A monoterpene cyclopropanated derivative and its preparation method

Abstract

The present invention discloses a monoterpene cyclopropanated derivative and a preparation method thereof, belonging to the field of biosynthesis. In the present invention, perillyl alcohol, carveol, carvone or perillartine is used as a substrate, and the trifluoromethyl cyclopropanation modification is carried out by catalysis with the artificial metalloenzyme BM3I357AL86G-Fe(TPP)Cl to obtain the monoterpene cyclopropanated derivative; wherein, the artificial metalloenzyme BM3I357AL86G-Fe(TPP)Cl is prepared by titration reaction of the synthase BM3I357AL86G and Fe(TPP)Cl and then purified. The present invention realizes the trifluoromethyl cyclopropanation modification of four natural monoterpenes, namely L-carveol, carvone, perillyl alcohol and perillartine, by enzymatic catalysis under green and mild conditions, and obtains four natural monoterpene derivatives with new structures. Compared with chemical catalysis, the yield selectivity is improved, and the yield is significantly increased, and the monoterpene derivative 3b is increased by nearly 28 times.

Description

Technical Field

[0001] The present invention relates to the field of biosynthesis, and particularly to a monoterpene cyclopropanated derivative and a preparation method thereof. Background Art

[0002] The trifluoromethyl cyclopropyl group is very important in the field of pharmaceuticals. The correct introduction of some cyclopropane or fluorine-containing groups can increase bioavailability or improve the metabolic stability of drugs. However, due to the unique electronegativity of fluorine atoms, the introduction of fluorine atoms into specific positions has always been quite challenging, and the modification of important potential drug groups by biocatalytic methods has great potential.

[0003] Monoterpenes are a class of compounds composed of 2 isoprene units with 10 carbon atoms. Monoterpene derivatives containing heteroatoms (usually oxygen atoms) are also called monoterpenoids. In recent years, with the exploration of the biological activities of natural products in the field of drug development, monoterpenes and their derivatives have gradually shown new biological activities such as analgesic, antibacterial, anticonvulsant, and anticancer effects. It has been reported that the unnatural iron porphyrin Fe(TPP)Cl with four benzene rings can be used as a chemical catalyst for the chemical preparation of styrene trifluoromethyl cyclopropanated products. However, there are currently problems such as low yield and low selectivity in the trifluoromethyl cyclopropanation modification of natural monoterpenes. Therefore, there is an urgent need for the emergence of more effective chemical catalysts and catalytic methods to solve the existing technical problems. Summary of the Invention

[0004] The purpose of the present invention is to provide a monoterpene cyclopropanated derivative and a preparation method thereof to solve the problems existing in the above-mentioned prior art, and four new monoterpene derivatives are obtained by catalyzing monoterpenes with artificial metalloenzymes.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a monoterpene cyclopropanated derivative, and the structural formula of the monoterpene cyclopropanated derivative is any one of the following shown as 2b - 5b:

[0007]

[0008] The present invention also provides a preparation method of the monoterpene cyclopropanated derivative. Using perillyl alcohol, carveol, carvone or perillartine as a substrate, trifluoromethyl cyclopropanation modification is carried out by catalyzing with the artificial metalloenzyme BM3 I357AL86G - Fe(TPP)Cl to obtain the monoterpene cyclopropanated derivative; wherein, the artificial metalloenzyme BM3 I357AL86G - Fe(TPP)Cl is obtained by titration reaction of the synthase BM3 I357AL86G and Fe(TPP)Cl and then purified.

[0009] The present invention also provides that the molar ratio of the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl to the substrate is 1:(450 - 550).

[0010] Preferably, the conditions for the trifluoromethyl cyclopropanation modification catalyzed by the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl are as follows: reacting at room temperature for 12 - 18 h in the presence of a diazo compound gas.

