Application of monoamine oxidase as catalyst in preparation of quinoline nitrogen heterocyclic compounds

Abstract

The invention provides application of monoamine oxidase serving as a catalyst in preparation of quinoline nitrogen heterocyclic compounds. The monoamine oxidase is derived from proteus, pseudomonas, saccharospirillum, marine bacteria RHCI1, photophilic bacteria, vibrio Vibrio sp.JCM 19236 or pseudomonas monteilii. The monoamine oxidase is mild in reaction condition, only recombinant VsMAO whole cells from Vibrio sp.JCM 19236 are used as a biocatalyst, air is used as a molecular oxygen source, pure water is used as a solvent, no toxic reagent is used, the yield of a substrate in a substrate spectrum can reach 99% or above at most, and when VsMAO is used for preparation scale reaction, the VsMAO still shows a good catalytic effect, so that the monoamine oxidase has good application prospects. And an efficient and sustainable way is provided for biological manufacturing of quinoline.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to the application of monoamine oxidase as a catalyst in the preparation of quinoline nitrogen heterocyclic compounds. Background Art

[0002] Aromatic nitrogen heterocycles usually have a wide range of physiological and pharmacological activities. Quinoline is a special nitrogen heterocycle scaffold. Quinoline and its derivatives have a wide range of applications in many fields, including: (1) Pharmaceutical field: Quinoline and its derivatives are mainly used in the manufacture of three major types of drugs: nicotinic acid, 8-hydroxyquinoline and quinine. Nicotinic acid drugs include nicotinamide, cardiotonic agents, stimulants and drugs for treating tapeworms; 8-hydroxyquinoline can be used to manufacture drugs for treating amoebiasis and wound disinfectants, as well as antifungal agents and textile auxiliaries; Quinine, also known as cinchona alkaloids, is a common alkaloid that was first widely used in the treatment of malaria. (2) Pesticide field: The discovery of agricultural chemicals based on quinoline scaffold structure has made great progress. Some new quinoline pesticides have been commercialized or are under development, such as oxine-copper, tebufloquin, quinoxaline, pyrroloquinone, ipflufenoquin, quinoline and quinclorac. These compounds show significant biological activity in bactericidal, insecticidal, acaricidal, anti-plant virus and weed control. (3) Quinoline compounds can also be used to prepare dyes and photosensitive materials, rubber and solvents, and chemical reagents. In summary, quinoline and its derivatives have a wide range of applications in many fields such as medicine, pesticides, dyes, rubber, solvents and chemical reagents.

[0003] Quinoline structural motifs are easily obtained by chemical synthesis. However, most of these traditional synthesis methods have harsh conditions, such as the consumption of strong acidic solutions and toxic reagents, high temperature, and the generation of a large number of by-products, which may cause serious environmental problems and do not meet the development needs of green chemistry. Biocatalysis has gradually become an attractive synthetic tool in modern organic synthesis due to its advantages such as mild conditions, high efficiency, high selectivity and sustainability. The catalytic process is green and pollution-free, meeting the requirements of large-scale production. Among them, monoamine oxidase (MAO) can efficiently realize the oxidative aromatization of tetrahydroquinoline to generate quinoline with the assistance of cofactor flavin adenine dinucleotide (FAD) and oxygen. It stands out in the synthetic route of quinoline. Its simple process and high catalytic efficiency have attracted widespread attention from researchers. For example, 7-chloroquinoline is an important intermediate in the synthesis of chloroquine (a drug for the treatment of malaria). It can be generated in one step by the oxidative aromatization of 7-chloro-1,2,3,4-tetrahydroquinoline catalyzed by monoamine oxidase. The synthetic process catalyzed by monoamine oxidase has high sustainability and atomic utilization.

[0004] However, only a few biocatalytic methods have been reported for the synthesis of quinoline. Although these methods have promoted the development of biocatalytic synthesis of quinoline compounds, there are still problems such as relatively low substrate concentration and conversion rate, which will increase the actual production cost and are far from sufficient for the effective preparation of quinoline compounds. Therefore, it is an urgent problem to screen monoamine oxidases with high catalytic activity that can meet the requirements of industrial biocatalysis, and on this basis, to construct a green production process for quinoline products. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides the use of monoamine oxidase as a catalyst in the preparation of quinoline nitrogen heterocyclic compounds.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first object of the present invention is to provide the use of monoamine oxidase as a catalyst in the preparation of quinoline nitrogen heterocyclic compounds, wherein the monoamine oxidase is derived from Gammaproteobacteria bacterium, Pseudomonas sp., Saccharospirillum sp., Thalassomonassp. RHCI1, Luminiphilus sp., Vibrio sp. JCM 19236 or Pseudomonas monteilii ZMU-T01.

[0008] Preferably, the monoamine oxidase is derived from Vibrio sp. JCM 19236.

[0009] Preferably, the amino acid sequence of the monoamine oxidase is SEQ ID NO.1 in the sequence list.

[0010] Preferably, the gene sequence encoding the monoamine oxidase is SEQ ID NO.2 in the sequence list.

[0011] The second object of the present invention is to provide a method for preparing quinoline nitrogen heterocyclic compounds, using the THQs compound of formula (I) as a substrate and the above-mentioned monoamine oxidase as a catalyst to carry out an oxidative aromatization reaction in a buffer solution to prepare quinolines nitrogen heterocyclic compounds of formula (I);

[0012] Formula (I)

[0014] In formula (I), R1, R2, R3, R4, R5, R6, R7 are independently selected from hydrogen, methyl, hydroxy, methoxy, fluorine, chlorine or bromine;

[0015] Preferably, in formula (I), one or two of R1, R2, R3, R4, R5, R6, R7 are selected from methyl, hydroxy, methoxy, fluorine, chlorine or bromine.

