Peroxidase fluorescent substrate, its preparation method and application

Abstract

This invention belongs to the field of fluorescent compound technology, specifically relating to a peroxidase fluorescent substrate, its preparation method, and its applications. This invention constructs a new class of HRP fluorescent substrates by modifying the halogenated skeleton, with AQHR and AQHR-1 as representative compounds. Compared with the traditional substrate ADHP, AQHR and AQHR-1 exhibit stronger hydrophilicity and significantly red-shifted absorption and emission spectra. AQHR and AQHR-1 demonstrate excellent reactivity in the HRP / H2O2 system, while their anti-interference ability is significantly improved compared to ADHP. This invention further demonstrates the advantages of AQHR through multi-enzyme cascade fluorescence detection and high-throughput screening experiments of bacterial carboxylesterase mutants based on FADS. AQHR and its analogues are expected to become a promising class of HRP substrates, finding wide application in FADS and other analytical fields.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent compound technology. More specifically, it relates to a peroxidase fluorescent substrate, its preparation method, and its application. Background Technology

[0002] Horseradish peroxidase (HRP) and its fluorescent substrates have become one of the most widely used techniques in biochemical analysis and immunodiagnostics. Since this enzymatic reaction depends on H2O2, the HRP / fluorescent substrate system can be directly used for H2O2 detection. Furthermore, this system can also be used for indirect analysis of H2O2 generation reactions through multi-enzyme cascades, including glucose oxidase (GOx) / glucose (Glu) reactions and xanthine oxidase (XOx) / xanthine reactions. Currently, 10-acetyl-3,7-dihydroxyphenazine (Amplex Red or ADHP) is the most commonly used HRP fluorescent substrate and is used as a fluorescent reporter molecule in various commercial diagnostic kits. Although ADHP is suitable for most HRP detection scenarios, its non-specific response and poor signal stability limit its application in certain scenarios, especially when the detection system contains complex biological matrices.

[0003] In recent years, fluorescence-activated droplet sorting (FADS) has attracted widespread attention in the fields of single-cell analysis and enzyme engineering. HRP / fluorescent substrate reactions are also considered one of the optical detection methods that can be incorporated into FADS. In FADS, the enzyme reaction typically occurs within the microdroplet. If the HRP substrate or its fluorescent product cannot be effectively retained within the droplet, the fluorescence signal will leak across the droplet, causing increased background and reducing analytical sensitivity and accuracy. Therefore, FADS urgently requires fluorescent reporter molecules with strong droplet retention capabilities. Furthermore, FADS analysis often deals with complex biological samples, such as live cell or live bacterial cultures, requiring the fluorescent reporter system to have strong anti-interference capabilities. However, current HRP substrates, such as ADHP, suffer from esterase non-specific responses and poor molecular water solubility leading to cross-droplet leakage, making the HRP / ADHP system insufficient for FADS requirements. Although research over the past decade has focused on developing improved HRP substrates, previous studies have not adequately explored key properties such as substrate solubility and enzyme selectivity. In their previous research (CN115677612A), the inventors prepared the HRP substrate AR-2 by introducing chlorine atoms into the benzene ring of ADHP and replacing the acetyl group with a cyclopropionyl group, which essentially solved the problem of non-specific esterase reactions. However, AR-2 still suffers from insufficient water solubility and microdroplet leakage. Based on these considerations, developing new HRP fluorescent substrates with higher water solubility and both high reactivity and high selectivity is of great significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing HRP substrates or their fluorescent products, such as insufficient water solubility and microdroplet leakage, and to provide a halogenated derivative or a pharmaceutically acceptable salt thereof.

[0005] The purpose of this invention is to provide a method for preparing the aforementioned halogenated derivative.

[0006] Another object of the present invention is to provide a fluorescent probe.

[0007] Another object of the present invention is to provide the application of the halogenated derivative or the fluorescent probe.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention protects a halogenated derivative or a pharmaceutically acceptable salt thereof, having the structure shown in formula (I) or formula (I'): ; Where n is any integer from 0 to 6.

[0010] Preferably, n is any integer from 0 to 3.

[0011] More preferably, the halogenated derivative has any one of the following structures: .

[0013] Furthermore, the salts of the halogen derivatives include triethylamine salts, sodium salts, or potassium salts.

[0014] This invention also protects a method for preparing the halogenated derivative or a pharmaceutically acceptable salt thereof, comprising the following steps: S1. Resorcinol was dissolved in concentrated sulfuric acid, and compound 2 was added at 80–90°C. The reaction was then carried out completely at 100–120°C to obtain compound 3. S2. Dissolve compound 3 obtained in step S1 in an acylation reagent, add an organic base, and react completely at room temperature to obtain compound 4; S3. Under a protective atmosphere, the compound 4 obtained in step S2 is dissolved in an organic solvent, and an alkaline reagent and a mercaptoalkyl sulfonic acid or its salt are added. The reaction is carried out completely at room temperature to obtain the target compound (I). S4. Dissolve the target compound (I) obtained in step S3 in an organic solvent, add a reducing agent and react until complete, add cyclopropane carbonyl chloride and a basic reagent, and react completely at room temperature to obtain the target compound (I'): ; The definition of n is consistent with that described above.

[0015] Furthermore, the parameters of the preparation method include one or more of the following: (1) In step S2, the acylation reagent is selected from acetic anhydride or acetyl chloride; (2) In step S2, the organic base is selected from at least one of pyridine, triethylamine, and diisopropylethylamine; (3) In step S3 or step S4, the alkaline reagent is independently selected from at least one of triethylamine, diisopropylethylamine, NaHCO3, KHCO3, Na2CO3, and K2CO3.