[0011] Preferably, the preparation method of the synthase BM3 I357AL86G includes the following steps:

[0012] Transfer the gene encoding the BM3 I357AL86G protein into a vector to construct a recombinant vector;

[0013] Transfer the recombinant vector into Escherichia coli competent cells to construct a recombinant bacterium;

[0014] Induce and culture the recombinant bacterium with isopropyl-β-D-thiogalactoside and 5-aminolevulinic acid hydrochloride, collect the thalli, and then purify to obtain the synthase BM3 I357AL86G.

[0015] Preferably, the coding sequence and amino acid sequence of the BM3 I357AL86G protein are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

[0016] Preferably, the medium for the induction culture is 2×YT medium, and the culture conditions are culturing in a shaker at 37°C and 200 rpm until OD 600 = 0.8 - 1.0.

[0017] Preferably, the final concentrations of isopropyl-β-D-thiogalactoside and 5-aminolevulinic acid hydrochloride are both 1 mM.

[0018] Preferably, the titration reaction is as follows: drop the synthase BM3 I357AL86G into the Fe(TPP)Cl solution and stir for 1 - 3 h;

[0019] The purification is as follows: after the titration reaction is completed, collect the supernatant by centrifugation, dialyze the supernatant with a buffer solution, and then centrifuge to remove the precipitate, followed by desalting treatment to obtain the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl.

[0020] The present invention discloses the following technical effects:

[0021] The artificial metalloenzyme I357AL86G-Fe(TPP)Cl constructed in the present invention realizes the trifluoromethyl cyclopropanation modification of four natural monoterpenes, namely L-carveol, carvone, perillyl alcohol, and perillartine, under green and mild conditions, obtaining four natural monoterpene derivatives with new structures. The monoterpenes generated by the artificial metalloenzyme catalysis in the present invention have higher selectivity and significantly increased yields compared to chemical catalysis. Among them, the yield of monoterpene derivative 3b is increased by nearly 28 times, which lays a scientific foundation for large-scale production. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 Plasmid construction map of pET22b-BM3 I357L86G;

[0024] Figure 2 SDS-PAGE diagram of each gradient eluate for the purification of protein I357AL86G; 1: I357AL86G precipitate; 2: I357AL86G supernatant; 3: I357AL86G penetration solution; 4: I357AL86G 10 mM eluate; 5: I357AL86G 50 mM eluate; 6: I357AL86G 100 mM eluate; 7: I357AL86G 200 mM eluate;

[0025] Figure 3 UV-Vis spectrum of the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl;

[0026] Figure 4 Natural monoterpenes undergoing cyclopropanation reaction with ethyl diazoacetate; in the figure, 1a-7a are successfully converted into the expected cyclopropanated products;

[0027] Figure 5 GC-MS mass spectrum of the cyclopropanation of monoterpenes and ethyl diazoacetate;

[0028] Figure 6 GC-MS-TIC ion chromatogram of the cyclopropanation of monoterpenes and ethyl diazoacetate;

[0029] Figure 7 Schematic diagram of the reaction device for the enzymatic catalysis of the trifluoromethyl cyclopropanation of natural monoterpenes;

[0030] Figure 8The structure for the successful catalytic monoterpene trifluoromethylcyclopropanation by BM3 I357AL86G-Fe(TPP)Cl;

[0031] Figure 9 The GC-MS-TIC ion current diagram for the trifluoromethylcyclopropanation catalyzed by BM3 I357AL86G-Fe(TPP)Cl;

[0032] Figure 10 The GC-MS mass spectrum diagram for the trifluoromethylcyclopropanation reaction of natural monoterpenes;

[0033] Figure 11 The HPLC diagram for the trifluoromethylcyclopropanation product 5b of perillartine;

[0034] Figure 12 The LC-MS diagram for the trifluoromethylcyclopropanation product 5b of perillartine;

[0035] Figure 13 The LC-MS diagram for the trifluoromethylcyclopropanation product 4b of carvone;

[0036] Figure 14 The GC-TOF-MS diagram for the trifluoromethylcyclopropanation product 2b of perilla alcohol;

