Method for preparing nitrone compound by using non-heme double-iron-ion enzyme

Abstract

The invention discloses a method for preparing nitrone compounds by using a non-heme double-iron-ion enzyme, and belongs to the field of biological enzyme catalysis. A substituted styrene compound and a substituted aniline compound are used as substrates and react in a PBS buffer solution in the presence of non-heme double iron ion type amino oxidase AzoC to obtain the nitrone compound. According to the present invention, the enzyme catalysis nitrone synthesis system verifies the feasibility of the non-heme double iron ion type amino oxidase mediated bimolecular nitrone compound synthesis, the solid foundation is laid for the subsequent research and application, and the strategy provides the basis for the subsequent enzyme modification and optimization.

Description

Technical Field

[0001] The invention belongs to the field of biological enzyme catalysis, relates to a method for preparing nitrone compounds, and specifically relates to a biological method for preparing nitrone compounds by enzyme catalysis. Background Art

[0002] As a nitrogen-containing biological compound, nitrone occupies an important position in the field of biochemistry and organic synthesis due to its unique biological activity. It is an attractive synthetic starting material for many nitrogen-containing biologically active compounds, such as amino acids, alkaloids, β-lactam antibiotics and other clinically important compounds. However, the traditional nitrone synthesis method has the limitations of harsh reaction conditions, cumbersome steps, and serious environmental pollution, making it difficult to achieve large-scale synthesis. In recent years, enzymes, as a natural catalyst, have played an increasingly important role in green and sustainable synthesis due to their mild action conditions, low cost, and natural degradation. Especially in asymmetric catalysis, due to the unique spatial structure of its active pocket, it can reduce the reaction activation energy and then regulate the reaction process, thereby achieving higher chemical selectivity, regioselectivity and stereoselectivity under mild action conditions, playing an increasingly important role in both industrial production and academic research.

[0003] However, compared with traditional chemical catalysts, enzyme catalysis still has many limitations. For example, the reaction range is limited because they have the specificity of natural selection and limited catalytic function space; the stability is poor and they are easily deactivated at high temperature, strong acid and alkali, and organic solvents, which greatly limits their application in chemical reactions.

[0004] Therefore, it is of great significance to explore the non-native reactivity of natural enzymes and expand the catalytic function of enzymes to achieve more and wider biosynthesis of nitrone. Summary of the invention

[0005] In order to overcome the above technical defects, the present invention uses non-heme diferric ion amino oxidase as a biocatalyst to achieve a more efficient and green biosynthetic pathway of nitrone. This discovery broadens the reaction range of natural biological enzymes and provides a new synthesis method for the application of nitrone in the fields of drug development, material science, etc.

[0006] The present invention adopts the following technical solution to solve the above technical problems, a method for preparing nitrone compounds using non-heme diiron ion enzyme, the reaction equation is expressed as follows:

[0007]

[0008] Where: R 1 , R 2Each is independently selected from C1-C4 alkyl, C1-C4 alkoxy, nitro, trifluoromethyl or halogen.

[0009] The present invention discloses a method for preparing nitrone compounds by using a non-heme diferric ion type enzyme, comprising the following steps: using a substituted styrene compound 1 and a substituted aniline compound 2 as substrates, reacting in a PBS buffer solution in the presence of a non-heme diferric ion type amino oxidase AzoC, to obtain a nitrone compound 3.

[0010] Furthermore, under the preferred conditions of the above technical solution, the molar ratio of the substituted styrene compound 1 to the substituted aniline compound 2 is 1:1-1.2.

[0011] Furthermore, under the preferred conditions of the above technical solution, the molar ratio of the substituted styrene compound 1, PBS and non-heme diiron ion type amino oxidase AzoC is 1:0.01-0.02:0.005-0.01.

[0012] Furthermore, under the preferred conditions of the above technical solution, the pH of the PBS buffer solution is 4-6.

[0013] Furthermore, under the preferred conditions of the above technical solution, the reaction temperature is 25-30°C, and the temperature is controlled by a constant temperature shaker.

