ISOLATION OF 3 O-ACETATESTACHYBOTRIDIAL FROM THE TOXIC MUSHROOM Stachybotrys chartarum Uz-21 AND STUDY OF ITS STRUCTURE

ABSTRACT

The purpose of the study is to determine the composition and fungus Stachybotrys chartarum Uz-21, a toxic micromycete isolated from the root rhizosphere of crops and soil, as well as structure of physico-chemical and biological properties of isolated secondary metabolites.

Moreover, separation of 3 O-acetatestachybothridial secondary metabolite with fresh sesquiterpenoid structure from a local fungus, Stachybotrys chartarum Uz-21, development of O-acylation synthesis methods of new derivatives, and study using structure by means of modern physical research methods as IR, ¹H, ¹³C NMR, 2M NMR ¹H-¹H correlation ROESY methods, 2M NMR ¹H-¹³C correlation HMQC, НМВС methods, mass spectrometry methods and high performance thin layer Chromatography, high-performance gas-liquid adsorption chromatography methods.

АННОТАЦИЯ

Цель исследования - определение состава и гриба Stachybotrys chartarum Uz-21- токсичного микромицета, выделенного из корневой ризосферы сельскохозяйственных культур и почвы, а также структуры физико-химических и биологических свойств выделенных вторичных метаболитов.

Кроме того, выделено 3 O-ацетатестахиботридиальное вторичное метаболитное соединение со свежей сесквитерпеноидной структурой из местного гриба Stachybotrys chartarum Uz-21, разработка методов О-ацилирования синтеза новых производных и изучение структуры проводили с помощью современных физических методов исследования, таких как IR, ¹H, ¹³C. ЯМР, 2М ЯМР ¹H-¹H корреляционные методы ROESY, 2М ЯМР ¹H-¹³C корреляционные методы HMQC, НМВС, методы масс-спектрометрии и высоко эффективной тонкослойной хроматографии, методы высоко эффективной газожидкостной адсорбционной хроматографии.

Ключевые слова: стахиботриотоксикоз, морфологический, таксономический, этиологический, экстракция, колонка, спектр, силуфол, хроматография, штамм, микрогриб.

Keywords: Stachybotriotoxicosis, morphological, taxonomic, etiological, extraction, column, spectrum, silufol, chromatography, strain, microfungus.

INTRODUCTION

Phenylspirodrimanes are a diverse family of fungal metabolites with a common drimane skeleton and a meroterpenoid, consisting of a benzene ring linked through a spirofuran. Phenylspirodrimane-type metabolites are secondary metabolites of Stachybotrys fungi and can be used as a chemotaxonomic marker for such structural metabolites [1]. To date, more than 80 compounds of this class of metabolites have been isolated from Stachybotrys fungi.

In addition to trichothecenes, as mentioned above, four new phenylspirodrimane sesquiterpenoids stachybotrin, stachybotrolide, stachybothral and stachybotrin A were also identified from the biomass and culture liquid of S. alternans strain (spores collected from the soil collected from the Tashkent region of Uzbekistan and grown and isolated in the mycology laboratory of the Institute of Botany). Their structures were studied by spectroscopic methods and it was determined for the first time that the structure of stachybotrin is a molecule with a phenylspirodrimane structure based on X-ray crystallographic studies [1-8].

This K-76 metabolite Stachybotrys sp. is the best-known phenylspirodriman metabolite isolated from the culture fluid of the fungus. K-76 (isolated from a spore collected from soil in Okinawa, Japan) was obtained as a result of an extensive effort involving the screening of over 3000 actinomycete and fungal strains for their inhibitory activity. [9].

Later, stachybotryn was found in another fungus, Stachybotrys sp. isolated from strain F462 [10 ].

MATERIALS AND METHODS

Stachybotrys chartarum Uz-21was isolated and purified from the plant rhizosphere at the Institute of Microbiology of the Academy of Science of Republic of Uzbekistan, strain was taken from Mandel's nutrient medium in 10 l volume and grown in pots at room temperature for 20 days. When the secondary metabolites were quantitatively analyzed, it was found that the amount of secondary metabolites grown for 14 days was high. Stachybotrys chartarum Uz-21 in order to isolate secondary metabolites strain biomass was separated from the culture fluid by filtration and the culture fluid and biomass composition was studied using the following method.

