CN116199564A - Novel diterpenoid compound and preparation method and application thereof - Google Patents
Novel diterpenoid compound and preparation method and application thereof Download PDFInfo
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- CN116199564A CN116199564A CN202310213295.XA CN202310213295A CN116199564A CN 116199564 A CN116199564 A CN 116199564A CN 202310213295 A CN202310213295 A CN 202310213295A CN 116199564 A CN116199564 A CN 116199564A
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Images
Classifications
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- C07C35/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring
- C07C35/22—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system
- C07C35/44—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system with a hydroxy group on a condensed ring system having more than three rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C13/00—Cyclic hydrocarbons containing rings other than, or in addition to, six-membered aromatic rings
- C07C13/28—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof
- C07C13/32—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof with condensed rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C13/00—Cyclic hydrocarbons containing rings other than, or in addition to, six-membered aromatic rings
- C07C13/28—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof
- C07C13/32—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof with condensed rings
- C07C13/47—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof with condensed rings with a bicyclo ring system containing ten carbon atoms
- C07C13/52—Azulenes; Completely or partially hydrogenated azulenes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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Abstract
The invention belongs to the technical field of biochemistry and molecular biology, and particularly relates to a novel diterpenoid compound, and a preparation method and application thereof. A novel diterpenoid compound is a compound containing 5-5-6-7tetracyclic diterpenoid skeleton, a compound containing 6-5-6-6 tetracyclic diterpenoid skeleton, a compound containing 5-7 bicyclo diterpenoid skeleton or a compound containing 5-11 bicyclo diterpenoid skeleton. The diterpenoid compound provided by the invention has novel structure and function. In addition, the invention constructs the self-sufficient engineering bacteria for producing the novel diterpenoid compounds by utilizing methods of enzyme engineering and genetic engineering. The whole operation process is simple, the process is mature, the cost is low, the whole process does not contain harmful impurities, and the preparation method is nontoxic and is very environment-friendly. The obtained novel diterpenoid compound is expected to be applied to the fields of pharmaceutical chemical industry, agricultural production and the like.
Description
Technical Field
The invention belongs to the technical field of biochemistry and molecular biology, and particularly relates to a novel diterpenoid compound, and a preparation method and application thereof.
Background
Terpene compounds are a class of structural units (C) containing an integer multiple of isoprene derived from polyisoprenyl pyrophosphates of limited carbon chain length catalyzed by terpene synthases 5 H 8 ) Is a hydrocarbon of (Dickschat, J.S. bacterial cycles, nat. Prod. Rep.33,87-110 (2016). Zeng, T., et al, terokit: a database-driven web server for terpenome research, J.chem. Inf. Model.60,2082-2090 (2020), rudolf, J.D., alsup, T.A., xu, B., li, Z.bacterial cycles, nat. Prod. Rep.38,905-980 (2021). Terpenes can be classified into a half terpene (containing 1C 5H8 unit), a monoterpene (containing 2C 5H8 units), a sesquiterpene (containing 3C 5H8 units), a diterpene (containing 4C 5H8 units), and so on, depending on the number of carbon atoms. Terpene compounds and derivatives thereof modified with hetero atoms such as oxygen, nitrogen, halogen, etc. are collectively called terpenoids. They are the most numerous natural products in nature, and more than one hundred thousand terpenoids have been reported. The terpenoid is used as primary or secondary metabolite in human, animal, plant and microorganism, has various structure types and wide distribution, and can be biosynthesized in more than 14000 organisms in 1109 families in nature. Meanwhile, terpenoids have rich biological functions such as aromatic odor/flavor, sterilization, anti-tumor, antiviral, anti-inflammatory, immunosuppression, regulation of physiological metabolic processes in human and animal bodies, and the like, and have important application values and potentials in the industries of medicine, food and essence and perfume (gershzon, j., dudariva, n.the function of terpene natural products in the natural world. Nat. Chem. Biol.3,408-414 (2007)). Representative terpenoids, such as the sesquiterpene lactone derivative artemisinin isolated from Artemisia annua taught by Nobel prize, tu Youyou, are not only anti-cancer and