Hydrophilic bifunctional probe and synthesis and application thereof

Abstract

This invention provides a hydrophilic bifunctional probe and its synthesis and application. Specifically, this invention provides a hydrophilic bifunctional probe (6-(1-aminoethyl)naphthyl-2-yl)glycine (N1) and its corresponding fluorescent molecule (6-acetylnaphthyl-2-yl)glycine (N2). This pair of molecules in this invention exhibits high photostability and water solubility, and can be used in aqueous luminescent materials, biofluorescent probes, and biofluorescent imaging. It can be applied to fluorescence-based high-throughput screening systems to identify strains of highly active enzymes through quantitative detection in trace systems. The structures of the hydrophilic bifunctional probe and its corresponding fluorescent molecule are as follows: [structures omitted].

Description

Technical Field

[0001] This invention belongs to the fields of fluorescent dyes and bioengineering technology, specifically relating to a hydrophilic bifunctional probe and its synthesis and application. It is a synthesis and application of a hydrophilic bifunctional probe and its corresponding fluorescent molecule. Background Technology

[0002] Fluorescence detection technology, due to its inherent advantages including ease of operation, reliable detection signals, fast analysis speed, and strong in-situ imaging capabilities, has broad prospects in environmental monitoring and clinical diagnosis. In recent years, various fluorescent probes based on different fluorophores have been developed for the analysis and detection of various analytes, achieving significant results.

[0003] PRODAN-based fluorescent molecules, as a class of high-performance fluorescent dyes, possess excellent fluorescence properties due to their naphthalene ring skeleton, including stable luminescence, high molar extinction coefficient, and fluorescence quantum yield. They also exhibit a degree of biocompatibility, showing potential for applications in fields such as bioimaging. However, existing PRODAN derivatives generally suffer from insufficient water solubility and short emission wavelengths, resulting in poor dispersibility in aqueous solutions and susceptibility to interference from biological autofluorescence, making it difficult to meet the requirements of high-precision detection. Furthermore, their optical performance modulation methods are limited; there is a lack of systems that can achieve significant signal changes through simple group transformations, restricting their application expansion in complex biological environments. Therefore, developing PRODAN-based fluorescent molecules with both good water solubility and tunable optical properties has become an important direction for promoting their practical application in related fields.

[0004] Chiral drugs constitute a significant portion of drug molecules, with 90% possessing chiral amine structures. Chiral amines are an important class of pharmaceutical intermediates, flexibly applied in drug molecules and various fine chemical products, such as sitagliptin (a highly selective DPP-4 inhibitor), Vyvanse (a central nervous system stimulant), and Idelalisib (a PI3K-delta inhibitor). Since the successful industrial production of sitagliptin using engineered transaminases, enzymatic synthesis of chiral amines has accelerated the introduction of enzyme-catalyzed synthesis into the pharmaceutical industry, as seen with transaminases and imine reductases. Ketones are also important pharmaceutical and fine chemical intermediates, widely used in food, flavorings, and other fields. The enzymatic synthesis of both types of compounds has developed rapidly, meeting the demands of green chemistry development with mild reaction conditions.

[0005] However, the chemical complexity of non-natural substrates often exceeds the catalytic capacity of natural enzymes. Therefore, natural enzymes need to be modified using appropriate screening methods to adapt them to different non-natural substrates and harsh industrial conditions. Efficiently discovering new enzymes or directionally evolving known enzymes requires the development of high-throughput screening methods. However, ultra-high-throughput screening methods for amines / ketones are rarely reported. Summary of the Invention

[0006] The purpose of this invention is to provide a hydrophilic bifunctional probe and its corresponding fluorescent molecule, wherein the probe and its corresponding fluorescent molecule have better hydrophilicity.

[0007] This invention provides a hydrophilic bifunctional probe and its corresponding fluorescent molecule, having the following structure: , I II.

[0008] R1 is selected from hydrophilic groups, specifically including carboxyl (-COOH), sulfonic acid (-SO3H), hydroxyl (-OH), phosphate (-PO3H2), and amino (-NH2, -NHR, -NR2), where R is C 1‒6 alkyl), quaternary ammonium (-N) + R a RᵦRᵧX - , where R a Rᵦ and Rᵧ are independently selected from C 1‒6 Alkyl or benzyl, X - (e.g., halide ions, sulfate ions, or phosphate ions), amide groups (-CONH2, -CONHR, -CONR2, where R is C) 1‒6 alkyl or aryl), sulfonate group (-SO3R, where R is C 1‒6 alkyl, aryl, or benzyl groups), carboxylic acid ester groups (-COOR, where R is C). 1‒6 Alkyl, aryl, or benzyl), ether (-OR, where R is C) 1‒6 Alkyl, aryl, or substituted alkyl groups), aldehydes (-CHO), pyridyl groups (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, and their N-oxides or C-oxides), and pyridyl groups (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, and their N-oxides or C-oxides). 1‒6 Alkyl-substituted derivatives), R2 is hydrogen (H), C 1‒6 Alkyl, substituted C 1‒6 Alkyl groups (substituents selected from halogen, hydroxyl, amino, carboxyl, sulfonic acid, ether, or C) 1‒6 alkoxy), C 3‒8 cycloalkyl, substituted C 3‒8 Cycloalkyl (substituents selected from C) 1‒6 (alkyl, halogen, hydroxyl, amino or carboxyl), C 5‒10 Aryl (such as phenyl, naphthyl), substituted C6‒10 Aryl (substituents selected from C) 1‒6 Alkyl, halogen, hydroxyl, amino, carboxyl, sulfonic acid, or C 1‒6 alkoxy), R3 is C 1‒6 One of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxy; n is an integer from 0 to 12.

