Mononuclear non-heme iron enzyme catalytic product indole compound and preparation method thereof

Abstract

The invention relates to a mononuclear non-heme iron enzyme catalytic product indole compound and a preparation method thereof, and relates to the technical field of enzyme engineering. According to the method, a natural substrate is obtained through fermentation of a bean dreg culture medium, target protein is induced, expressed and purified by using an escherichia coli chassis, and then large-scale biochemical reaction of the protein, the substrate and cofactors is carried out in vitro. The compound is prepared by combining modern chromatographic separation and purification technologies such as analytical, semi-preparative and preparative high performance liquid chromatographs, medium-pressure liquid chromatographs and the like. The structure is determined to be a novel enzymatic product of OkaE in-vitro catalysis by adopting a spectroscopy structure identification method. According to the invention, the molecular structure of okaramine is modified by adopting an enzymatic modification strategy, an important way is provided for widening the structural diversity of okaramine alkaloids, and the method has remarkable scientific significance and application value.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to an indole compound catalyzed by a mononuclear nonheme iron enzyme and its preparation method. Background Technology

[0002] Natural products are an important source of green pesticides. Natural product pesticides mainly refer to pesticides developed from insecticidal secondary metabolites produced by plants, animals, and microorganisms. Compared with traditional chemically synthesized pesticides, natural product pesticides have many advantages, including high activity, high selectivity, environmental friendliness, human friendliness, and low likelihood of inducing biological resistance. To date, many natural product pesticides, such as pyrethroids, rotenone, and abamectin, have been widely used.

[0003] Fungal indole alkaloids possess a complex core structural framework and a wide range of biological activities, including antibacterial, antitumor, antimalarial, and anti-inflammatory effects. Okaramine-type indole alkaloids, isolated and identified from species of *Penicillium* and *Aspergillus*, are a class of natural products associated with crop disease resistance. These alkaloids share the same target as the commercially available insecticide ivermectin and its derivatives, acting on glutamate-gated chloride channels (GluCls) in insects, and hold promise as lead compounds for novel insecticides. Furthermore, okaramine B, a representative natural product of the okaramine class, is inactive against common chloride channels in humans and exhibits lower toxicity to mammals compared to ivermectin, demonstrating greater safety for humans. Therefore, okaramine B and its derivatives can be used to develop novel, highly effective, and environmentally friendly insecticides that target insect GluCls specifically.

[0004] Okaramine B, a representative compound of okaramine alkaloids, possesses an octagonal multichiral central structural framework comprising a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered azacyclic ring, an indole ring, and a four-membered azacyclic butane. Structure-activity relationship studies have shown that azacyclic butane significantly enhances the insecticidal activity of okaramine alkaloids. In the biosynthetic pathway of okaramine alkaloids, azacyclic butane is formed by the catalysis of the α-ketoglutarate-dependent mononuclear nonheme iron enzyme OkaE. In the field of natural product synthesis, OkaE is the first α-ketoglutarate-dependent mononuclear nonheme iron enzyme to catalyze the formation of azacyclic butane, and its unique chemical function and novel catalytic mechanism are of significant research value.

[0005] Currently, research on the structure of okaramine alkaloids mostly focuses on the isolation of natural products or chemical synthesis. In vivo okaE knockout experiments yield relatively limited products. Furthermore, because aziridines are four-membered ring structures, they exhibit significant ring strain. During the formation of the four-membered ring, the bond angles between the ring-forming atoms undergo significant distortion, deviating considerably from the ideal bond angles. This leads to increased molecular energy, decreased stability, and thermodynamically unfavorable reactions, making reverse reactions more likely and reducing yields. Moreover, multiple reaction sites may exist in the synthesis of aziridines, making it difficult to control the stereoselectivity of the reaction. Therefore, the chemical synthesis of aziridine structures of okaramine alkaloids is challenging. Thus, modifying the okaramine molecular structure using enzymatic modification methods is of great significance for broadening the chemical cosmology of okaramine alkaloids. Summary of the Invention

[0006] Technical Objective: This invention provides indole compounds catalyzed by mononuclear non-heme iron enzymes and their preparation methods. The objective is to modify okaramine molecules using enzymatic modification methods to produce novel structures, and to provide a novel okaramine-type indole alkaloid and its preparation method.

[0007] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0008] A method for preparing indole compounds, the catalytic products of mononuclear non-heme iron enzymes, involves mixing OkaE purified enzyme, substrate Okaramine A, and Fe. Ⅱ α-KG and Vc are added to an oxygen-saturated solution to activate the enzymatic reaction; the reaction product is extracted and separated to obtain the catalytic product, an indole compound.

[0009] Preferably, the specific steps are as follows:

[0010] Step 1: Prepare a mixture containing okaramine A, OkaE, and Fe. Ⅱ α-KG, ascorbic acid and DMSO, the solvent used is a buffer solution: 100mM Tris-HCl and 50mM NaCl, pH 7.5;

[0011] Step 2: Seal the mixed reaction mixture in a flask, add 10 mL of oxygen-saturated buffer solution, and the dissolved oxygen concentration is 1.25 mM;

[0012] Step 3: After incubation for 30 minutes, add 14 mL of ethyl acetate to quench the enzyme reaction, centrifuge at 13,000 rpm for 10 min to remove the precipitated protein, and separate the ethyl acetate layer;

[0013] Step 4: Continue to extract the reaction mixture with 14 mL of ethyl acetate; combine the two ethyl acetate layers and concentrate the extract using a rotary evaporator;

[0014] Step 5: Redissolve the residue in a 1:1 mixture of CH2Cl2 and ACN.

[0015] Preferably, the mixture system used in step 1 comprises 4 mL and contains 210 μMokaramine A, 70 μMokaramine E, and 2,100 μMFe. Ⅱ The final concentration was 3.6%, consisting of 700 μM α-KG, 210 μM ascorbic acid, and DMSO.

[0016] Preferably, the mixed reactants in step 3 must be incubated at room temperature for 30 minutes.

[0017] Preferably, the specific steps of the extraction and separation method include:

[0018] Step 1: The mixed solution was separated using reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column was selected, and the flow rate was set to 3.0 mL / min. Specifically, the concentration of acetonitrile in water was gradually increased from 34% to 53.2% within 0 to 32 minutes, and then eluted with a 99% acetonitrile aqueous solution for another 5 minutes to obtain four components.

[0019] Step 2: Component 1 was purified using semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column was selected, and the flow rate was set to 1.0 mL / min. Specifically, the concentration of acetonitrile in 0.1% formic acid aqueous solution was gradually increased from 25% to 35% within 0 to 20 minutes; and the concentration of acetonitrile in 0.1% formic acid aqueous solution was gradually increased from 35% to 40% within 20 to 30 minutes, yielding in vitro catalytic product 4.