[0016] Preferably, the THQs compound in formula (I) is selected from 2-methyl-1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroquinoline, 6-fluoro-1,2,3,4-tetrahydroquinoline, 6-chloro-1,2,3,4-tetrahydroquinoline, 6-bromo-1,2,3,4-tetrahydroquinoline, 6-methoxy-1,2,3,4-tetrahydroquinoline, 6-hydroxy-1,2,3,4-tetrahydroquinoline, 3-methyl-1,2,3,4-tetrahydroquinoline, 4-methyl-1,2,3,4-tetrahydroquinoline, 5-methyl-1,2,3,4-tetrahydroquinoline, 6-methyl-1, 2,3,4-tetrahydroquinoline, 7-methyl-1,2,3,4-tetrahydroquinoline, 8-methyl-1,2,3,4-tetrahydroquinoline, 7-methoxy-1,2,3,4-tetrahydroquinoline, 7-bromo-1,2,3,4-tetrahydroquinoline, 7-chloro-1,2,3,4-tetrahydroquinoline, 4-hydroxy-1,2,3,4-tetrahydroquinoline, 8-hydroxy-1,2,3,4-tetrahydroquinoline, 2-methyl-6-fluoro-1,2,3,4-tetrahydroquinoline, 2-methyl-6-bromo-1,2,3,4-tetrahydroquinoline or 4-methyl-6-bromo-1,2,3,4-tetrahydroquinoline.

[0017] Preferably, the catalyst comprises wet monoamine oxidase bacteria, freeze-dried Escherichia coli cells containing monoamine oxidase, and crude enzyme solution of monoamine oxidase;

[0018] The monoamine oxidase wet bacterial cell is a recombinant strain obtained by connecting the monoamine oxidase gene sequence to a vector to obtain a recombinant expression vector, and then introducing it into a host bacterium for inducing expression, wherein the host bacterium includes Escherichia coli;

[0019] The freeze-dried Escherichia coli cells containing monoamine oxidase are obtained by freeze-drying the wet cells of monoamine oxidase;

[0020] The crude enzyme solution of monoamine oxidase is obtained by crushing the wet bacterial cells of monoamine oxidase, centrifuging and taking the supernatant;

[0021] Preferably, the catalyst is freeze-dried Escherichia coli cells containing monoamine oxidase.

[0022] Preferably, the concentration of the substrate is 5-50 mM; and / or

[0023] The amount of the catalyst is 4-40 g / L; and / or

[0024] During the reaction, the reaction temperature is 20-40°C; and / or

[0025] During the reaction, the reaction time is 3h-7d; and / or

[0026] The pH value of the buffer solution is 7.0-9.0.

[0027] Preferably, the concentration of the substrate is 10-50 mM; most preferably, the concentration of the substrate is 50 mM; and / or

[0028] The amount of the catalyst is 16 g / L; and / or

[0029] During the reaction, the reaction temperature is 30-40°C; preferably the reaction temperature is 30°C; and / or

[0030] During the reaction, the reaction time is 3h or 7d; and / or

[0031] The buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution with a pH of 7.0-8.0 or a glycine-sodium hydroxide buffer solution with a pH of 8.0-9.0; preferably, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution with a pH of 7.5.

[0032] Preferably, during the reaction, no additional cofactor flavin adenine dinucleotide (FAD) and / or hydrogen peroxide is added.

[0033] In the present invention, the FAD required for the reaction is inherent in the monoamine oxidase structure of the present invention, and no additional addition is required; during the reaction, the reaction is carried out by relying on the dissolved oxygen in the reaction buffer system and the air in the blank part of the reaction container, and no additional addition of hydrogen peroxide is required; a control reaction was performed at the end of the reaction condition optimization for the reaction with the addition of catalase solution, and the results showed that it had little effect on the yield improvement.

[0034] The beneficial effects of the technical solution of the present invention are as follows:

[0035] (1) The optimum temperature of the monoamine oxidase of the present invention is 30°C, and the reaction conditions are mild.

[0036] (2) After incubation in phosphate at pH = 10.0 for 48 hours, the monoamine oxidase of the present invention can still maintain about 50% of its activity, indicating that the monoamine oxidase of the present invention has good alkaline resistance.

[0037] (3) The monoamine oxidase substrate spectrum of the present invention is relatively broad.

[0038] (4) The monoamine oxidase of the present invention has the highest activity towards the substrate 6-fluoro-1,2,3,4-tetrahydroquinoline, and can achieve complete conversion within 3 hours.

[0039] (5) The monoamine oxidase of the present invention realizes the large-scale preparation of 6-fluoroquinoline at a substrate concentration of 10 mM, wherein 0.756 g of substrate 6-fluoro-1,2,3,4-tetrahydroquinoline is fed, the liquid phase yield is 82%, and after simple separation and purification, the separation yield is 86%, and 0.519 g of product can be finally obtained.

[0040] (6) When the monoamine oxidase of the present invention catalyzes the conversion of the substrate 6-fluoro-1,2,3,4-tetrahydroquinoline, the substrate concentration that can be tolerated is 50 mM, which is much higher than the 5 mM reported in the literature (Green Chem., 2023, 25, 5296-5303). This provides an efficient and sustainable approach for the biomanufacturing of quinoline.

[0041] (7) The present invention only uses recombinant VsMAO whole cells derived from Vibrio sp. JCM19236 as a biocatalyst, air as a molecular oxygen source, and pure water as a solvent. No toxic reagents are used, and no additional cofactor flavin adenine dinucleotide (FAD) and oxygen are required. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0043] Figure 1 This is the HPLC detection result of the monoamine oxidase VsMAO in Example 1 of the present invention in catalyzing the oxidation of the substrate 2-methyl-1,2,3,4-tetrahydroquinoline.

[0044] Figure 2 The present invention shows the optimal pH and optimal buffer solution system experimental results when the monoamine oxidase VsMAO of the present invention catalyzes the oxidative aromatization of the substrate 2-methyl-1,2,3,4-tetrahydroquinoline.

[0045] Figure 3 The optimum temperature experimental result of the monoamine oxidase VsMAO of the present invention when catalyzing the oxidative aromatization of the substrate 2-methyl-1,2,3,4-tetrahydroquinoline.

[0046] Figure 4 The figures are the thermal stability test results of the monoamine oxidase VsMAO of the present invention at different temperatures.