anti-inflammatory, but also antimalarial special effects that save millions of lives (Mu, X., wang, C.artemsins-a promising new treatment for systemic lupus erythematosus: a descriptive review.curr.Rheumatoid.Rep.20, 1-10 (2018)); paclitaxel isolated from taxus genus plants as a marketed "anticancer star molecule" was successfully applied to the clinical treatment of ovarian and breast cancers (Gueritte-Voegelein, f., guenard, d., lavelle, f., le Goff, m.t., mangatal, l., potier, p.relayionships between the structure of taxol analogs and their antimitotic activity.j.med.chem.34,992-998 (1991); tanshinone extracted from radix Salviae Miltiorrhizae has antibacterial, antiinflammatory, blood circulation promoting, and blood stasis dispelling effects, and can be widely used for treating cardiovascular and cerebrovascular diseases (Gao, S., liu, Z., li, H, little, P.J., liu, P., xu, S.cardioascularis actions and therapeutic potential of tanshinone IIA.atherosclerosis 220,3-10 (2012)); steroid hormones (fernandez-Cabez, l., galan, b., garcria, j.l. new insights on steroid biotechnology.front.microbiol.9,958 (2018)) which are indispensable for humans and mammals and are widely involved in the regulation of growth and metabolism in vivo; active steroid saponins in traditional Chinese medicine are widely available (Liang, Y., zhao, S.Process in understanding of ginsenoside biosynthesis.plant biol.10,415-421 (2008)). In order to meet urgent demands of people on healthy life, the excavation of novel active terpenoids or terpene skeletons is always an important link in the development of new drugs.
However, due to resource and technical limitations, especially the fact that plants are used as main sources of terpenoids, the disadvantages of long growth cycle, low yield of terpenoids and high acquisition cost exist, so that researchers are more and more difficult to directly excavate terpenoids from the nature. Moreover, the novel terpenoid obtained by the chemical synthesis strategy has the defects of more reaction steps, harsh conditions, poor catalytic selectivity, environmental pollution and the like, and more importantly, the novel terpenoid has limited capability of obtaining a brand new structural framework. In recent years, with the rapid development of structural biology and enzyme engineering, the biosynthesis of novel terpenes is realized by replacing important amino acid sites of terpene synthases by site-directed mutagenesis technology to change the stability of hydrogen bond networks or carbocation intermediates of active pockets thereof through analysis of crystal structures and catalytic mechanisms of terpene synthases, which has become an important means for excavating novel terpenes for basic and application science research (Christianson, d.w. structural and chemical biology of terpenoid cycles.chem.rev.117, 11570-11648 (2017)).
Diterpene synthase VenA from Streptomyces venezuelans (Streptomyces venezuelae ATCC 15439) catalyzes the biosynthesis of the new backbone diterpene venezuelan A (venezuelaene A) (Li, Z., et al, fragrans venezuelaenes A and B with a 5-5-6-7tetracyclic skeleton:discovery,biosynthesis,and mechanisms of central catalysts.ACS Catal.10,5846-5851 (2020)) containing 5-5-6-7tetracyclic groups from geranylgeranyl pyrophosphate (geranylgeranyl pyrophosphate, GGPP). VenA is well expressed in Escherichia coli, has good catalytic efficiency and good substrate broad property, and is expected to develop into bacterial diterpene synthase model enzyme. However, the search found that there is no report on the production of novel diterpenoids venezuelene A, venezuelene B, (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene and dichotomene C by engineering diterpenoid synthase VenA, or the production of the corresponding venezuelene A, venezuelene B, (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene and dichotomene C by expressing VenA mutants in E.coli by a heterologous expression method, and isolation and purification thereof at home and abroad.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel diterpenoid compound, and a preparation method and application thereof.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a novel diterpenoid compound is a compound containing a 5-5-6-7tetracyclic diterpenoid skeleton, and the structural formula of the compound is shown as a formula (1):
or the compound is a compound containing a 6-5-6-6 tetracyclic diterpenoid skeleton, and the structural formula of the compound is shown as the formula (2):
or the compound is a compound containing 5-7 bicyclo diterpenoid frameworks, and the structural formula of the compound is shown as formula (3):
or the compound is a compound containing 5-11 bicyclo diterpenoid frameworks, and the structural formula of the compound is shown as formula (4):
the invention also provides a mutant protein, the amino acid sequence of which is shown in SEQ ID NO:2 or is identical to SEQ ID NO:2 above 80%; the mutant protein is separated and purified to obtain a compound shown as a formula (1) or a formula (2).