Stachybotrys chartarum Uz-21strain was grown in 10 l Mandel's medium for 14 days. The grown biomass was 23.86 g when separated from the culture liquid. Filtered 23.86 g of biomass was placed in a 500 ml flask containing 150 ml of ether and heated in a water bath to 40-45⁰C with a reflux condenser installed at the mouth of the flask. Extraction process of fungal biomass repeated three times and 3.70 gr extraction amount received. The remnant watery part was extracted with chloroform, and when the extract was dried with vacuum rotor equipment, 0.93 gr extraction sum was formed. Extraction amount common weight was 4.63 g (Table 1).

Table 1.

Results of quantitative analysis of secondary metabolites of Stachybotrys chartarum Uz-21strain

The highest advantage of the above-mentioned methods is the quantitative and qualitative clean and easy separation of identifiable secondary metabolites, and isolated secondary metabolites were separated by ether extraction of fungal biomass and culture fluid. This method is characterized by easy purification of substances from additives and low amount of primary metabolites.

Stachybotrys chartarum Uz-21 strain were identified by column chromatography and the following secondary metabolites (Table 2) were isolated.

Table 2.

Stachybotrys chartarum Uz-21 secondary metabolites obtained from the strain

RESULTS

Isolation of 3O-acetatestachybotrydial (IX). 32.8 g dry extract amount of the strain of Stachybotrys chartarum Uz-21 was adsorbed on 800 g Silpearl silica gel. The adsorbent was placed in a chromatographic column. Chromatographic column 5-18 mg of 3 O-acetatestachybothridial (IX) was isolated when washed with the system. The isolated substance was found to have crystalline properties. The structure of the obtained substance was determined using ¹ H NMR spectroscopy methods. C₂₅H₃₂O₆, liquid. temp. 148°С (methanol), R_f=0.65 (YuQX, silufol, 5 - system), [α] _D²⁴ =11.6 ± 2° (σ 0.8; CHCl₃ -MeOH, 1:1) .

Synthesis of 3O, 13O- diacetatestachybothridial (X). 12 mg of 3-acetacetoxytridial was taken, 1 ml of absolute pyridine was added to it, and it was acetylated with 0.5 ml of acetic anhydride and kept at room temperature for 15 minutes. The solvent of the reaction mixture was evaporated and the residue was subjected to column chromatography. When the chromatographic column was washed with system eluent 2, 8.45 mg (yield 70.41%) of 3 O, 13 O-diacetatetaxybothridial (X) was obtained. taken, C₂₇H₃₄O₇ , liquid tem. 257°C, [α]_D²⁴ =13.3 (s 0.8; CHCl ₃ -MeOH, 1:1), Rf=0.71 (YuQX, silufol, system 2).

Synthesis of 3O,13O-diacetatetaxybotricarbonic acid (XI). Take 15 mg of 3O,13O-diacetatetaxybothridial (X) and dissolve in 5 ml of acetone and cool to -5°. Add 2 drops of Jones reagent to the reaction mixture and stir for 10 minutes at this temperature. The reaction was stopped by adding a few drops of methanol. After the reaction mixture was worked up, the solvent was evaporated and the residue was subjected to advanced chromatography and washed with system 1 . Chromatography yielded 7.29 mg (yield 48.78%) of 3O,13O - diacetatetaxybotricarbonic acid (XI), C₂₇N₃₄O₉ , liquid. tem. 210°C, [α]_D²⁴ =13.4 (s 0.8; CHCl ₃ -MeOH, 1:1), R_f =0.78 (YuQX, silufol, 1 – system).