[0020] Step 3: Component 3 was purified using semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column was selected, and the flow rate was set to 1.0 mL / min. Specifically, from 0 to 27 minutes, the concentration of acetonitrile in H2O was 33%. From 27 to 40 minutes, the concentration of acetonitrile in H2O gradually increased from 36% to 53%, yielding in vitro catalytic product 5.

[0021] Step 4: Component 4 was purified using semi-preparative reversed-phase high-performance liquid chromatography; a C18 column was selected and the flow rate was set to 1.0 mL / min; specifically, the concentration of MeOH in H2O was 62% from 0 to 28 minutes; from 28 to 40 minutes, the concentration of MeOH in H2O gradually increased from 62% to 65%, yielding in vitro catalytic product 6 and in vitro catalytic product 7.

[0022] Preferably, the mobile phase elution program used in step 1 is gradient elution; the mobile phase elution program used in step 2 is gradient elution; the mobile phase elution program used in step 3 is gradient elution; and the mobile phase elution program used in step 4 is gradient elution.

[0023] Preferably, the catalytic product indole compounds include,

[0024] The in vitro catalytic product 4 of α-ketoglutarate-dependent mononuclear non-heme ironase is a compound with an octyl ring multichiral central skeleton containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an octyl nitrogen heterocycle, and a carboxyl-modified four-membered nitrogen heterocycle butane. Its molecular formula is C4. 32 H 30 N4O5, the structural formula is shown in Formula 4;

[0025] The in vitro catalytic product 5 of α-ketoglutarate-dependent mononuclear non-heme ironase is a compound with a nine-ring multichiral central structure containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, a four-membered nitrogen heterocycle butane, and a five-membered internal ether ring. Its molecular formula is C5. 32 H 30 N4O4, the structural formula is shown in Formula 5;

[0026] The in vitro catalytic product 6 of α-ketoglutarate-dependent mononuclear non-heme ironase is a compound with a heptaneous multichiral central skeleton containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, and an isopentenyl side chain. Its molecular formula is C60. 32 H 32 N4O4, the structural formula is shown in Formula 6;

[0027] The in vitro catalytic product 7 of α-ketoglutarate-dependent mononuclear non-heme ironase is a compound with a seven-ring multichiral central skeleton containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, and a three-membered oxygen ring. Its molecular formula is C7. 32 H 30 N4O5, the structural formula is shown in Formula 7.

[0028]

[0029] Preferably, the method for preparing and separating the substrate okaramine A includes the following steps:

[0030] Step 1: The fungal strain Penicillium simplicissimum ATCC 90288 was streaked in three zones onto PDA solid medium and incubated at 28°C for 3 days to obtain the fungal solid medium.

[0031] Step 2: Cut the fungal solid culture medium into blocks, inoculate into 100mL of PDB liquid culture medium, and culture at 28℃ and 220rpm for 3 days to obtain fungal seed liquid; the fermentation culture method is to inoculate 5mL of fungal seed liquid into 50g of fresh soybean residue culture medium and incubate at 28℃ for 14 days.

[0032] Step 3: The fermented cells were extracted four times with 10L of acetone. The organic phase was removed by vacuum concentration. The remaining aqueous phase was extracted four times with ethyl acetate to obtain the crude extract.

[0033] Step 4: The crude extract obtained from fermentation was initially separated using normal-phase silica gel column chromatography; CH2Cl2—MeOH was used as the elution mobile phase for silica gel column chromatography, and gradient elution was adopted: 100:0, 50:1, 20:1, 10:1 and 0:100 to obtain five elution fractions A, B, C, D and E;

[0034] Step 5: Dissolve component B in a 1:1 mixed solution of CH2Cl2 and MeOH, and separate it by reversed-phase silica gel column chromatography. Use MeOH-H2O as the elution mobile phase for silica gel column chromatography, and use a gradient elution of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% by volume for B1-B8.

[0035] Step 6: Component B5 was separated by semi-preparative reversed-phase high-performance liquid chromatography; further purification was performed using a C18 column with 68% MeOH-H2O as the mobile phase and isocratic elution at a flow rate of 3.0 mL / min to finally obtain okaramine A, the natural catalytic substrate of α-ketoglutarate-dependent mononuclear nonheme iron enzyme OkaE.

[0036] Beneficial effects:

[0037] This invention provides the extraction, separation, and identification of catalytic products following the in vitro catalytic reaction of α-ketoglutarate-dependent mononuclear nonheme iron enzyme OkaE.

[0038] This invention provides a method for enzymatically modifying okaramine molecules, which efficiently modifies novel okaramine molecular structures.

[0039] This invention expands the chemical space of okaramine-type indole alkaloids by modifying the okaramine molecule, which is of great significance for adding more indole compounds with potential green insecticide value. Attached Figure Description

[0040] Figure 1Chemical structure diagrams and HPLC chromatograms of the original product (1-3) and novel enzymatic product (4-7) of the α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE provided in the embodiments of the present invention;

[0041] Figure 2 The HR-ESI-MS image of compound 1 in acetone-d6 provided in the embodiments of the present invention;

[0042] Figure 3 Compound 1 provided in the embodiments of the present invention contains acetone-d6. 1 H NMR spectrum;

[0043] Figure 4 Compound 1 provided in the embodiments of the present invention contains acetone-d6. 13 C NMR spectrum;

[0044] Figure 5 The HR-ESI-MS image of compound 2 in acetone-d6 provided in the embodiments of the present invention;

[0045] Figure 6 Compound 2 provided in the embodiments of the present invention in acetone-d6 1 H NMR spectrum;

[0046] Figure 7 Compound 2 provided in the embodiments of the present invention in acetone-d6 13 C NMR spectrum;

[0047] Figure 8 The above is an HR-ESI-MS image of compound 3 in methanol-d4 provided in the embodiments of the present invention;

[0048] Figure 9 Compound 3 provided in the embodiments of the present invention in methanol-d4 1 H NMR spectrum;

[0049] Figure 10 Compound 3 provided in the embodiments of the present invention in methanol-d4 13 C NMR spectrum;

[0050] Figure 11 The relative configurations of compounds 1-3 provided in the examples of this invention;

[0051] Figure 12 The HR-ESI-MS image of compound 4 in acetone-d6 provided in the embodiments of the present invention;

[0052] Figure 13 Compound 4 provided in the embodiments of the present invention contains acetone-d6. 1 H NMR spectrum;

[0053] Figure 14 Compound 4 provided in the embodiments of the present invention contains acetone-d6. 13 C NMR spectrum;

[0054] Figure 15 The HSQC diagram of compound 4 in acetone-d6 provided in the embodiments of the present invention;

[0055] Figure 16 HMBC diagram of compound 4 in acetone-d6 provided in the embodiments of the present invention;

[0056] Figure 17 The COSY diagram of compound 4 in acetone-d6 provided in the embodiments of the present invention;

[0057] Figure 18 The NOESY diagram of compound 4 in acetone-d6 provided in the embodiments of the present invention;