The invention also provides another mutant protein, the amino acid sequence of which is SEQ ID NO:4 or is identical to SEQ ID NO:4, the sequence identity is higher than 80%; the mutant protein is separated and purified to obtain a compound shown as a formula (3).
The invention also provides another mutant protein, the amino acid sequence of which is SEQ ID NO:6 or is identical to SEQ ID NO:6, the sequence identity is higher than 80%; the mutant protein is separated and purified to obtain a compound shown as a formula (4).
The invention further provides a venA mutant gene for encoding the mutant protein, and the nucleotide sequence of the mutant gene is shown as SEQ ID NO:1 or to encode a sequence corresponding to said SEQ ID NO:2 above 80%.
The invention further provides another venA mutant gene encoding said mutant protein, characterized in that: the nucleotide sequence of the mutant gene is shown as SEQ ID NO:3 is shown in the figure; or to encode a sequence corresponding to SEQ ID NO:4 above 80%.
The invention further provides another venA mutant gene encoding said mutant protein, characterized in that: the nucleotide sequence of the mutant gene is shown as SEQ ID NO:5 is shown in the figure; or to encode a sequence corresponding to SEQ ID NO:6 is higher than 80%.
The invention also provides a preparation method of the novel diterpenoid compound, and the mutant protein is obtained by expressing the venA mutant gene in escherichia coli through a heterologous expression strategy; the mutant protein is separated and purified to obtain a compound shown as a formula (1), a formula (2), a formula (3) or a formula (4).
The invention also provides application of the novel diterpenoid compounds in pharmaceutical chemical industry and agricultural production.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a novel diterpenoid compound, and a preparation method and application thereof. The inventor modifies key amino acids in a diterpenoid synthase VenA active pocket from S.venezuelae ATCC 15439 in a point mutation mode to obtain mutant genes, and realizes the expression of diterpenoid compounds in escherichia coli. Finally, 4 novel diterpenoid compounds are obtained through separation and purification, and the diterpenoid compounds have novel structures and functions. In addition, the innovation point of the invention is also embodied in the construction of self-sufficient engineering bacteria for producing novel diterpenoid compounds by utilizing methods of enzyme engineering and genetic engineering. The whole operation process is simple, the process is mature, the cost is low, the whole process does not contain harmful impurities, and the preparation method is nontoxic and is very environment-friendly. The obtained novel diterpenoid compound is expected to be applied to the fields of pharmaceutical chemical industry, agricultural production and the like.