[0037] Figure 15 The GC-TOF-MS diagram for the trifluoromethylcyclopropanation product 3b of L-carveol;

[0038] Figure 16 For the trifluoromethylcyclopropanation product 5b of perillartine 1 1H NMR;

[0039] Figure 17 For the trifluoromethylcyclopropanation product 5b of perillartine 13 13C NMR;

[0040] Figure 18 The 19F NMR diagram for the trifluoromethylcyclopropanation product 5b of perillartine. Detailed implementation manners

[0041] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0042] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0043] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0044] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0045] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0046] Example 1

[0047] 1. Test method

[0048] 1.1 Construction of artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl

[0049] The gene of P450 BM3 I357AL86G mutant (nucleotide sequence shown in SEQ ID NO: 1, amino acid sequence shown in SEQ ID NO: 2) was synthesized and cloned into the pET22b vector to construct pET22b-BM3 I357L86G( Figure 1 ), and then expressed and purified.

[0050] SEQ ID NO: 1

[0051]

[0052] SEQ ID NO: 2

[0053] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGGFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLAPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRASIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPL.

[0054] To introduce the non-native metal porphyrin Fe(TPP)Cl of the catalytic center into the protein BM3, the plasmid pET22b-I357AL86G of the P450 BM3 mutant protein I357AL86G was constructed. The plasmid pET22b-I357AL86G was transformed into the Escherichia coli competent cell BL21(DE3). It was heat-shocked at 42 °C for 90 s and then immediately placed on ice for 2 min, and spread on an ampicillin-resistant LB plate and cultured overnight in a 37 °C incubator. The next day, the transformants were picked into a liquid medium of LB ampicillin resistance and cultured in a shaker at 37 °C and 200 rpm for 12 h, and then transferred at 1% of the medium volume to 1 L of 2×YT medium containing ampicillin resistance and cultured in a shaker at 37 °C and 200 rpm until 600 OD = 0.8 - 1.0, and 1.0 mM IPTG (isopropyl-β-D-thiogalactoside) and 1.0 mM ALA (5-aminolevulinic acid hydrochloride) were added, and then it was continued to be cultured in a shaker at 30 °C and 200 rpm for 16 h.

[0055] The expressed medium was centrifuged at 8000 rpm and 4 °C to collect the bacterial cells, and then the protein was purified according to the instructions of the Ni-NTA 6FF His-tag Protein Purification Kit of Sangon Biotech. Then, the protein bands were verified according to the instructions of the Sangon SDS-PAGE Kit, and the protein I357AL86G was successfully obtained. It was identified that the target protein component of I357AL86G (52 kDa) was successfully eluted in the eluent containing 50, 100, and 250 mM imidazole ( Figure 2 ).

[0056] Then, it was titrated and recombined with the unnatural porphyrin Fe(TPP)Cl to obtain the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl. The specific operation steps were as follows:

[0057] (1) Under the condition of 4 °C, the cofactor-free I357AL86G protein (10 - 50 μmol / L) obtained above was added dropwise to the metal porphyrin (>1 mmol / L) dissolved in 0.01 mol / L NaOH solution, and stirred for 1 - 3 h;

[0058] (2) The supernatant was collected in a low-temperature centrifuge at 8000 rpm for 30 min, and dialyzed 4 times with 10 mmol / L KPi phosphate buffer at pH 7.0 at 4 °C for 4 h each time;

[0059] (3) After dialysis, the precipitate was removed by centrifugation, and the excess heme or metal complex was removed by a desalting column;

[0060] (4) Concentrate to an appropriate volume, and replace the buffer with a 100 mmol / L KPi phosphate buffer at pH 7.0 using an ultrafiltration concentrator or a desalting column.

[0061] The artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl was identified by UV-Vis spectroscopy. The results showed that the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl was successfully obtained, the solution showed a brownish red color, and a characteristic absorption peak after binding porphyrin was shown at 417 nm ( Figure 3 ), proving that the unnatural porphyrin Fe(TPP)Cl was successfully assembled into the BM3 I357AL86G protein.

