A probe compound for coa detection, fluorescent probe and application

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a probe compound for CoA detection, a fluorescent probe and application, comprising a modified rhodamine series fluorescent dye, a pantothenate kinase inhibitor, a HaloTag, a Nanoluc luciferase and a pantothenate kinase fusion protein; through protein labeling, a series of biosensors are obtained, coenzyme A concentration in living cells can be rapidly detected, and rapid screening of coenzyme A drugs is regulated; through rational design and modification of the chemical probe molecules, and engineering modification of the sensor proteins, the chemical probes can label the sensor proteins in living cells, and then the bioluminescent sensors working in living cells are obtained, the requirements of rapid and high-sensitivity detection of CoA can be met, and the drugs capable of regulating the change of coenzyme A concentration can be high-throughput screened.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a probe compound, a fluorescent probe, and its application for CoA detection. Background Technology

[0002] Coenzyme A (CoA) is one of the most important coenzymes in the human body, participating in numerous biochemical reactions, such as the tricarboxylic acid cycle, amino acid metabolism, post-translational protein modification, and gene expression regulation. Disorders of CoA metabolism are closely related to the occurrence and development of neurodegenerative diseases, obesity, and tumors.

[0003] Currently, there are very limited detection technologies that can accurately detect the concentration and distribution of CoA in living cells. There is only one known CoA biosensor, but it relies on fluorescence sensing technology and requires expensive fluorescence microscopy to detect CoA. Moreover, its detection sensitivity, efficiency, and throughput cannot yet meet the requirements for rapid drug screening. Summary of the Invention

[0004] The purpose of this invention is to provide a probe compound, a fluorescent probe, and its application for CoA detection, overcoming the shortcomings of existing technologies. By constructing a biosensor through chemical probe molecular labeling and sensor protein, modifying the chemical probe, and engineering the sensor protein, the sensitivity of the fluorescent sensor is greatly improved. It can be located in different subcellular structures of cells and used for quantitative analysis of CoA in subcellular cells and drug screening.

[0005] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0006] A probe compound for CoA detection, the compound having the structural formula shown in Formula 1:

[0007]

[0008] in:

[0009] L represents: C6 or PEG2 linker;

[0010] R1 is methyl or amino;

[0011] R2 is substituted o-trifluoromethylbenzoic acid.

[0012] Furthermore, the structural formula of L in the compound is either Formula 2 or Formula 3, and the structural formulas of Formula 2 and Formula 3 are as follows:

[0013]

[0014] The structural formula of R2 in the compound is any one of Formulas 4-7, and the structural formulas of Formulas 4-7 are as follows:

[0015]

[0016] The present invention also protects a fluorescent probe comprising the above-mentioned compound.

[0017] The present invention also protects a sensor composed of the aforementioned fluorescent probe, fusion-expressed protein, and auxiliary agents.

[0018] Furthermore, the fusion-expressed protein consists of HaloTag, Nanoluc luciferase, pantothenic acid kinase, and inactivated pantothenic acid kinase.

[0019] The present invention also protects a kit comprising the above-described compound, a fluorescent probe, a sensor, and an acceptable carrier.

[0020] Finally, this invention protects the application of the above-mentioned compound, fluorescent probe, sensor, and kit, specifically including any one of the following:

[0021] (1) Fluorescent labeling;

[0022] (2) Fluorescent labeling of subcellular proteins;

[0023] (3) Detection of coenzyme A concentration by bioluminescence;

[0024] (4) Screening for drugs with modifiable coenzyme A concentration changes.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention rationally designs and modifies chemical probe molecules, while simultaneously engineering sensor proteins, enabling these chemical probes to label sensor proteins within living cells, thereby obtaining bioluminescent sensors that can function within living cells. This meets the requirements for rapid and highly sensitive detection of CoA and can be used for high-throughput screening of drugs with adjustable coenzyme A concentration changes. Attached Figure Description

[0027] Figure 1 This is a comparison diagram of the fluorescence intensity of the probes prepared in Examples 1-11 of the present invention.

[0028] Figure 2 This is a graph showing the ratio of the sensor CoA concentration to the luminescence intensity of luciferase and rhodamine in this invention.

[0029] Figure 3 This is a confocal imaging image of the sensor protein in a living cell in this invention.

[0030] Figure 4This is a schematic diagram of the process by which the sensor detects CoA concentration in living cells in this invention.