[0009] Based on the properties and reaction requirements of the fluorescent probe, this invention specifically provides a hydrophilic bifunctional probe (6-(1-aminoethyl)naphthyl-2-yl)glycine (N1) and its corresponding fluorescent molecule (6-acetylnaphthyl-2-yl)glycine (N2), the structural formula of which is as follows: .

[0010] Another object of the present invention is to provide a method for synthesizing the above-mentioned hydrophilic bifunctional probe and its corresponding fluorescent molecule pair, the synthetic route of which is as follows: The chemical name of compound 1 is 6-methoxy-2-acetylnaphthalene; The chemical name of compound 2 is 6-acetyl-2-naphthol; The chemical name of compound 3 is N–(6–acetylnaphthyl–2–yl)–2–hydroxy–2–methylpropionamide; The chemical name of compound 4 is 2-acetyl-6-aminonaphthalene; The chemical name of the compound N2 is (6-acetylnaphthyl-2-yl)glycine; The chemical name of the compound N1 is (6–(1–aminoethyl)naphthyl-2–yl)glycine.

[0011] The specific synthesis steps are as follows: (1) Synthesis of compound 2: Compound 1 and concentrated hydrochloric acid (con. HCl) were added to a round-bottom flask and reacted at 90 °C. After the raw materials were completely reacted, the mixture was filtered while hot, cooled in an ice-water bath, filtered again, and dried to obtain compound 2. (2) Synthesis of compound 3: Compound 2, sodium hydroxide (NaOH) and N,N-dimethylacetamide (DMA) were added to a flask and stirred at room temperature. Then, bromoisobutyramide was added and reacted overnight at room temperature. The mixture was extracted with dichloromethane and water, dried over anhydrous magnesium sulfate, and evaporated to dryness to obtain a solution of compound 3 containing N,N-dimethylacetamide. (3) Synthesis of compound 4: Anhydrous ethanol and concentrated hydrochloric acid were added to an N,N-dimethylacetamide solution containing compound 3, and the mixture was incubated overnight at 90 °C. Saturated sodium hydroxide was added dropwise until the pH was > 10. The ethanol was removed by evaporation, and the mixture was filtered, retaining the residue. The filtrate was extracted with ethyl acetate, dried with saturated brine and anhydrous magnesium sulfate. After drying, the residue and organic phase were separated by silica gel column chromatography to obtain a yellow solid. The solid was then slurried with chloroform to obtain a yellow powder, which is compound 4. (4) Synthesis of compound N2: Compound 4, ethyl bromoacetate (BrCH2COOEt) and sodium acetate (AcONa) were added to a double-necked flask. Anhydrous ethanol (EtOH) was added under nitrogen protection and the mixture was refluxed overnight. After cooling to room temperature, the mixture was dried by rotary evaporation. Under nitrogen protection, 2N sodium hydroxide solution (2N NaOH) was added and the mixture was refluxed. After the reaction was completed, the mixture was cooled to room temperature and the pH was adjusted to 3 with 2N hydrochloric acid. The mixture was filtered, washed with the filtrate, dried, and recrystallized from ethanol to obtain compound N2. (5) Synthesis of compound N1: Compound N2, ammonium acetate (AcONH4) and sodium cyanoborohydride (NaBH3CN) were added to a double-necked round-bottom flask. Anhydrous methanol (MeOH) was added under nitrogen atmosphere and the reaction was carried out overnight under reflux. After the reaction was completed, water was added to quench the reaction, the mixture was dried by rotary evaporation, and compound N1 was obtained by silica gel column chromatography.

[0012] In step (1), the ratio of reagent usage is 3 g of compound 1 added to 500 mL of concentrated hydrochloric acid, filtered, and the funnel and product are dried together.

[0013] In step (2), the ratio of each reagent is 1 g of compound 2 and 840 mg of NaOH added to 15 mL of DMA, reacted at room temperature for 1 h, and then 1.5 g of bromoisobutyramide was added.

[0014] In step (3), the ratio of reagents used is 37.5 mL of anhydrous ethanol and 37.5 mL of concentrated hydrochloric acid added to the crude solution of compound 3 in the previous step; the silica gel column separation uses petroleum ether:tetrahydrofuran = 20:1–1.5:1 as the eluent.

[0015] In step (4), the ratio of each reagent is as follows: 186 mg of compound 4, 167 mg of ethyl bromoacetate, and 83 mg of sodium acetate are dissolved in 10 mL of anhydrous ethanol, followed by the addition of 15 mL of 2N sodium hydroxide solution.

[0016] In step (5), the ratio of each reagent is 243 mg of compound N2, 310 mg of ammonium acetate, and 26 mg of sodium cyanoborohydride added to 15 mL of anhydrous methanol; the silica gel column separation is performed using chloroform:methanol = 50:1–10:1 (0.1% triethylamine).

[0017] The inorganic base used in the step is at least one of sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, potassium bicarbonate, and sodium acetate, and the reducing agent used is one of sodium cyanoborohydride and sodium borohydride.

[0018] The synthesis method provided by this invention has the advantages of inexpensive and readily available raw materials, simple synthesis steps, and easy purification.

[0019] Another object of the present invention is to provide the application of the hydrophilic bifunctional probe and its corresponding fluorescent molecule in a fluorescence-based high-throughput screening system. The hydrophilic bifunctional probe and its corresponding fluorescent molecule are compound N1 and compound N2. Compound N1 is mainly indigo blue, while compound N2, when converted or used directly, is green fluorescent (emitted at 508 nm) and has a wide Stokes shift (104 nm).

[0020] This invention utilizes the aforementioned hydrophilic bifunctional probes and their corresponding fluorescent molecules to develop a fluorescence-based high-throughput screening system. This system is suitable for reaction systems capable of converting amino groups to carbonyl groups or vice versa, such as for high-throughput screening of transaminases using microplate readers or fluorescence-activated microdroplet sorting (FADS). It can be applied to the determination of amine / ketone compound content, screening of highly active enzymes, ultra-high-throughput enzyme evolution, and the discovery of novel transaminases. It features high speed, high throughput, and low cost. Quantitative detection in trace systems makes the identification of highly active bacterial strains possible.

