Synthesis and application of novel cell membrane and multi-type organelle probes

Abstract

The present invention provides a probe that can be used for chemical modification of protein lysine residues, and its structure is shown below: The present invention also provides a method for preparing the above-mentioned probe pyridinium salt that can be used for chemical modification of protein lysine residues. The present invention also provides the use of the above-mentioned probe that can be used for chemical modification of protein lysine residues in fluorescence imaging, labeling, tracing or biotherapy. The active ester probe for pyridinium salt of the present invention can specifically target lysosomes, endoplasmic reticulum, mitochondria and cell membranes, and is used in fluorescence imaging, labeling, tracing and biotherapy.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry and relates to a probe, specifically an ester derivative containing a pyridinium salt, which can be used to modify protein lysine (Lys, K) residues and is used in fluorescence imaging, labeling, tracing and biotherapy. Background Art

[0002] With the vigorous development of life sciences, people have been studying cell membranes and subcellular organelles in depth. The cell membrane is the outer layer of the cell, which maintains the shape and stability of the cell. It also plays a vital role in controlling the entry and exit of substances, transmitting information, and participating in cell adhesion and movement. Lysosomes, endoplasmic reticulum, and mitochondria, as the main organelles of eukaryotic cells, undertake necessary and important life activities. Lysosomes are the "digestive organs" of organelles. The various acidic hydrolases contained in the lysosomes keep the pH value of their internal environment within the range of 4.5-5.0. As important acidic organelles in eukaryotic cells, lysosomes have important functions of intracellular digestion and secretion regulation. At the same time, abnormalities in their number and distribution may cause various diseases such as lung diseases, lysosomal storage diseases, and tumors. The endoplasmic reticulum is a bridge connecting the nucleus, cell membrane, and cytoplasm, and is responsible for the synthesis and transport of sugars, lipids, and proteins. Physiological and pathological conditions can interfere with the homeostasis of the endoplasmic reticulum, thereby negatively affecting protein folding and leading to the accumulation of unfolded proteins. Dysfunction usually accumulates in the ER, which in turn induces neurodegenerative diseases, cancer and other diseases. Therefore, maintaining the homeostasis and function of the ER is necessary for normal cell function. Real-time tracking of the ER is of great significance for understanding the complex physiological function of the ER and the pathological mechanism of ER-related diseases. As a direct provider of ATP, mitochondria participate in the differentiation, regulation and apoptosis of cells. Changes in their number, structure and function are closely related to neurodegenerative diseases such as Alzheimer's disease and Parkinson's syndrome.

[0003] Nucleophilic amino acids make important contributions to protein function, including playing a key role in catalysis and serving as sites for post-translational modification. The electrophilic groups of targeted amino acid nucleophiles have been used to make covalent ligands and drugs, but so far, they are mainly limited to cysteine ​​and serine. Protein lysine (Lys) is one of the essential amino acids for organisms and plays a vital role in maintaining various physiological functions. The amino group of the lysine side chain residue plays many important roles in protein function. The amino side chain is a good nucleophilic group, and chemical modification of it is expected to change the chemical structure of the protein and change its spatial structure, thereby improving its own biological activity and function. Therefore, chemical modification of lysine in biological samples is an important means to study the three-dimensional structure and physiological function of proteins, and it is also a method for directional modification of protein properties. It has broad application prospects in protein engineering and omics research.

[0004] In recent years, due to its high sensitivity, high resolution and fast response time, fluorescence imaging technology has been widely used in many fields such as biology and medicine. Organelle fluorescent probes can penetrate into cells and selectively bind to organelles. Using confocal laser scanning microscope (CLSM), not only can clear fluorescent images of organelles such as cell membranes, lysosomes, endoplasmic reticulum, mitochondria, and nuclei be obtained, but also the morphological changes of living cells can be dynamically observed. At present, there are few types of organelle fluorescent probe reagents that are easily available on the market, and the prices are generally expensive. The compound structure is also relatively complex, and the photostability and toxicity need to be improved, which has a greater impact on the side effects of the measured object. At the same time, the synthesis process of most commercially available organelle labeling reagents is difficult and the production cost is high, which is not conducive to industrialization.