[0058] Figure 19 The relative configuration of compound 4 and key two-dimensional NMR correlations provided for examples of the present invention;

[0059] Figure 20 The HR-ESI-MS image of compound 5 in acetone-d6 provided in the embodiments of the present invention;

[0060] Figure 21 Compound 5 provided in the embodiments of the present invention contains acetone-d6. 1 H NMR spectrum;

[0061] Figure 22 Compound 5 provided in the embodiments of the present invention contains acetone-d6. 13 C NMR spectrum;

[0062] Figure 23 The HSQC diagram of compound 5 in acetone-d6 provided in the embodiments of the present invention;

[0063] Figure 24 HMBC diagram of compound 5 in acetone-d6 provided in the embodiments of the present invention;

[0064] Figure 25The COSY diagram of compound 5 in acetone-d6 provided in the embodiments of the present invention;

[0065] Figure 26 The NOESY diagram of compound 5 in acetone-d6 provided in the embodiments of the present invention;

[0066] Figure 27 The relative configuration and key two-dimensional NMR correlation of compound 5 provided for examples of the present invention;

[0067] Figure 28 The above is an HR-ESI-MS chromatogram of compound 6 in pyridine-d5 provided in the embodiments of the present invention;

[0068] Figure 29 Compound 6 provided in the embodiments of the present invention contains pyridine-d5. 1 H NMR spectrum;

[0069] Figure 30 Compound 6 provided in the embodiments of the present invention contains pyridine-d5. 13 C NMR spectrum;

[0070] Figure 31 The HSQC diagram of compound 6 in pyridine-d5 provided in the embodiments of the present invention;

[0071] Figure 32 The HMBC diagram of compound 6 in pyridine-d5 provided in the embodiments of the present invention;

[0072] Figure 33 The COSY diagram of compound 6 in pyridine-d5 provided in the embodiments of the present invention;

[0073] Figure 34 The NOESY diagram of compound 6 in pyridine-d5 provided in the embodiments of the present invention;

[0074] Figure 35 The relative configuration and key two-dimensional NMR correlation of compound 6 provided for examples of the present invention;

[0075] Figure 36 The HR-ESI-MS chromatogram of compound 7 in pyridine-d5 provided in the embodiments of the present invention;

[0076] Figure 37 Compound 7 provided in the embodiments of the present invention contains pyridine-d5. 1 H NMR spectrum;

[0077] Figure 38 Compound 7 provided in the embodiments of the present invention contains pyridine-d5. 13 C NMR spectrum;

[0078] Figure 39 The HSQC diagram of compound 7 in pyridine-d5 provided in the embodiments of the present invention;

[0079] Figure 40 The HMBC diagram of compound 7 in pyridine-d5 provided in the embodiments of the present invention;

[0080] Figure 41 The COSY diagram of compound 7 in pyridine-d5 provided in the embodiments of the present invention;

[0081] Figure 42 The NOESY diagram of compound 7 in pyridine-d5 provided in the embodiments of the present invention;

[0082] Figure 43 The relative configuration of compound 7 provided in this invention and its key two-dimensional NMR correlation. Detailed Implementation

[0083] The following is in conjunction with the appendix Figure 1 To be continued Figure 43 The principles and features of the present invention are described, and the examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0084] This invention employs various separation materials such as normal-phase silica gel, reverse-phase silica gel, and dextran gel, combined with modern chromatographic separation and purification techniques including analytical, semi-preparative, and preparative high-performance liquid chromatography (HPLC) and medium-pressure liquid chromatography (HPLC), and obtains the natural substrates okaramine A, 12-deshydroxyl okaramine E, and the product okaramine E catalyzed by OkaE from the fermentation extract of the source strain P. simplecissimum ATCC 90288 through structural identification methods such as spectroscopic analysis.

[0085] After obtaining the cDNA of okaE from the NCBI website, a pASK-3plus-okaE plasmid containing the okaE gene sequence was synthesized by the company. This plasmid was then transformed into an E. coli expression system. Transformants that were verified to be correct by sequencing were used for protein expression and purification to obtain the OkaE protein. The purified enzyme protein, substrate, and cofactor were mixed, and then an oxygen-saturated solution was added to activate the enzymatic reaction. The reaction product was extracted and detected by high-resolution liquid chromatography-mass spectrometry to identify the reaction type and product of the catalytic substrate. New enzyme-catalyzed products were also separated and their structures identified.

[0086] Example 1

[0087] In this embodiment, the α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE was screened from Penicillium spp. P. simplecissimum ATCC 90288, and the amino acid sequence of the α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE is shown in SEQ ID NO.1.

[0088] SEQ ID NO.1(Amino acid sequences of OkaE with the C-terminal strep-tag)

[0089] TLTENVMKPQIQRFTRDHDPKVLWQVVEEDGAVIIEGFLPHEVIQKFDCELDVRSKATKGGEMNQEFYQMPVPTTKWMNDLTATCPTFRHEILNNDILHSLCNVAFEPHGDYWLLNGMAMEMMPGNP TQQIHNDHGTHPILQYLRPDAPAPVFSIITAVTEFTESNGATRVILGSHRWPQGQKAKDDQAVRAALQPGDALVMHRSTKHGGAAHDADNQDHRRLLLTCMGTCQLAPYETNVTVPRPIVESMTPLAQK MIGWRSTRPVISNVTGLNTVRMKHLENQIELKSNVPLNVGGCWSHPQFEK

[0090] Among them, Penicillium simplicissimum ATCC 90288 underwent first-generation subculture, and its first-generation subculture strain is deposited in BeNa Culture Collection (BNCC) with accession number BNCC279046, located at Beijing BeNa Innovation Biotechnology Research Institute, No. 15 Jianguo Road, Chaoyang District, Beijing, China.

[0091] After obtaining the cDNA of okaE from the NCBI website, we commissioned Anshengda Life Sciences to synthesize the pASK-3plus-okaE plasmid containing the okaE gene sequence.

[0092] 10 ng of plasmid was transformed into E. coli BL21(DE3). After screening with a plate containing 0.1% ampicillin, single clones were picked and sequenced. The correct sequence was selected for subsequent induction of expression in E. coli BL21(DE3). The α-ketoglutarate-dependent mononuclear nonheme ironase OkaE was obtained by isolation.

[0093] This embodiment provides some indole compounds catalyzed by α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE, including:

[0094] The catalytic product 4 of the α-ketoglutarate-dependent mononuclear nonheme iron enzyme is a compound with an octyl ring multichiral central structure containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an octyl azo ring, and a carboxyl-modified four-membered azobutane, with the molecular formula C. 32 H 30 N4O5, with the structural formula shown in Formula 1, is named Neuokaramine I.

[0095] Product 5 is a compound with a nine-ring multi-chiral central structure containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, a four-membered nitrogen heterocycle butane, and an oxygen-containing bridged ring. Its molecular formula is C10. 32 H 30 N4O4, with the structural formula shown in Formula 2, is named Neuokaramine II.