[0047] Figure 5 The results are the pH stability test results of the monoamine oxidase VsMAO of the present invention at the optimum temperature.

[0048] Figure 6 This is the HPLC detection result of the monoamine oxidase VsMAO catalytic conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline in Example 8 of the present invention.

[0049] Figure 7 The figure is the nuclear magnetic resonance result of the monoamine oxidase VsMAO catalyzing the conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline.

[0050] Figure 8 The figure is a process curve diagram of the monoamine oxidase VsMAO catalyzing the conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline with a substrate concentration of 50 mM.

[0051] Fig. 9 This is the high performance liquid chromatography detection result of the monoamine oxidase VsMAO catalyzing the conversion of 6-methoxy-1,2,3,4-tetrahydroquinoline in Example 14 of the present invention.

[0052] Fig.10 This is the high performance liquid chromatography detection result of the monoamine oxidase VsMAO catalyzing the conversion of 5-methyl-1,2,3,4-tetrahydroquinoline in Example 18 of the present invention.

[0053] Fig.11 This is the high performance liquid chromatography detection result of the monoamine oxidase VsMAO catalyzing the conversion of 8-hydroxy-1,2,3,4-tetrahydroquinoline in Example 26 of the present invention. DETAILED DESCRIPTION

[0054] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0055] In the present invention, the formula of LB culture medium is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and if it is solid LB culture medium, 17 g / L agar is added.

[0056] The formula of TB medium is 12 g / L tryptone, 24 g / L yeast extract, 17 mM potassium dihydrogen phosphate, 72 mM potassium hydrogen phosphate, and 4 g / L glycerol.

[0057] Example 1 Construction of monoamine oxidase genetically engineered bacteria

[0058] (1) According to literature reports, monoamine oxidase MAO5 from Pseudomonas monteilii ZMU-T01 produces quinoline as a byproduct in the dynamic kinetic resolution reaction of chiral 2-substituted-1,2,3,4-tetrahydroquinoline (Deng Guozhong, et al. Deracemization of Phenyl-Substituted 2-Methyl-1,2,3,4-Tetrahydroquinolines by a Recombinant Monoamine Oxidase from Pseudomonasmonteilii ZMU-T01[J]. ChemCatChem, 2018, 10(11): 2374-2377.), so its amino acid sequence was used as a template for homologous sequence search and a "protein" to "protein" blast screening was performed in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). In order to increase the probability of soluble expression of the candidate enzyme in Escherichia coli, only the amino acid sequence from "bacteria" was retained. In order to reduce the workload of experimental screening, firstly, sequences with identity greater than 80% and less than 50% with the template sequence were eliminated; then only one candidate enzyme from the same bacterial species was retained; multiple sequence alignment was performed between the candidate enzyme and the template sequence, and sequences that were too long or too short compared with the template were removed. Cluster X was used to perform multiple sequence alignment between the template and the remaining candidate enzyme sequences, and MEGA constructed a phylogenetic tree. After the evolutionary relationship and source analysis, solubility prediction and other steps, 23 sequences were selected to reconstruct the phylogenetic tree, and finally 6 sequences were randomly selected for heterologous expression. At the same time, MAO5 described in the literature was heterologously expressed (as shown in Table 1).

[0059] Table 1 Monoamine oxidase from seven different sources

[0060] Protein name source NCBI accession number / serial number G JZ Gammaproteobacteria MCL5043555.1 PsMAO Pseudomonas sp HCW97116.1 S m Saccharospirillum sp. MCH8530221.1 TZ Marine bacteria genus (Thalassomonas sp.) WP_281556337.1 LqCy Luminiphilus sp. MDB4491165.1 VxD Vibrio sp. JCM 19236 GAM70821.1 MAO5 Pseudomonas monteri WP_013972159.1

[0061] (2) Obtain and synthesize each target gene according to the above NCBI accession number / serial number (can be directly synthesized by a biological company), insert the above target gene into the plasmid vector pET-28a(+) through the NcoI and XhoI restriction sites to construct a recombinant expression plasmid, and transform the vector pET-28a(+) into E. coli BL21(DE3) to prepare E. coli BL21(DE3) / pET-28a(+). The specific steps are as follows: take a sterilized 2mL EP tube, add 50μL of competent cells E. coli BL21(DE3) melted in an ice bath and 1-5μL of recombinant plasmid, mix gently, and let stand in an ice bath for 30 minutes. Then heat shock in a 42℃ water bath for 45-60s, and then quickly transfer the EP tube to an ice bath for 2 minutes. Do not shake the EP tube during this process. Add 500 μL of sterile LB medium (without antibiotics) to the EP tube, mix well and culture at 37°C, 200 rpm for 1 hour to allow the cells to recover. Pipette 50-200 μL of transformed competent cells and add them to LB agar medium (containing 50 μg / mL of kanamycin), and spread the cells evenly. Place the plate at 37°C until the liquid is absorbed, invert the plate, and culture at 37°C overnight. Pick a single colony from the coated plate and inoculate it in 10 mL of LB culture solution (containing kanamycin, final concentration 50 ug / mL), culture overnight at 37°C, 180 rpm for 12-16 hours to obtain recombinant engineered bacteria E. coli BL21 (DE3) / pET-28a (+), add glycerol with a final concentration of 30% volume fraction to the obtained engineered bacterial solution to prepare glycerol bacteria, and store at -80°C.

[0062] Example 2 Screening of monoamine oxidase

[0063] (1) Induced expression of monoamine oxidase: The recombinant engineered bacteria E. coli BL21 (DE3) / pET-28a (+) obtained in step (2) of Example 1 was inoculated into a liquid LB medium containing 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37 ° C overnight for 14 hours. Then, the inoculum concentration was inoculated into a liquid TB medium containing 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37 ° C for 2.5 hours. Then, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium to a final concentration of 0.2mM, and the expression was induced at 25 ° C for 20 hours to obtain a fermentation broth. The obtained fermentation broth was placed at 4 ° C and 8000 rpm for centrifugation for 5 minutes, and the precipitate was collected as bacterial cells to obtain wet bacterial cells (i.e., recombinant engineered bacteria containing monoamine oxidase genes).