[0005] Based on the good water solubility of pyridinium salt, no other organic solvents need to be used in the interaction with living organisms, which is environmentally friendly and efficient. The key structural unit of the pyridine ring as the probe has better biocompatibility, lower biological toxicity, higher internal absorption and higher selectivity, which can solve the problem of high cell toxicity of other amino acid probes such as iodoacetamide as cysteine ​​probes, so the probe containing pyridinium salt that can be used for chemical modification of protein lysine residues has an important and very broad application space in the chemical modification of protein side chains. And the purpose of the present invention is not only limited to the efficient and rapid synthesis of a certain organelle-specific targeted probe, but it kills many birds with one stone, and a method is used to predictably obtain specific probes for lysosomes, endoplasmic reticulum, mitochondria and cell membranes. Summary of the invention

[0006] In view of the above-mentioned technical problems in the prior art, the present invention provides a probe that can be used for the chemical modification of protein lysine residues, a preparation method and an application. The probe that can be used for the chemical modification of protein lysine residues, a preparation method and an application are intended to solve the technical problems in the prior art of poor selectivity for chemical modification of protein lysine residues containing pyridinium salts as active functional groups, poor organelle localization and difficult preparation.

[0007] In order to achieve the above-mentioned purpose, the technical solution of the present invention is as follows:

[0008] The present invention provides a probe that can be used for chemical modification of protein lysine residues, and its structure is shown below:

[0009]

[0010] R1 is any one of an azide derivative group, an alkyl derivative group, an alkenyl derivative group, an alkynyl derivative group, a fluorescent group, a proteolysis targeting chimera group or biotin;

[0011] R2 is an organelle targeting group or an organelle localization group, wherein the organelle targeting group is composed of an organelle localization group and a linker group connecting the organelle localization group and the fluorophore, and the organelle localization group is selected from a nucleus localization group, a mitochondrial localization group, an endoplasmic reticulum localization group or a lysosome localization group;

[0012] X is substituted or unsubstituted and is independently selected from Cl, Br, I, NO3, ClO4, BF4, PF6, CF3CO2 or CF3SO3;

[0013] Y is O, S or NH;

[0014] n is 0-16.

[0015] Furthermore, the fluorescent group is coumarin, 2-methyl Tokyo Green, a 2-methyl Tokyo Green derivative, fluorescent yellow, a 1,8-naphthalimide derivative, resorufin, aminocyanine fluorescein isothiocyanate or hydroxyfluorescein.

[0016] Furthermore, R2 is selected from any one of the following groups:

[0017]

[0018] Furthermore, the probe that can be used for chemical modification of protein lysine residues is selected from any one of the following structural formulas:

[0019]

[0020]

[0021] The present invention also provides a method for preparing the above-mentioned probe pyridinium salt that can be used for chemical modification of protein lysine residues, comprising the following steps:

[0022] (1) Preparation of compound III: using a monosubstituted or polysubstituted pyridine derivative I as a raw material, a carboxylic acid derivative II and an esterification condensation agent to react to produce compound III;

[0023] (2) Preparation of compound IV: compound III and alkylating agent R 2 (CH2) n X, react in DCM, and after the reaction is complete, add an equal volume of ether to promote the precipitation of the product to obtain compound IV;

[0024]

[0025] The present invention also provides the use of the above probe in fluorescence imaging, labeling, tracing or biological treatment.

[0026] The present invention also provides the use of the above-mentioned probe or its intermediate in protein lysine modification.