[0062] 1.2 BM3 I357AL86G-Fe(TPP)Cl Catalyzes the Cyclopropanation of Natural Monoterpenes and Ethyl Diazoacetate

[0063] In order to achieve trifluoromethylcyclopropanation modification of natural monoterpenes, a natural monoterpene substrate library was first established, and enzymatic reactions were carried out with ethyl diazoacetate donors. Ethyl diazoacetate was first used as a carbene donor because the fluorine atom in diazotrifluoroethane itself is very active and difficult to control in the reaction. In addition, ethyl diazoacetate exists in liquid form, which is more suitable for preliminary screening than gaseous diazotrifluoroethane donors. Monoterpene substrates that can be catalyzed by artificial enzymes were screened and further used for trifluoromethylcyclopropanation modification.

[0064] Selection of natural monoterpene substrates: Natural monoterpenes with terminal olefins are preferred, and the structure should not be too large to prevent the substrate from entering the enzyme active center. The substrate is dissolved in anhydrous ethanol to prepare a 400mmol / L substrate solution, and ethyl diazoacetate is also dissolved in anhydrous ethanol to prepare a 600mmol / L solution. The total reaction system is 400μL. Under argon deoxygenation, first add the artificial metal enzyme BM3 I357AL86G-Fe(TPP)Cl with a final concentration of 20μmol / L, dissolved in 50mmol / L phosphate buffer with a pH value of 7.0; then add a sodium dithionite solution with a final concentration of 10mmol / L, and use a double-row tube device to continue to remove the air in the bottle. Then add a substrate with a final concentration of 10mmol / L, and continue to blow in argon for 1min. And use a micro-syringe pump to add 15μL of 600mmol / L ethyl diazoacetate solution within 4h, and react at room temperature for 12h. After the reaction, the mixture was extracted with 400 μL×3 volume of 1:1 dichloromethane, centrifuged at 13000 rpm for 10 min, and the organic layer was taken for GC-MS detection.

[0065] The results showed that natural monoterpene compounds such as perillyl alcohol, L-carveol, carvone, myrcene, valenic acid tangerine, perillaldehyde, carvyl acetate, limonene, ocimene, and forskolin were selected as substrates for enzymatic cyclopropanation ( Figure 4 ). 4-Chlorostyrene has been reported to react with ethyl diazoacetate under the catalysis of myoglobin to produce a carbene transfer product, which exists in the GC-MS spectral library. Therefore, a catalytic experiment with 4-chlorostyrene as a substrate was added, and the GC-MS results of the product were compared with those reported in the literature to determine the feasibility of the experimental operation method and the expected idea. The target product can be identified by the molecular ion peak value by viewing the GC-MS spectrum. The positive ion formed by the loss of an electron by an organic compound molecule is called a molecular ion (M +· ), its mass-to-charge ratio (m / z) represents the relative molecular mass value of the target molecule. The retention time of 1a detected by gas chromatography is 15.198min, and the molecular ion peak of the mass-to-charge ratio (m / z) of the mass spectrum is 224 ( Figure 5 ), which is consistent with the relative molecular mass (M) of the expected product 1a of 224, and M:M +2The relative abundance ratio of is equal to 3:1, indicating the presence of Cl element in the structure. It is also consistent with the 1a structure in the NIST mass spectrometry library through similarity search. Based on the realization of the catalysis of 4-chlorostyrene, the expected idea was verified to be feasible. Therefore, the enzymatic reaction of cyclopropanation of terpenoid natural products 2-12 with ethyl diazoacetate was continued using BM3 I357AL86G-Fe(TPP)Cl. As Figure 6 shown, it shows the expected cyclopropanation product structures of each monoterpene. The retention time of 2a is 17.415 min; the retention time of 3a is 16.573 min; the retention time of 4a is 16.014 min; the retention time of 5a is 19.125 min; the retention time of 6a is 12.716 min; the retention time of 7a is 19.354 min. They were detected by GC-MS. Under the catalysis of the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl, the expected products were generated with conversion rates of 3.1%, 50.1%, 8.5%, 0.4%, 3.9%, and 0.3% respectively. Their molecular ion peaks are all consistent with the expected products. The molecular ion peaks of 2a and 3a are both 238; the molecular ion peak of 4a is 236; the molecular ion peak of 5a is 251; the molecular ion peak of 6a is 222; the molecular ion peak of 7a is 290( Figure 5 ). After preliminary screening, several natural monoterpene substrates, perillyl alcohol 2, L-carveol 3, carvone 4, perillartine 5, myrcene 6, and valencene 7, were used as substrates for the subsequent trifluoromethylcyclopropanation reaction.