[0064] (2) Preparation of freeze-dried Escherichia coli cell powder of recombinant engineered bacteria: The collected wet bacteria containing monoamine oxidase were washed and placed in a -20°C refrigerator for overnight freezing, and then placed in a freeze dryer for 24 hours to obtain freeze-dried Escherichia coli cell powder.

[0065] (3) Preparation of crude monoamine oxidase enzyme solution: The collected wet bacteria containing monoamine oxidase were resuspended in 50 mL of 50 mM phosphate buffer solution at pH 7.5, and crushed using a high-pressure homogenizer. The crushing conditions were 3 mL / s, 800 MPa, and 60 s. After the crushing was completed, the crushed liquid was placed at 4°C, 10,000 rpm, centrifuged for 20 min, and the supernatant was collected, which was the crude monoamine oxidase enzyme solution. The protein concentration was determined to be 1.0 mg / mL using the Coomassie Brilliant Blue method.

[0066] (4) Isolation and purification of monoamine oxidase: The target protein containing His6-tag was isolated and purified by Ni-NTA affinity chromatography. After equilibrating the Ni-NTA column with protein loading buffer solution, the filtered crude enzyme solution was loaded into the Ni-NTA column. Subsequently, gradient elution was performed using protein purification elution buffer solutions with imidazole concentrations of 30mM, 60mM, 300mM and 500mM, respectively. The eluate was desalted using a desalting column to remove excess imidazole, and the pure enzyme was obtained and stored at 4°C.

[0067] (5) The yield of the reaction system was determined using a high performance liquid chromatograph (HPLC): the total reaction volume was 5 mL, which contained the purified enzyme solution (2.0 mg / mL), 3.7 mg of 2-methyl-1,2,3,4-tetrahydroquinoline substrate (final concentration of 5.0 mM), and a phosphate buffer solution of pH 7.5 and 50 mM. The reaction system was placed in a shaker at 30°C and 200 rpm for 24 h. A pure enzyme solution prepared from empty Escherichia coli cells without the monoamine oxidase gene was used as a blank control reaction. The upper organic phase was extracted with three volumes of ethyl acetate, and dried with anhydrous magnesium sulfate. The product was concentrated by reduced pressure evaporation, and the conversion rate and yield were determined using high performance liquid chromatography.

[0068] (6) After testing, compared with the control experiment, the prepared monoamine oxidase GbMAO, PsMAO, SsMAO, TsMAO, LsMAO, VsMAO, and MAO5 enzyme solutions can catalyze the oxidative aromatization of 2-methyl-1,2,3,4-tetrahydroquinoline to produce quinoline, but different monoamine oxidases have different catalytic efficiencies (see Table 2). Among them, VsMAO obtained a conversion rate of 60% and a yield of 48%, showing the highest activity compared with other candidate enzymes and template enzyme MAO5, so VsMAO was selected for further research.

[0069] Table 2 Catalytic effects of different monoamine oxidases

[0070]

[0071]

[0072] The amino acid sequence of monoamine oxidase VsMAO is as follows (SEQ ID NO.1 in the sequence listing):

[0073]

[0074] The nucleotide sequence of monoamine oxidase VsMAO (codon optimized) is as follows (SEQ ID NO.2 in the sequence listing):

[0075] Example 3 Application of monoamine oxidase in the synthesis of 2-methylquinoline

[0076] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the conversion rate and yield.

[0077] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, a substrate 2-methyl-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate buffer with a pH of 7.550 mM is added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for reaction for 24 hours, extracted with three volumes of ethyl acetate, and the upper organic phase is collected, dried with anhydrous magnesium sulfate, and the product is concentrated by reduced pressure evaporation, and then the conversion rate and yield are determined by high performance liquid chromatography.

[0078] High performance liquid chromatography was used for detection, using CHIRALCEL OJ-H column, mobile phase conditions were isopropanol / n-hexane (V / V) = 10:90, detection conditions were flow rate 1ml / min, detection wavelength 254nm. Results are shown in Figure 1 .

[0079] Figure 1 The results of high performance liquid chromatography (HPLC) of the monoamine oxidase VsMAO of Example 1 of the present invention in catalyzing the oxidation of the substrate 2-methyl-1,2,3,4-tetrahydroquinoline. Among them, Figure a is a substrate standard (because the substrate has chirality, the substrate standard has two peaks, and the use of a chiral chromatographic column can separate the two configurations of the product and produce two peaks), Figure b is a product standard, and Figure c is a liquid chromatogram of the reaction solution of Example 1 of the present invention. Among them, 2-methylquinoline peaks at 6.1min, (S)-2-methyl-1,2,3,4-tetrahydroquinoline and (R)-2-methyl-1,2,3,4-tetrahydroquinoline peak at 10.1min and 11.1min respectively.

[0080] The test results show that the monoamine oxidase prepared in Example 2 can catalyze the oxidative aromatization of 2-methyl-1,2,3,4-tetrahydroquinoline to produce 2-methylquinoline, wherein both (R) and (S) configurations can be converted. After the reaction is completed, the conversion rate of the substrate can reach 91%, and the yield of the product can reach 76%.

[0081] Example 4 Determination of the optimal pH and optimal buffer solution system for the enzymatic properties of monoamine oxidase

[0082] (1) Enzyme activity determination method: The enzyme activity was determined by high performance liquid chromatography. The total volume of the enzyme activity determination reaction was 1 mL, including 100 μL of pure monoamine oxidase VsMAO prepared in Example 2, 50 μL of a substrate solution with an initial concentration of 100 mM (the substrate is 2-methyl-1,2,3,4-tetrahydroquinoline), and 850 μL of a dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer solution with an initial concentration of 50 mM at pH 7.5. The change in the peak area of ​​the liquid chromatography at 254 nm after the above system reacted for 15 min was detected, and the peak area change was brought into the liquid phase standard curve of the product 2-methylquinoline to calculate the product concentration.