[0027] The present invention provides a pyridinium active ester probe that can specifically target lysosomes, endoplasmic reticulum, mitochondria and cell membranes, and is used for fluorescence imaging, labeling, tracing and biotherapy. The present invention also provides a method for modifying the amino group of proteins on specific organelles. The method uses cheap and readily available hydroxypyridine and its analogs to efficiently and quickly synthesize pyridinium active ester derivatives with diverse structures. Compared with traditional reaction systems, this method not only achieves high-throughput rapid synthesis of a variety of pyridinium active ester derivatives, but also has the advantages of simple operation, mild conditions, low cost, etc. At the same time, this method can quickly establish a powerful functional molecule library targeting different organelles, which has huge potential commercial value.

[0028] The present invention develops a multi-type organelle-specific targeted probe reagent molecular library based on pyridinium salt active probes, quickly and efficiently synthesizes compounds 3, 4, 5, 6, and 7, and applies them to specific labeling of lysosomes, endoplasmic reticulum, mitochondria and protein lysine on cell membranes, and applies them to the fields of in vivo imaging, tracing and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the mass spectrum of compound 1.

[0030] Figure 2 is the mass spectrum of compound 2.

[0031] Figure 3 is the mass spectrum of compound 3.

[0032] Figure 4 is the mass spectrum of compound 4.

[0033] Figure 5 is the mass spectrum of compound 5.

[0034] Figure 6 is the mass spectrum of compound 6.

[0035] Figure 7 is the mass spectrum of compound 7.

[0036] Figure 8 This is the secondary mass spectrum of the reaction between compound 6 and peptide LP9.

[0037] Fig. 9 This is the primary mass spectrum of the reaction between compound 6 and protein Lysozyme.

[0038] Fig.10 This is a gel image of the reaction between compound 6 and cell lysate.

[0039] Fig.11 This is the fluorescence confocal co-imaging image of compound 6 and cell membrane.

[0040] Fig.12 This is the fluorescence confocal co-imaging image of compounds 3, 4, 5 and cell organelles. DETAILED DESCRIPTION

[0041] The present invention is described clearly and completely below through specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the protection scope of the present invention.

[0042] Example 1: Preparation of Compound 1:

[0043]

[0044] A solution of 4-hydroxybenzoic acid (6.9 g, 50 mmol), 3-bromoprop-1-yne (5.1 ml, 60 mmol) and NaOH (4.0 g, 100 mmol) in 200 ml MeOH was stirred at room temperature for 48 hours. Then, 1 M HCl aqueous solution (100 mL) was added to neutralize the reaction mixture, and the precipitated white solid was collected by filtration, washed with water, and dried in vacuo to obtain compound 1 (3.74 g, 43%). The mass spectrum data of the compound are as follows: MS m / z calcd for C 10 H7O3[MH] + 175.04, found 175.30 (see Figure 1 ).

[0045] Example 2: Preparation of Compound 2:

[0046]

[0047] 4-dimethylaminopyridine (122 mg, 1 mmol), 4-(prop-2-ynyl-1-yloxy)benzoic acid (1.76 g, 10 mmol), then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.92 g, 10 mmol). After stirring for 10 minutes, add p-hydroxypyridine (0.95 g, 10 mmol) and stir at room temperature for 16 hours under nitrogen atmosphere. After the reaction is completed by TLC monitoring, wash with saturated brine 3 times, dry with Na2SO4 and then column chromatography to obtain compound 2 (1.97 g, 78%). The mass spectrum data of the compound are as follows: MS m / z calcd for C 15 H 12 NO3[M+H] + 254.08, found 254.00 (see Figure 2 ).

[0048] Example 3: Preparation of Compound 3:

[0049]

[0050] Compound 2 (253 mg, 1 mmol) and (6-bromohexyl)triphenylphosphonium bromide (507 mg, 1 mmol) were added to 5 ml of acetonitrile and refluxed for 12 hours. After the reaction was completed, the column chromatography was used to obtain compound 3 (207 mg, 30%). The mass spectrum data of the compound is as follows: MS m / z calcd for C 39 H 38 NO3P2 + [M] 2+ 299.63, found 299.65 (see Figure 3 ).