[0021] This high-throughput screening system uses one or both of the following: a hydrophilic bifunctional probe (6–(1–aminoethyl)naphthyl-2–yl)glycine (N1) and its corresponding fluorescent molecule (6–acetylnaphthyl-2–yl)glycine (N2).

[0022] First, it is provided that when using the hydrophilic bifunctional probe N1 as a reaction substrate, it can be used for at least one of the following (a1)-(a4): (a1) Detection of ketone compound content; (a2) Detection of enzyme activity; (a3) Screening of highly active enzymes; (a4) Directed evolutionary screening of enzymes; The enzyme is an enzyme that can catalyze the generation of the corresponding fluorescent molecule N2 or consume N1.

[0023] This invention also provides, when using fluorescent molecule N2 as a reaction substrate, its use in at least one of the following (a1)-(a5): (a1) Detection of amine compound content; (a2) Detection of enzyme activity; (a3) Screening of highly active enzymes; (a4) Directed evolutionary screening of enzymes; (a5) Biofluorescence imaging; The enzyme is an enzyme that can catalyze the generation of probe N1 or consume N2. The present invention also protects a reagent kit containing N1 or N2; the application of the reagent kit includes at least one of the above.

[0024] This high-throughput screening system can use N1 as an amino donor for the detection of ketone compounds or enzyme activity. The system includes the following steps: N1 and the sample to be tested are added to a buffer solution and mixed. The mixture is then reacted at a corresponding temperature. The fluorescence values ​​of the test system and the control system are compared under excitation light at 404 nm and emission light at 508 nm. The higher the fluorescence value of the test system compared to the control system, the higher the content of ketone compounds in the test sample or the greater the consumption of ketone compounds. The control system is a reaction system without the addition of the test sample.

[0025] In the method described above, during quantitative detection, the content or consumption of ketone compounds in the sample to be tested is determined as follows to infer the reaction rate: after the reaction is completed, the measured fluorescence value is substituted into the standard curve equation to calculate the content or consumption of ketone compounds in the sample to be tested; the standard curve equation is obtained as follows: a series of N2 standard solutions of known concentrations are used for detection, and the fluorescence values ​​corresponding to each concentration are measured to obtain the standard curve equation between N2 concentration and fluorescence value. In the method described above, the concentration of N1 in the reaction system is 2. M–200 mM. Specifically, N1 can be added to the reaction system in the form of a mother liquor. When preparing the mother liquor, water, DMSO, or other organic solvents can be used for dissolution. The preferred final concentration in the reaction system is 2%. M–200 M is set within the linear range of N2 concentration and corresponding fluorescence value.

[0026] The pH of the buffer solution in the reaction system of the method is 3.0–14.0, and the pH of the buffer solution is preferably 6.5–9.5. There are no specific requirements for the type of buffer solution.

[0027] The reaction system to be tested in the method can be carried out in an aqueous phase, an organic phase, or a heterogeneous system.

[0028] The reaction time in the method is 0.5–24 h.

[0029] This invention protects the application of this system in two instruments that can detect fluorescence changes: microplate reader and fluorescence activated microdroplet sorting instrument (FADS system).

[0030] This invention also protects the application of any of the methods described in enzyme activity detection or screening of highly active enzymes; the enzyme is an enzyme capable of catalyzing the generation of the corresponding fluorescent molecule N2 or consuming N2. When the system is applied in enzyme activity detection or screening of highly active enzymes, if the enzyme to be tested is an enzyme capable of catalyzing the generation of N2, the enzyme to be tested is used to carry out the catalytic reaction, and the generation of N2 in the reaction system is detected using the method described above. If the content of generated N2 is higher, the fluorescence signal is stronger, and the activity of the enzyme to be tested is higher. When the system is applied in enzyme activity detection or screening of highly active enzymes, if the enzyme to be tested is an enzyme that consumes N2, the enzyme to be tested is used to carry out the catalytic reaction, and the consumption of N2 in the reaction system is detected using the method described above. If the content of ketone compounds is higher, the fluorescence signal is weaker, and the activity of the enzyme to be tested is higher.

[0031] This invention also protects the application of any of the methods described in enzyme-directed evolution screening; the enzyme is an enzyme capable of catalyzing the generation of the corresponding fluorescent molecule N2 or consuming N2. When this system is applied in enzyme-directed evolution screening, after enzyme engineering modification of natural enzymes, high-throughput screening is performed using this system to obtain superior mutants with significantly enhanced activity.

[0032] In any of the applications, methods, or kits described above, the ketone compounds include aliphatic ketones, aromatic ketones, hydroxyl ketones, and heterocyclic ketones, while the amine compounds include aliphatic amines, aromatic amines, hydroxylamines, and heterocyclic amines. Attached Figure Description

[0033] Figure 1 The 1H NMR spectrum of compound N2 prepared in Example 1.

[0034] Figure 2 The carbon NMR spectrum of compound N2 prepared in Example 1.

[0035] Figure 3 The 1H NMR spectrum of compound N1 prepared in Example 1.

[0036] Figure 4 Carbon NMR spectrum of compound N1 prepared in Example 1.

[0037] Figure 5 Normalized spectra of the absorption and fluorescence emission spectra of compounds N2 and N1 in 10 mM pH 8.5 PB solution in Example 2.

[0038] Figure 6 The standard curves for detecting fluorescence intensity at different concentrations of N2 are shown.

[0039] Figure 7 The figure shows the results of enzyme activity identification based on sodium pyruvate and N1.