[0083] The enzyme activity was calculated as follows:

[0084]

[0085] The specific enzyme activity calculation formula is:

[0086]

[0087] Among them, c 产物 It represents the concentration of the product produced by the system within 15 minutes (in mM), V represents the volume of the reaction, which is 1 mL in this case, and t represents the time for measuring enzyme activity, which is 15 minutes in this case.

[0088] (2) According to the reaction system and reaction method in step (1), the potassium dihydrogen phosphate and potassium dihydrogen phosphate buffers with an initial concentration of 50 mM at pH 7.5 were replaced with different buffer solution systems (citric acid-sodium citrate buffer system, potassium dihydrogen phosphate-potassium hydrogen phosphate buffer system and Gly-NaOH buffer system) with an initial concentration of 50 mM in different pH ranges (5.0-10.5), and the optimal pH value and optimal buffer solution system of monoamine oxidase in catalyzing the oxidative aromatization of substrate 2-methyl-1,2,3,4-tetrahydroquinoline were measured for enzyme activity, and the enzyme activity of the monoamine oxidase of the present invention at 30°C was measured using high performance liquid chromatography. The experimental results are as follows: Figure 2 shown.

[0089] Figure 2The present invention shows the optimal pH and optimal buffer solution system experimental results when the monoamine oxidase VsMAO of the present invention catalyzes the oxidative aromatization of the substrate 2-methyl-1,2,3,4-tetrahydroquinoline.

[0090] After testing, when the buffer solutions were citric acid-sodium citrate buffer solutions of pH 5.0, pH 5.5, and pH 6.0, respectively, the enzyme activities obtained were 30%, 58%, and 70% of the maximum enzyme activity, respectively; when the buffer solutions were potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solutions of pH 6.0, pH 6.5, pH 7.0, pH 7.5, and pH 8.0, respectively, the enzyme activities obtained were 74%, 90%, 96%, 100%, and 90% of the maximum enzyme activity, respectively; when the buffer solutions were Gly-NaOH buffer solutions of pH 8.0, pH 8.5, pH 9.0, pH 9.5, pH 10.0, and pH 10.5, respectively, the enzyme activities obtained were 82%, 74%, 65%, 56%, 50%, and 45% of the maximum enzyme activity, respectively. The results showed that the buffer solution was potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution with a pH of 7.5, which was the optimal pH value and the optimal buffer solution system. At this time, the enzyme activity was the largest, which was 17.43 U / g 蛋白 The enzyme activity at this time was defined as 100%.

[0091] Example 5 Determination of the optimum temperature for the enzymatic properties of monoamine oxidase VsMAO

[0092] Method for determining the optimum temperature of monoamine oxidase VsMAO: total reaction volume 1mL: 100μL of the monoamine oxidase VsMAO pure enzyme solution prepared in Example 2 was placed at 20℃, 30℃, 35℃, 40℃, 50℃, 60℃, and 70℃ for incubation for 5min, and then 50μL of a substrate solution with an initial concentration of 100mM was added, and 850μL of a pH 7.5 initial concentration of 50mM dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer were added for reaction. The enzyme activity was determined by high performance liquid chromatography to detect the enzyme activity of the monoamine oxidase prepared in Example 2 on the substrate 2-methyl-1,2,3,4-tetrahydroquinoline at different temperatures.

[0093] After testing, the enzyme activities of monoamine oxidase at 20℃, 30℃, 35℃, 40℃, 50℃, 60℃, and 70℃ were 70%, 100%, 93%, 90%, 57%, 18%, and 4% of the maximum enzyme activity, respectively. Figure 3 As shown, the enzyme activity of monoamine oxidase is the highest at 30℃, which is 17.43U / g 蛋白 That is, the optimum conversion temperature is 30°C.

[0094] Figure 3The optimum temperature experimental result of the monoamine oxidase VsMAO of the present invention when catalyzing the oxidative aromatization of the substrate 2-methyl-1,2,3,4-tetrahydroquinoline.

[0095] Example 6 Determination of the thermal stability of monoamine oxidase enzymatic properties

[0096] The monoamine oxidase VsMAO pure enzyme solution prepared in Example 2 was incubated at 30°C and 50°C for different time periods, and the enzyme activity at the optimal conversion temperature in different time periods was determined by high performance liquid chromatography. The total reaction volume was 1 mL, which included 100 μL of the monoamine oxidase VsMAO pure enzyme solution prepared in Example 2, 50 μL of a substrate solution with an initial concentration of 100 mM (the substrate was 2-methyl-1,2,3,4-tetrahydroquinoline), and 850 μL of a pH 7.5 dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM. The reaction temperature was 30°C.

[0097] After the monoamine oxidase enzyme solution was incubated at 30°C for 0, 1, 2, 4, 8, 12, and 24 hours, the enzyme activity at 30°C was 100%, 98%, 95%, 89%, 81%, 69%, and 51% of the initial enzyme activity, respectively. Figure 4 shown.

[0098] After the monoamine oxidase enzyme solution was incubated at 50°C for 0, 1, 2, 4, and 8 hours, the enzyme activity at 50°C was 100%, 94%, 75%, 65%, and 27% of the initial enzyme activity, respectively. Figure 4 shown.

[0099] Figure 4 The figures are the thermal stability test results of the monoamine oxidase VsMAO of the present invention at different temperatures.

[0100] Depend on Figure 4 It can be seen that the monoamine oxidase VsMAO of the present invention has good thermal stability under the condition of 30°C.

[0101] Example 7 Determination of pH stability of monoamine oxidase enzymatic properties

[0102] The monoamine oxidase VsMAO pure enzyme solution prepared in Example 2 was incubated at pH = 7.5 and pH = 10.0 for different time periods, and the enzyme activity at the optimal conversion temperature in different time periods was determined by high performance liquid chromatography. The total reaction volume was 1 mL, which included 100 μL of the monoamine oxidase VsMAO pure enzyme solution prepared in Example 2, 50 μL of a substrate solution with an initial concentration of 100 mM (the substrate was 2-methyl-1,2,3,4-tetrahydroquinoline), and 850 μL of a potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer solution with an initial concentration of 50 mM at pH 7.5 or pH 10.0. The reaction temperature was 30°C.