[0016] Further, in step S3 or step S4, the organic solvent is independently selected from at least one of ethanol, N,N-dimethylformamide, tetrahydrofuran, and dioxane.

[0017] Furthermore, the reducing agent includes at least one of stannous chloride, zinc chloride, zinc powder, and sodium borohydride.

[0018] Furthermore, compound 2 is obtained commercially or in its own preparation.

[0019] Furthermore, when prepared in-house, the reaction pathway of compound 2 is as follows: ; The specific preparation method of compound 2 includes the following steps: compound 1 is added to an ethanol solution containing an inorganic base, isoamyl nitrite is added under ice bath conditions, and the reaction is completed at room temperature to obtain compound 2.

[0020] Preferably, the inorganic base is potassium hydroxide or sodium hydroxide. Preferably, the ethanol solution is 75 vol% to 85 vol% ethanol.

[0021] Preferably, the molar ratio of compound 1, inorganic base and isoamyl nitrite is 1:(1-2):(1-2), more preferably 1:(1-1.5):(1-1.5).

[0022] Preferably, in the preparation method of compound 2, the reaction time is 0.5 to 2 h, more preferably 0.8 to 1.2 h.

[0023] Furthermore, in the preparation method of compound 2, the complete reaction also includes post-treatment, which includes precipitation, filtration, washing and drying, specifically including the following steps: the pH of the reaction solution after complete reaction is adjusted to 2 with dilute hydrochloric acid, a light yellow solid is precipitated, the crude product is collected by filtration, washed with cold water 1 to 5 times, and then vacuum dried at room temperature to obtain compound 2.

[0024] Preferably, in step S1, the molar ratio of resorcinol to compound 2 is 1:(0.5~2), more preferably 1:(1~1.5).

[0025] Furthermore, as a preferred embodiment, compound 2 is added in batches.

[0026] Preferably, in step S1, the temperature at which the reaction is completed is 105~115 °C.

[0027] Preferably, in step S1, the reaction takes 10-30 h to complete, more preferably 15-25 h.

[0028] Furthermore, in step S1, the complete reaction also includes post-treatment, which includes precipitation, filtration, washing and drying, specifically including the following steps: after the reaction solution is cooled to room temperature, cold water is added to precipitate a brown precipitate, the crude product is collected by suction filtration, washed three times with cold water, and then dried under vacuum at room temperature to obtain compound 3.

[0029] Preferably, in step S2, the molar ratio of compound 3 to acylation reagent is 1:(5~25), more preferably 1:(20~25).

[0030] Preferably, in step S2, the reaction takes 8 to 20 hours to complete, more preferably 10 to 15 hours.

[0031] Furthermore, in step S2, the complete reaction also includes post-processing, which includes filtration, washing and drying, specifically including the following steps: filtering the reaction solution after the complete reaction, washing it three times with cold water, and drying it under vacuum to obtain compound 4.

[0032] Preferably, in step S3, the molar ratio of compound 4, alkaline reagent and mercaptoalkyl sulfonic acid or its salt is 1:(0.8~2):(3~8), more preferably 1:(1~1.5):(4~6).

[0033] Furthermore, the salts in the mercaptoalkyl sulfonic acid or its salts include sodium or potassium salts.

[0034] Preferably, in step S3, the reaction takes 8 to 20 hours to complete, more preferably 10 to 15 hours.

[0035] Furthermore, in step S3, the protective atmosphere gas is selected from at least one of nitrogen, argon, neon, and helium.

[0036] Furthermore, in step S3, the complete reaction also includes post-treatment, which includes solvent removal and reversed-phase column chromatography purification, specifically including the following steps: after removing the solvent from the reaction solution after the reaction is complete, the solution is purified by reversed-phase column chromatography (methanol / water = 1:50) to obtain the target compound (I).

[0037] Preferably, in step S4, the molar ratio of compound 4, reducing agent, cyclopropane carbonyl chloride and alkaline reagent is 1:(2~4):(3~5):(2~4), more preferably 1:(2.5~3.5):(3.5~4.5):(2.5~3.5).

[0038] Preferably, in step S4, the reaction takes 1 to 5 hours to complete, more preferably 2.5 to 3.5 hours.

[0039] Furthermore, in step S4, the complete reaction also includes post-treatment, which includes solvent removal and silica gel column chromatography purification, specifically including the following steps: after removing the solvent from the reaction solution after the reaction is complete, the crude product is purified by silica gel column chromatography (dichloromethane / methanol = 5 / 1) to obtain the target compound (I').

[0040] The present invention also protects a fluorescent probe comprising one or more of the aforementioned halogenated derivatives.

[0041] This invention protects the application of the halogenated derivative or the fluorescent probe in fluorescence detection and analysis.

[0042] Furthermore, the application in the fluorescence analysis is as a peroxidase substrate.

[0043] Specifically, the applications as peroxidase substrates include fluorescence-activated droplet sorting, reactive oxygen species and nitric oxide radicals, cell imaging, Western blot hybridization analysis, and fluorescence detection analysis of enzyme-linked immunosorbent assay (ELISA) or hydrogen peroxide generation systems.

[0044] Preferably, the peroxidase includes horseradish peroxidase (HRP).