Drawings
FIG. 1 is an ellipsoidal diagram of a three-dimensional structure of a compound according to an embodiment of the present invention; wherein (a) is venezuelaenol a; (b) Is (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene;
FIG. 2 is a gas mass spectrum of venezuelaenol A provided in an embodiment of the invention;
FIG. 3 is a diagram of venezuelaenol A according to an embodiment of the present invention 1 H- 1 Key related signals for H COSY, HMBC and NOESY;
FIG. 4 shows a Venezuelaenol A in CD according to an embodiment of the present invention 3 In CN 1 H NMR chart;
FIG. 5 is a schematic view of a displayThe venezuelaenol A provided by the embodiment of the invention is arranged on the CD 3 In CN 13 C NMR chart;
FIG. 6 shows a Venezuelaenol A in CD according to an embodiment of the present invention 3 HSQC map in CN;
FIG. 7 shows a Venezuelaenol A in CD according to an embodiment of the present invention 3 HMBC graph in CN;
FIG. 8 shows a Venezuelaenol A in CD according to an embodiment of the present invention 3 In CN 1 H- 1 H COSY pattern;
FIG. 9 shows a Venezuelaenol A in CD according to an embodiment of the present invention 3 NOESY diagram in CN;
FIG. 10 is a gas mass spectrum of venezuelaenol B provided in an embodiment of the invention;
FIG. 11 shows venezuelaenol B according to an embodiment of the invention 1 H- 1 Key related signals for H COSY, HMBC and NOESY;
FIG. 12 shows a Venezuelaenol B in CD according to an embodiment of the present invention 3 In CN 1 H NMR chart;
FIG. 13 shows a Venezuelaenol B in CD according to an embodiment of the present invention 3 In CN 13 C NMR chart;
FIG. 14 shows a Venezuelaenol B in CD according to an embodiment of the present invention 3 In CN 13 C-DEPT 135 DEG map;
FIG. 15 shows a Venezuelaenol B in CD according to an embodiment of the present invention 3 HSQC map in CN;
FIG. 16 shows a Venezuelaenol B in CD according to an embodiment of the present invention 3 HMBC graph in CN;
FIG. 17 shows a Venezuelaenol B in CD according to an embodiment of the present invention 3 In CN 1 H- 1 H COSY pattern;
FIG. 18 shows a Venezuelaenol B in CD according to an embodiment of the present invention 3 NOESY diagram in CN;
FIG. 19 is a diagram of a gas mass spectrum of a Dictyriene C provided by an embodiment of the present invention;
FIG. 20 is a diagram of a Dictyriene C according to an embodiment of the present invention 1 H- 1 Key related messages of H COSY, HMBC and NOESYA number;
FIG. 21 shows a Dictyriene C in CD according to an embodiment of the present invention 3 In Cl 1 HNMR diagram;
FIG. 22 shows a Dictyriene C in CD according to an embodiment of the present invention 3 In CN 13 C NMR chart;
FIG. 23 shows a Dictyriene C in CD according to an embodiment of the present invention 3 In Cl 13 C-DEPTQ diagram;
FIG. 24 shows a Dictyriene C in CD according to an embodiment of the present invention 3 HSQC pattern in Cl;
FIG. 25 shows a Dictyriene C in CD according to an embodiment of the present invention 3 HMBC plot in Cl;
FIG. 26 shows a Dictyriene C in CD according to an embodiment of the present invention 3 In Cl 1 H- 1 H COSY pattern;
FIG. 27 shows a Dictyriene C in CD according to an embodiment of the present invention 3 NOESY diagram in Cl;
FIG. 28 is an ECD analysis chart of the Dictyriene C provided by the embodiments of the present invention;
FIG. 29 is a graph of the gas phase of (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene provided by the example of the present invention;
FIG. 30 shows (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene provided by an embodiment of the present invention 1 H- 1 Key related signals for H COSY, HMBC and NOESY;
FIG. 31 shows the (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene in CD according to an embodiment of the present invention 3 In Cl 1 HNMR diagram;
FIG. 32 shows the (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene in CD according to an embodiment of the present invention 3 In Cl 13 CNMR map;
FIG. 33 shows the (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene in CD according to an embodiment of the present invention 3 In Cl 13 C-DEPT 135 DEG map;
FIG. 34 shows the (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene in CD according to an embodiment of the present invention 3 HSQC pattern in Cl;
FIG. 35 is a diagram of an embodiment of the present invention (1S, 3E,7E,11R, 1)2S) -3,7,18-dolabellatriene in CD 3 HMBC plot in Cl;
FIG. 36 shows the (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene in CD according to an embodiment of the present invention 3 In Cl 1 H- 1 HCOSY diagram;
FIG. 37 shows the (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene in CD according to an embodiment of the present invention 3 NOESY plot in Cl.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and any simple modification, equivalent changes and modification made to the embodiments according to the technical substance of the present invention are within the scope of the technical solution of the present invention.