[0096] Product 6 is a compound with a heptcyclic multichiral central skeleton containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, and an isopentenyl group, and its molecular formula is C60. 32 H 32 N4O4, with the structural formula shown in Formula 3, is named NeuokaramineⅢ.

[0097] Product 7 is a compound with a seven-ring multi-chiral central structure containing an indole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, and a three-membered oxygen ring, and its molecular formula is C. 32 H 30 N4O5, with the structural formula shown in Formula 4, is named Neuokaramine Ⅳ.

[0098]

[0099] This embodiment provides a method for preparing indole compounds, which are α-ketoglutarate-dependent mononuclear non-heme iron enzyme catalyzed products, including the following steps:

[0100] Mixed OkaE pure enzyme, substrate okaramine A, Fe Ⅱα-KG and Vc were added to an oxygen-saturated solution to activate the enzymatic reaction. The reaction products were extracted and then analyzed by high-resolution liquid chromatography-mass spectrometry to identify the reaction type of the catalytic substrate and the products.

[0101] okaramine A is a natural substrate of OkaE and needs to be isolated and prepared from the OkaE source strain P. simplecissimum ATCC90288. The specific steps are as follows:

[0102] The fungal strain was streaked into three zones on PDA solid medium (4 g / L potato extract, 20 g / L glucose, 15 g / L agar) and incubated at 28°C for 3 days to obtain the fungal solid medium.

[0103] The solid fungal culture medium was cut into blocks (approximately 1 cubic centimeter) and inoculated into 100 mL of PDB liquid medium (4 g / L potato extract, 20 g / L glucose). The mixture was then incubated at 28°C with shaking at 220 rpm for 3 days to obtain the fungal seed culture. For fermentation culture, 5 mL of the seed culture was inoculated into 50 g of fresh soybean residue culture medium and incubated statically at 28°C for 14 days.

[0104] The preparation conditions for soybean residue culture medium are as follows:

[0105] Each sterile bag contains 50g of soybean residue and is sterilized in an autoclave at 115℃ for 30 minutes.

[0106] The fermented cells were extracted four times with 10L of acetone. The organic phase was removed by vacuum concentration. The remaining aqueous phase was extracted four times with ethyl acetate to obtain 164.0g of crude extract.

[0107] The crude extract of fermented bacterial cells was purified and prepared using silica gel column chromatography and semi-preparative liquid chromatography (HPLC), including the following steps:

[0108] The crude extract obtained from fermentation was initially separated using normal-phase silica gel column chromatography. CH2Cl2—MeOH was used as the elution mobile phase for silica gel column chromatography, and gradient elution (100:0, 50:1, 20:1, 10:1, and 0:100) was employed to obtain five eluted fractions (A-E).

[0109] Component B (24.0 g) was dissolved in a 1:1 mixture of CH2Cl2 and MeOH. The components were then separated again using reversed-phase silica gel column chromatography with MeOH-H2O as the elution mobile phase. Gradient elution (30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, v / v) was employed to obtain eight eluents. Component B5 contained okaramine A (1), and component B6 contained 12-deshydroxyl-okaramine E (2).

[0110] Component B5 was separated by semi-preparative reversed-phase high-performance liquid chromatography. Further purification was performed using a C18 column (Agilent-ZORBAX-SB, 9.4×250 mm, 5 μm) with isocratic elution at a flow rate of 3.0 mL / min using 68% MeOH-H2O as the mobile phase to obtain okaramine A(1) with a retention time of 37.2 min.

[0111] Component B6 was separated by semi-preparative reversed-phase high-performance liquid chromatography. Further purification was performed using a C18 column (Agilent-ZORBAX-SB, 9.4×250 mm, 5 μm) with isocratic elution using 66% MeOH-H2O containing 0.1% formic acid as the eluent to obtain 12-deshydroxyl-okaramine E(2) with a retention time of 24.8 min.

[0112] Component C was separated by reversed-phase silica gel column medium-pressure liquid chromatography with gradient elution of MeOH-H2O at volume ratios from 30:70 to 100:0; then it was separated by semi-preparative reversed-phase high-performance liquid chromatography with isocratic elution of 55% MeOH-H2O containing 0.1% formic acid to obtain okaramine E(3) with a retention time of 27.5 min.

[0113] This embodiment also provides a method for inducing α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE expression, the specific steps of which are as follows:

[0114] The sequence of the okaE gene was inserted into the pASK-3plus vector, which placed the okaE gene under the control of the tet promoter, thereby generating the C-terminal Strep-tag-tagged OkaE protein.

[0115] The recombinant plasmid containing okaE was transformed into *E. coli* BL21(DE3) cells. After selection using resistance plates (LB solid medium with 100 μg / mL ampicillin), single colonies were picked and inoculated into 10 mL of LB medium supplemented with 100 μg / mL ampicillin, and cultured at 37°C with shaking at 220 rpm for 12 h. Subsequently, 10 mL of seed culture and 39.2 mg of Fe(NH4)2·(SO4)2·6H2O were inoculated into 1 L of LB medium supplemented with 100 μg / mL ampicillin, and cultured at 37°C until OD500. 600 After adjusting the concentration to within the range of 0.6–0.8, the mixture was cooled to 20°C, and tetracycline at a final concentration of 500 μg / L was added to induce OkaE protein overexpression. The mixture was then cultured at 20°C and 220 rpm for 16 h to obtain wet Escherichia coli fermentation cells.

[0116] This embodiment also provides a method for purifying α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE protein, the specific steps of which are as follows:

[0117] Resuspend the wet E. coli fermentation cells in buffer A (100 mM Tris-HCl and 50 mM NaCl, pH 7.5). Add lysozyme, EDTA, and vitamin C to the cell suspension to a final concentration of 0.5 mg / mL, 5 mM, and 5 mM, respectively. Incubate the mixture on ice with gentle stirring for 30 min, then use Sonics Vibra-Cell. TM The VCX130 Ultrasonic Processor is used to sonicate cells (43 times, 3 seconds each time).

[0118] The lysed bacterial culture was centrifuged at 18,000 rpm for 40 min at 4°C using a high-performance centrifuge. The supernatant was mixed with Strep-Tactin resin and incubated on ice for 30 min. After the cell lysate was removed by gravity, the column was washed with buffer B (100 mM Tris-HCl, 50 mM NaCl, pH 7.5) until OD500 was reached. 280 Less than 0.05. After removing excess liquid again using gravity, OkaE protein was eluted with elution buffer (2.5 mM desthiobiotin, 100 mM Tris-HCl, 50 mM NaCl, pH 7.5). After confirming the purified protein was correct by SDS-PAGE, it was concentrated using an ultrafiltration vessel and then flash-frozen in liquid nitrogen at -80°C.