[0045] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a new class of HRP fluorescent substrates by modifying the halogenated skeleton, with AQHR and AQHR-1 as representative compounds. Compared with the traditional substrate ADHP, AQHR and AQHR-1 exhibit stronger hydrophilicity and significantly red-shifted absorption and emission spectra. AQHR and AQHR-1 demonstrate excellent reactivity in the HRP / H2O2 system, while their anti-interference ability is significantly improved compared to ADHP. This invention further demonstrates the advantages of AQHR through multi-enzyme cascade fluorescence detection and high-throughput screening experiments of bacterial carboxylesterase mutants based on FADS. AQHR and its analogues are expected to become a promising class of HRP substrates, finding wide application in FADS and other analytical fields. Attached Figure Description

[0046] Figure 1 The absorption and fluorescence spectra of Resorufin (C) and its derivatives (Resorufin-Cl, D), AQR (A), and AQR-1 (B) were obtained. The spectra were measured in PBS solution (pH 7.4).

[0047] Figure 2 Figure A shows the structure of each substrate and the structure of the fluorescent product after reaction with HRP / H2O2; Figures B and C show the fluorescence kinetics curves of the fluorescent substrates AQHR, AQHR-1, ADHP, and AR-2 reacting with HRP / H2O2, respectively.

[0048] Figure 3 Figure A shows the synthetic pathway of AR-2g molecule; Figure B shows the fluorescence kinetics curves of AR-2g reacting with H2O2 and HRP / H2O2, respectively; Figure C shows the fluorescence kinetics curves of AQHR reacting with H2O2 and HRP / H2O2, respectively; Figure D shows the fluorescence kinetics curves of AQHR-1 reacting with H2O2 and HRP / H2O2, respectively.

[0049] Figure 4 Figure A shows the effect of the non-specific reaction of the commercial substrate ADHP with CES on HRP fluorescence analysis; Figure B shows the statistical data of fluorescence kinetic changes of each substrate with CES; Figure C shows the reaction kinetics of AQHR / HRP / H2O2 under different pH conditions; Figures DF show the statistical data of fluorescence characteristics and anti-protein interference ability tests of the fluorescent products corresponding to each substrate AQR, Resorufin, and Resorufin-Cl.

[0050] Figure 5A schematic diagram (A) shows how AQHR / HRP can be coupled with various enzymes that generate H2O2 to achieve quantitative analysis of the relevant enzymes or their substrates. The enzyme reaction system includes, but is not limited to: formate oxidase (FOX) / sodium formate (B), pyruvate oxidase (POX) / sodium pyruvate (C), monoamine oxidase (MAO) / S-phenylethylamine (D), D-amino acid oxidase (AAO) / D-alanine (E), alcohol oxidase (AOX) / ethanol (F), glucose oxidase (GOX) / β-D-glucose (G), and galactose oxidase (GAO) / D-galactose (H).

[0051] Figure 6 Figure A shows the statistical distribution of residues within 5 Å of the ligand in the Ecu0554 protein; Figure B is a schematic diagram of the fluorescence coupling strategy for Ecu0554 activity assay; Figure C is a standard curve of substrate concentration versus fluorescence intensity; Figure D is a statistical diagram of real-time fluorescence intensity in PBS at different pH values; final concentrations of each substance: (1S, 5R)-sobrerol formate 2 mM, AQHR 100 μM, CES 0.1 mg / mL, AoFOx 0.1 mg / mL, HRP 50 ng / mL.

[0052] Figure 7 The graph shows the microdroplet retention characteristics of various substrates and their fluorescent products. AQHR and AQHR-1 and their products can be retained in microdroplets for a long time without leakage; commercial substrate ADHP and previously reported substrate AR-2 both showed rapid leakage.

[0053] Figure 8 Figure A shows the Ecu0554 site saturated mutant library and FADS sorting schematic diagram; Figure B shows the bright field and fluorescence images of the first-generation mutant NNK-R0 and the second-generation mutant NNK-R1 after incubation in a microdroplet reactor for 1 hour, with fluorescence from the multi-enzyme coupling system of AQHR / HRP with Ecu0554 and AoFOx (scale bar: 50 μm); Figure C is a histogram of the number of droplets with different fluorescence intensities in the NNK-R0 and NNK-R1 mutant libraries, and the inset in the upper right corner of Figure C shows the distribution of droplets with fluorescence intensities exceeding 1000 in the NNK-R0 and NNK-R1 mutant libraries; Figure D is a statistical graph of the relative enzyme activities of variants identified from positive droplets.

[0054] Figure 9 A schematic diagram of the design strategy for hydrophilic HRP fluorescent substrates: water solubility and droplet retention are improved by introducing sulfonic acid groups, and enzyme reaction specificity is enhanced by replacing acetyl groups with cyclopropionyl groups. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0056] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0057] Figure 1 A represents Figure 1 Figure A in the text, Figure 1 B indicates Figure 1 The naming of the other figures follows the same pattern, starting with Figure B.

[0058] Example 1: Synthesis of AQR / AQR-1 The synthesis path for AQR / AQR-1 is shown below:

[0059] The specific preparation method includes the following steps: Compound 2: KOH (72.93 mg, 1.3 mmol) was dissolved in a mixed solvent (4 mL ethanol + 1 mL deionized water). After adding Compound 1 (144.5 mg, 1.0 mmol), the reaction solution was cooled in an ice bath, followed by the dropwise addition of isoamyl nitrite (148 μL, 1.1 mmol). The ice bath was removed, and the reaction was stirred at room temperature for 1 h. The pH of the reaction solution was adjusted to 2 with dilute hydrochloric acid, resulting in the precipitation of a pale yellow solid. The crude product was collected by suction filtration, washed three times with cold water, and dried under vacuum at room temperature to obtain Compound 2 (pale yellow solid), with a yield of 85%.