[0031] Figure 5 This is a comparative graph showing the changes in CoA concentration in the cytoplasm and mitochondria after drug treatment in living cells, as described in this invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] This invention discloses a fluorescent probe for CoA detection, comprising: a fluorescent dye portion, a linker L, a dye-modifying group R1, and a pantothenic acid kinase inhibitor-modifying group R2. The fluorescent dye is a rhodamine derivative, which is a polar-sensitive dye. The fluorescence "on" and "off" properties of the molecule can be controlled by modifying the substituents on the polarity-regulating group R1. The pantothenic acid kinase inhibitor-modifying group R2 is linked to the inhibitor main framework via an amide bond. The linker L is linked to rhodamine and the inhibitor via an amide bond and a carbamate, respectively, separating the two portions. Optionally, the above-mentioned fluorescent probe has the following characteristics:

[0034] The structural features shown in Equation 1

[0035]

[0036] Linker L is linked to a rhodamine carboxyl group via an amide group and to a pantothenic acid kinase inhibitor via a carbamate group, as shown in Formula 2 or Formula 3.

[0037]

[0038] The dye-modifying group R1 is methyl or amino, and is linked to the carboxyl group of the rhodamine spirocyclic ring via sulfonamide.

[0039] The pantothenic acid kinase inhibitor modification group R2 is achieved by adding a substituent to the benzene ring of o-trifluoromethylbenzoic acid, wherein the substituent is a fluorine atom or a chlorine atom.

[0040] The structures shown in Equations 4 to 7 can be selected:

[0041]

[0042] Optionally, the fluorescent probe described above is selected from compounds of the following formula:

[0043]

[0044]

[0045]

[0046] On the other hand, a method for preparing the above-mentioned chemical probe is also provided, including a chemical reaction step involving a fluorescent dye of Formula 1 or the fluorescent dye shown with an active reactive group and an optional linker.

[0047] This invention provides an engineering method for constructing a fusion protein for a sensor, comprising the fusion expression of HaloTag, Nanoluc luciferase (NLuc), pantothenic acid kinase (PanK), and inactivated pantothenic acid kinase (PanK*), having a fusion expression protein as shown in Formulas 20 to 25.

[0048] Halo-PanK*-NLuc-cpPanK, Equation 20

[0049] Halo-cpPanK*-NLuc-cpPanK, Formula 21

[0050] Halo-PanK[213-cpNLuc-214], Equation 22

[0051] Halo-PanK*-cpPanK-NLuc, Equation 23

[0052] Halo-NLuc-cpPanK*-PanK, Equation 24

[0053] Halo-PanK-cpPanK*-NLuc, Equation 25

[0054] This invention provides a method for constructing a bioluminescent biosensor, comprising the following steps: co-incubating the chemical probe with a sensor fusion protein, wherein the HaloTag substrate reactive group of the chemical probe forms a covalent bond with HaloTag, thereby labeling the chemical probe onto the protein.

[0055] The present invention also provides a method for constructing a sensor within cells, comprising the following steps: co-incubating the chemical probe with animal cells, wherein the chemical probe penetrates the cell membrane and enters the cell to form a covalent bond with the protein expressed in situ, and thereby labeling the chemical probe onto the protein.

[0056] The present invention also provides the use of the biosensor in coenzyme A detection, cell research and drug screening.

[0057] The present invention also provides a probe kit including the chemical probe. Optionally, the probe kit further includes a biocompatible medium; optionally, the biocompatible medium is selected from at least one of dimethyl sulfoxide, buffer solution, and physiological saline; optionally, the buffer solution includes, but is not limited to, phosphate buffer solution.

[0058] Those skilled in the art can select biosensors with different detection ranges.

[0059] According to one implementation plan, after the chemical probe labels the sensor protein, it binds to the biomolecule, its polarity increases, its fluorescence brightness increases, and it has good fluorescent molecular activation properties.

[0060] According to one implementation plan, chemical probe labeling is fast both in vitro and intracellularly.

[0061] According to one implementation scheme, the chemical probe is highly specific, labeling only intracellular sensor proteins.

[0062] According to one implementation plan, the chemical probe molecules have high brightness, excellent photobleaching resistance, and good photostability.

[0063] According to one implementation scheme, the sensor molecules have high sensitivity and can be used at extremely low concentrations.

[0064] According to one implementation scheme, the sensor molecules are highly specific and can selectively detect coenzyme A concentration.