[0119] This embodiment also provides a method for biochemical reactions using α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE protein, the specific steps of which are as follows:

[0120] The total volume of the reaction mixture was 200 μL, and its composition included 70 μM Mokaramine A, 70 μM Mokaramine E, and 140 μM Fe. Ⅱ The reaction mixture was prepared using 70 μM α-KG and a buffer system consisting of 100 mM Tris-HCl, 50 mM NaCl, and pH 7.5. A reaction mixture containing boiled OkaE was used as a negative control. The mixture was sealed in a double-necked flask, and 600 μL of oxygen-saturated buffer with a dissolved oxygen concentration of 1.25 mM was added to activate the reaction. After reacting at room temperature for 15 minutes, 800 μL of ethyl acetate was added to quench the enzyme reaction. The mixture was then centrifuged at 12,000 rpm for 2 minutes to remove the precipitated protein and separate the ethyl acetate layer for further analysis. The reaction mixture was then extracted again with 800 μL of ethyl acetate. The two ethyl acetate layers were combined and concentrated under vacuum using a rotary evaporator. The concentrated residue was redissolved in 60 μL of acetonitrile and detected on a C18 column (ACE C18-PFP, 4.6 × 250 mm, 5 μm). The detection conditions were set as follows: flow rate of 1 mL / min; acetonitrile concentration in pure water was gradually increased from 34% to 53.2% within 0–32 minutes; and finally, elution was performed using a 99% acetonitrile aqueous solution for 5 minutes. The products generated in vitro by α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE and the corresponding HPLC chromatograms are shown below. Figure 1 As shown.

[0121] This embodiment also provides a method for the aerobic multi-equivalent biochemical reaction of α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE protein, the specific steps of which are as follows:

[0122] The total volume of the reaction mixture was 200 μL, and its composition included 210 μMokaramine A, 70 μMokaE, and 420 μMFe. ⅡThe reaction mixture consisted of 210 μM α-KG, 210 μM ascorbic acid, and 3.6% DMSO, in a buffer system of 100 mM Tris-HCl, 50 mM NaCl, and pH 7.5. A reaction mixture containing boiled OkaE was used as a negative control. The mixture was sealed in a two-necked flask, and 600 μL of oxygen-saturated buffer with a dissolved oxygen concentration of 1.25 mM was added to activate the reaction. After reacting at room temperature for 15 minutes, 800 μL of ethyl acetate was added to quench the enzyme reaction. The mixture was then centrifuged at 12,000 rpm for 2 minutes to remove the precipitated protein and separate the ethyl acetate layer for further analysis. The reaction mixture was then extracted again with 800 μL of ethyl acetate. The two ethyl acetate layers were combined and concentrated under vacuum using a rotary evaporator. The concentrated residue was redissolved in 60 μL of acetonitrile and detected on a C18 column (ACE C18-PFP, 4.6 × 250 mm, 5 μm). The detection conditions were set as follows: flow rate of 1 mL / min; acetonitrile concentration in pure water was gradually increased from 34% to 53.2% within 0–32 minutes; finally, elution was performed using a 99% acetonitrile aqueous solution for 5 minutes. The products generated in vitro by α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE and the corresponding HPLC chromatograms are shown below. Figure 1 As shown.

[0123] This embodiment also provides a method for amplifying the biochemical reaction of α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE protein, the specific steps of which are as follows:

[0124] The biochemical reactions in this study were conducted in an inert atmosphere chamber. The total volume of the reaction mixture was 4 mL, and its composition included 210 μM Mokaramine A, 70 μM Mokaramine E, and 1, 2, 100 μM Fe. Ⅱ The reaction mixture consisted of 700 μM α-KG, 210 μM ascorbic acid, and 3.6% DMSO, in a buffer system of 100 mM Tris-HCl, 50 mM NaCl, and pH 7.5. The mixture was sealed in a double-necked flask, and 10 mL of 1.25 mM dissolved oxygen saturated buffer was added to activate the reaction. After reacting at room temperature for 30 minutes, 14 mL of ethyl acetate was added to quench the enzyme reaction. The mixture was then centrifuged at 13,000 rpm for 10 minutes to remove the precipitated protein and separate the ethyl acetate layer for further analysis. The reaction mixture was then extracted again with 14 mL of ethyl acetate. The two ethyl acetate layers were combined and concentrated under vacuum using a rotary evaporator. The residue was redissolved in a 1:1 mixture of CH2Cl2 and ACN.

[0125] This embodiment also provides a method for separating the catalytic products of the multifunctional α-ketoglutarate-dependent mononuclear non-heme iron enzyme OkaE, the specific steps of which are as follows:

[0126] The mixed solutions were separated using reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column (Agilent-ZORBAX-SB, 9.4 × 250 mm, 5 μm) was used, with a flow rate of 3.0 mL / min and a gradient elution method employed. Specifically, the concentration of acetonitrile in water was gradually increased from 34% to 53.2% over 0 to 32 minutes, followed by elution with a 99% acetonitrile aqueous solution for another 5 minutes to obtain four fractions.

[0127] Component 1 was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (ACE C18-PFP, 4.6 × 250 mm, 5 μm) at a flow rate of 1.0 mL / min with a gradient elution method. Specifically, the concentration of acetonitrile in 0.1% formic acid aqueous solution was gradually increased from 25% to 35% within 0 to 20 minutes; and the concentration of acetonitrile in 0.1% formic acid aqueous solution was gradually increased from 35% to 40% within 20 to 30 minutes. This purification process yielded compound 4 with a retention time of 29.2 min.

[0128] Component 3 was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (ACE C18-PFP, 4.6 × 250 mm, 5 μm) at a flow rate of 1.0 mL / min. A gradient elution method was employed: the concentration of acetonitrile in water was maintained at 33% from 0 to 27 minutes; from 27 to 40 minutes, the concentration gradually increased from 36% to 53%. This purification process yielded compound 5, with a retention time of 37.3 min.

[0129] Component 4 was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (ACE C18-PFP, 4.6 × 250 mm, 5 μm) at a flow rate of 1.0 mL / min and a gradient elution method. Specifically, the concentration of MeOH in water was maintained at 62% from 0 to 28 minutes; from 28 to 40 minutes, the concentration of MeOH in water gradually increased from 62% to 65%. This purification process yielded compounds 6 and 7, with a retention time of 39.3 min for compound 6 and 25.8 min for compound 7.

[0130] Compounds 1-3 are known compounds, while compounds 4-7 are compounds not reported in the literature.

[0131] Compound 1 is a yellow powder. Its structure was determined by comparing its MS and NMR data with those reported in the literature. HR-MS (ESI+) showed its quasi-molecular ion peak at m / z 521.2547 [M+H]. + (Calculated value is 521.2547), degree of unsaturation is 19, molecular formula is C 32 H 32 O3N4( Figure 2 —4, Figure 11 (Table 1).