[0103] The enzyme activity of monoamine oxidase solution at the optimum temperature (i.e. 30°C) after incubation at pH = 7.5 for 0, 1, 2, 4, 8, 12, 24, and 48 hours was 100%, 97%, 93%, 89%, 84%, 80%, 74%, and 51% of the initial enzyme activity, respectively. Figure 5 shown.

[0104] The enzyme activity of monoamine oxidase solution at the optimum temperature (i.e., 30°C) after incubation at pH = 10.0 for 0, 1, 2, 4, 8, 12, 24, and 48 hours was 100%, 92%, 87%, 83%, 74%, 70%, 66%, and 44% of the initial enzyme activity, respectively. Figure 5 shown.

[0105] Figure 5 The results are the pH stability test results of the monoamine oxidase VsMAO of the present invention at the optimum temperature.

[0106] Depend on Figure 5 It can be seen that the monoamine oxidase VsMAO of the present invention has good alkali resistance.

[0107] Example 8 Application of monoamine oxidase in the synthesis of 6-fluoroquinoline

[0108] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0109] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, a substrate 6-fluoro-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 is added to make up to 5 mL, and placed in a shaker at 30° C. and 200 rpm for reaction for 3 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated the product by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography.

[0110] The results were detected by high performance liquid chromatography using a CHIRALCEL OJ-H column, with a mobile phase of isopropanol / n-hexane (V / V) = 10:90, a flow rate of 1 ml / min, and a detection wavelength of 254 nm. Figure 6 .

[0111] Figure 6 The results of high performance liquid chromatography (HPLC) of the conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline catalyzed by monoamine oxidase VsMAO in Example 8 of the present invention are shown in Figure a, Figure b is a standard product, the product 6-fluoroquinoline peaks at 6.3 min, the substrate 6-fluoro-1,2,3,4-tetrahydroquinoline peaks at 9.5 min, and Figure c is a liquid chromatogram of the reaction solution in Example 8 of the present invention.

[0112] The test results show that the monoamine oxidase of the present invention can catalyze the conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline into the product 6-fluoroquinoline, the conversion rate of the substrate can reach 100%, and the yield of the product can reach 96%.

[0113] Example 9 Application of monoamine oxidase in large-scale preparation of 6-fluoroquinoline

[0114] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0115] The total volume of the conversion reaction system is 500 mL, and the system includes: 16 g / L monoamine oxidase VsMAO frozen stem cell powder, a substrate 6-fluoro-1,2,3,4-tetrahydroquinoline with a final concentration of 10 mM, 10% volume fraction of DMSO as a cosolvent, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 500 mL, and placed in a 30°C, 200 rpm water bath for 72 hours. During the reaction, 8M sodium hydroxide is added to control the pH of the reaction system to 7.5. After the reaction is completed, three volumes of ethyl acetate are used for extraction, the upper organic phase is collected, dried with anhydrous magnesium sulfate, and the product is concentrated by reduced pressure evaporation, and then the conversion rate and yield are determined by high performance liquid chromatography.

[0116] The detection was performed using a CHIRALCEL OJ-H column, the mobile phase conditions were isopropanol / n-hexane (V / V) = 10:90, the detection conditions were a flow rate of 1 ml / min, and the detection wavelength was 254 nm. After 72 hours of reaction, the freeze-dried Escherichia coli cell powder of the monoamine oxidase VsMAO prepared in Example 9 had a conversion rate of 90% when catalyzing the conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline, and the yield of the generated product 6-fluoroquinoline was 82%. After a simple column separation and purification, the mobile phase component of the column was n-hexane: ethyl acetate (v:v) = 10:1, and the separation yield was 86%, to obtain 519 mg of 6-fluoroquinoline product, and the product was characterized and analyzed by nuclear magnetic resonance. The characterization results are as follows Figure 7 .

[0117] Figure 7 The NMR results of the conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline catalyzed by the monoamine oxidase VsMAO of the present invention are shown in Figure a, which is the hydrogen spectrum of the product 6-fluoroquinoline, and Figure b is the carbon spectrum of the product 6-fluoroquinoline.

[0118] Example 10 Monoamine oxidase is used in the reaction with a substrate concentration of 50 mM to synthesize 6-fluoroquinoline

[0119] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0120] The total volume of the conversion reaction system is 5 mL, and the system includes: 48 g / L monoamine oxidase VsMAO frozen stem cell powder, a substrate 6-fluoro-1,2,3,4-tetrahydroquinoline with a final concentration of 50 mM, 10% volume fraction of DMSO as a cosolvent, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 7 days. During the reaction, 8M sodium hydroxide is added to control the pH of the reaction system to 7.5. Use three volumes of ethyl acetate for extraction, collect the upper organic phase, dry it with anhydrous magnesium sulfate, and concentrate the product by reduced pressure evaporation. Then use high performance liquid chromatography to determine the conversion rate and yield, monitor the process curve, and the results are as follows: Figure 8 .

[0121] Figure 8 The figure is a process curve diagram of the monoamine oxidase VsMAO catalyzing the conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline with a substrate concentration of 50 mM.

[0122] Depend on Figure 8 It can be seen that the monoamine oxidase VsMAO of the present invention can catalyze the conversion of 6-fluoro-1,2,3,4-tetrahydroquinoline into the product 6-fluoroquinoline. At a substrate concentration of 50 mM, the conversion rate can reach 85% and the product yield can reach 83%.

[0123] Example 11 Application of monoamine oxidase in the synthesis of quinoline

[0124] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0125] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The test results showed that the conversion rate was 95% and the yield was 84%.

[0126] Example 12 Application of monoamine oxidase in the synthesis of 6-bromoquinoline

[0127] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0128] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 6-bromo-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 48 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 94% and the yield was 81%.