[0051] Example 4: Preparation of Compound 4:

[0052]

[0053] Compound 2 (253 mg, 1 mmol) and N-(3-bromopropyl)-4-methylbenzenesulfonamide (290 mg, 1 mmol) were added to 5 ml of acetonitrile and refluxed for 12 hours. After the reaction was completed, the column chromatography was used to obtain compound 4 (152 mg, 28%). The mass spectrum data of the compound are as follows: MS m / z calcd for C 25 H 25 N2O5S +[M] + 465.14, found. 465.05 (see Figure 4 ).

[0054] Example 5: Preparation of Compound 5:

[0055]

[0056] Compound 2 (253 mg, 1 mmol) and 4-(3-bromopropyl)morpholine (207 mg, 1 mmol) were added to 5 ml of acetonitrile and refluxed for 12 hours. After the reaction was completed, the column chromatography was used to obtain compound 5 (98 mg, 21%). The mass spectrum data of the compound are as follows: MS m / z calcd for C 22 H 25 N2O4 + [M] + 381.18, found 381.20 (see Figure 5 ).

[0057] Example 6: Preparation of Compound 6:

[0058]

[0059] Compound 2 (253 mg, 1 mmol) and 1,3-propane sultone (244 mg, 2 mmol) were added to 5 ml of dichloromethane and reacted at room temperature for 12 hours. After the reaction was completed, the column chromatography was used to obtain compound 6 (276 mg, 74%). The mass spectrum data of the compound are as follows: MS m / z calcd for C 18 H 18 NO6S + [M+H] + 376.08, found 376.10 (see Figure 6 ).

[0060] Example 7: Preparation of Compound 7:

[0061]

[0062] N-(6-hydroxyhexyl)-4-methylbenzenesulfonamide (271 mg, 1 mmol) and pyridine (121 μl, 1.5 mmol) were added to 5 ml of dichloromethane and stirred at -20°C for 5 min. Then trifluoromethanesulfonic anhydride (250 μl, 1.5 mmol) was dissolved in 5 ml of dichloromethane and added dropwise to the reaction solution and reacted for 2 h. The organic phase was then washed with 1M HCl and saturated NaHCO3 and dried over anhydrous sodium sulfate. The organic phase was filtered with cotton and compound 7 (253 mg, 1 mmol) was added. The reaction was continued at room temperature for 12 h. After the reaction was completed, TLC was monitored and column chromatography was performed to obtain compound 7 (169 mg, 26%). The mass spectrum data of the compound are as follows: MS m / z calcd for C 28 H 31 N2O5S + [M+H] + 507.19, found 507.15 (see Figure 7 ).

[0063] Example 8: Reaction results of the probe 6 for chemical modification of polypeptide lysine residues of the present invention with polypeptide:

[0064]

[0065] In order to verify the reactivity of the probe that can be used for chemical modification of lysine residues in polypeptides and covalently bind to lysine (Lys) at the polypeptide labeling level, a secondary mass spectrometry experiment of the reaction between LP9 polypeptide and probe 6 was conducted to further prove that the main reaction site of the probe 6 that can be used for chemical modification of lysine residues in polypeptides of the present invention with polypeptides is lysine, and it can be further studied as a highly selective lysine modification probe ( Figure 8 ).

[0066] Example 9: Research results of probe 6 of the present invention that can be used for chemical modification of protein lysine residues:

[0067] 50 μM (50 μl 0.1 mM stock solution, pH 7.4 phosphate buffer) protein substrate was treated with 0, 50, 250, 500 μM probe 6 (0, 0.5, 2.5, 5 μl of 10 mM stock solution), respectively, and then a constant volume of buffer was added to 100 μl. In phosphate buffer at pH 7.4 and 37°C for 1 hour. The mixture was centrifuged (5000 rpm) using an ultrafiltration centrifuge tube (with a molecular weight cutoff of 3K or 10K) pretreated with water. The protein substrate was washed (3×200 μl water) to remove the remaining 6 and characterized using a ThermoScientificTM Q Exactive HF-X mass spectrometer. The results showed that the modification of lysozyme had a dose-dependent effect on 6 (see Fig. 9 ).