[0065] According to one implementation scheme, the sensor molecule is highly tunable and can detect coenzyme A concentrations over a wide concentration range.

[0066] According to one implementation scheme, the sensor molecules have the characteristic of subcellular localization.

[0067] According to one implementation scheme, the sensor molecule can be used to detect coenzyme A concentration in living cells.

[0068] According to one implementation scheme, sensor molecules can be used to screen drugs related to the regulation of coenzyme A concentration in living cells.

[0069] In Examples 1 to 8 and the verification examples of this invention, the raw materials and reagents used can be purchased from the market or synthesized according to the references.

[0070] The present invention will be further illustrated below with reference to the embodiments:

[0071] Example 1

[0072]

[0073] The general synthetic method for compounds 1-4: Trifluoromethylbenzoic acid (1.0 eq, 1.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 2.0 eq, 2.0 mmol, 0.38 g), 1-hydroxybenzotriazole (2.0 eq, 2.0 mmol, 0.27 g), and N,N-diisopropylethylamine (DIPEA, 3.0 eq, 3.0 mmol, 0.53 mL) were dissolved in 2 mL of N,N-Dimethylformamide (DMF). The mixture was stirred at room temperature for 30 minutes. Then, a DMF solution containing triazoleamine (1.0 eq, 1.0 mmol, 0.37 g) was added to this solution. After stirring at room temperature for 12 hours, the reaction was quenched with 0.5 mL of glacial acetic acid. The products were purified by reversed-phase high-performance liquid chromatography and freeze-dried to obtain compounds 1-4.

[0074] Compound 1 (white powder, 0.41 g, yield 72%). HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time 20 min. HRMS (ESI, pos. mode) m / z calc. for C 26 H 23 F5N5O2S + 564.1493, found 564.1489 [M+H] + .

[0075]

[0076] The synthesis of compound 2 was performed according to Example 1. Compound 2 (white powder, 0.41 g, yield 72%). HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time 21 min. HRMS (ESI, pos.mode) m / zcalc.for C 26 H 23 F5N5O2S + 564.1493, found 564.1489 [M+H] + .

[0077]

[0078] The synthesis of compound 3 was performed according to Example 1. Compound 3 (white powder, 0.39 g, yield 70%). HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time 21 min. HRMS (ESI, pos.mode) m / zcalc.for C 26 H 23 F5N5O2S +564.1493, found 564.1491[M+H] + .

[0079]

[0080] The synthesis of compound 4 was performed according to Example 1. Compound 4 (white powder, 0.44 g, yield 76%). HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time 22 min. HRMS (ESI, pos.mode) m / zcalc.for C 26 H 23 ClF4N5O2S + 580.1197, found 580.1195 [M+H] + .

[0081] Example 2

[0082]

[0083] The general synthetic method for compounds 5-12 was as follows: In 1 mL of dichloromethane solution containing compound 1 (1.0 eq, 0.5 mmol, 0.28 g), pyridine (3.0 eq, 1.5 mmol, 0.12 g) and 4-dimethylaminopyridine (1.0 eq, 0.5 mmol, 61 mg) were added. After stirring at room temperature for 2 minutes, p-nitrophenyl chloroformate (3.0 eq, 1.5 mmol, 0.3 g) was added, and the reaction was allowed to proceed for 3 hours at room temperature. The above solution was then slowly added dropwise to a dichloromethane solution containing 10 equivalents of diamines of different carbon chain lengths, and the reaction was continued for 30 minutes. After the reaction was complete, the products were purified by reversed-phase high-performance liquid chromatography and lyophilized to obtain compounds 5 and 6.

[0084] Compound 5:

[0085] HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time: 20 min, yield: 45%. HRMS (ESI, pos.mode) m / z calc.for C 33 H 37 F5N7O3S + 706.2599, 706.2602 [M+H] + .

[0086] Compound 6:

[0087] HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time: 20.5 min, yield: 51%. HRMS (ESI, pos.mode) m / z calc. for C33H37F5N7O5S + 738.2497,738.2498[M+H] + .

[0088]

[0089] Compound 7:

[0090] HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time: 20, yield: 46%. HRMS (ESI, pos.mode) m / z calc.for C 33 H 37 F5N7O3S + 706.2599, 706.2603 [M+H] + .