[0040] Figure 8 This is a graph showing the corresponding activity results of the laboratory transaminase library and substrate library.

[0041] Figure 9 High-throughput evolution of fluorescence-activated microdroplet sorting At The process of TA.

[0042] Figure 10 To validate the production of mutants by HPLC ( R The result graph of )-BPA. Detailed Implementation

[0043] The following embodiments will illustrate the present invention in detail, but are not intended to limit the present invention. Any improvements or substitutions based on the basic content of these embodiments shall still fall within the scope of protection of the claims of the present invention.

[0044] Example 1: Synthesis of fluorescent molecules N2 and N1.

[0045] Includes the following steps: (1) Synthesis of 6-acetyl-2-naphthol (compound 2 for short): Add 3 g of 6-methoxy-2-acetylnaphthalene and 500 mL of concentrated HCl to a 1 L round-bottom flask, add a condenser, and cover the condenser with a balloon. Incubate at 90 °C for 2-3 h. Monitor the reaction of the starting material by TLC (PET:EA = 1:1) to ensure complete reaction. Filter while hot to remove black impurities, then cool to room temperature, followed by cooling in an ice-water bath. Filter again, wash with water until the filtrate is neutral, and then dry the solid along with the funnel (direct transfer to a petri dish would be very sticky), obtaining a white solid.

[0046] (2) Synthesis of N–(6–acetylnaphthyl–2–yl)–2–hydroxy–2–methylpropionamide (referred to as compound 3): Weigh 1 g of compound 2 and 840 mg of NaOH into a 100 mL round-bottom flask, add 15 mL of DMA, and stir at room temperature (25-30℃) for 1 h. The solution color darkens. Add 1.5 g of bromoisobutyramide and react at room temperature (25-30℃) overnight. The rearrangement product is directly generated. Extract with DCM (3 x 50 mL) and 150 mL of water. Remove the DCM by rotary evaporation to obtain a crude product solution (dark brown) containing DMA.

[0047] (3) Synthesis of 2-acetyl-6-aminonaphthalene (referred to as compound 4): Add 37.5 mL of anhydrous ethanol and 37.5 mL of con.HCl to the crude DMA product solution, incubate overnight at 90°C. After the reaction is complete, monitor the reaction by TLC (PET:EA = 1:1). Add saturated NaOH dropwise with stirring until pH > 10. Remove the ethanol by evaporation, filter, and retain the residue. Extract the filtrate with EA, dry with saturated NaCl and anhydrous Na₂SO₄. After drying, purify the residue and organic phase together by silica gel column chromatography (PET:THF = 1.5:1) to obtain a yellow solid. Pulping with chloroform yields a yellow powder, compound 4.

[0048] (4) Synthesis of (6-acetylnaphthyl-2-yl)glycine (compound N2): 186 mg of compound 4, 167 mg of ethyl bromoacetate, and 82.03 mg of AcONa were placed in a 25 mL double-necked flask, evacuated, and protected with nitrogen. 10 mL of anhydrous ethanol was added, and the mixture was refluxed. After reacting overnight, the mixture was cooled to room temperature and evaporated to dryness. After evaporation, 15 mL of 2N NaOH solution was added under nitrogen protection, and the mixture was refluxed for 2 h. After the reaction was complete, the mixture was cooled to room temperature, the pH was adjusted to 3 with 2N hydrochloric acid, filtered, and washed with the filtrate. The solid was dried and recrystallized from ethanol to give compound N2. Its 1H NMR data (see [link to flask description]). Figure 1 )for: 1 H NMR (500 MHz, DMSO- d 6) δ 12.75 (s, 1H), 8.39 (d, J = 1.4 Hz,1H), 7.83 – 7.78 (m, 2H), 7.60 (d, J = 8.7 Hz, 1H), 7.13 (dd, J = 8.9, 2.3Hz, 1H), 6.70 (d, J = 2.1 Hz, 1H), 3.96 (s, 2H), 3.39 (s, 1H), 2.60 (s, 3H). Its carbon NMR data (see Figure 2 )for: 13 C NMR (125 MHz, DMSO- d 6) δ 196.99, 172.12, 148.60,137.62, 130.46, 129.92, 125.52, 125.18, 124.07, 118.80, 102.36, 44.37, 26.39.HRMS (ESI-ion trap) m / z: Calculated for [C 14 H14 NO3] + : 244.0968, found 244.0967.

[0049] (5) Synthesis of (6-(1-aminoethyl)naphth-2-yl)glycine (compound N1 for short): 243 mg of compound N2, 310 mg of NH4OAc, and 26 mg of NaBH3CN were weighed and added to a 25 mL double-necked round-bottom flask under nitrogen protection. Then, 15 mL of anhydrous methanol was added, and the mixture was reacted overnight under reflux. After the reaction was complete, the mixture was evaporated to dryness and purified by silica gel column chromatography (CHCl3: MeOH = 10:1, 0.1% triethylamine) to obtain compound N1. Its 1H NMR data (see...) Figure 3 )for: 1 HNMR (500 MHz, Methanol- d 4) δ 7.59 – 7.54 (m, 3H), 7.33 (dd, J = 8.6, 1.8 Hz, 1H), 6.97 (dd, J = 8.8, 2.4 Hz, 1H), 6.72 (d, J = 2.4 Hz, 1H), 4.10:(q, J =6.7 Hz, 1H), 3.73 (s, 2H), 1.44 (d, J = 6.7 Hz, 3H). Its carbon NMR data (see Figure 4 )for: 13 C NMR (125 MHz, Methanol- d 4) δ HRMS (ESI-ion trap) m / z: Calculated for [C 14 H 14 NO2] + : 228.1019, found 228.1015.

[0050] Example 2: Measurement of absorption and fluorescence emission spectra in 10 mM pH 8.5 PB solution.