[0066] 1.3 Trifluoromethylcyclopropanation of natural monoterpenes catalyzed by BM3 I357AL86G-Fe(TPP)Cl

[0067] Since the trifluoromethyl donor for the enzymatic trifluoromethylcyclopropanation of natural monoterpenes is diazotrifluoroethane, but diazotrifluoroethane is a gas and needs to be prepared on-site from 2,2,2-trifluoroethylamine hydrochloride, the double reaction chamber method was used for this experiment( Figure 7) That is, the CF3CHN2 gas generated in reaction chamber one is transported to reaction chamber two through a double-headed solvent transfer needle. The reaction is carried out under anaerobic conditions. 244 mg of 2,2,2-trifluoroethylamine hydrochloride, 20 mg of anhydrous sodium acetate, and 12.4 mg of 4-dimethylaminopyridine are added to reaction chamber one, and 3 mL of degassed water is added; then 6.6 μL of concentrated H2SO4 dissolved in 0.5 mL of degassed water is added, and it is degassed for 1 min; a solution of 149 mg of NaNO2 dissolved in degassed distilled water is added within 4 h; the total reaction system in reaction chamber two is 400 μL, and artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl with a final concentration of 20 μmol / L is added, dissolved in 50 mmol / L phosphate buffer at pH 7.0; then sodium dithionite with a final concentration of 10 mmol / L is added; the air in the bottle is removed using a double-tube device, and then a 10 mmol / L substrate (the substrate is pre-prepared as a 400 mmol / L stock solution) is added, and argon is continuously blown in for 1 min. The diazo compound gas generated in reaction chamber one is continuously introduced into reaction chamber two through the double-headed solvent transfer needle using argon, and the reaction is carried out at room temperature under anaerobic conditions for 18 h. In the control group, the enzyme is replaced with 50 mmol / L phosphate buffer at pH 7.0, and the other conditions remain unchanged. After the reaction is completed, it is extracted three times with an equal volume of dichloromethane, centrifuged at 13000 rpm for 10 min, and the organic layer is taken for GC-MS detection.

[0068] Using artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl as a protein catalyst, perillyl alcohol 2, L-carvomenthol 3, carvone 4, perillartine 5, myrcene 6, and valencene 7 are used as substrates for trifluoromethylcyclopropanation reaction. After GC-MS detection, the corresponding trifluoromethylcyclopropanation products are detected for 2-5. 2b, 3b, 4b, and 5b are successfully converted with conversion rates of 3.4%, 44.2%, 4.1%, and 1.1% respectively. Figure 8 The structures of the expected cyclopropanation products of each monoterpene are shown.

[0069] As Figure 9As shown, the target peaks of the 2b product appear at 12.130, 12.355, and 12.515 min respectively, and the peak area ratio is 13.3:47.2:39.5; since the substrate 3 of 3b is a cis-trans isomer, more groups of product peaks will be detected compared to other substrates. The target peaks of 3b appear at 10.650, 10.950, 11.090, 11.145, 11.200, 11.475, and 11.575 min respectively, and the peak area ratio is 11.6:2.2:27.1:1.7:26.4:21.9:19.2; the target peaks of 4b appear at 11.545 and 11.745 min respectively, and the peak area ratio is 34.5:65.5; the target peaks of 5b appear at 14.190, 14.270, 14.500, and 14.570 min respectively, and the peak area ratio is 3.1:6.8:48.0:42.1. Their molecular ion peaks are all consistent with the expected products.