[0091] Compound 8:

[0092] HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time: 22 min, yield: 36%. HRMS (ESI, pos.mode) m / z calc.for C 33 H 37 F5N7O5S + 738.2497, 738.2500 [M+H] + .

[0093]

[0094] Compound 9:

[0095] HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time: 20 min, yield: 44%. HRMS (ESI, pos.mode) m / z calc.for C 33 H 37 F5N7O3S + 706.2599, 706.2603 [M+H] + .

[0096] Compound 10:

[0097] HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time: 25.7 min, yield: 48%. HRMS (ESI, pos.mode) m / z calc.for C 33 H 37 F5N7O5S + 738.2497, 738.2500 [M+H] + .

[0098]

[0099] Compound 11:

[0100] HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time: 22 min, yield: 41%. HRMS (ESI, pos.mode) m / z calc.for C 33 H 37 ClF4N7O3S + 722.2303, 722.2301 [M+H] + .

[0101] Compound 12:

[0102] HPLC (0.1% TFA / water / acetonitrile 15-95%, 35 min), retention time: 21.8 min, yield: 50%. HRMS (ESI, pos.mode) m / z calc.for C 33 H 37 ClF4N7O5S + 754.2202, 754.2210 [M+H] + .

[0103] Example 3

[0104]

[0105] In a 0.5 mL DMF solution of rhodamine dye (1.0 eq, 1.0 μmol, 5.6 mg), DIPEA (3.0 eq, 3.0 μmol, 5.3 μL) was added. After reacting for 2 minutes, 0.2 mL of TSTU dissolved in DMF (1.1 eq, 1.1 μmol, 3.3 mg) was slowly added dropwise. The resulting mixture was stirred at room temperature for 10 minutes. The above solution was then slowly added dropwise to a 0.5 mL DMF solution of 2-(2-(6-chlorohexyloxy)ethoxy)ethylamine (2.0 eq, 2.0 μmol, 4.5 mg) and DIPEA (3.0 eq, 3.0 μmol, 5.3 μL). After stirring the mixture at room temperature for 1 hour, 100 μL of glacial acetic acid was added to quench the reaction. The product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and lyophilized to give compound 13 in 85% yield. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time was 25 min. HRMS(ESI,pos.mode)m / zcalc.for C 42 H 55 ClN3O8 + 764.3672, found 764.3673 [M] + .

[0106] Example 4

[0107]

[0108] Synthesis of Compound 14: Compound 13 (1.0 μmol, 7.6 mg) was dissolved in 1.0 mL of trifluoroacetic acid. The mixture was stirred at room temperature for 1 hour, then the solvent was removed under vacuum. The product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and lyophilized to obtain Compound 14, with a yield of 92%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time was 18 min. HRMS (ESI, pos.mode) m / zcalc.for C 38 H 47 ClN3O8 + 708.3046, found 708.3050 [M] + .

[0109] Example 5

[0110]

[0111] Synthetic methods of compounds 15-16:

[0112] In a 0.5 mL DMF solution of compound 14 (1.0 eq, 1.0 μmol, 7.1 mg), DIPEA (3.0 eq, 3.0 μmol, 5.3 μL) was added. After reacting for 2 minutes, 0.2 mL of TSTU dissolved in DMF (1.1 eq, 1.1 μmol, 3.3 mg) was slowly added dropwise. The resulting mixture was stirred at room temperature for 10 minutes. The above solution was then slowly added dropwise to a 0.5 mL DMF solution of compound 5 or 6 (2.0 eq, 2.0 μmol, 4.5 mg) and DIPEA (3.0 eq, 3.0 μmol, 5.3 μL). After stirring the mixture at room temperature for 1 hour, 100 μL of glacial acetic acid was added to quench the reaction. The product was purified by reversed-phase high-performance liquid chromatography and freeze-dried to obtain compounds 15 and 16 (red powder), respectively.

[0113] Compound 15: Red powder, yield 75%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 19 min. HRMS (ESI, pos.mode) m / z calc. for C 71 H 81 ClF5N 10 O 10 S + 1395.5461, found 1395.5470 [M] + .

[0114] Compound 16: Red powder, yield 80%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 18.5 min. HRMS (ESI, pos.mode) m / z calc. for C 71 H 81 ClF5N 10 O 12 S + 1427.5359, found1427.5355[M] + .