[0051] The prepared fluorescent molecules N2 and N1 were dissolved in a 10 mM pH 8.5 PB solution to prepare a 1 mM stock solution. Their absorption and emission spectra were measured and normalized. Figure 5 As shown, in PB solution, the maximum excitation of fluorescent molecule N1 is at 350 nm and the fluorescence emission is at 418 nm, while the maximum excitation of fluorescent molecule N2 is at 404 nm and the fluorescence emission is at 508 nm.

[0052] The high-throughput detection method for ketones provided by this invention can react with ketone compounds to generate detectable fluorescent substances. The following examples will illustrate this invention in detail, but are not intended to limit the invention. Any improvements or substitutions based on the basic content of these examples still fall within the scope of protection of the claims of this invention.

[0053] In the following examples, the hydrophilic bifunctional probe (6–(1–aminoethyl)naphthyl-2–yl)glycine (N1) and its corresponding fluorescent molecule (6–acetylnaphthyl-2–yl)glycine (N2) used for screening were both synthesized within the group.

[0054] In the following examples, unless otherwise specified, the strains, plasmids, reagents, materials, gene synthesis, and gene sequencing used are all commercially available.

[0055] Example 3: Detection of the activity of different transaminases on pyruvate based on an enzyme-linked immunosorbent assay (ELISA) reader.

[0056] First, a certain amount of N1 and N2 were weighed using a precision balance, and a 100 mM stock solution was prepared using 10 mM pH 8.50 PB solution. This stock solution was then stored at -20 °C. N2 was diluted to 1, 2, 5, 10, 20, 50, 100, 200, and 500 μM. A linear relationship between fluorescence intensity and concentration was observed in the range of 1–200 μM. (See figure). Figure 6 .

[0057] In this embodiment, a hydrophilic bifunctional probe N1 is used as the ammonia source and pyruvate as the substrate. After amination by transaminase, the corresponding fluorescent molecule N2 and the corresponding product D / L-alanine are generated, which are used to detect the activity of different transaminases for pyruvate. Due to the reversibility of transaminase, the fluorescence of N1 is detected, which is used to detect the activity of different amine sources. The chemical equation is as follows: .

[0058] The reaction system consisted of 100 μL of 10 mM pH 8.50 PB solution, to which 10 μL of purified enzyme, 0.2 mM NM1, 0.1 mM PLP, and 1 mM pyruvate were added. This mixture could be prepared centrally and aliquoted into 96-well plates. The purified enzyme concentration was 0.1 mg / mL. The reaction was monitored over 30 minutes at 37 °C using a microplate reader, with the plate shaken for 15 seconds every 1 minute and the result measured once. The excitation wavelength was 404 nm, and the emission wavelength was 508 nm. For N1 detection, the excitation wavelength was 350 nm, and the emission wavelength was 418 nm.

[0059] In this embodiment, five R-type and five S-type transaminases were selected from the laboratory transaminase library to detect the difference in their activity with pyruvate. The results are shown in […]. Figure 7 These enzymes are R1 (from Aspergillus terreus NIH2624, NCBI: XP_001209325.1), R2 (from Phialemonium atrogriseum, NCBI: XP_060279526.1), R3 (from Mycolicibaterium wolinskyi, NCBI: WP_067853383.1), R4 (from Shinella lacus, NCBI: WP_256114772.1), R5 (from Exophiala xenobiotica, NCBI: XP_013320890.1), S1 (from Brucella pseudintermedia, NCBI: WP_011982390.1), S2 (from Chromobacterium violaceum, NCBI: WP_152637556.1), and S3 (from Thermomicrobia). S4 (from Thermomicrobia bacterium, NCBI:MCA1723115.1), S5 (from Sphaerobacter thermophilus, NCBI:WP_012872904.1). As shown in the figure, this invention has universal applicability to a variety of transaminases and can be used for activity detection.

[0060] Example 4: Detection of corresponding activities between laboratory substrate libraries and transaminase libraries based on ELISA reader.

[0061] This embodiment uses a hydrophilic bifunctional probe N1 as the ammonia source and 31 compounds (1-1 to 1-31) from the substrate library as substrates. After amination by transaminase, the corresponding fluorescent molecule N2 and the corresponding product are generated, which are used to detect the activity of multiple enzymes in the laboratory transaminase library on the substrate library. The reaction system consists of 100 μL of 10 mM pH 8.50 PB solution, to which 10 μL of purified enzyme, 0.2 mM N1, 0.1 mM PLP, and 1 mM substrate are added. This solution can be prepared centrally and aliquoted into 96-well plates. The purified enzyme concentration is 0.1 mg / mL. The reaction process is monitored within half an hour at 37 °C using a microplate reader, with the plate shaken for 15 s every 1 min and the detection performed once. The excitation wavelength is 404 nm and the emission wavelength is 508 nm.

[0062] In this example, 19 transaminases were selected (RTA1-RTA5 correspond to R1-R5 in Example 3, STA1-STA5 correspond to S1-S5 in Example 3; RTA6 was from Luminiphilus syltensis, NCBI: WP_009018841; RTA7 was from Arthrobacter sp. KNK168, NCBI: 3WWH_A; STA6-STA12 were commercial transaminases ES-ATA-101~ES-ATA-107 purchased from EnzymeScience, Inc.), including 7 R-type transaminases and 12 S-type transaminases. A total of 31 carbonyl compounds were included in the substrate library (purchased from MCE Bioactive Compound Library HY-L001, selecting the first 31 carbonyl compounds). Using this screening method, a one-to-one activity relationship was obtained, and the results are shown in […]. Figure 8 As can be seen from the figure, this method has good universality and efficiency, and all samples can be screened in just 3 days.

[0063] Example 5: FADS-based ultra-high-throughput evolution of transaminases.