[0132] Compound 2 is a yellow powder. Its structure was determined by comparing its MS and NMR data with those reported in the literature. HR-MS (ESI+) showed its quasi-molecular ion peak at m / z 521.2527 [M+H]. + (Calculated value is 521.2547), degree of unsaturation is 19, molecular formula is C 32 H 32 O3N4( Figure 5 —7, Figure 11 (Table 2).

[0133] Compound 3 is a yellow powder. Its structure was determined by comparing its MS and NMR data with those reported in the literature. HR-MS (ESI+) showed a quasi-molecular ion peak at m / z 537.2474 (calculated value 537.2496), an unsaturation degree of [value missing], and a molecular formula of C[value missing]. 32 H 32 O4N4( Figure 8 —11, Table 2).

[0134] Compound 4 is a yellow powder. HR-MS (ESI+) showed its quasi-molecular ion peak at 551.2271 [M+H]. + (Calculated value is 551.2289), degree of unsaturation is 20, molecular formula is C 32 H 30 O5N4( Figure 12 Compound 4 1 H-NMR, 13 C-NMR and HSQC spectral data analysis showed that this compound has 10 olefin quaternary carbons (δ¹⁰). C 141.6; 150.5; 167.4; 171.1; 128.1; 148.3; 135.1; 131.3; 106.2; 164.2), 4 sps 3 Quaternary carbon (δ C 87.7; 93.7; 64.9; 36.8), 11 olefin tertiary carbons (δ C / H125.1 / 7.49; 124.4 / 7.06; 129.9 / 7.23; 119.2 / 6.79; 115.4 / 7.62; 123.6 / 5.75; 140.3 / 5.93; 112.5 / 7.39; 122.8 / 7.14; 121.6 / 7.16; 118.1 / 7.68), 2 sps 3 Tertiary carbon (δ C / H 58.3 / 4.63; 47.5 / 3.94), 1 methylene (δ C / H 41.1 / 2.85&2.74), 4 singlet methyl groups (δ C / H 27.9 / 2.06; 27.7 / 0.95; 26.8 / 1.71; 28.2 / 1.65)( Figure 13 —15, Table 3). These spectral features are highly similar to those of okaramine E(3) reported in the literature, thus it can be inferred that compound 4 is an okaramine alkaloid. However, after detailed analysis, it was found that the carbonyl group (δ) of compound 4 C The 171.1, C-12) signal replaces the oxidized methylene (δ) of compound 3. H 4.15, dd, J=11.0, 6.9Hz, H-12a; δ H 4.32, dd, J=11.0, 7.8Hz, H-12b; δ C The signal at position 60.6 (C-12) indicates that the hydroxyl group at position C-12 in compound 3 was oxidized to a carboxylic acid in compound 4. According to the HMBC spectrum, H-11 (δ... H 3.94) and C-14 (δ) C 27.7), C-13 (δ) C 27.9), C-10 (δ) C 64.9), C-3a(δ) C 87.7), C-8a(δ) C 93.7), C-12 (δ) C The correlation signal between 171.7) confirms the above hypothesis. Figure 16 (Table 3) The NOE effect exists between H-2 and H-3, H-5' and H-14', H-7 and H-14, H-4 and H-11, and H-1' and H-11' in the NOESY spectrum, indicating that these protons have the same orientation, which can be used to determine the relative configuration of compound 4. Figure 18 —19, Table 3).

[0135] Compound 5 is a yellow powder. HR-MS (ESI+) showed its quasi-molecular ion peak at 535.2323 [M+H]. +(Calculated value is 535.2340), degree of unsaturation is 20, molecular formula is C 32 H 30 O4N4( Figure 20 Compound 5 1 H-NMR, 13 C-NMR and HSQC spectral data analysis showed that this compound has 9 olefin quaternary carbons (δ¹⁸). C 142.5; 153.2; 167.7; 127.3; 149.3; 135.3; 131.4; 106.1; 164.7), 4 sps 3 Quaternary carbon (δ C 96.9; 100.8; 62.5; 36.9), 12 tertiary carbons of olefins (δ C / H 124.9 / 7.49; 125.7 / 7.13; 130.5 / 7.29; 121.1 / 6.85; 103.9 / 5.58; 116.1 / 7.80; 123.8 / 5.76; 140.1 / 5.94; 112.6 / 7.40; 123.0 / 7.16; 121.8 / 7.19; 117.9 / 7.76), 2 sps 3 Tertiary carbon (δ C / H 60.2 / 4.84; 56.2 / 3.31), 1 methylene (δ C / H 35.6 / 2.79 & 2.98), 4 singlet methyl groups (δ) C / H 20.2 / 0.81; 32.3 / 1.91; 27.2 / 1.74; 28.3 / 1.67)( Figure 21 —23, Table 4). These spectral features are highly similar to those of okaramine E(3) reported in the literature, thus it can be inferred that compound 5 is an okaramine alkaloid. However, according to 1D NMR alignment, the oxidized methylene signal (δ) at C-12 in compound 3 is not significantly different. C 60.6, C-12; δ H 4.15,dd,J=11.0,6.9Hz,H-12&δ H 4.32, dd, J = 11.0, 7.8 Hz, H-12) is converted to oxidized methine (δ) in compound 5. C 103.9, C-12; δ H 5.58, br s, H-12) signal. Compound 5 also exhibits one more degree of unsaturation than compound 3. Combined with mass spectrometry and 1D NMR data analysis, the C-12 and H-12 in compound 5 show a significant lower field shift, indicating the presence of a bridging oxygen between C-12 and C-3a in compound 5. Simultaneously, the H-12 (δ) signal in compound 5... H 5.58) and C-11(δC 56.2), C-10(δ) C 62.5), C-8a(δ) C 100.8), C-3a(δ) C The HMBC correlation signal and related mass spectrometry data between 96.9) also confirmed the above inference. Figure 20 , Figure 24 (Table 4). The NOE effect exists between H-2 and H-3, H-5' and H-14', H-3 and H-4, H-11 and H-13, H-12 and H-14, H-11 and H-12, and H-1' and H-11' in the NOESY spectrum, indicating that these protons have the same orientation, thus allowing determination of the relative configuration of compound 5. Figure 26 —27, Table 4).