[0115] Synthesis methods of probes 1-3:

[0116] In a 5 mL acetonitrile solution of a mixture of compounds 15 or 16 (1.0 eq, 0.41 mmol), benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (PyBOP, 2.0 eq, 0.82 mmol, 430.9 mg), 1-hydroxybenzotriazole (HOBt) (2.0 eq, 0.82 mmol, 111.9 mg), 4-dimethylaminopyridine (0.2 eq, 0.082 mmol, 10.1 mg), and methanesulfonamide or sulfonamide (5 eq, 2.0 mmol), DIPEA (10.0 eq, 4.2 mmol, 721.1 μL) was added. After reacting for 3 hours at room temperature, the reaction was quenched with 1.0 mL of glacial acetic acid. The product was purified by reversed-phase high-performance liquid chromatography and lyophilized to obtain probes 1-3.

[0117] Probe 1: Red powder, yield 55%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 25 min. HRMS (ESI, pos.mode) m / z calc.for C 72 H 84 ClF5N 11 O 11 S2 + 1472.5396, found 1472.5400 [M] + .

[0118] Probe 2: Red powder, yield 48%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 20.5 min. HRMS (ESI, pos.mode) m / z calc.for C 72 H 84 ClF5N 11 O 13 S2 + 1504.5295, found 1504.5299 [M] + .

[0119] Probe 3: Red powder, yield 35%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 22.5 min. HRMS (ESI, pos.mode) m / z calc.for C 71 H 83 ClF5N 12 O 11 S2 2+ 1473.5349, found 1473.5342 [M] + .

[0120] Example 6

[0121]

[0122] The synthesis steps of probes 4-6 are the same as those of probes 1-3.

[0123] Probe 4: Red powder, yield 43%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 23.4 min. HRMS (ESI, pos.mode) m / z calc.for C 72 H 84 ClF5N 11 O 11 S2 + 1472.5396, found 1472.5402 [M] + .

[0124] Probe 5: Red powder, yield 51%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 22.6 min. HRMS (ESI, pos.mode) m / z calc.for C 72 H 84 ClF5N 11 O 13 S2 + 1504.5295, found1504.5291[M] + .

[0125] Probe 6: Red powder, yield 33%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 22.7 min. HRMS (ESI, pos.mode) m / z calc.for C 71 H 83 ClF5N 12 O 11 S2 2+ 1473.5349, found 1473.5351 [M] + .

[0126] Example 7

[0127]

[0128] The synthesis steps of probes 7-9 are the same as those of probes 1-3.

[0129] Probe 7: Red powder, yield 54%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 23.2 min. HRMS (ESI, pos.mode) m / z calc.for C72 H 84 ClF5N 11 O 11 S2 + 1472.5396, found 1472.5496 [M] + .

[0130] Probe 8: Red powder, yield 41%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 22 min. HRMS (ESI, pos.mode) m / z calc.for C 72 H 84 ClF5N 11 O 13 S2 + 1504.5295, found 1504.5297 [M] + .

[0131] Probe 9: Red powder, yield 37%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 22.7 min. HRMS (ESI, pos.mode) m / z calc.for C 71 H 83 ClF5N 12 O 11 S2 2+ 1473.5349, found 1473.5355 [M] + .

[0132] Example 8

[0133]

[0134] The synthesis steps of probes 10-12 are the same as those of probes 1-3.

[0135] Probe 10: Red powder, yield 47%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 23.9 min. HRMS (ESI, pos.mode) m / z calc.for C 72 H 84 Cl2F4N 11 O 11 S2 2+ 1488.5101,found1488.5102[M] + .

[0136] Probe 11: Red powder, yield 42%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 19.9 min. HRMS (ESI, pos.mode) m / z calc.for C 72 H 84 Cl2F4N 11 O 13 S2 2+ 1520.4999, found 1520.4990 [M] + .

[0137] Probe 12: Red powder, yield 36%. HPLC (0.1% TFA / water / acetonitrile 20-95%, 35 min). Retention time 23.6 min. HRMS (ESI, pos.mode) m / z calc.for C 71 H 83 Cl2F4N 12 O 11 S2 2+ 1489.5053, found 1489.5055 [M] + .