[0064] This embodiment still uses the high-throughput screening system, which is combined with FADS technology. Using picoliter droplets as microreactors, highly active transaminase mutants are screened out, which can be used for the directed evolution of transaminases and other enzymes that can convert N1 to N2.

[0065] In this embodiment, 1-benzyl-3-piperidinone (BPO) was used as the substrate, and its product ( R )-3-amino-1-benzylpiperidine (( R )-BPA is an important intermediate for drug molecules such as Linagliptin, Alogliptin, and Trelagliptin. R The precursor of 3-aminopiperidine has significant value in the pharmaceutical field, and the specific technical route is as follows: .

[0066] (1) Fabrication of microfluidic chips: The microfluidic chip is fabricated on a pre-designed silicon wafer using a soft material such as polydimethylsiloxane (PDMS) (using the commercially available SYLGARD 184Silicone Elastomer Kit). Subsequently, a glass slide and a perforated PDMS chip block (with openings and exits created using a 0.75 mm diameter punch) are placed in a Vacuum Plasma SystemCOVANCE (Femto Science) to remove surface organic contaminants and modify the surface. The two treated surfaces are then bonded together and placed at 60 °C for 4 hours to ensure a strong bond. Afterward, a modifier (5% trichloroisocyanurate added to Novec 7500 electronic fluorinated oil) is added. H , 1 H , 2 H , 2 H – Perfluorooctyl silane (hereinafter referred to as the modifier), treat for 3 min, then clean with Novec 7500 electronic fluorinated oil (3M). The electrodes are subsequently filled into the electrode channels by low-temperature solder melting.

[0067] (2) Establishment of the mutation library: The initiating enzyme for site-directed saturation mutagenesis comes from Aspergillus terreus NH2624 R Type I transaminase At TA, constructed on the pET-28a (+) plasmid (Tsingke Biotechnology Co., Ltd.), with the restriction enzyme site. Based on 10 pairs of NNK primers, full plasmid PCR was performed using PrimeSTAR Max Premix (2×) (TakaRa) according to the manufacturer's instructions. After adding DpnⅠ (TakaRa) and digesting at 37 °C for 1 h, the purified full plasmid PCR product was mixed in equal volumes and recombined using the ClonExpressII One Step Cloning Kit (Vazyme). After recombination, the product was purified using the DNA Clean & Concentrator Kit (Zymo Research) and then electroporated. E coli 10G of electrotransfer competent cells were added to 1 mL of antibiotic-free LB at 37°C, and then the whole cell was added to 15 mL of LB containing 0.1 mg / mL kanamycin. The cells were cultured overnight at 37°C, and the plasmid was extracted to form a site-specific saturated library.

[0068] The starting enzyme for the random mutant library was the single-point mutant M11 obtained from site-controlled saturation library screening. Primers were designed at the N and C ends to introduce two restriction sites, BamHI and HindIII, into the N and C ends of the original R128W plasmid, respectively, to facilitate subsequent experiments. Subsequently, an appropriate number of random mutations were introduced into the target sequence using eq-PCR (NEB Taq enzyme). The mutated target sequence was digested with BamHI and HindIII restriction endonucleases (Thermo Fisher Scientific) at 37 °C for 1 h. After gel recovery, the sequence was ligated using DNA Ligation Mix (2×) (TakaRa) at 16 °C for 1 h. After purification using a DNA Clean & Concentrator kit (Zymo Research), the sequence was electroporated. E coli In 10G electrotransfer competent cells, 1 mL of antibiotic-free LB was added and incubated at 37°C. The entire contents were then added to 15 mL of LB containing 0.1 mg / mL kanamycin. The cells were incubated overnight at 37°C, and plasmids were extracted to form the original random mutant library. (Subsequent microdroplet screening followed by plasmid recovery and PCR library construction followed the same method, except that the template changed from a single plasmid to multiple positive plasmids).

[0069] (3) Induced expression of the mutant library: Electroporation of the plasmid of the mutant library E coli In BL21 (DE3) electrocompetent cells, 1 mL of antibiotic-free LB was added and incubated at 37°C. This was then added to 15 mL of LB containing 0.1 mg / mL kanamycin, and the cells were incubated overnight at 37°C to obtain the seed culture. Subsequently, the seed culture was inoculated at a ratio of 2% into 15 mL of LB containing 0.1 mg / mL kanamycin, incubated at 37°C for 3 h, and then 0.1% 1 M IPTG solution was added. Induction was then performed at 16°C for 16 h.

[0070] (4) Generation of microdroplets: Take 1 mL of the induced mutant library culture, wash three times with 10 mM phosphate buffer (PB, pH 8.50), and then dilute to OD. 600 The concentration should be 0.2–0.3 (this condition maximizes the single-encapsulation of E. coli by the droplets). Prepare a 90 μL 10 mM PB reaction mixture (containing 1 mM N2, 0.2 mM PLP, 2 mM BPO, and 60% BugBuster protein extraction reagent) and transfer it to a 200 μL PCR tube.

[0071] Place the chip under a microscope system and adjust the objective lens to precisely focus on the interface between the oil and aqueous phases. Introduce the oil phase, open the oil phase pressure valve, and close the valve after the chip is completely filled with the oil phase. To prepare for droplet generation, introduce the above reaction system as one aqueous phase and the diluted bacterial solution as the other aqueous phase, and connect the outlet. Adjust the pressure to stably generate droplets with a diameter of approximately 30 μm (under the microscope, the two aqueous phases can be seen converging and forming a central streamline, which basically indicates that the two are mixed in a 1:1 ratio). Collect the droplets, observe them under a fluorescence microscope, and incubate them in a 37 ℃ incubator for 2 h.

