CN112694406B - Preparation method and application of two dimeric hexyl itaconic acid derivatives - Google Patents

Abstract

The invention discloses two novel dihexyl itaconic acid derivatives prepared by fermentation and separation of Aspergillus sp MF180262 and application of a compound in preparation of a herbicide, wherein the compound has good inhibition activity on growth of chlorella.

Description

Preparation method and application of two dimeric hexyl itaconic acid derivatives

Technical Field

The invention relates to the technical field of agricultural biology, in particular to two compounds with herbicidal activity.

Background

Due to the special ecological environment of the ocean, such as high pressure, high salt, light resistance, oxygen deficiency and the like, the secondary metabolite of the marine microorganism has good chemical diversity and biological activity. In agricultural production, with the use of a large amount of traditional chemical herbicides, the traditional chemical herbicides are continuously accumulated in human bodies, so that nervous system injury is induced, the human body hormone balance is disturbed, the immunity of the human bodies is reduced, and the health of the human bodies is gradually influenced. Natural products are always important sources of medicines and have the characteristic of low toxic and side effects. In the research aspect of secondary metabolites of aspergillus, alkaloid, polyketone, mixed source terpene and naphthopyrone compounds are mainly found. The discovery of new small molecule compounds with herbicidal effect from aspergillus provides a material resource for the development of natural herbicides.

Disclosure of Invention

A first object of the embodiments of the present invention is to provide two compounds having herbicidal activity, which have the following structures shown in formulas (I) and (II):

it is a second object of embodiments of the present invention to provide a process for the preparation of compounds of formulae (I) and (II), comprising the steps of:

fermenting Aspergillus (Aspergillus sp.) MF180262 in rice solid culture medium, extracting with ethyl acetate and methanol to obtain crude extract, separating and purifying by normal phase silica gel column chromatography, gel Sephadex LH-20 column chromatography and high performance liquid chromatography to obtain the compounds of formula (I) and formula (II).

The embodiment of the invention has the following advantages: the compound provided by the invention has good growth activity of inhibiting chlorella and is suitable for preparing natural herbicides.

Drawings

FIG. 1 is a structural formula of compounds of formula (I) and (II) provided in an embodiment of the present invention.

FIG. 2 is a high resolution mass spectrum of a compound of formula (I) provided in an embodiment of the present invention

FIG. 3 is a diagram of compounds of formula (I) provided in an embodiment of the present invention¹H nuclear magnetic resonance spectrum.

FIG. 4 shows a compound of formula (I) according to an embodiment of the present invention¹³C nuclear magnetic resonance spectrum.

FIG. 5 is a high resolution mass spectrum of a compound of formula (II) provided in an example of the present invention.

FIG. 6 shows a compound of formula (II)¹H nuclear magnetic resonance spectrum.

FIG. 7 is a diagram of a compound of formula (II) provided in an embodiment of the present invention¹³C nuclear magnetic resonance spectrum.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were obtained from a conventional biochemical reagent store.

Example 1 isolation and identification of Strain MF180262

Collection and separation of strain MF180262

A strain is separated from Xiamen coastal sea mud in 12 months in 2018 and is named as a strain MF 180262.

II, identification of strain MF180262

The beta-tubulin gene sequence of the strain MF180262 is shown in a sequence table, and has the highest sequence similarity with GENBANK ACCESSION NO. MH614471.1, wherein the similarity is 100%.

According to the above identification results, the strain MF180262 belongs to Aspergillus (Aspergillus sp.).

Third, preservation of Strain MF180262

The strain MF180262 belongs to Aspergillus sp, and has been deposited in China general microbiological culture Collection center (CGMCC, address No.3 Xilu 1 Beijing Kogyo Beichen) at 21.10.2020, and the registration number of the collection center is CGMCC No. 3.20110.

EXAMPLE 2 fermentation culture of fungi

PDA culture medium: mixing potato 200g, glucose 20g, agar 15g and water 1000mL, and sterilizing with 115 deg.C high pressure steam for 30 min.