[0070] During the detection process, multiple target peaks were found. This is because the formation of trifluoromethyl cyclopropanation products will have at least 2 chiral centers. At the same time, the substrate L-carvone 3 of 3b itself is a cis-trans isomer mixture, so the product peaks of 3b are the most, with 7 detected. Due to the low conversion rate or the existence of multiple mixed isomers, a single configuration product has not been separated and prepared. The ratios of each peak are listed here to present the proportion of each configuration. For example Figure 10 , which shows the GC-MS mass spectra of the peak with the largest peak height for each product. The molecular ion peak of 2b is 234, GC-MS m / z (% relative intensity): 234(1.86), 138(17.9), 107(100.0), 138(17.9), 203(10.8), 79(62.9); the molecular ion peak of 3b is 234, GC-MS m / z (% relative intensity): 234(38.5), 137(85.1), 109(100.0), 84(69.4), 138(25.7), 201(10.8), 219(11.6); the molecular ion peak of 4b is 232, GC-MS m / z (% relative intensity): 232(2.1), 136(90.1), 108(46.4), 82(45.4), 190(2.8), 149(2.0), 93(100.0); the molecular ion peak of 5b is 247, GC-MS m / z (% relative intensity): 247(75.4), 248(11.0), 230(55.9), 213(16.6), 150(27.2), 133(99.2), 122(77.0), 106(94.1), 91(99.3), 79(100.0).

[0071] Meanwhile, the synthesis of trifluoromethyl cyclopropanation products using a chemical catalyst was used as a control (for the purpose of comparing the products synthesized by artificial metalloenzymes). The specific steps are as follows:

[0072] Add Fe(TPP)Cl (0.0066 mmol = 4.65 mg), 4-dimethylaminopyridine (DMAP) (0.022 mmol = 2.69 mg), anhydrous sodium acetate (0.044 mmol = 3.61 mg), and 2,2,2-trifluoroethylamine hydrochloride (0.33 mmol = 44.72 mg) to a round-bottom flask. Add degassed distilled water (1 mL) and concentrated H2SO4 (1.2 μL, 0.022 mmol), and degas the solution by blowing argon for 1 min. Subsequently, add the substrate (0.22 mmol = 33.44 mg) to the round-bottom flask, and add the NaNO2 solution (27 mg, 0.399 mmol, dissolved in 1 mL of degassed distilled water) to the round-bottom flask via a syringe pump within 10 h. After reacting for an additional 4 h, add dichloromethane and water, and extract the aqueous phase with dichloromethane three times. Collect the organic layer and concentrate it under reduced pressure. Filter the crude reaction mixture through silica gel and elute with dichloromethane to collect the fractions for GC-MS analysis. (This experimental design is used as a control for the catalytic effect of artificial enzymes).

[0073] The four unreported trifluoromethyl cyclopropanation reaction products, 2b, 3b, 4b, and 5b, were separated and purified respectively. Since the products were not of a single configuration and the conversion rate was low, only the perillartine derivative 5b accumulated and approximately 2 mg of the sample was isolated. The purity of the prepared 5b sample was detected by HPLC, and the retention time was 37.848 min ( Figure 11 ).