[0072] (5) Sorting of microdroplets: Droplets are introduced into the detection / sorting device, and sorting is performed based on the emitted fluorescence intensity (405 nm–508 nm). Upon detection of a positive droplet, it is separated by electrode action. The upper aqueous phase is used as a template for PCR amplification using PrimeSTARMax Premix (2×). The PCR product is then subjected to restriction endonuclease B... amH I and H ind III. After enzyme digestion, the target fragment was recovered by agarose gel electrophoresis, cloned into the pET-28a (+) vector, transformed into E. coli 10G electrocompetent cells, and a plasmid library was obtained for the next round of screening.

[0073] In the Fluorescence Activated Droplet Sorting (FADS) process, the site-specific saturated mutant library was first screened. After one round of sorting and deep-well plate verification, the superior single-site mutant M11 was identified. Subsequently, the random mutant library of M11 was sorted three times. Combined with deep-well plate screening and mutant splitting and combination verification, the highly active two-site mutant M21 was finally identified.

[0074] The specific screening process is as follows: At TA-based fixed-point saturated library screening: Original At The TA site-directed saturated library was constructed using 10 primer pairs, with a theoretical library capacity of 6560 elements (including 1440 NNK elements and 5120 TT elements). Therefore, only one round of sorting was needed to meet the 5-fold coverage requirement. After preparing droplets and incubating them using the above method, fluorescence microscopy showed significant effects from the mutant library; therefore, the sorting threshold was increased to 1500. More than 10 elements were sorted within just 2 hours. 6 The selection rate was 2.04% (based on a 10% encapsulation rate), which is still at a relatively high level. Subsequently, the plasmids in the positive droplets were amplified by PCR using the method described above to reconstruct a new mutant library; after plating, some clones were used to prepare droplets for microdroplet sorting, while the remainder were screened using deep-well plates. The results show... Figure 9 A and 9B show that after site-directed saturation mutation, there is a significant increase in positive droplets, while Figure 9 The sorting data for C indicate that the sorting process enriched positive droplets.

[0075] M11 random mutation library screening: At After one round of screening of the TA site-specific saturated library, the superior single-site mutant R128W was obtained. Based on this mutant, a random mutant library was constructed by eq-PCR, with a library size of approximately 2.15 × 10⁻⁶. 5 Theoretically, a 5-fold library capacity coverage requires a capacity of millions of samples, which would be time-consuming and labor-intensive using traditional deep-well plate screening. However, using FADS technology, each round of sorting only requires 2–3 hours to meet the requirements. For the weakly enhanced four-point mutants obtained through screening, the negative effects of multiple random mutant combinations were analyzed and verified, revealing that M21 is a two-point mutant with overlapping dominant sites. From the results... Figure 9 As can be seen from D, the number of positive droplets increases with the end of each round of sorting; Figure 9 E indicates that the FADS sorting effect on the M11 random mutant library is significant, with an increase in the proportion of positive droplets above the threshold.

[0076] (6) 96-well plate verification: The mutant library plasmids screened by microdroplets were electroporated into Escherichia coli BL21 (DE3) electroporated competent cells, 1 mL of antibiotic-free LB medium was added, and the cells were revived and cultured at 37 ℃. Subsequently, the bacterial culture was diluted 10 times and spread on LB solid agar plates containing kanamycin (prepared in advance), and incubated overnight at 37 ℃.

[0077] Add 500 μL of LB medium containing 0.1 mg / mL kanamycin to each well of a deep-well plate using a multi-pipette. Single colonies are picked using sterile toothpicks and inoculated into the corresponding wells of the deep-well plate (with single colonies of the starting enzyme inoculated diagonally between A1–H8 as controls). Incubate overnight at 37 ℃ with shaking at 1000 rpm. The next day, transfer the culture to deep-well plates containing fresh LB medium at a 2% inoculation rate. Incubate at 37 ℃ with shaking at 1000 rpm for 3 h. Then, add 10 μL of 0.02 M IPTG to each well using a multi-pipette and induce expression at 16 ℃ with 1000 rpm for 16 h. After induction, centrifuge the entire deep-well plate, discard the supernatant, and collect the bacterial cells.

[0078] The bacterial cells were resuspended in 100 μL of 10 mM PB containing 30% BugBuster protein extraction reagent and lysed on ice for 15 min. Simultaneously, a reaction mixture was prepared: 100 μL of 10 mM PB containing 10 μL of crude enzyme solution, 0.2 mM N1, 0.1 mM PLP, and 1 mMBPO. After lysis, the reaction mixture was placed in a Synergy H1 microplate reader (Bio-Tek Instruments, Inc.) and detected at 37°C for 30 min; the detection parameters were set to 404 nm excitation and 508 nm emission. Thus, excellent mutants M11 and M21 were identified in each round, and their specific activities were 45.58 U / g and 67.30 U / g, respectively, representing increases of 133 and 197 times compared to the starting enzyme.

[0079] (7) Liquid phase data determination: In this embodiment, D-alanine, commonly used in industrial production, was selected as the ammonia source, and conversion rate tests were conducted. At high substrate concentrations, M21 achieved a 70.03% conversion rate of BPO within 4 hours, which is significantly higher than... At TA increased by 22 times, its ee The value is also greater than 99%, see Figure 10 Therefore, the conversion rates of M21 and M11 are consistent with those obtained by fluorescence assay. These results demonstrate that the FADS high-throughput evolution method based on this invention is suitable for the directed evolution of transaminases.