The basic molecular biology experimental techniques such as PCR amplification, plasmid extraction, transformation, etc., used in the examples of the present invention, unless otherwise specified, are usually performed according to conventional methods, and can be specifically described in "molecular cloning Experimental guidelines" (third edition) (Sambrook J, russell DW, janssen K, argentine J Huang Peitang, et al, 2002, beijing: scientific Press), or according to the specifications provided by the relevant manufacturers.
Plasmids pET28b-venA and pET28b-venD used in the examples of the present invention were stored in the laboratory; escherichia coli BL21 (DE 3) competent cells were purchased from Qingdao Optimaceae. The construction method of the engineering bacterium Eco-P (Escherichia coli BL (DE 3)/pACYC-mavEmavS & pTrc-low) of the escherichia coli is shown in Microb Cell face 15,74 (2016).
The one-step cloning kit used in the examples of the present invention was purchased from the company Nanjinouzan; agarose gel DNA recovery kit was purchased from Omega company; coli expression vector pET28b was purchased from Invitrogen; high fidelity DNA polymerase was purchased from Takara; restriction enzymes were purchased from sirolimus; PCR primer synthesis and DNA sequencing were done by qinghao.
The seed culture medium of the enterobacteria related to the embodiment of the invention is LB liquid culture medium, and the formula is as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of NaCl; the fermentation culture medium is TB cultureThe formula of the nutrient medium is as follows: 12g/L tryptone, 24g/L yeast extract, 40g/L glycerol, K 2 HPO 4 9.4g/L，KH 2 PO 4 2.2g/L。
Example 1 Gene expression vector construction
The diterpene synthase VenA mutant encoding gene venA is obtained by using pET28b-venA as a template and respectively using primers W107A-F/W107A-R, V111W-F/V111W-R and F185A-F/F185A-R (Table 1) W107A 、venA V111W And venA F185A The reaction system is as follows: 5X PrimeSTAR GXL Buffer. Mu.L, 200. Mu.M dNTPs, 4. Mu.L DMSO, 0.3. Mu.M each of the upstream and downstream primers, an appropriate amount of DNA template (10-100 ng), high-fidelity polymerase (PrimeSTAR GXL DNA polymerase) 2.5U, ddH 2 O is added to 50 mu L; the reaction conditions are as follows: pre-denaturation at 98℃for 5min, denaturation at 98℃for 30s, annealing at 60℃for 15s, extension at 68℃for 1kb/min, set time according to the fragment length of interest, 35 cycles of reaction, and finally extension at 68℃for 10min. Amplified linearized plasmid pET28b-venA W107A 、pET28b-venA V111A 、pET28b-venA F185A After purification by the nucleic acid purification kit, the template DNA was further removed by dpnl digestion.
In this example, the mutant encoding gene venA F185A 、venA V111W And venA W107A The nucleotide sequences of (a) are respectively shown as SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO:5, the amino acid sequences of the encoded mutant proteins are respectively shown in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: shown at 6. In other embodiments of the invention, the sequence of the mutant encoding gene may also be a sequence encoding a sequence as set forth in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO:6 or a nucleotide sequence corresponding to an amino acid sequence having a sequence identity of more than 80% to the amino acid sequence described above.