[0060] Compound 3: Resorcinol (110.1 mg, 1 mmol) was dissolved in concentrated sulfuric acid (2 mL) and heated to 85 °C. Compound 2 (total addition 1.1 mmol) was then added in portions. The reaction mixture was heated to 110 °C and stirred at this temperature for 20 h. After cooling to room temperature, cold water was added, resulting in a brown precipitate. The crude product was collected by suction filtration, washed three times with cold water, and dried under vacuum at room temperature to give compound 3 (brown solid), with a yield of 95%.

[0061] Compound 4: Compound 3 (247.6 mg, 1 mmol) was dissolved in 2.1 mL of acetic anhydride (2.25 g, 22 mmol), and 0.3 mL of anhydrous pyridine was added. The mixture was stirred overnight (12 h) at room temperature. The crude product was filtered, washed three times with cold water, and dried under vacuum to obtain compound 4. The crude product yield was 95%, and it was used directly in the next reaction without further purification.

[0062] AQR and AQR-1: Compound 4 (289.7 mg, 1 mmol) was dissolved in 5 mL of anhydrous methanol, and triethylamine (101.2 mg, 1 mmol) was added. The reaction was maintained under a nitrogen atmosphere for 10 min. Sodium 2-mercaptoethanesulfonate (830.9 mg, 5 mmol) was dissolved in 1 mL of PBS (pH 7.4) and added to the reaction mixture using a syringe. The reaction was allowed to proceed overnight (12 h) at room temperature, monitored by thin-layer chromatography (TLC). After solvent removal, the mixture was purified by reversed-phase column chromatography (methanol / water = 1:50) to give the triethylamine salt of compound AQR (purple solid, 368 mg, yield 81%). NMR and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO- d 6) δ 7.70(d, J = 8.7 Hz, 1H), 7.12 (s, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.82 (s, 1H), 6.34(s, 1H), 3.15 (dd, J = 9.9, 6.2 Hz, 2H), 3.08 (q, J = 7.3 Hz, 6H), 2.77 (dd, J =10.1, 6.3 Hz, 2H), 1.16 (t, J = 7.4 Hz, 9H). 13 C NMR (100 MHz, DMSO- d 6) δ 181.45,163.08, 149.96, 145.84, 145.41, 142.74, 131.75, 127.98, 122.95, 115.77,104.67, 102.16, 49.67, 46.19, 25.58, 9.15. HRMS (ESI) m / z: [MH] - Calcd forC 14 H 10 NO6S2351.9955; Found 351.9954. The synthesis of AQR-1 followed the same steps as above, except that sodium 2-mercaptoethanesulfonate was replaced with sodium 3-mercaptopropanesulfonate (891 mg, 5 mmol), yielding 388 mg of triethylamine salt of AQR-1 (purple solid), with a yield of 83%. NMR and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO- d6) δ 7.69 (d, J = 8.7 Hz, 1H), 7.11 (s, 1H), 6.90 (d, J = 8.7 Hz, 1H), 6.81 (s, 1H), 6.31 (s, 1H), 3.11 (dd, J = 9.9, 6.2 Hz, 2H), 3.08 (q, J = 7.3 Hz, 6H), 2.57 (dd, J = 10.1, 6.3 Hz, 2H), 1.83 (m, 2H),1.16 (t, J = 7.4 Hz, 9H). 13 C NMR (100 MHz, DMSO- d 6) δ 181.45, 163.08, 149.96,145.84, 145.41, 142.74, 131.75, 127.98, 122.95, 115.77, 104.67, 102.16,49.67, 46.19, 25.58, 25.22, 9.15. HRMS (ESI) m / z: [MH] - Calcd forC 15 H 12 NO6S2366.0106; Found 366.0107. Example 2 Synthesis of AQHR / AQHR-1 The synthesis pathway for AQHR / AQHR-1 is shown below:

[0063] The specific preparation method includes the following steps: AQR triethylamine salt (454.6 mg, 1 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran (THF), and stannous chloride (Tin(II) chloride, 676.9 mg, 3 mmol) was added. The mixture was stirred at 60 °C for 1 h, followed by the sequential addition of cyclopropane carbonyl chloride (418.1 mg, 4 mmol) and triethylamine (TEA, 556 μL, 3 mmol). The reaction was carried out at room temperature for 3 h, monitored by TLC. After solvent removal, the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 5 / 1) to give 210 mg of triethylamine salt of compound AQHR (yield 40%). NMR and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO- d6) δ 10.27 (s, 1H), 9.87 (s, 1H), 7.45 (s, 1H), 7.38 (d, J = 8.5 Hz, 1H), 6.71 (s, 1H), 6.65-6.52 (m, 2H), 3.16 (s, 1H), 3.05 (q, J = 7.3 Hz, 6H), 3.01-2.93 (m, 2H), 2.69-2.54 (m, 2H), 2.05-1.89 (m, 1H), 1.17 (t, J = 7.4 Hz, 9H), 1.01-0.76 (m, 4H). 13 C NMR (100 MHz, DMSO-d6) δ 172.01, 156.66, 155.49, 151.30, 150.41, 127.89, 126.02, 121.57,120.99, 115.55, 110.77, 103.85, 103.63, 51.56, 45.91, 28.83, 12.83, 9.65,8.94. HRMS (ESI) m / z: [MH] - Calcd for C 18 H 16 NO7S2422.0374; Found 422.0379. The synthesis of AQHR-1 followed the same method described above, yielding 225 mg of the triethylamine salt of compound AQHR-1 (yield 42%). NMR and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO- d 6) δ 10.26 (s, 1H), 9.85 (s, 1H), 7.43 (s, 1H), 7.35 (d, J = 8.5 Hz, 1H), 6.70 (s, 1H), 6.64-6.51 (m, 2H), 3.11 (s, 1H), 3.05 (q, J = 7.3 Hz, 6H), 3.01-2.93 (m, 2H), 2.54-2.44 (m, 2H), 2.05-1.89 (m, 1H), 1.82 (m, 2H), 1.16 (t, J = 7.4 Hz, 9H), 1.01-0.76 (m, 4H). 13CNMR (100 MHz, DMSO-d6) δ 172.01, 156.66, 155.49, 151.30, 150.41, 127.89,126.02, 121.57, 120.99, 115.55, 110.77, 103.85, 103.63, 51.56, 45.91, 28.83,25.22, 12.83, 9.65, 8.94. HRMS (ESI) m / z: [MH] - Calcd for C 19 H 18 NO7S2436.0525; Found 436.0527. Example 3 Experimental Case 1 Experimental Methods 1.1 Measurement and Calculation of Photophysical Properties AQR, AQR-1, and Resorufin-Cl were diluted with Resorufin in phosphate buffer (PBS pH 7.4) and their absorbance (OD) was measured at 530 nm. 530 The value was approximately 0.05. UV-Vis spectra were measured using a spectrophotometer, and fluorescence spectra were recorded under 530 nm excitation. The OD value was... 530 The relative quantum yield (φ) was calculated using the absorbance and the area under the fluorescence spectrum curve as parameters, according to the methods reported in the literature. The fluorescence lifetimes of AQR, AQR-1, dichlorohalogen, and halogen were determined using a FluoroMax® Plus fluorescence spectrometer.

[0064] 1.2 Fluorescence Detection In fluorescence intensity assays, the AQHR, AQR-1, ADHP, and AR-2 reaction systems were excited at 584 nm, 584 nm, 570 nm, and 581 nm, respectively, and fluorescence intensities were recorded at 640 nm, 640 nm, 588 nm, and 602 nm, respectively. Assays were performed in 96-well plates, with 100 μL of solution added to each well. Unless otherwise specified, fluorescence assays were performed using PBS buffer at room temperature (25 °C). In a typical HRP reaction, the fluorescent substrate was first mixed with HRP, followed by the addition of H2O2. In multi-enzyme cascade reactions, the substrate was mixed with a specific oxidase (Ox) and HRP, and then the corresponding oxidase substrate was added to the set concentration. The final concentrations of the fluorescent substrate and HRP were 100 μM and 50 ng / mL, respectively. Fluorescence was measured using a microplate reader after reacting at room temperature for 10 min. For the substrate / CES / AoFOx / HRP / AQHR (or AQHR-1) reaction, AQHR (or AQHR-1) was mixed with CES, AoFOx, and HRP, and then (1S,5R)-sobrerol formate was added to achieve a final concentration of 0.1 mM–50 mM. The final concentrations of AQHR, CES, AoFOx, and HRP were 100 μM, 0.1 mg / mL, 0.1 mg / mL, and 50 ng / mL, respectively. Fluorescence intensity was measured after reacting at room temperature for 30 min.

[0065] 1.3 Microdroplet formation and fluorescence observation Collect cells from 1 mL of the mutant bacterial culture and wash three times with 10 mM PBS (pH 7.4). Dilute the bacterial suspension to OD0.05. 600 = 0.04 - 0.06, to achieve monodisperse encapsulation of single *E. coli* cells within a single droplet. A 500 μL reaction mixture containing 60% BugBuster protein extraction reagent, 20 mM (1S, 5R)-sobrerol formate, 0.2 mg / mL AoFOx, 100 ng / mL HRP, and 100 μM AQHR (or AQHR-1) was prepared using 50 mM PBS. The droplet generation chip was fixed to the microscope stage, precisely aligned with the tar-water interface. First, the oil phase pressure valve was opened to introduce the oil phase, then the reaction mixture and diluted bacterial suspension were loaded separately, adjusting the pressure to stabilize the generation of droplets approximately 30 μm in diameter. The collected droplets were observed under a fluorescence microscope or used for subsequent FADS sorting.

[0066] 1.4 Microdroplet sorting The first-generation microdroplets (NNK-R0) were prepared according to the above method and incubated at 37 °C for 1 h. Microdroplet sorting was performed according to previously reported methods. The reaction was then terminated by placing the microdroplets on ice, and the droplets were injected into a sorting chip. Sorting was performed based on fluorescence intensity (510 nm excitation, 610 ± 10 nm collection and emission). The bacteria contained in the sorted positive droplets (with fluorescent signals) were detected by PCR, digested with restriction endonucleases, cloned into the pET28a vector, and transformed into E. coli 10G for the next round of microdroplet preparation and FADS screening.

[0067] 2. Experimental Results 2.1 Photophysical properties of the obtained compounds Table 1. Photophysical parameters of halogenated ...