[0080] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A hydrophilic bifunctional probe N1, characterized in that: The chemical name of this probe is (6–(1–aminoethyl)naphthyl-2–yl)glycine, and its molecular structure is as follows: 。 2. A fluorescent molecule N2 corresponding to claim 1, characterized in that: The fluorescent molecule, chemically named (6-acetylnaphthyl-2-yl)glycine, is a product of the hydrophilic bifunctional probe N1 after catalysis, transformation, and transamination, or a precursor for the synthesis of N1, and has the following structure: 。 3. The method for synthesizing probe N1 and fluorescent molecule N2 according to claims 1 and 2, characterized in that: The synthesis route is as follows: ； Compound 1 is 6-methoxy-2-acetylnaphthalene, compound 2 is 6-acetyl-2-naphthol, compound 3 is N-(6-acetylnaphthalene-2-yl)-2-hydroxy-2-methylpropionamide, compound 4 is 2-acetyl-6-aminonaphthalene, compound N2 is (6-acetylnaphthalene-2-yl)glycine, and compound N1 is (6-(1-aminoethyl)naphthalene-2-yl)glycine; The specific synthesis steps are as follows: (1) Synthesis of compound 2: Compound 1 and concentrated hydrochloric acid were added to a round-bottom flask and reacted at 90 °C. After the reactants had completely reacted, the mixture was filtered while hot and cooled in an ice-water bath. After filtration and drying, compound 2 was obtained. (2) Synthesis of compound 3: Compound 2, sodium hydroxide and N,N-dimethylacetamide were added to a flask and stirred at room temperature. Then bromoisobutyramide was added and reacted overnight at room temperature. The mixture was extracted with dichloromethane and water, dried over anhydrous magnesium sulfate, and evaporated to dryness to obtain a solution of compound 3 containing N,N-dimethylacetamide. (3) Synthesis of compound 4: Anhydrous ethanol and concentrated hydrochloric acid were added to an N,N-dimethylacetamide solution containing compound 3, and the mixture was incubated overnight at 90 °C. Saturated sodium hydroxide was added dropwise until the pH was > 10. The ethanol was removed by evaporation, and the mixture was filtered, retaining the residue. The filtrate was extracted with ethyl acetate, dried with saturated brine and anhydrous magnesium sulfate. After drying, the residue and organic phase were separated by silica gel column chromatography to obtain a yellow solid. The solid was then slurried with chloroform to obtain a yellow powder, which is compound 4. (4) Synthesis of compound N2: Compound 4, ethyl bromoacetate and sodium acetate were added to a double-necked flask. Anhydrous ethanol was added under nitrogen protection and the mixture was refluxed. The reaction was allowed to proceed overnight. After cooling to room temperature, the mixture was dried by rotary evaporation. 2N sodium hydroxide solution was added under nitrogen protection and the mixture was refluxed. After the reaction was completed, the mixture was cooled to room temperature and the pH was adjusted to 3 with 2N hydrochloric acid. The mixture was filtered, washed with the filtrate, dried, and recrystallized from ethanol to obtain compound N2. (5) Synthesis of compound N1: Compound N2, ammonium acetate and sodium cyanoborohydride were added to a double-necked round-bottom flask, anhydrous methanol was added under nitrogen, and the reaction was carried out overnight under reflux. After the reaction was completed, water was added to quench the reaction, the mixture was dried by rotary evaporation, and compound N1 was obtained by silica gel column chromatography.

4. The application of the hydrophilic bifunctional probe N1 and the corresponding fluorescent molecule N2 as described in claims 1 and 2 in a fluorescence-based high-throughput screening system, characterized in that, The hydrophilic bifunctional probe and its corresponding fluorescent molecules are compound N1 ((6–(1–aminoethyl)naphthyl-2–yl)glycine) and compound N2 ((6–acetylnaphthyl-2–yl)glycine). Compound N1 is mainly indigo blue, while compound N2, when converted or used directly, is green fluorescent and has a Stokes shift of 104 nm.

5. The application according to claim 4, characterized in that, The application of N1 as an amino group donor in biological systems that convert amino groups to carbonyl groups in fluorescence-based enzyme screening or evolution-based high-throughput screening systems.

6. The application according to claim 4, characterized in that, When using the hydrophilic bifunctional probe N1 as a reaction substrate, it is used for: (a1) Detection of ketone compound content; (a2) Detection of enzyme activity; (a3) Screening of highly active enzymes; (a4) Directed evolutionary screening of enzymes; The enzyme catalyzes the generation of the corresponding fluorescent molecule N2 or the consumption of N1. When using fluorescent molecule N2 as a reaction substrate, it is used for: (a1) Detection of amine compound content; (a2) Detection of enzyme activity; (a3) Screening of highly active enzymes; (a4) Directed evolutionary screening of enzymes; (a5) Biofluorescence imaging; The enzyme catalyzes the generation of probe N1 or an enzyme that consumes N2; 。 7. The application according to claim 5, characterized in that: Using N1 as an amino donor, this method is used for the detection of ketone compounds or enzyme activity. The steps include: adding N1 and the sample to be tested to a buffer solution, mixing, and reacting at room temperature; comparing the fluorescence values ​​of the test system and the control system under excitation light at 404 nm and emission light at 508 nm; the higher the fluorescence value of the test system compared to the control system, the higher the content of ketone compounds in the sample or the more ketone compounds are consumed. The control system is a reaction system in which no test sample is added.

8. The application according to claim 7, characterized in that: The concentration of N1 in the reaction system is 20. M–200 mM; The pH of the buffer solution in the reaction system is 3.0–14.0; The reaction time is 0.5–24 h; The method is used in two instruments that can detect fluorescence changes: microplate reader and fluorescence activated microdroplet sorting system (FADS system).

9. The application according to any one of claims 6-7, characterized in that, The enzyme described is an enzyme that catalyzes the generation of the corresponding fluorescent molecule N2 or consumes N1, and is used in enzyme activity detection or enzyme directed evolution screening.

10. The application according to any one of claims 6-7, characterized in that: The ketones include aliphatic ketones, aromatic ketones, hydroxy ketones, and heterocyclic ketones; the amines include aliphatic amines, aromatic amines, hydroxylamines, and heterocyclic amines.

Images