[0014] Furthermore, under the preferred conditions of the above technical solution, GDH and NAD are added during the reaction. + And Glucose recycling.

[0015] The present invention has the following advantages:

[0016] A. The enzyme-catalyzed nitrone synthesis system of the present invention verifies the feasibility of bimolecular synthesis of nitrone compounds mediated by non-heme diiron ion amino oxidase, laying a solid foundation for subsequent research and application.

[0017] B. This strategy also provides a basis for the subsequent modification and optimization of enzymes. It reveals that the catalytic activity pocket of non-heme diiron enzymes has plasticity characteristics. Through the targeted modification of the microenvironment of the substrate binding pocket, it is possible to endow natural catalysts and non-natural free radicals with chemical transformation capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a simulation diagram of the AzoC substrate binding model. DETAILED DESCRIPTION

[0019] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0020] Typical operation steps: In a 1.5mL sample tube, aniline (0.2μmol, 2μL, 0.1mmol / L) and styrene (0.2μmol, 2μL, 0.1mmol / L) were placed in a PBS solution (pH=5, 100μL), and then enzymes AzoC (0.5% mol), GDH (0.5mol%), and NAD + (0.04μmol, 0.2eq) and Glucose (4μmol, 20eq) circulation system, and finally add the electron transfer system PMS (5-methylphenazine methyl sulfate, 1% mol). After fixing the reaction sample tube, put it in a 25℃ constant temperature shaker for 5 hours. After the reaction is completed, add ethyl acetate solution for extraction, rotate, and purify by column chromatography to obtain the product.

[0021] AzoC: Chemoenzymatic Synthesis of Phenol Di arylamine Using Non-HemeDiiron N-Oxygenase[J]. ACS Catalysis, 2023, 13(2):1412-7.

[0022] Example 1

[0023]

[0024] Compound 1a (0.2 μmol), compound 2a (0.2 μmol), PBS buffer solution (pH = 5, 100 μL), enzyme (0.5% mol), GDH (0.5 mol%), NAD + (0.04μmol, 0.2eq) and Glucose (4μmol, 20eq) were circulated in the system, and finally the electron transfer system PMS (5-methylphenazine methyl sulfate, 1% mol) was added, the lid was sealed, and the mixture was stirred at 25°C for 5 hours. After the reaction was completed, the extraction was dried and separated by silica gel column to obtain the product 3a.

[0025] By changing the reaction buffer solution type, buffer solution pH value, additives and reaction temperature, the reaction results are as follows:

[0026]

[0027]

[0028] Note: The mutation site of AzoC-II is AzoC-T98G-L101G; the mutation site of AzoC-III is AzoC-T98A-L101A.

[0029] Example 2

[0030] Compound 1 (0.2 μmol), compound 2 (0.2 μmol), PBS buffer solution (pH = 5, 100 μL), enzyme AzoC (0.5% mol), GDH (0.5 mol%), NAD + (0.04μmol, 0.2eq) and Glucose (4μmol, 20eq) circulation system, finally add the electron transfer system PMS (5-methylphenazine methyl sulfate, 1% mol), cover with a lid and seal, place in a 25℃ constant temperature box and stir for 5 hours. After the reaction is completed, extract and spin dry, and separate the product 3 with a silica gel column (n-hexane / ethyl acetate = 5:1). The reaction results are as follows:

[0031]

[0032] The representative product characterization data are as follows:

[0033] Compound 3a: 1 H NMR(600MHz, CDCl3)δ8.42-8.38(m,2H),7.93(s,1H),7.80-7.76(m,2H),7.51-7.45(m,6H).HRMS[M+H] + calculated for198.0913, found as 198.0914.

[0034] Compound 3b: 1 H NMR(600MHz, CDCl3)δ8.37-8.34(m,2H),7.91(s,1H),7.78-7.75(m,2H),7.50-7.43(m,5H).HRMS[M+H] + calculated for 232.0524, found as 232.0534.