[0136] Compound 6 is a yellow powder. HR-MS (ESI+) showed its quasi-molecular ion peak at 537.2487 [M+H]. + (Calculated value is 537.2496), degree of unsaturation is 19, molecular formula is C 32 H 32 O4N4( Figure 28 Compound 6 1 H-NMR, 13 C-NMR and HSQC spectral data analysis showed that this compound has 10 olefin quaternary carbons (δ¹⁰). C 133.5; 143.6; 181.1; 167.8; 126.7; 149.1; 135.8; 131.7; 106.5; 165.2), 3 sps 3 Quaternary carbon (δ C 75.5; 60.9; 37.1), 12 tertiary carbons of olefins (δ C / H 124.8 / 7.75; 123.4 / 7.12; 129.5 / 7.25; 115.1 / 7.16; 145.1 / 5.96; 124.1 / 6.10; 140.3 / 5.98; 112.6 / 7.41; 122.8 / 7.24; 121.7 / 7.27; 118.3 / 7.96; 115.1 / 8.26), 1 sp 3 Tertiary carbon (δ C / H 52.9 / 5.26), 1 olefin secondary carbon (δ C / H 113.4 / 5.03), 1 sp 3 Secondary carbon (δ C / H 40.9 / 2.80&3.12), 4 singlet methyl groups (δ) C / H 28.3 / 1.83; 25.7 / 1.51; 27.8 / 1.76; 28.4 / 1.69)( Figure 29—31, Table 5). These spectral features are highly similar to those of okaramine A(1) reported in the literature, therefore it can be inferred that compound 6 is an okaramine alkaloid. According to 1D NMR data, the carbonyl group (δ) in compound 6 C The 181.1, C-8a) signal replaces the methine (δ) in 1. C 85.7, C-8a; δ H The 5.52, s, H-8a) signal indicates that C-8a in compound 1 is oxidized to the amide carbonyl group in 6. Furthermore, according to HMBC and corresponding 1D NMR data, 1-NH(δ) H 8.89) and C-2(δ C 59.2), C-2'(δ) C 126.7), C-9 (δ) C 167.8) There is a related signal ( Figure 29 —30, Figure 32 Table 5 shows that the indole ring portion of the hexahydropyrroloindole ring in compound 1 undergoes ring-opening in compound 6. The NOESY spectrum shows NOE effects between H-1' and H-11', H-5' and H-14', H-1 and H-2, H-2 and H-3a-OH, H-2 and H-3, H-3 and H-3a-OH, H-1 and H-3a-OH, H-3 and H-4, H-12 and H-7, H-11 and H-7, and H-13 and H-7, indicating that these protons have the same orientation, thus allowing determination of the relative configuration of compound 6. Figure 34 —35, Table 5).

[0137] Compound 7 is a yellow powder. HR-MS (ESI+) showed its quasi-molecular ion peak at 551.2271 [M+H]. + (Calculated value is 551.2289), degree of unsaturation is 20, molecular formula is C 32 H 30 O5N4( Figure 36 Compound 7 1 H-NMR, 13 C-NMR and HSQC spectral data analysis showed that this compound has 11 olefin quaternary carbons (δ¹⁸). C 200.9; 140.0; 138.3; 151.8; 164.8; 123.8; 149.2; 135.3; 130.8; 105.4; 161.1), 2 sps 3 Quaternary carbon (δ C 61.6; 36.6), 11 tertiary carbons of olefins (δ C / H1130.3 / 7.75; 128.6 / 7.37; 132.1 / 7.41; 128.5 / 7.56; 116.5 / 7.91; 122.2 / 5.54; 139.9 / 5.91; 112.2 / 7.36; 122.5 / 7.21; 121.4 / 7.25; 117.6 / 7.83), 2 sps 3 Tertiary carbon (δ C / H 53.8 / 5.33; 56.3 / 3.64), 2 olefin secondary carbons (δ C / H 41.1 / 3.79&3.91; 46.9 / 2.87&3.22), 4 singlet methyl groups (δ C / H 23.7 / 1.46; 23.2 / 1.37; 26.8 / 1.75; 28.2 / 1.54)( Figure 37 —39, Table 6). These spectral features are highly similar to those of okaramine A(1) reported in the literature, therefore it can be inferred that compound 7 is an okaramine alkaloid. According to 7 13 C-NMR data show that: C-7a(δ C 135.3), C-3b(δ) C 140.0), C-3a(δ C 200.9), C-8a(δ) C 151.8) is located in the low field portion of the spectrum, and according to HMBC data: H-7 (δ H 7.56) and C-5(δ) C 128.6), C-3b(δ) C There is a correlation signal between 140.0 and ( Figure 40 Table 6), combined with mass spectrometry data, shows that C-8a (δ) C 151.8), C-3a(δ) C 200.9) is the carbonyl carbon, C-7a (δ C 135.3), C-3b(δ) C 140.0) is an aromatic carbon ( Figure 36 That is, the indole ring portion of the hexahydropyrroloindole ring in 1 undergoes ring-opening between C-8a and C-3a, and C-8a (δ C 151.8) is oxidized to a carbonyl group. Simultaneously, the carbonyl group (δ) on C-3a... C The 200.9) signal replaced the hydroxyl group (δ) in 1. C The signal (85.0) indicates that the chemical bond breaking between C-8a and C-3a in compound 1 leads to the oxidation of the hydroxyl group at the C-3a position to a ketone carboxyl group. Furthermore, a comparison of the 1D NMR data of compounds 7 and 1 shows that the methine group at C-11 in compound 7 (δ) C 56.3, C-11; δH 3.64, dd, J = 2.6 Hz, H-11) signal and C-12 methylene (δ C 46.9, C-12; δ H 2.87, t, J = 4.2 Hz & δ H The 3.22, dd, J = 2.6, 4.2 Hz, H-12) signal replaced the olefin carbon signal (δ) in 1. C 149.2, C-11; δ H 6.40, dd, J=17.7, 10.8Hz, H-11; δ C 111.5, C-12; δ H 5.04, dd, J=10.7, 1.3Hz&δ H 5.17, dd, J=17.8, 1.3Hz, H-12)( Figure 37 —39, Table 6), while H-12a and H-12b are related to C-11 (δ C 56.3) and C-10(δ C The presence of HMBC correlation signals between 61.6 indicates that the double bond between C-12 and C-11 in compound 1 is oxidized to the three-membered oxygen ring in 7. The presence of NOE effects between H-5' and H-13', H-1' and H-11', H-14 and H-3, H-2 and H-3, H-11 and H-12, H-11 and H-14, H-3 and H-11, and H-14' and H-2 in the NOESY spectrum indicates that these protons have the same orientation, thus allowing determination of the relative configuration of compound 7. Figure 42 —43, Table 6).

[0138] Table 1. 1D NMR data of compound 1

[0139]

[0140]

[0141] Table 2. 1D NMR data of compounds 2 and 3

[0142]

[0143]

[0144]

[0145] Table 3. 1D and 2D NMR data for compound 4

[0146]

[0147]

[0148]

[0149] Table 4. 1D and 2D NMR data for compound 5

[0150]

[0151]

[0152]

[0153] Table 5. 1D and 2D NMR data for compound 6

[0154]

[0155]

[0156]

[0157] Table 6. 1D and 2D NMR data for compound 7

[0158]

[0159]

[0160]

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing indole compounds catalyzed by mononuclear non-heme iron enzymes, characterized in that, Mixed OkaE pure enzyme, substrate okaramine A, Fe Ⅱ α-KG and Vc are added to an oxygen-saturated solution to activate the enzymatic reaction; the reaction product is extracted and separated to obtain the catalytic product, an indole compound.