[0129] Example 13 Application of monoamine oxidase in the synthesis of 6-chloroquinoline

[0130] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0131] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 6-chloro-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 98% and the yield was 91%.

[0132] Example 14 Application of monoamine oxidase in the synthesis of 6-methoxyquinoline

[0133] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0134] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 6-methoxy-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up the system volume to 5 mL, and placed in a shaker at 30°C and 200 rpm for 5 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The test results are shown in Fig. 9 .

[0135] Fig. 9 The results of high performance liquid chromatography (HPLC) of the conversion of 6-methoxy-1,2,3,4-tetrahydroquinoline catalyzed by monoamine oxidase VsMAO in Example 14 of the present invention are shown in Figure a. The standard product of the substrate is shown in Figure b. The product 6-methoxyquinoline peaks at 7.6 min, and the substrate 6-methoxy-1,2,3,4-tetrahydroquinoline peaks at 14.4 min. Figure c is the liquid chromatogram of the reaction solution in Example 14 of the present invention. The results show that the substrate conversion rate is 95% and the product yield is 91%.

[0136] Example 15 Application of monoamine oxidase in the synthesis of 6-hydroxyquinoline

[0137] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0138] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 6-hydroxy-1,2,3,4-tetrahydroquinoline with a final concentration of 10 mM, potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up the system volume to 5 mL, and placed in a shaker at 30°C and 200 rpm for 72 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 88% and the yield was 80%.

[0139] Example 16 Application of monoamine oxidase in the synthesis of 3-methylquinoline

[0140] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0141] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 3-methyl-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 96% and the yield was 72%.

[0142] Example 17 Application of monoamine oxidase in the synthesis of 4-methylquinoline

[0143] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0144] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 4-methyl-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 94% and the yield was 83%.

[0145] Example 18 Application of monoamine oxidase in the synthesis of 5-methylquinoline

[0146] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0147] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 5-methyl-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The test results are shown in Fig.10 .

[0148] Fig.10 The results of high performance liquid chromatography (HPLC) of the conversion of 5-methyl-1,2,3,4-tetrahydroquinoline catalyzed by monoamine oxidase VsMAO in Example 18 of the present invention are shown in Figure a. The standard product of the substrate is shown in Figure b. The product 5-methylquinoline peaks at 6.0 min, and the substrate 5-methyl-1,2,3,4-tetrahydroquinoline peaks at 7.5 min. Figure c is the liquid chromatogram of the reaction solution in Example 18 of the present invention. The results show that the substrate conversion rate is 74% and the product yield is 56%.

[0149] Example 19 Application of monoamine oxidase in the synthesis of 6-methylquinoline

[0150] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0151] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 6-methyl-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 99% and the yield was 78%.

[0152] Example 20 Application of monoamine oxidase in the synthesis of 7-methylquinoline

[0153] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0154] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 7-methyl-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 48 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 80% and the yield was 63%.

[0155] Example 21 Application of monoamine oxidase in the synthesis of 8-methylquinoline

[0156] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0157] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 8-methyl-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 99% and the yield was 94%.

[0158] Example 22 Application of monoamine oxidase in the synthesis of 7-methoxyquinoline

[0159] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0160] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO freeze-dried cell powder, a substrate 7-methoxy-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 90% and the yield was 85%.

[0161] Example 23 Application of monoamine oxidase in the synthesis of 7-chloroquinoline

[0162] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0163] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 7-chloro-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 78% and the yield was 61%.

[0164] Example 24 Application of monoamine oxidase in the synthesis of 7-bromoquinoline

[0165] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0166] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 7-bromo-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 83% and the yield was 79%.

[0167] Example 25 Application of monoamine oxidase in the synthesis of 4-hydroxyquinoline

[0168] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0169] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 4-hydroxy-1,2,3,4-tetrahydroquinoline with a final concentration of 10 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 54% and the yield was 43%.

[0170] Example 26 Application of monoamine oxidase in the synthesis of 8-hydroxyquinoline

[0171] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0172] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 8-hydroxy-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 48 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The test results are shown in Fig.11 .

[0173] Fig.11 The results of high performance liquid chromatography (HPLC) of the conversion of 8-hydroxy-1,2,3,4-tetrahydroquinoline catalyzed by monoamine oxidase VsMAO in Example 26 of the present invention are shown in Figure a. The standard product of the substrate is shown in Figure b. The product 8-hydroxyquinoline peaks at 6.4 min, and the substrate 8-hydroxy-1,2,3,4-tetrahydroquinoline peaks at 13.9 min. Figure c is the liquid chromatogram of the reaction solution in Example 26 of the present invention, and the results show that the conversion rate is 81% and the yield is 68%.

[0174] Example 27 Application of monoamine oxidase in the synthesis of 2-methyl-6-fluoroquinoline

[0175] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0176] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 2-methyl-6-fluoro-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 64 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 97% and the yield was 95%.

[0177] Example 28 Application of monoamine oxidase in the synthesis of 2-methyl-6-bromoquinoline

[0178] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0179] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 2-methyl-6-bromo-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 is added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 70% and the yield was 70%.

[0180] Example 29 Application of monoamine oxidase in the synthesis of 4-methyl-6-bromoquinoline

[0181] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0182] The total volume of the conversion reaction system is 5 mL, and the system includes: 16 g / L monoamine oxidase VsMAO lyophilized cell powder, substrate 4-methyl-6-bromo-1,2,3,4-tetrahydroquinoline with a final concentration of 5 mM, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaker at 30°C and 200 rpm for 24 hours, extracted with three volumes of ethyl acetate, collected the upper organic phase, dried with anhydrous magnesium sulfate, and concentrated by reduced pressure evaporation, and then the conversion rate and yield were determined by high performance liquid chromatography. The results showed that the conversion rate was 67% and the yield was 51%.

[0183] Example 30 Monoamine oxidase is used in the reaction with a substrate concentration of 50 mM to synthesize 2-methylquinoline

[0184] The freeze-dried E. coli cell powder containing monoamine oxidase VsMAO prepared in Example 2 was used to carry out a transformation experiment and determine the transformation rate and yield.