Subsequently, PCR was performed using the vector pET28b-venD expressing geranylgeranyl pyrophosphate synthase as a template and the primer T7venD-F/T7venD-R to obtain a DNA fragment containing the T7 promoter and terminator sequences and venD, followed by digestion with DpnI to remove the template DNA. Finally, venD was cloned into the above prepared linearized pET28b-venA W107A 、pET28b-venA V111W And pET28b-venA F185A The co-expression vector pET28b-venA containing 2 venA mutant genes of T7 promoters and venD was obtained W107A D、pET28b-venA V111W D and pET28b-venA F185A D。
Table 1: primer sequences for amplification of genes encoding diterpene synthase VenA mutant in example 1
EXAMPLE 2 construction of recombinant E.coli, fermentation production of novel diterpenoid Compounds
pET28b-venA W107A D、pET28b-venA V111W D and pET28b-venA F185A D is transformed into engineering bacteria Eco-P (Escherichia coli BL (DE 3)/pACYC-mavEmavS of escherichia coli which is prepared in advance and can produce high yield of dimethylpropenyl pyrophosphate and isopentenyl pyrophosphate&pTrc-low) chemical competence to obtain recombinant escherichia coli engineering bacterium Eco-P/pET28b-venA for self-sufficient production of novel diterpenoid compounds W107A D、Eco-P/pET28b-venA V111W D and Eco-P/pET28b-venA F185A D。
Eco-P/pET28b-venA was picked from LB solid plates (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L,15g/L agar powder) F185A D was inoculated into 100mL of LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl 10 g/L) containing 50. Mu.g/mL kanamycin, 25. Mu.g/mL chloramphenicol, and 100. Mu.g/mL ampicillin, and cultured overnight at 37℃and 220 rpm. Then, the seed solution was transferred to 10L of TB medium (tryptone 12g/L, yeast extract 24g/L, glycerol 40g/L, K) containing 50. Mu.g/mL kanamycin, 25. Mu.g/mL chloramphenicol and 100. Mu.g/mL ampicillin in a volume ratio of 1:100 2 HPO 4 9.4g/L，KH 2 PO 4 2.2 g/L) at 37℃and 220 rpm. When OD is 600 Reaching 1.0-1.5, adding isopropyl-beta-D-thiogalactoside with the final concentration of 0.2mM, inducing protein expression at 18 ℃ and 220rpm, and biosynthesizing venezuelaenol A and venezuelaenol B. The same method adopts recombinant engineering strain Eco-P/pET28b-venA V111W D biosynthesis of Dictyriene C; using Eco-P/pET28b-venA W107A D biosynthesis (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatrien.
Example 3 isolation and purification and Structure identification of novel diterpene Compounds
Coli Eco-P/pET28b-venA to be recombined W107A D、Eco-P/pET28b-venA V111W D and Eco-P/pET28b-venA F185A D, after fermenting for 3 days, adding an equal volume of ethyl acetate for extraction for three times, and evaporating the extract by using a rotary evaporator to obtain an extract. Then, the extract was extracted four times with 50-100mL of n-hexane to remove the polar components. Finally, the n-hexane extract was evaporated to dryness and dissolved in acetonitrile for semi-preparation of the diterpene compound by reversed phase C18 chromatographic column.
The specific column was YMC-18 column (10X 250mm,5 μm) and the elution procedure for preparing venezuelaenol A and venezuelaenol B was: 100% acetonitrile at a flow rate of 1.5mL/min (0-17 min); the flow rate is 2.0mL/min (7.5-23 min); the flow rate is 2.5mL/min (23.5-29 min); the flow rate was 3mL/min (29.5-35 min). The elution procedure for the preparation of Dictyriene C and (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatrien was as follows: 100% acetonitrile was eluted for 30min at a flow rate of 3mL/min.
Finally, the obtained venezuelaenol A is colorless transparent oil-like after being dried by nitrogen, and has specific rotation
(c 0.083,CH 3 CN). Further, in order to complete the structure identification and analysis of venezuelaenol a, 2mg of the compound was dissolved in 0.2mL of chromatographically pure acetonitrile, naturally volatilized at 4 ℃ and needle-like crystals were formed after about one week for X-ray single crystal diffraction analysis to obtain the three-dimensional structure thereof, as shown in fig. 1 (a).
Compound venezuelaenol A (formula: C) 20 H 34 The chemical structure of O) is shown as a formula (1):
the chemical shifts were assigned to table 2 by gas phase mass spectrometry as shown in fig. 2 and by nuclear magnetic resonance spectroscopy as shown in fig. 3-9.