DISCUSSION

The doublet signal of the AB system (2H-11) proton gives ² J = 16 Hz SSCC signal at δ_H 3.27 and 3.32 and the singlet signal of the aromatic proton was shown at δ_H 7.26. These data indicate that the new metabolite is a sesquiterpenoid drimane spirobenzofuran series metabolite. δ_H 0.85 – 1.24 signals of two methyl groups were observed. The appearance of a single H-5 proton-specific doublet-doublet signal at ³ J ₁ = 13 and ³ J ₂ = 2.4 Hz SSCC values at δ_H 2.56 indicates that rings A and B are in the trans-configuration state. Also, the formation of a multiplet signal at δ_H 3.61 characteristic of one proton indicates the presence of a proton located geminal to the acetate group and indicates the β -orientation direction of the proton located on the C-3 carbon. Therefore, the acetate group on the C-3 carbon is considered to have an α- orientation. δ_с 74.76 in the ¹³ C NMR spectrum of the acetate group located on the secondary carbon in metabolite IX. formation of signals at , the appearance of a triplet signal in the ¹ H NMR spectrum of monoacetate IX at δ_H 3.61 at J = 2.7 Hz SSСС value (CDCl_3), confirms the conclusion about the configurational location of the acetate group in question (Table 3).

Table 3.

3 O-acetatstaxybothrodial (IX) and 3O,13O-diacetate stachybothrodial (X)  (C₅D₅N, 0 – ТМC)

Note: IX and the spectrum of diacetate X was recorded in deuteropyridine solvents. Chemical shifts, multipletity and spin-spin interaction constant (SSСС) were determined in 2M NMR ¹H- ¹H COSY and HMQC correlation spectra. Abbreviations: s-singlet, d-doublet, t-triplet, dd-doublet-doublet, td-triplet-doublet, dt-doublet-triplet, tt-triplet-triplet, kd-quartet-doublet, m-multiplet .

The fifth and sixth oxygen atoms belong to the dialdehyde group, whose carbon atoms were observed in the resonance state at δ_c 189.72 and 190.47 in the ¹³ C NMR spectrum of metabolite IX. Signals of protons of dialdehyde group are δ_H 9.5 and 9.7 was observed in the singlet signal. Based on biogenesis, it can be assumed that the dialdehyde function involves the C-20-21 atoms, since the reduction of the lactone group of 3O-acetatstaxybotrodial results in the formation of a dialdehyde group. The acylation reactions specific to the hydroxyl group in the aromatic ring of the obtained 3O-acetacetoxybothrodial (IX) follow the following equation:

When studying the acylation reactions specific to hydroxyl groups in the molecule, δ_H 7.40 of the proton located in the aromatic ring in the ¹H NMR spectrum. The appearance of singlet signals, the shift of the signals to the weak region, indicates that the reaction went to the hydroxyl group located in the aromatic ring:

As a nucleophilic agent, the oxygen atom of the hydroxyl group located in the aromatic ring of the 3O-acetacetoxybothridial molecule interacts with the positive carbon atom of the acetic anhydride molecule and forms an intermediate compound. During the reaction, the covalent bond between oxygen and carbon creates a new intermediate compound. Due to the displacement of the hydrogen atom in the hydroxyl group, acetic acid is released and 3O,13O-diacetatetaxybothridial (X) is obtained.

3O,13O-diacetatastachybothridial (X) molecule was oxidized with Jones reagent to synthesize 3O,13O-diacetatstachybothricarbonic acid (XI) .

Thus, the compound corresponding to the formula XI was synthesized for the first time in the work. When ¹H NMR spectra were studied, the signals of protons located on C-20-21 carbon were shifted to the weak region at δ_H 11.2 and 11.4 shift to the area indicates the transfer of aldehyde groups to carboxyl groups.

CONCLUSION

The data show that the structure of 3O-acetacetachybotrodial obtained from the sum extracted from the culture liquid of the fungus Stachybotrys chartarum Uz-21with ethylacetate solvent was determined by chemical and modern physical research methods namely NMR ¹H, ¹³C, ¹H-¹H COSY, HMQC and HMBC. It was determined that the new sesquiterpnoid drimane is a new compound with a benzofuran structure using correlation spectrum methods.

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