondrial probe, purchased from Invitrogen), and then washed to remove the free Mito-tracker Deep Red. Then, IDA-2 was used for labeling, and the free IDA-2 was washed away. The cells were imaged under a 60x oil lens using a Leica TCS SP8 STED 3X confocal imaging system, with 405 nm excitation light and 480-600 nm collection of IDA-2 fluorescence signal, and 635 nm excitation light and greater than 650 nm collection of Mito-tracker Deep Red fluorescence signal. The co-localization coefficient was calculated using the imaging system software.
[0084] As shown in the results Figure 5 , it is shown that the adjacent thiol proteins labeled by IDA-2 in living cells are mainly distributed in the mitochondrial part.
[0085] Example 7 Analysis of changes of adjacent thiol proteins in different oxidation states of living cells using probe IDA-2 by flow cytometry
[0086] HepG2 cells were harvested at 70-90% confluency in exponential phase. The culture medium was removed. Then the cells were washed with 1 mL PBS buffer for 3 times, 1 mL fresh serum-free DMEM was added and redox stimulators were added. After 30 min incubation, the cells were washed with PBS buffer for 1 time, 1 mL fresh serum-free DMEM (containing IDA-2) was added and incubated for another 30 min. The cells were washed with 1 mL PBS buffer for 1 time. The samples were analyzed on ACEA NovoCyte flow cytometer (ACEA / Agilent, USA) under 405 nm laser. Emission signal collection channel: 500-550 nm (525 / 50 band-pass filter). The obtained data were analyzed by NovoCyte software and FlowJo software.
[0087] The results, as shown in Figure 6 , indicated that IDA-2 was successfully applied to flow cytometry fluorescence analysis of adjacent thiol proteins in living cells under different redox states.
[0088] The above merely illustrates the technical idea of the present application, and cannot be used to limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.
Claims
1. A fluorescent probe for adjacent thiol protein labeling and bioimaging, characterized in that, The structural formula of the fluorescent probe is as follows: 。 2. The fluorescent probe for adjacent thiol protein labeling and bioimaging according to claim 1, characterized in that, This fluorescent probe exhibits reversible labeling of adjacent thiol peptides, with the characteristic of reversible labeling that can be restored to the initial peptide after decoupling with dithiothreitol.
3. The use of the fluorescent probe for adjacent thiol protein labeling and bioimaging as described in claim 1 or 2 in the detection of adjacent thiol proteins for non-disease diagnostic purposes.
4. The application as described in claim 3, characterized in that, The adjacent thiol protein is labeled and a bio-imaging fluorescent probe is applied to the target object, and the in vitro detection of adjacent thiol proteins is achieved through spectroscopic signal response.
5. The use of the fluorescent probe for adjacent thiol protein labeling and bioimaging as described in claim 1 or 2 in cell localization for non-disease diagnostic purposes.
6. The application as described in claim 5, characterized in that, The adjacent thiol protein labeling and bioimaging fluorescent probes were co-incubated with mitochondrial and lysosomal localization dyes, and subcellular localization of adjacent thiol proteins in living cells was achieved through in situ fluorescent labeling in living cells.