[0068] Example 10: Research results of the probe 6 of the present invention that can be used for chemical modification of lysine residues in cell lysate:

[0069] For probe labeling assay, 100 μg of protein extracted from MCF-7 cells was treated with the indicated concentration of probe for 1 hour at room temperature. Then fresh premixed click chemistry reaction mixture (50 μM TAMRA-N3 in DMSO, 100 μM TBTA in DMSO:t-butanol 1:4, 1 mM tris(2-carboxyethyl)phosphine hydrochloride(TCEP)in water, and 1 mM CuSO4 in water) was added to the above mixture. The reaction was further incubated for another 1 hour at room temperature. Then 4× SDS-PAGE loading buffer was boiled at 95°C for 10 minutes. The samples were analyzed by 10% SDS-PAGE gel (polyacrylamide gel electrophoresis) and visualized by in-gel fluorescence using a BioRad ChemiDocTM imager. The gel was then stained with Coomassie stain (CBB) and scanned. For competition assay, proteins were treated with IAA and NHS-Ac at the indicated concentrations, respectively, as described above. In the subsequent competitive labeling lysate experiments, the fluorescence band became weaker with pretreatment with NHS-Ac, but not IAA, which further confirmed the lysine selectivity of 6 ( Fig.10 AB).

[0070] Example 11: Compounds and commercially available cell membrane dye DIO and mitochondrial dye Mito-Tracker TM Green, lysosomal dye Lyso-Tracker TM Green, endoplasmic reticulum dye ER-Tracker TM Green fluorescence confocal imaging of MCF7 cells

[0071] First, MCF7 cells were cultured at 37°C (DMEM with 10% fetal bovine serum, 5% CO2) for 24 hours. Then, the culture medium was removed and 10 μM acetonitrile solutions of probes 3 (mitochondrial probe-Mito), 4 (endoplasmic reticulum probe-ER), 5 (lysosome probe-Lyso) and 6 (cell membrane probe-DIO) were added to different culture dishes and cultured at 37°C for 1 hour, and then washed twice with PBS. Then, Hoechst nuclear dye and the corresponding organelle and cell membrane Tracker were added and incubated for 45 minutes. The dye was discarded and freshly prepared Click cocktail (1 μM TAMRA-N3; 1 μM TBTA; 10 μM TCEP; 10 μM CuSO4) was added and incubated for 1 hour. Finally, the cells were washed 2-3 times with PBS and photographed (see Fig.11 and Fig.12 ), wherein compound 7 provides an example of a triflate anion.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A probe that can be used for chemical modification of protein lysine residues, characterized in that: Its structure is as follows: ， IV, The structure of general formula IV is selected from any one of the following structural formulas: 。 2. The method for preparing a probe that can be used for chemical modification of protein lysine residues according to claim 1, characterized in that The steps include: (1) Preparation of compound III: using a monosubstituted or polysubstituted pyridine derivative I as a raw material, a carboxylic acid derivative II and an esterification condensation agent to react to produce compound III; (2) Preparation of compound IV: compound III and alkylating agent R 2 (CH2) n X, react in DCM, and after the reaction is complete, add an equal volume of ether to promote the precipitation of the product to obtain compound IV; 。 3. Use of the probe for chemical modification of protein lysine residues as claimed in claim 1 in the preparation of products for fluorescence imaging, labeling, tracing or biotherapy.

4. Use of the probe for chemical modification of protein lysine residues according to claim 1 in the preparation of protein lysine-modified products.

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