[0068] The HRP substrates ADHP, AR-2, AQHR, and AQHR-1 correspond to the fluorescent molecules halogen, dichlorohalogen, AQR, and AQR-1, respectively, and their photophysical parameters are shown in Table 1 and 2. Figure 1 As shown. AQR ( Figure 1 A) and AQR-1 ( Figure 1 The fluorescence emission peak of B) is located at 640 nm, compared to that of halogen (B). Figure 1 C) and dichlorohalogen ( Figure 1 D) A significant red shift, placing its spectrum in the deep red to near-infrared region; its Stokes shift is 56 nm, significantly greater than that of halogen (18 nm) and dichlorohalogen (20 nm). Generally, both a fluorescence red shift and an increased Stokes shift are beneficial for improving the anti-interference capability of fluorescence detection. Although the quantum yields of AQR and AQR-1 are lower than those of halogen and dichlorohalogen, a fluorescence quantum yield close to 0.08 is still acceptable for near-infrared fluorescent molecules, basically meeting the requirements for high-sensitivity detection.

[0069] Table 2. Calculated lipid-water partition coefficients (cLogP) of halogenated derivatives and their corresponding HRP substrates.

[0070] Enzymatic reactions typically occur in an aqueous environment, thus requiring sufficient water solubility in the enzyme's fluorescent substrate. Amplex red (ADHP), a classic HRP substrate, was developed in 1994 and remains considered the most sensitive HRP fluorescent substrate. ADHP largely satisfies HRP-based fluorescence analysis requirements and is therefore the primary fluorescent probe molecule used in current HRP fluorescence analysis kits. However, ADHP exhibits a non-specific response to carboxylesterases (CES) independent of HRP, severely hindering its application in the analysis of complex biological samples. This is because natural CES can remove the acetyl group on ADHP, leading to spontaneous oxidation of the substrate into the fluorescent product halogen. The inventors previously prepared a new HRP substrate, AR-2, by replacing the naturally occurring acetyl group with the less common cyclopropionyl group, largely overcoming the CES non-specific response problem. However, ADHP, AR-2, and their fluorescent products all suffer from poor water solubility, making their fluorescence signals susceptible to interference from the non-specific adsorption of biomolecules. Furthermore, the core of FADS for single-cell analysis and enzyme-directed evolution relies on microdroplets composed of both oil and aqueous phases. Substrates with poor water solubility and strong lipophilicity can easily permeate through the oil phase into other microdroplets, resulting in high background and affecting the accuracy of the analysis.

[0071] As shown in Table 2, the lipid-water partition coefficients (cLogP) of the HRP substrates AQHR and AQHR-1 obtained in this invention are -0.0968 and 0.236, respectively, which are significantly lower than those of the commercial substrate ADHP (1.028), indicating that the former has better water solubility. Furthermore, the corresponding fluorescent products AQR and AQR-1 have lipid-water partition coefficients of 0.0370 and 0.3698, respectively, which are also significantly lower than those of halogenated ...

[0072] 2.2 Reaction test of AQHR and AQHR-1 as HRP substrates Figure 2 A shows the structural diagrams of each substrate and the structures of the fluorescent products after their reaction with HRP / H2O2; from Figure 2 The kinetic curves show that AQHR ( Figure 2 B) and AQHR-1 ( Figure 2 C) The reaction rate is higher than that of ADHP ( Figure 2 D) and AR-2 ( Figure 2 E) The fluorescence intensity decreased slightly, possibly due to steric hindrance caused by the increased substrate molecule size. However, AQHR and AQHR-1 are also excellent substrates for the HRP / H2O2 reaction, exhibiting rapid fluorescence onset, reaching a peak and stabilizing within 30 minutes. Figure 3As shown in Figure A, for comparison, the inventors prepared another type of water-soluble halogenated fluorescent molecule 2g (Qingwei T, Shuxuan Z, Yuyao L, et al. Regioselective Difluoromethane sulfonylation and Triflylation of Resorufin Derivatives.[J]. Organic letters, 2021, 23(21):DOI:10.1021 / ACS.ORGLETT.1C03192.) using the same acylation method, but the obtained AR-2g was extremely unstable and easily oxidized and deteriorated during purification and storage. Preliminary tests showed that in the absence of HRP, AR-2g could be non-specifically oxidized by H2O2, producing gradually enhanced background fluorescence ( Figure 3 B). When HRP / H2O2 coexist, the fluorescence onset of AR-2g is relatively slow, and it has not yet reached equilibrium after 30 minutes of reaction. However, AQHR and AQHR-1 are stable in the presence of only H2O2, and when HRP / H2O2 coexist, the fluorescence onset is rapid. Figure 3 C and Figure 3 D).

[0073] As mentioned above, ADHP's nonspecific response to CES is one of its main drawbacks. Figure 4 A) Introducing a non-natural cyclopropionyl group modification is expected to suppress this non-specific reaction. Therefore, the inventors evaluated and compared the response of the obtained representative substrate AQHR to CES, and compared it with ADHP and AR-2. The results are as follows: Figure 4 As shown in Figure B, ADHP can be rapidly converted by CES in the absence of HRP and H2O2, while AQHR and AR-2 show no response to CES. This indicates that the introduction of the cyclopropionyl group can effectively overcome the non-specific reaction of the HRP substrate to CES and improve the specificity of fluorescence analysis. Subsequently, the inventors evaluated the optimal pH of the AQHR / HRP H2O2 system. The results showed that the reaction proceeded smoothly under all tested pH conditions, with the highest fluorescence intensity at pH 8.0. Figure 4 (C), mainly due to the superior fluorescence properties of AQR under weakly alkaline conditions.