2. The method for preparing the indole compound catalyzed by mononuclear non-heme ironase as described in claim 1, characterized in that, The specific steps are as follows: Step 1: Prepare a mixture containing okaramine A, OkaE, and Fe. Ⅱ α-KG, ascorbic acid and DMSO, the solvent used is a buffer solution: 100mM Tris-HCl and 50mM NaCl, pH 7.5; Step 2: Seal the mixed reaction mixture in a flask, add 10 mL of oxygen-saturated buffer solution, and the dissolved oxygen concentration is 1.25 mM; Step 3: After incubation for 30 minutes, add 14 mL of ethyl acetate to quench the enzyme reaction, centrifuge at 13,000 rpm for 10 min to remove the precipitated protein, and separate the ethyl acetate layer; Step 4: Continue to extract the reaction mixture with 14 mL of ethyl acetate; combine the two ethyl acetate layers and concentrate the extract using a rotary evaporator; Step 5: Redissolve the residue in a 1:1 mixture of CH2Cl2 and ACN.

3. The method for preparing indole compounds catalyzed by mononuclear non-heme ironase as described in claim 2, characterized in that, The mixture used in step 1 consists of 4 mL of substances, containing 210 μMokaramine A, 70 μMokaramine E, and 2,100 μMFe. Ⅱ The final concentration was 3.6%, consisting of 700 μM α-KG, 210 μM ascorbic acid, and DMSO.

4. The method for preparing indole compounds catalyzed by mononuclear non-heme iron enzymes as described in claim 2, characterized in that, In step 3, the mixed reactants must be incubated at room temperature for 30 minutes.

5. The method for preparing indole compounds, the mononuclear non-heme ironase catalytic product, as described in claim 2, is characterized in that, The specific steps of the extraction and separation method include: Step 1: The mixed solution was separated using reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column was selected, and the flow rate was set to 3.0 mL / min. Specifically, the concentration of acetonitrile in water was gradually increased from 34% to 53.2% within 0 to 32 minutes, and then eluted with a 99% acetonitrile aqueous solution for another 5 minutes to obtain four components. Step 2: Component 1 was purified using semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column was selected, and the flow rate was set to 1.0 mL / min. Specifically, the concentration of acetonitrile in 0.1% formic acid aqueous solution was gradually increased from 25% to 35% within 0 to 20 minutes; and the concentration of acetonitrile in 0.1% formic acid aqueous solution was gradually increased from 35% to 40% within 20 to 30 minutes, yielding in vitro catalytic product 4. Step 3: Component 3 was purified using semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column was selected, and the flow rate was set to 1.0 mL / min. Specifically, from 0 to 27 minutes, the concentration of acetonitrile in H2O was 33%. From 27 to 40 minutes, the concentration of acetonitrile in H2O gradually increased from 36% to 53%, yielding in vitro catalytic product 5. Step 4: Component 4 was purified using semi-preparative reversed-phase high-performance liquid chromatography; a C18 column was selected and the flow rate was set to 1.0 mL / min; specifically, the concentration of MeOH in H2O was 62% from 0 to 28 minutes; from 28 to 40 minutes, the concentration of MeOH in H2O gradually increased from 62% to 65%, yielding in vitro catalytic product 6 and in vitro catalytic product 7.

6. The method for preparing the indole compound catalyzed by mononuclear non-heme ironase as described in claim 5, characterized in that, The mobile phase elution program used in step 1 is gradient elution; the mobile phase elution program used in step 2 is gradient elution; the mobile phase elution program used in step 3 is gradient elution; the mobile phase elution program used in step 4 is gradient elution.

7. The method for preparing the indole compound catalyzed by mononuclear non-heme ironase as described in claim 1, characterized in that, The preparation and separation method of the substrate okaramine A includes the following steps: Step 1: The fungal strain Penicillium simplicissimum ATCC 90288 was streaked in three zones onto PDA solid medium and incubated at 28°C for 3 days to obtain the fungal solid medium. Step 2: Cut the fungal solid culture medium into blocks, inoculate into 100mL of PDB liquid culture medium, and culture at 28℃ and 220rpm for 3 days to obtain fungal seed liquid; the fermentation culture method is to inoculate 5mL of fungal seed liquid into 50g of fresh soybean residue culture medium and incubate at 28℃ for 14 days. Step 3: The fermented cells were extracted four times with 10L of acetone. The organic phase was removed by vacuum concentration. The remaining aqueous phase was extracted four times with ethyl acetate to obtain the crude extract. Step 4: The crude extract obtained from fermentation was initially separated using normal-phase silica gel column chromatography; CH2Cl2—MeOH was used as the elution mobile phase for silica gel column chromatography, and gradient elution was adopted: 100:0, 50:1, 20:1, 10:1 and 0:100 to obtain five elution fractions A, B, C, D and E; Step 5: Dissolve component B in a 1:1 mixed solution of CH2Cl2 and MeOH, and separate it by reversed-phase silica gel column chromatography. Use MeOH-H2O as the elution mobile phase for silica gel column chromatography, and use a gradient elution of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% by volume for B1-B8. Step 6: Component B5 was separated by semi-preparative reversed-phase high-performance liquid chromatography; further purification was performed using a C18 column with 68% MeOH-H2O as the mobile phase and isocratic elution at a flow rate of 3.0 mL / min to finally obtain okaramine A, the natural catalytic substrate of α-ketoglutarate-dependent mononuclear nonheme iron enzyme OkaE.

8. A catalytic product indole compound, characterized in that, The compound is prepared by the preparation method described in claims 1-7.

9. The indole compound as the catalytic product according to claim 8, characterized in that... The compounds include, among others, the in vitro catalytic product 4 of α-ketoglutarate-dependent mononuclear nonheme iron enzyme, which is a compound with an octyl ring multichiral central structure containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, and a carboxyl-modified four-membered nitrogen heterocycle butane, and its molecular formula is C. 32 H 30 N4O5, the structural formula is shown in Formula 4; The in vitro catalytic product 5 of α-ketoglutarate-dependent mononuclear non-heme ironase is a compound with a nine-ring multichiral central structure containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, a four-membered nitrogen heterocycle butane, and a five-membered internal ether ring. Its molecular formula is C5. 32 H 30 N4O4, the structural formula is shown in Formula 5; The in vitro catalytic product 6 of α-ketoglutarate-dependent mononuclear non-heme ironase is a compound with a heptaneous multichiral central skeleton containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, and an isopentenyl side chain. Its molecular formula is C60. 32 H 32 N4O4, the structural formula is shown in Formula 6; The in vitro catalytic product 7 of α-ketoglutarate-dependent mononuclear non-heme ironase is a compound with a seven-ring multichiral central skeleton containing an indole ring, a hexahydropyrroloindole ring, a diketopiperazine ring, an eight-membered nitrogen heterocycle, and a three-membered oxygen ring. Its molecular formula is C7. 32 H 30 N4O5, the structural formula is shown in Formula 7.

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