[0035] Compound 3c: 1 H NMR (600MHz, CDCl3) δ8.32-8.26(m,2H),7.89(s,1H),7.78-7.74(m,2H),7.64-7.58(m,2H),7.51-7.43(m,3H). 13 CNMR(151MHz, CDCl3)δ148.90,133.43,131.85,130.23,130.08,129.46,129.17,124.76,121.62,77.16,76.95,76.74.HRMS[M+H]+ Calculated for 276.0019, found as 276.0019.

[0036] Compound 3d: 1 H NMR (600 MHz, CDCl3) δ 8.42 - 8.34 (m, 2H), 7.84 (s, 1H), 7.76 - 7.71 (m, 2H), 7.45 - 7.38 (m, 3H), 6.98 - 6.93 (m, 2H), 3.84 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 161.62, 148.82, 134.51, 131.30, 129.66, 129.13, 123.68, 121.65, 114.06, 77.35, 77.14, 76.93, 55.42. HRMS [M+H] + Calculated for 228.1019, found as 228.1033.

[0037] Compound 3e: 1 H NMR (600 MHz, CDCl3) δ 8.50 (d, J = 8.2 Hz, 2H), 8.00 (s, 1H), 7.80 - 7.75 (m, 2H), 7.72 (d, J = 8.3 Hz, 2H), 7.54 - 7.47 (m, 3H). 13 C NMR (151 MHz, CDCl3) δ 148.96, 133.71, 133.17, 132.02, 131.81, 130.44, 129.33, 128.96, 125.60, 121.77, 77.25, 77.04, 76.83. 19 F NMR (565 MHz, CDCl3) δ -62.92. HRMS [M+H] + Calculated for 266.0787, found as 266.0783.

[0038] Compound 3f: 1 H NMR (600 MHz, CDCl3) δ 8.32 - 8.27 (m, 2H), 7.88 (s, 1H), 7.79 - 7.73 (m, 2H), 7.49 - 7.42 (m, 3H), 7.28 (d, J = 8.0 Hz, 2H), 2.41 (s, 3H). 1313C NMR(151MHz, CDCl3) δ 148.97, 141.79, 135.09, 129.85, 129.42, 129.27, 129.17, 127.97, 121.77, 77.25, 77.04, 76.82, 21.83. HRMS [M+H] + calculated for 212.1070, found as 212.1071

[0039] Compound 3g: 1 1H NMR(600MHz, CDCl3) δ 8.35 - 8.32(m, 2H), 7.90(s, 1H), 7.78 - 7.74(m, 2H), 7.51 - 7.47(m, 2H), 7.47 - 7.41(m, 3H), 1.35(s, 9H). 13 13C NMR(151MHz, CDCl3) δ 154.65, 149.02, 134.66, 129.81, 129.15, 129.09, 128.00, 125.62, 121.75, 76.90, 35.12, 31.13. HRMS [M+H] + calculated for 254.1539, found as 254.1556.

[0040] Compound 3i: 1 1H NMR(600MHz, CDCl3) δ 8.40 - 8.35(m, 2H), 7.90(s, 1H), 7.70 - 7.65(m, 2H), 7.63 - 7.58(m, 2H), 7.48(ddt, J=6.0, 4.3, 2.4Hz, 3H). 13 13C NMR(151MHz, CDCl3) δ 147.92, 134.64, 132.31, 131.26, 130.43, 129.15, 128.74, 123.88, 123.32. HRMS [M+H] + calculated for 276.0019, found as 276.0024.

[0041] Compound 3m: 1 1H NMR(600MHz, CDCl3) δ 8.41 - 8.35(m, 2H), 7.90(s, 1H), 7.72 - 7.66(m, 2H), 7.50 - 7.44(m, 3H), 7.34 - 7.31(m, 2H), 2.98(hept, J=6.9Hz, 1H), 1.28(d, J=7.0Hz, 6H).13 C NMR (151MHz, CDCl3) δ151.14,147.02,134.43,130.87,130.77,129.06,128.66,127.14,121.69,33.91,23.88.HRMS[M+H] + calculated for 240.1383,found as240.1384.