[0074] In addition, the inventors investigated the interference of proteins with the fluorescence signals of the fluorescent products of each substrate, and the results were as follows: Figure 4As shown in Figures D-F, the fluorescence intensity of the AQHR fluorescent product AQR was not affected by mixing with high concentrations of bovine serum albumin (BSA). However, the fluorescence of the ADHP product halogen and the AR-2 product dichlorohalogen was significantly affected by protein interference, with increasing BSA concentration noticeably reducing their fluorescence intensity. This effect primarily stems from the non-specific adsorption between BSA and fluorescent molecules; enhancing the water solubility of the molecules can reduce this non-specific effect. Therefore, it can be said that AQHR and AQHR-1 have stronger anti-interference capabilities and better accuracy than ADHP and AR-2 when used for analyzing complex biological samples.

[0075] 2.3 AQHR / HRP reacts with various enzymes to form a multi-enzyme cascade fluorescence analysis system HRP / fluorescent substrate-based detection systems have become the mainstream method for tracking H2O2 and its generation reactions due to their high sensitivity and ease of operation. As a fundamental biochemical event, H2O2 is mainly generated by various oxidases (Oxs), participating in various physiological and pathological processes such as immune responses and cell metabolism. To verify the reliability of the substrate obtained in this invention, a series of enzyme-coupled assays were performed using AQHR as a representative. Several representative oxidases and their substrates (Ox / substrate) were selected and coupled with AQHR / HRP (… Figure 5 A) Construct a multi-enzyme cascade system by combining: formate oxidase (FOX) / sodium formate ( Figure 5 B), Pyruvate oxidase (POX) / Sodium pyruvate ( Figure 5 C), Monoamine oxidase (MAO) / S-phenylethylamine ( Figure 5 D), D-amino acid oxidase (AAO) / D-alanine ( Figure 5 E), alcohol oxidase (AOX) / ethanol ( Figure 5 F), glucose oxidase (GOX) / β-D-glucose ( Figure 5 G), galactose oxidase (GAO) / D-galactose ( Figure 5 H). The reaction principle is as follows: a specific oxidase reacts with its substrate to produce H2O2, which further participates in the AQHR / HRP reaction and produces the fluorescent substrate AQR, thereby enabling qualitative and quantitative analysis of the oxidase or its substrate. Figure 5 The results showed that, in the seven oxidase-coupled systems, the fluorescence enhancement of AQHR was well correlated with the increase in substrate concentration. Therefore, the AQHR / HRP system can be coupled with a variety of enzymes to achieve quantitative analysis of related enzymes and their substrates, demonstrating good versatility and reliability.

[0076] 2.4 The AQHR / HRP system was successfully used for FADS-based directed enzyme evolution research. The results above indicate that the AQHR / HRP system has significant application potential in FADS-based high-throughput analysis. The inventors demonstrate the application potential of the AQHR / HRP system using the directed evolution of a bacterial carboxylesterase (Ecu0554) as an example. It is well known to those skilled in the art that CES can hydrolyze carboxylic esters, thioesters, and amide bonds, and has important applications in various environmental engineering and pharmaceutical industries. Given the importance of CES, its catalytic mechanism research and activity-guided protein engineering have received widespread attention, and FADS technology provides a high-throughput screening platform for related research. The inventors' previous research found that the bacterial CES subtype Ecu0554 can selectively hydrolyze (1... S , 5 R )-sobrerol formate, forming (1 S , 5 R Ecu0554, a naturally occurring enzyme, has limited hydrolytic activity. To improve its hydrolytic activity, the inventors first performed molecular docking, screening 14 potential key sites (G105, G106, A107, L110, E188, A190, M193, F271, L273, L313, F314, M358, L362, F363) within a 5 Å range from the substrate, and constructed a site-saturated mutant library. Figure 6 A).

[0077] Subsequently, the inventors further optimized the fluorescence coupling strategy (Ecu0554-AoFOx-HRP) for detecting the enzyme activity of Ecu0554 wild-type and mutant: Ecu0554 hydrolyzes its substrate (1 S , 5 R )-sobrerol formate is converted to formic acid, which is then used as an intermediate by formate oxidase AoFOx to generate H2O2. HRP / H2O2 then catalyzes the oxidation of AQHR to produce a fluorescent signal. Figure 6 B). Since AQHR has no nonspecific response to CES (including Ecu0554), AQHR / HRP can perfectly match the needs of FADS-based CES-directed evolution. Figure 6 As shown in Figure C, the fluorescence growth rate exhibits a significant linear relationship with the substrate concentration, indicating that the system has high signal transduction efficiency and can meet the requirements of high-throughput screening of mutant libraries. Further analysis confirms that the system has good pH tolerance and can provide good fluorescence indication under both weakly acidic and weakly alkaline conditions. Figure 6 D), is not easily affected by pH changes.

[0078] Given that droplet retention is crucial to the success of FADS, the inventors first compared the retention of fluorescence signals from four fluorescent substrates—AQHR, AQHR-1, ADHP, and AR-2—in microdroplets. In the test, the inventors placed the reacted (1) S 5 R Fluorescent droplets of the )-sobrerol formate / Ecu0554-AoFOx-HRP / fluorescent substrate coupling system were mixed with non-fluorescent blank droplets of the enzyme-free reaction system at a ratio of 2:3 and incubated under light-protected conditions. After a period of time, imaging was performed using a fluorescence microscope. Figure 7 As shown, the ADHP and AR-2 systems exhibited varying degrees of fluorescence leakage from the start of incubation; while the AQHR and AQHR-1 systems remained completely leak-free even after 48 hours of incubation, indicating that the strong hydrophilicity of the HRP fluorescent substrate enables it to meet the long-term droplet retention requirements of FADS. The commercial substrate ADHP cannot be retained in droplets for extended periods, thus failing to meet the relevant requirements of FADS.