Rice culture medium: 200g of rice was soaked in 200mL of water overnight and autoclaved at 121 ℃ for 30 min.

Cutting two pieces of 1cm Aspergillus fungus activated in PDA culture medium²Inoculating the large and small bacterial blocks together with PDA culture medium into rice solid culture medium, and culturing at 28 deg.C for 36 d.

Example 3 extraction of fermentation product

(1) After the fermentation is finished, an organic reagent (ethyl acetate: methanol ═ 4:1) is added into the solid fermentation product, ultrasonic extraction is carried out for 20min at room temperature, and the first supernatant is collected.

(2) Adding an organic reagent (ethyl acetate: methanol ═ 4:1) into the solid fermentation product in the step (1), carrying out ultrasonic extraction at room temperature for 20min, and collecting the second supernatant.

(3) Adding an organic reagent (ethyl acetate: methanol ═ 4:1) into the solid fermentation product in the step (2), carrying out ultrasonic extraction at room temperature for 20min, and collecting the third supernatant.

And combining the first supernatant, the second supernatant and the third supernatant, and distilling under reduced pressure until the mixture is dried to obtain a crude extract.

EXAMPLE 4 isolation and purification of the Compound

(1) Reduced pressure forward silica gel column chromatography

Mixing the crude fermented extract with silica gel (100-200 mesh, 11.58g), separating by reduced pressure normal phase silica gel column chromatography (thin layer chromatography silica gel H; silica gel column size L100 mm,

) Gradient elution is carried out by using mixed solvents of n-hexane/dichloromethane/methanol with different volume ratios, 300mL of the mixed solvent is used for each time, and dichloromethane/methanol elution fractions with the ratio of 19:1 are collected.

(2) Sephadex LH-20 gel chromatographic separation

The fraction collected in step (1) is dissolved by dichloromethane/methanol solvent with the ratio of 2:1, and Sephadex LH-20 gel chromatographic separation is carried out (the size of a gel column is L600 mm,

) Eluting with 2:1 dichloromethane/methanol solvent as mobile phase, and detecting by TLC to obtain combined fractions.

(3) Separating and purifying by high performance liquid chromatography

Separating dichloromethane/methanol eluent of 19:1 of decompression forward silica gel column chromatography by gel chromatography, and then separating by high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography are as follows: an Agilent Zorbax SB-C18 reversed phase chromatographic column (250 multiplied by 9.4mm, 5 μm) is adopted, the flow rate is 3.0mL/min, the acetonitrile concentration is eluted for 30min from 25-80% gradient, the detection wavelength is 254nm, eluents with peak retention time of 12.480min and 12.772min are collected, the collected HPLC eluents are distilled under reduced pressure to be dried, and 25.1mg of the compound (I) and 19.8mg of the compound (II) are respectively obtained.

EXAMPLE 5 structural characterization of the Compounds

Performing structure identification by using mass spectrum and nuclear magnetic resonance technology, and measuring nuclear magnetic resonance by using a Bruker AVANCE DRX-500 NMR Sectrometer; high resolution Mass Spectrometry Agilent Accurate-Mass-Q-TOF LC/MS 6520instrument assay.

The characterization data of the product are as follows:

compound (I) is a colorless, colloidal solid; molecular weight is 442, molecular formula is C₂₂H₃₄O₉(ii) a Process for preparing compound (I)¹H NMR and¹³c NMR analysis results (see Table 1).

Compound (II) is a colorless colloidal solid; molecular weight is 442, molecular formula is C₂₂H₃₄O₉(ii) a Of the compound (II)¹H NMR and¹³c NMR analysis results (see Table 1).

Based on the above characterization data, the physicochemical data and the map of the above compound identify the structures of the compounds (I) and (II), and the two compounds are searched by the database to be new structural compounds.

TABLE 1 NMR data (500MHz, Acetone-d) for compounds (I) and (II)₆)

EXAMPLE 5 analysis of herbicidal Activity of Compounds

And (3) detecting the growth inhibition concentration of the compounds (I) and (II) on chlorella by adopting a microdilution method.