Table 2: examples of venezuelaenol A 1 H NMR(600MHz,CD 3 CN) and 13 C NMR(151MHz,CD 3 CN) chemical shift assignment
The finally obtained venezuelaenol B is dried by nitrogen to be white powder, and has specific rotation
(c 0.133,CH 3 CN). From gas phase mass spectrum (figure 10) and nuclear magnetic resonance spectrum analysis (figures 11-18), it was confirmed that it was diterpene compound (molecular formula: C) containing 6-5-6-6 new skeleton 20 H 34 O) and its chemical shift is assigned to table 3.
The chemical structural formula of the compound venezuelaenol B is shown as a formula (2):
table 3: examples of venezuelaenol B 1 H NMR(600MHz,CD 3 CN) and 13 C NMR(151MHz,CD 3 CN) chemical shift assignment
The finally obtained Dictyriene C is colorless transparent oil-like after being dried by nitrogen, and has specific rotation
(c 0.125,CH 2 Cl 2 ). From the gas phase mass spectrum (FIG. 19), nuclear magnetic resonance spectrum and ECD analysis (FIGS. 20-28), it was confirmed that it was a diterpene compound having a 5-7 skeleton (molecular formula: C) 20 H 32 ) And its chemical shift is assigned to table 4.
The chemical structural formula of the compound Dictyriene C is shown as a formula (3):
table 4: in examples, dictyriene C 1 H NMR(600MHz,CDCl 3 ) And 13 C NMR(151MHz,CDCl 3 ) Chemical shift assignment
The finally obtained (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene is dried by nitrogen to be colorless transparent oil, and has specific rotation [ alpha ]] 2 D 0 ＝-27.5(c 0.417,CH 2 Cl 2 ). Further, to complete the structural identification and resolution of (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene, 8mg of the compound was dissolved in 0.5mL of chromatographically pure acetonitrile, naturally volatilized at 4℃for about one week, and needle-like crystals were formed for X-ray single crystal diffraction analysis to obtain the three-dimensional structure thereof, as shown in FIG. 1 (b). From the gas phase mass spectrum (FIG. 29, nuclear magnetic resonance spectroscopy (FIGS. 30-37), it was confirmed that it was a diterpene compound having a 5-11 skeleton (molecular formula: C) 20 H 32 And its chemical shift is assigned to table 5.
The chemical structural formula of the novel compound (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene is shown as a formula (4):
table 5: examples (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene 1 H NMR(600MHz,CDCl 3 ) And 13 C NMR(151MHz,CDCl 3 ) Chemical shift assignment
EXAMPLE 4 evaluation of the Compounds venezuelaenol A, venezuelaenol B, dictyriene C and (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatrien for use in medicine and agriculture
To investigate the antibacterial activity of compounds venezuelaenol a, venezuelaenol B, dichotomene C and (1 s,3e,7e,11r,12 s) -3,7,18-dolabella triene, novel diterpenoid compounds were tested against seven gram positive bacteria (Ampicillin-resistant Bacillus subtilis), bacillus subtilis RIK1285 (B. Subtitle 1285), bacillus subtilis 168 (B. Subtitle 168), bacillus cereus (Bacillus cereus), potato ring rot pathogen (Clavibacter michiganense subsp. Sepedonicus), staphylococcus aureus (Staphylococcus aureus ATCC 25923) and mycobacterium smegmatis (Mycolicibacterium smegmatis MC 155)) and four gram negative plant pathogenic bacteria (carrot pectobacterium), cucumber bacterial horn (54 p 83), cucumber bacterial horn (67 p.37) and rice plant pathogenic bacteria (67.37.67.67.yellow rot) were determined according to a trace broth dilution method.