[0079] The inventors encapsulated the constructed site-saturated mutant library in microdroplets for FADS (Fragmentation-Assisted Discharge). Figure 8 A). The initial first-generation droplet pool was named NNK-R0. The positive mutant library enriched in the first round of sorting was then encapsulated to generate a new droplet pool, NNK-R1. Fluorescence imaging and quantitative signal analysis both showed that the number of positive droplets and fluorescence intensity in NNK-R1 were significantly higher than those in NNK-R0. Figure 8 BC). An additional round of sorting was then performed on NNK-R1 to further enrich positive droplets and identify the Ecu0554 mutant; this mutant was expressed, purified, and its activity was re-tested to confirm the increased activity. The results showed that the mutant enzyme activity was significantly increased relative to the wild type, with F314H and F314N showing increases of 12.49-fold and 12.20-fold, respectively. Figure 8 D). In summary, the AQHR / HRP system can be used for Ecu0554 ultra-high throughput screening in droplet microreactors.

[0080] In summary, this invention prepared two novel HRP substrates, AQHR and AQHR-1, by modifying the fluorescent backbone of halogenated hydroxylamine. The key to substrate design lies in: introducing mercaptoethanesulfonic acid or mercaptopropanesulfonic acid to improve water solubility, and replacing the acetyl group with a cyclopropionyl group to enhance enzyme selectivity. Figure 9Compared to ADHP, AR-2, and their fluorescent products, AQHR and its fluorescent products AQR and AQHR-1, exhibit significantly lower lipid-water partition coefficients (cLogP), indicating enhanced water solubility. Experiments show that both AQHR and AQHR-1 can achieve sensitive detection of the HRP / H2O2 reaction, making them excellent HRP fluorescent substrates. Furthermore, compared to the commercial substrate ADHP, AQHR and AQHR-1 demonstrate significantly enhanced enzyme reaction specificity and resistance to interference. More importantly, AQHR, AQHR-1, and their fluorescent products AQR and AQR-1 exhibit long-term droplet retention, making them particularly suitable for the high-throughput analytical platform FADS. Subsequent in-depth studies using AQHR as a representative compound revealed that the AQHR / HRP system can be used for fluorescence analysis of various enzyme reaction systems through enzyme cascade design. More importantly, by utilizing the AQHR / HRP / H2O2 reaction system, this invention achieves high-throughput screening based on FADS, successfully identifying bacterial mutants expressing the highly active carboxylesterase (CES) isoform Ecu0554. This novel water-soluble HRP substrate will find applications in numerous fluorescence analyses, including FADS; high-throughput screening of esterases using FADS is just one of its applications.

[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A halogenated derivative or a pharmaceutically acceptable salt thereof, characterized in that, The halogenated derivative has the structure shown in formula (I) or formula (I'): ； Where n is any integer from 0 to 6.

2. The halogenated derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, n is any integer from 0 to 3.

3. The halogenated derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The halogen derivative has any one of the following structures: 。 4. A method for preparing the halogenated derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Resorcinol was dissolved in concentrated sulfuric acid, and compound 2 was added at 80–90°C. The reaction was then carried out completely at 100–120°C to obtain compound 3. S2. Dissolve compound 3 obtained in step S1 in an acylation reagent, add an organic base, and react completely at room temperature to obtain compound 4; S3. Under a protective atmosphere, the compound 4 obtained in step S2 is dissolved in an organic solvent, and an alkaline reagent and a mercaptoalkyl sulfonic acid or its salt are added. The reaction is carried out completely at room temperature to obtain the target compound (I). S4. Dissolve the target compound (I) obtained in step S3 in an organic solvent, add a reducing agent and react until complete, add cyclopropane carbonyl chloride and a basic reagent, and react completely at room temperature to obtain the target compound (I'): ； Wherein, the definition of n is consistent with that of any one of claims 1 to 3.

5. The preparation method according to claim 4, characterized in that, The parameters of the preparation method include one or more of the following: (1) In step S2, the acylation reagent is selected from acetic anhydride or acetyl chloride; (2) In step S2, the organic base is selected from at least one of pyridine, triethylamine, and diisopropylethylamine; (3) In step S3 or step S4, the alkaline reagent is independently selected from at least one of triethylamine, diisopropylethylamine, NaHCO3, KHCO3, Na2CO3, and K2CO3.

6. The preparation method according to claim 4, characterized in that, In step S3 or step S4, each organic solvent is independently selected from at least one of ethanol, N,N-dimethylformamide, tetrahydrofuran, and dioxane.

7. The preparation method according to claim 4, characterized in that, In step S4, the reducing agent is selected from at least one of stannous chloride, zinc chloride, zinc powder, and sodium borohydride.

8. A fluorescent probe, characterized in that, Includes one or more of the halogenated derivatives described in any one of claims 1 to 3.

9. The application of the halogenated derivative according to any one of claims 1 to 3 or the fluorescent probe according to claim 8 in fluorescence detection and analysis.

10. The application according to claim 9, characterized in that, The application in the fluorescence detection analysis is as a peroxidase substrate.

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