[0185] The total volume of the conversion reaction system is 5 mL, and the system includes: 48 g / L monoamine oxidase VsMAO frozen stem cell powder, a substrate 2-methyl-1,2,3,4-tetrahydroquinoline with a final concentration of 50 mM, 10% volume fraction of DMSO as a cosolvent, and potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer with an initial concentration of 50 mM at pH 7.5 are added to make up to 5 mL, and placed in a shaking table at 30°C and 200 rpm for 7 days. During the reaction, 8M sodium hydroxide is added to control the pH of the reaction system to 7.5. Three volumes of ethyl acetate are used for extraction, the upper organic phase is collected, dried with anhydrous magnesium sulfate, and the product is concentrated by reduced pressure evaporation, and the conversion rate and yield are determined by high performance liquid chromatography, and the results show that the conversion rate is 56% and the product yield is 52%.

[0186] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of monoamine oxidase as a catalyst in the preparation of quinoline nitrogen heterocyclic compounds, wherein the monoamine oxidase is derived from Gammaproteobacteria bacterium, Pseudomonas sp., Saccharospirillum sp., Thalassomonas sp. RHCI1, Luminiphilus sp., Vibrio sp. JCM 19236 or Pseudomonas monteilii ZMU-T01.

2. The use according to claim 1, characterized in that: The monoamine oxidase is derived from Vibrio sp. JCM19236.

3. The use according to claim 2, characterized in that: The amino acid sequence of the monoamine oxidase is SEQ ID NO.1 in the sequence list.

4. The use according to claim 3, characterized in that: The gene sequence encoding the monoamine oxidase is SEQ ID NO.2 in the sequence list.

5. A method for preparing a quinoline nitrogen heterocyclic compound, characterized in that: Using the THQs compound of formula (I) as a substrate and the monoamine oxidase described in any one of claims 1 to 4 as a catalyst, an oxidative aromatization reaction is carried out in a buffer solution to prepare quinolines nitrogen heterocyclic compounds of formula (I); In formula (I), R1, R2, R3, R4, R5, R6, R7 are independently selected from hydrogen, methyl, hydroxy, methoxy, fluorine, chlorine or bromine; Preferably, in formula (I), one or two of R1, R2, R3, R4, R5, R6, R7 are selected from methyl, hydroxy, methoxy, fluorine, chlorine or bromine.

6. A method for preparing quinoline nitrogen heterocyclic compounds according to claim 5, characterized in that: The THQs compound in formula (I) is selected from 2-methyl-1,2,3,4-tetrahydroquinoline, 1,2,3,4-tetrahydroquinoline, 6-fluoro-1,2,3,4-tetrahydroquinoline, 6-chloro-1,2,3,4-tetrahydroquinoline, 6-bromo-1,2,3,4-tetrahydroquinoline, 6-methoxy-1,2,3,4-tetrahydroquinoline, 6-hydroxy-1,2,3,4-tetrahydroquinoline, 3-methyl-1,2,3,4-tetrahydroquinoline, 4-methyl-1,2,3,4-tetrahydroquinoline, 5-methyl-1,2,3,4-tetrahydroquinoline, 6-methyl-1,2, 3,4-tetrahydroquinoline, 7-methyl-1,2,3,4-tetrahydroquinoline, 8-methyl-1,2,3,4-tetrahydroquinoline, 7-methoxy-1,2,3,4-tetrahydroquinoline, 7-bromo-1,2,3,4-tetrahydroquinoline, 7-chloro-1,2,3,4-tetrahydroquinoline, 4-hydroxy-1,2,3,4-tetrahydroquinoline, 8-hydroxy-1,2,3,4-tetrahydroquinoline, 2-methyl-6-fluoro-1,2,3,4-tetrahydroquinoline, 2-methyl-6-bromo-1,2,3,4-tetrahydroquinoline or 4-methyl-6-bromo-1,2,3,4-tetrahydroquinoline.

7. A method for preparing quinoline nitrogen heterocyclic compounds according to claim 5, characterized in that: The catalyst comprises wet monoamine oxidase bacteria, freeze-dried Escherichia coli cells containing monoamine oxidase, and crude enzyme solution of monoamine oxidase; The monoamine oxidase wet bacterial cell is a recombinant strain obtained by connecting the gene sequence of the monoamine oxidase described in any one of claims 1 to 3 to a vector to obtain a recombinant expression vector, and then introducing it into a host bacterium for inducing expression, wherein the host bacterium includes Escherichia coli; The freeze-dried Escherichia coli cells containing monoamine oxidase are obtained by freeze-drying the wet cells of monoamine oxidase; The crude enzyme solution of monoamine oxidase is obtained by crushing the wet bacterial cells of monoamine oxidase, centrifuging and taking the supernatant; Preferably, the catalyst is freeze-dried Escherichia coli cells containing monoamine oxidase.

8. A method for preparing a quinoline nitrogen heterocyclic compound according to any one of claims 5 to 7, characterized in that: The concentration of the substrate is 5-50 mM; and / or The amount of the catalyst is 4-40 g / L; and / or During the reaction, the reaction temperature is 20-40°C; and / or During the reaction, the reaction time is 3h-7d; and / or The pH value of the buffer solution is 7.0-9.

0.

9. A method for preparing a quinoline nitrogen heterocyclic compound according to claim 8, characterized in that: The concentration of the substrate is 10-50 mM; most preferably the concentration of the substrate is 50 mM; and / or The amount of the catalyst is 16 g / L; and / or During the reaction, the reaction temperature is 30-40°C; preferably the reaction temperature is 30°C; and / or During the reaction, the reaction time is 3h or 7d; and / or The buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution with a pH of 7.0-8.0 or a glycine-sodium hydroxide buffer solution with a pH of 8.0-9.0; preferably, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution with a pH of 7.

5.

10. A method for preparing a quinoline nitrogen heterocyclic compound according to any one of claims 5 to 9, characterized in that: During the reaction, no additional cofactor flavin adenine dinucleotide (FAD) and / or hydrogen peroxide was added.

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