Dissolving the above compound in dimethyl sulfoxide to obtain mother solution with concentration of 256 μg/mL, and adding into solution containing 10 6 The cfu/mL indicator bacteria are diluted in LB culture medium step by step to 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 mug/mL and the like, and the negative control is only added withAn equal volume of dimethyl sulfoxide was added. After the indicator bacteria are continuously cultured for 24 hours at 28 ℃ and 200rpm, the concentration of the compound corresponding to the clarified sample hole is the minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC). As shown in table 6, compound venezueleaenol a exhibited moderate inhibitory activity against bacillus subtilis 168, staphylococcus aureus ATCC25923, and cucumber angular leaf spot bacteria; wherein, the antibacterial spectrum of the compound Dictyriene C is wider, and the compound Dictyriene C shows the moderate inhibitory activity on 9 indicator bacteria to be detected. The compounds venezuelaenol A and the Dictyriene C are shown to have great application potential in the fields of medicine and agriculture.
Although the compound venezuelaenol B and the 5-11 dolastane containing backbone (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene did not exhibit significant antibacterial activity against the indicator bacteria. However, since many active natural products are reported to be modified by a sea-rabbit alkane skeleton, and venezuelaenol B contains a complex ring system similar to venezuelaenol A, the compounds venezuelaenol B and (1S, 3E,7E,11R, 12S) -3,7,18-dolabellatriene are expected to serve as precursors to be further modified by oxidation, glycosylation and the like to obtain corresponding active lead molecules, and the compounds are applied to the fields of medicine, agricultural production and the like.
Table 6: examples the invention provides novel diterpene compounds useful in the antibacterial activity test
Claims (9)
1. A novel diterpenoid compound, which is characterized in that: the compound is a compound containing 5-5-6-7tetracyclic diterpenoid skeletons, and the structural formula of the compound is shown as formula (1):
or the compound is a compound containing a 6-5-6-6 tetracyclic diterpenoid skeleton, and the structural formula of the compound is shown as the formula (2):
or the compound is a compound containing 5-7 bicyclo diterpenoid frameworks, and the structural formula of the compound is shown as formula (3):
or the compound is a compound containing 5-11 bicyclo diterpenoid frameworks, and the structural formula of the compound is shown as formula (4):
2. a mutant protein, characterized in that: the amino acid sequence is shown in SEQ ID NO:2 or is identical to SEQ id no:2 above 80%; the mutant protein is separated and purified to obtain a compound shown as a formula (1) or a formula (2).
3. A mutant protein, characterized in that: the amino acid sequence of which is SEQ ID NO:4 or is identical to SEQ id no:4, the sequence identity is higher than 80%; the mutant protein is separated and purified to obtain a compound shown as a formula (3).
4. A mutant protein, characterized in that: the amino acid sequence of which is SEQ ID NO:6 or is identical to SEQ id no:6, the sequence identity is higher than 80%; the mutant protein is separated and purified to obtain a compound shown as a formula (4).
5. A venA mutant gene encoding the mutant protein of claim 2, wherein: the nucleotide sequence of the mutant gene is shown as SEQ ID NO:1 or to encode a sequence corresponding to said SEQ ID NO:2 above 80%.
6. A venA mutant gene encoding the mutant protein of claim 3, wherein: the nucleotide sequence of the mutant gene is shown as SEQ ID NO:3 is shown in the figure; or to encode a sequence corresponding to SEQ ID NO:4 above 80%.
7. A venA mutant gene encoding the mutant protein of claim 4, wherein: the nucleotide sequence of the mutant gene is shown as SEQ ID NO:5 is shown in the figure; or to encode a sequence corresponding to SEQ ID NO:6 is higher than 80%.
8. The method for preparing the novel diterpenoid compound according to claim 1, which is characterized in that: expressing the venA mutant gene in escherichia coli by a heterologous expression strategy to obtain mutant protein; the mutant protein is separated and purified to obtain a compound shown as a formula (1), a formula (2), a formula (3) or a formula (4).
9. Use of a novel diterpenoid compound as claimed in claim 1 or 2 in pharmaceutical chemical or agricultural production.
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