[0138] Example 9

[0139] The CoA sensor protein of this invention is composed of a HaloTag tag protein, pantothenic acid kinase (PanK), and luciferase NanoLuc, wherein a rigid peptide linker (GGSLAEAAAKEAAAKEAAAKEAAAKEAAAKAAAGGS) is attached to the HaloTag tag. Since PanK is a dimer in its natural state, we designed six different methods for constructing the fusion protein by utilizing the dimerization characteristics of the PanK protein itself:

[0140] Fusion protein 1: By mutating at two sites, Y240F and N282A, PanK loses its ability to bind to substrates, resulting in PanK*. The original C-terminus and N-terminus of PanK are linked by GSGGTG. The amino acids at positions 213 and 214 of PanK are broken to form new C-terminus and N-terminus, forming cpPanK. The Halo-PanK*-NLuc-cpPanK sequence is constructed, in which PanK* and NanoLuc are linked by GGSGGTGGS.

[0141] Fusion protein 2: cpPanK is constructed in the same way as fusion protein 1, using the Halo-cpPanK*-NLuc-cpPanK sequence. The inactivated cpPanK* and NanoLuc are linked by GGS.

[0142] Fusion protein 3: The original C-terminus and N-terminus of NanoLuc are linked by GGTGGSGGTGGS. The 65th and 66th amino acids of NanoLuc are disconnected to form new C-terminus and N-terminus, forming cpNLuc. cpNLuc is inserted between the 213th and 214th amino acids of PanK to construct the Halo-PanK[213-cpNLuc-214] sequence.

[0143] Fusion protein 4: cpPanK is constructed in the same way as fusion protein 1, using the Halo-PanK*-cpPanK-NLuc sequence, with the two PanKs linked by GGS.

[0144] Fusion protein 5: cpPanK was constructed in the same manner as fusion protein 1, using the Halo-NLuc-cpPanK*-PanK sequence, with the two PanKs linked by GGSGGT.

[0145] Fusion protein 6: cpPanK is constructed in the same way as fusion protein 1, using the Halo-PanK-cpPanK*-NLuc sequence, with the two PanKs linked by GGS.

[0146] Example 10

[0147] This invention introduces mutations at the Y55F and H307A sites on the PanK protein, which can regulate the sensor's CoA binding ability to suit application scenarios with different CoA concentrations.

[0148] Fusion protein 7: Fusion protein 4 carries the Y55F mutation.

[0149] Fusion protein 8: Fusion protein 4 carries the Y55F and H307A mutations.

[0150] Example 11

[0151] This invention provides a sensor construction method:

[0152] In vitro construction: The fusion protein from Example 10 was dissolved in PBS to obtain a 1.0 μM solution. Then, any one of the chemical probes 1-12 was added to make the final concentration 4 μM. The solution was left to stand at room temperature for 2 hours to obtain the sensor.

[0153] Intracellular construction: Dissolve any one of probes 1-12 in complete DEME (10% FBS) culture medium to prepare a 1 μM solution. Incubate cells with this solution for 12 hours to obtain the in situ labeled sensor.

[0154] Verification Example

[0155] This invention utilizes the fluorescence activation phenomenon after the sensor protein solution is labeled as a probe.

[0156] Dissolve probes 1-12 separately in dimethyl sulfoxide to prepare a concentration of 1x10⁻⁶. -3 Solution M was added to phosphate buffer to prepare a final concentration of 5 x 10⁻⁶. -7 The fluorescence spectrum of solution M was detected under the same microplate reader parameters; 2 x 10⁻⁶ ppm was added to the solution. -6 The sensor protein of M is in the state after fluorescence activation, such as Figure 1 As shown.

[0157] Depend on Figure 1 The fluorescence intensity of the free probe in the example showed that the fluorescence intensity of the probe in phosphate buffer was low; after adding HaloTag protein, the chemical probe labeled the protein, and the fluorescence showed obvious activation and high brightness.

[0158] The sensor uses the bioluminescence of luciferase as its signal output. In the absence of CoA, the ligand on the probe binds to the PanK protein. At this time, the luciferase and the rhodamine fluorophore of the probe molecule are close together, resulting in a bioluminescent energy transfer (BRET) effect, and the sensor exhibits obvious rhodamine fluorescence. As the CoA concentration increases, the ligand on the probe is competed for by CoA. At this time, the luciferase and the rhodamine fluorophore of the probe molecule move further apart, the BRET effect weakens, the luciferase channel of the sensor emits more light, while the rhodamine channel emits less light. The ratio of the luciferase and rhodamine luminescence intensities, R = I, is measured. NLuc / I Rho This allows for accurate quantification of CoA concentration.