(1) Culture solution

(NH₄)_sSO₄0.2 g/L，Ca(H₂PO₄)₂0.03g/L，MgSO₄·7H₂O 0.08g/L， NaCO₃0.1 g/L，KCl 0.25g/L，FeCl₃(1%) 0.15ml/L, and nutrient solution 0.5 ml/L.

Nutrient solution: h₃BO₄2.85 g/L，MnCl₂·4H₂O 1.8g/L，ZnSO₄·7H₂O 0.22g/L， Na2MoO₄·2H₂O 0.39g/L，CuSO₄·5H₂O 0.08g/L。

(2) Cultivation of algal species

Inoculating Chlorella into Erlenmeyer flask containing sterile above culture medium, placing into illumination incubator, performing shake culture at 25 deg.C, centrifuging to collect algae, and adding NaHCO with concentration of 15mg/L₃After the solution is washed by suspension for 3 times, the solution is transferred into a culture solution for standby.

(3) EC of compound on chlorella₅₀Testing

Collecting Chlorella in logarithmic growth phase, filtering with sterile gauze to remove suspended substance, inoculating into sterile culture solution of (1), and adjusting Chlorella initial concentration to 10⁵one/mL, 1mL was added to a 24-well cell culture plate. Each sample was set to a final concentration of 1000. mu.g/mL, 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.25. mu.g/mL. After 96 hours, the OD is measured by using an ultraviolet-visible spectrophotometer₆₈₀。

Inhibition rate (control OD)₆₈₀Treatment of OD₆₈₀) Control OD₆₈₀. EC was calculated from a plot of log and inhibition of compound concentration₅₀。

(4) Activity result determination

The growth inhibitory effect test of the compounds of formulae (I) and (II) on Chlorella vulgaris was repeated three times.

EC of compounds of formula (I) and (II) on Chlorella₅₀168.4 and 189.8. mu.g/mL, respectively.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of geology in China (Beijing), institute of microbiology of Chinese academy of sciences

<120> preparation method and application of two dimeric hexyl itaconic acid derivatives

<130> 2020

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 771

<212> DNA

<213> Aspergillus sp.

<400> 1

atgtttgccc tccccgtccc tcgtccgtca ggagacgcgt cgttggttgg catctcttct 60

gctcgggacc ccaccggttc ttcgaccaac tcattcttgt gctaactgca tgtcttcttc 120

gcttcatagg ttcacctcca aaccggccag tgtgtaagtg ccaatatatg cttcggatga 180

ttgcccccaa gggtcttgat tggtgtttgg tggactaaac aatatatcat ggtggttagg 240

gtaaccaaat tggtgctgct ttctggtacg tatacaactg ccattggatt gggggatgga 300

acatcgtctc ttaggctatc tcagcttgag ttcagatgtt gtccattagg tacatgctat 360

cggtctaaga acacgtctaa caattcaaca ggcagaccat ctctggcgag cacggccttg 420

acggctccgg tgtgtaagtg caactttttc acacctctca attggtcaac aatgggcaaa 480

gggttgggtc ttctgacacg caggatagtt acaatggcac ctccgacctc cagctggagc 540

gcatgaacgt ctacttcaac gaggtgagat ccatcggacc ttggcttttt cacgacaata 600

tcatcaatgt cctaatcact tcagcaggct agcggtaaca agtatgttcc tcgtgccgtc 660

ctcgtcgacc tcgagcccgg taccatggac gccgtccgtg ccggtccttt cggccagctc 720

ttccgccccg acaacttcgt cttcggccag tccggtgctg gtaacaactg g 771

Claims (3)

1. Two compounds having the formula:

2. the use of a compound according to claim 1 for the preparation of herbicides.

3. Use according to claim 2, characterized in that the compound has an EC for chlorella₅₀168 and 189. mu.g/mL, respectively.

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