[0074] The LC-MS results of the trifluoromethylated product 5b of perillartine were confirmed again by LC-MS, showing [M+H] + value of 248.1257 ( Figure 12 ). The molecular weight of the trifluoromethylated product 4b of carvone was also determined again by LC-MS for [M+H] + value of 233.1148 ( Figure 13 ), both of which were exactly the same as the theoretical [M+H] + molecular weight of the product (accurate to the 4th decimal place). However, the structures of the alcohol hydroxyl groups in 2b and 3b were difficult to ionize with an ESI source, and the accurate molecular weight could not be obtained by LC-MS. Therefore, a GC-TOF-MS instrument was used to further identify the products 2b and 3b. The results showed that the molecular ion peak of 2b [M +· = 234.1233, which was consistent with the target product and within the error range of the theoretical value of 134.1231 ( Figure 14 ). The results showed that the molecular ion peak of 3b [M+· = 234.1224, which is consistent with the target product and within the error range of the theoretical value 134.1231 ( Figure 15 ). The trifluoromethylated product 5b of perillartine was 1 characterized by \(^1H\) NMR (700 MHz), 13 \(^{13}C\) NMR (700 MHz), 19 \(^{19}F\) NMR (645 MHz). The compound structure of the trifluoromethylated product 5b of perillartine was listed for convenient comparison with the results of NMR spectrum analysis ( Figures 16 - 18 ).

[0075] The results showed that the conversion rate of the product 3b catalyzed by the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl (44.2%, conversion rate % = [sum of peak areas of the target product / (sum of peak areas of all products + peak area of the remaining substrate)] × 100%) was nearly 28 times higher than that of the trifluoromethylcyclopropanation reaction of carvone catalyzed by the chemical catalyst Fe(TPP)Cl alone. The conversion rate of the trifluoromethylcyclopropanation reaction of carvone catalyzed by Fe(TPP)Cl to produce the product 3b was only 1.6%. As Figure 4 , it is the trifluoromethylcyclopropanation reaction of natural monoterpenes catalyzed by BM3 I357AL86G-Fe(TPP)Cl.

[0076] The above embodiments are only described for the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A monoterpene cyclopropanated derivative, characterized in that, The structural formula of the monoterpene cyclopropanated derivative is any one of the following shown in 2b-5b:

2. A method for preparing the monoterpene cyclopropanated derivative according to claim 1, characterized in that, Using perillyl alcohol, carveol, carvone or perillartine as a substrate, the monoterpene cyclopropanated derivative is prepared by catalyzing trifluoromethyl cyclopropanation modification with the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl; wherein, the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl is prepared by titration reaction of the synthase BM3 I357AL86G and Fe(TPP)Cl and then purified.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl to the substrate is 1:(450-550).

4. The preparation method according to claim 2, wherein The conditions for catalyzing trifluoromethyl cyclopropanation modification with the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl are: reacting at room temperature for 12-18 h in the presence of a diazo compound gas.

5. The preparation method according to claim 2, characterized in that, The preparation method of the synthase BM3 I357AL86G includes the following steps: Transfer the gene encoding the BM3 I357AL86G protein into a vector to construct a recombinant vector; Transfer the recombinant vector into Escherichia coli competent cells to construct a recombinant bacterium; The recombinant bacterium is induced and cultured with isopropyl-β-D-thiogalactoside and 5-aminolevulinic acid hydrochloride, the cells are collected and then purified to obtain the synthase BM3 I357AL86G.

6. The preparation method according to claim 5, wherein The coding gene sequence and amino acid sequence of the BM3 I357AL86G protein are respectively shown in SEQ ID NO: 1 and SEQ ID NO:

2.

7. The preparation method according to claim 5, characterized in that, The medium for induced culture is 2×YT medium, and the culture conditions are culturing in a shaker at 37°C and 200 rpm until OD 600 = 0.8 - 1.

0.

8. The preparation method according to claim 5, characterized in that, The final concentrations of isopropyl-β-D-thiogalactoside and 5-aminolevulinic acid hydrochloride are both 1 mM.

9. The preparation method according to claim 2, characterized in that, The titration reaction is: adding the synthase BM3 I357AL86G dropwise to the Fe(TPP)Cl solution and stirring for reaction for 1-3 h; The purification is: after the titration reaction is completed, the supernatant is collected by centrifugation, the supernatant is dialyzed with a buffer solution, and then the precipitate is removed by centrifugation, followed by desalting treatment to obtain the artificial metalloenzyme BM3 I357AL86G-Fe(TPP)Cl.

Images