[0159] Configure the sensor to a final concentration of 1x10. -9 The luminescence spectrum of M in PBS solution was measured as CoA was gradually added dropwise. The ratio of CoA concentration to the luminescence intensity of luciferase and rhodamine was plotted using the formula R = R min +(R max -R min ) / (1+C 50 By fitting the molecule to ([CoA]), the apparent binding constant C between the sensor molecule and CoA can be obtained. 50 And dynamic response range, see Figure 2 See Table 1.

[0160] In summary, the fluorescence of the chemical probes in the examples can be activated after binding to biomolecules, and the biosensor constructed using these probes can effectively detect CoA concentration. It features high sensitivity and can detect CoA concentration over a wide concentration range.

[0161] Taking HeLa cells as an example, the performance of the sensor in cells was tested. The labeling process was simple and easy: the probe molecules were directly dissolved in DMEM (10% FBS) medium, co-incubated with HeLa cells for 12 hours, washed once with HBSS solution, and then placed in HBSS solution containing 1 / 1000 furimazine for spectral measurement using a microplate reader. The sensor can localize to different subcellular structures through different localization signal peptides, and confocal imaging is performed as follows. Figure 3 As shown. The in-situ detection performance of CoA was tested using a microplate reader. In live cells, the sensor can rapidly and effectively detect CoA concentration in subcellular environments. (See...) Figure 4 and 5 .

[0162] Table 1 Sensing properties of the sensor

[0163]

[0164]

[0165] Table 1 shows that the sensors in the examples exhibit a signal dynamic response range (ΔR>3) in PBS solution, with several sensors showing a very ideal signal dynamic response range (ΔR>6). These sensors respond well to the C of CoA. 50 It has a very large range (0.6–416.5 μM) and can achieve quantitative detection of 60 nM–4 mM CoA.

[0166] Compared with existing technologies, compounds of Formula 1 exhibit very weak fluorescence intensity in the unlabeled state. Figure 1 After labeling the sensor protein, the fluorescence was significantly enhanced, with an increase in fluorescence intensity of more than 5.2 times. Figure 1 Among them, probe 6 showed a 43.5-fold fluorescence enhancement, and probe 3 showed a 35.4-fold fluorescence enhancement. These probe molecules exhibit high fluorescence quantum yields after fluorescence activation, which can be used for precise localization of subcellular organelles and detection of CoA on subcellular organelles.

[0167] Compared with existing technologies, sensor proteins can be used by BRET to detect CoA concentration, featuring high sensitivity. Among them, fusion proteins labeled with probes 1-10 yield sensors with multiple dynamic signal response ranges (ΔR>6), enabling rapid detection using a microplate reader (see...). Figure 2 ).

[0168] Compared to existing technologies, sensor proteins can be used within living cells (see...). Figure 4 and 5Furthermore, it can be located in different subcellular structures within cells. It features high resolution and can be used for rapid quantitative analysis of CoA in subcellular structures within living cells. Figure 3 ).

[0169] In summary, this invention provides a probe compound, a fluorescent probe, and its application for CoA detection.

[0170] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A sensor for CoA detection, characterized in that: The sensor consists of a fluorescent probe, a fusion-expressed protein, and an auxiliary agent; The fluorescent probe includes any one of the following compounds: ； ； ； The fusion-expressed protein consists of HaloTag, NanoLuc luciferase, pantothenic acid kinase, and inactivated pantothenic acid kinase. The method for constructing the fusion expression protein is as follows: By mutating the Y240F and N282A sites, PanK loses its ability to bind to substrates, resulting in PanK*. The original C-terminus and N-terminus of PanK are linked by GSGGTG. The amino acids at positions 213 and 214 of PanK are disconnected to form new C-terminus and N-terminus, forming cpPanK. The Halo-PanK*-cpPanK-NLuc sequence is constructed, and the two PanKs are linked by GGS.

2. The sensor for CoA detection according to claim 1, characterized in that: The fusion protein carries the Y55F mutation.

3. The sensor for CoA detection according to claim 1, characterized in that: The fusion protein carries the Y55F and H307A mutations.

4. The application of the sensor as described in claim 1, characterized in that: Specifically, it includes any of the following: (1) Fluorescent labeling; (2) Fluorescently labeled subcellular proteins; (3) Detection of coenzyme A concentration by bioluminescence; (4) Screening for drugs with adjustable coenzyme A concentration changes; the sensor is not used for disease diagnosis or treatment.

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