[0042] Compound 3n: 1 H NMR (600MHz, CDCl3) δ8.40-8.37(m,2H),7.91(s,1H),7.71-7.68(m,2H),7.50-7.45(m,5H),1.35(s,9H). 13 C NMR (151MHz, CDCl3) δ153.44,146.64,134.46,130.88,129.07,128.67,126.10,121.36,77.25,77.04,76.83,34.88,31.27.HRMS[M+H] + calculated for254.1539, found as 254.1539.

[0043] Compound 3o: 1 H NMR (600MHz, CDCl3) δ8.42-8.37(m,2H),7.90(s,1H),7.70-7.63(m,2H),7.50-7.45(m,3H),7.27(d,J=8.2Hz,2H),2.42(s,3H). 13 C NMR (151MHz, CDCl3) δ140.21,134.18,130.83,130.78,129.66,129.01,128.65,121.52,77.23,77.02,76.81,21.18.HRMS[M+H] + calculated for 212.1070, found as 212.1070

[0044] Compound 3p: 1H NMR (600MHz, CDCl3) δ8.40-8.35(m,2H),7.88(s,1H),7.58(d,J=1.8Hz,1H),7.51(dd,J=8.0,2. 2Hz,1H),7.44(dd,J=5.3,2.0Hz,3H),7.31(t,J=7.8Hz,1H),7.23(d,J=7.6Hz,1H),2.40(s,3H). 13 CNMR(151MHz,CDCl3)δ149.11,139.43,134.63,130.89,130.76,130.68,129 .09,128.93,128.64,122.45,118.77,77.39,77.17,76.96,21.39.HRMS[M+H] + calculated for 212.1070, found as 212.1070.

[0045] Compound 3q: 1 H NMR (600MHz, CDCl3) δ8.39 (dd, J=6.7, 2.8Hz, 2H), 7.92 (s, 1H), 7.48 (dd, J=5.4, 1.9Hz, 3H), 7.40 (d, J=2 .4Hz,1H),7.35(t,J=8.1Hz,1H),7.28(dd,J=7.8,1.9Hz,1H),7.00(dd,J=8.3,2.4Hz,1H),3.86(s,3H). 13 C NMR (151MHz, CDCl3) δ160.26,150.24,134.77,131.03,130.62,129.78,129. 15,128.68,116.29,113.51,107.53,77.27,77.05,76.84,55.66.HRMS[M+H] + calculated for 228.1019, found as 228.1020.

[0046] The above embodiments describe the main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing nitrone compounds using a non-heme diferric ion enzyme, characterized in that it comprises the following steps: using a substituted styrene compound 1 and a substituted aniline compound 2 as substrates, reacting in a PBS buffer solution in the presence of a non-heme diferric ion amino oxidase AzoC to obtain a nitrone compound 3; the reaction equation is expressed as follows: in: R 1 , R 2 Each is independently selected from C1-C4 alkyl, C1-C4 alkoxy, nitro, trifluoromethyl or halogen.

2. The method for preparing nitrone compounds using non-heme diferric ion enzyme according to claim 1, characterized in that: The molar ratio of the substituted styrene compound 1 to the substituted aniline compound 2 is 1:1-1.

2.

3. The method for preparing nitrone compounds using non-heme diferric ion enzyme according to claim 1, characterized in that: The molar ratio of the substituted styrene compound 1, PBS and non-heme diferric ion amino oxidase AzoC is 1:0.01-0.02:0.005-0.

01.

4. The method for preparing nitrone compounds using non-heme diferric ion enzyme according to claim 1, characterized in that: PBS buffer solution pH = 4-6.

5. The method for preparing nitrone compounds using non-heme diferric ion enzyme according to claim 1, characterized in that: The reaction temperature was 25-30°C and the temperature was controlled by a constant temperature shaker.

6. The method for preparing nitrone compounds using non-heme diferric ion enzyme according to claim 1, characterized in that: GDH and NAD were also added during the reaction. + And Glucose.

Images