Cornifronone: A cadinane-type sesquiterpene from a mason bee ( Osmia cornifrons )–derived Streptomyces sp

Abstract

A new cadinane-type sesquiterpene named cornifronone is isolated from the body surface of a mason bee (Osmia cornifrons)–derived Streptomyces sp. OC1611-8A. Its structure is identified by high-resolution electrospray ionization mass spectrometry data and nuclear magnetic resonance spectroscopic analysis. The absolute configuration of cornifronone was determined by electronic circular dichroism spectra calculations. Cornifronone inhibits hexokinase activity with a mean IC₅₀ of 124.3 μM.

Introduction

Natural products are organic compounds isolated and extracted from natural organisms and are important sources for the development of practical drugs. ¹ Microorganisms are the main source of natural products, of which actinomycetes are the largest contributors. ² These natural products have important effects on human health and life. ³

Studies in recent decades have shown that it is hard to isolate new actinomycetes from soil and to extract new bioactive secondary metabolites. ⁴ Thus, researchers have turned their studies toward insects. Insects can form special mutually beneficial relationships with their symbiotic microorganisms. Among them, secondary metabolites produced by some insect symbiotic actinomycetes can be used to help host insects protect larvae and cocoons from pathogens;^5–10 for example, the European beewolf (Philanthus triangulum) and Streptomyces philanthi; the Streptomyces can be absorbed by the larvae and transferred to the cocoon wall, thus protecting the cocoon from pathogenic fungi and producing a variety of antibiotics to significantly improve the survival rate of the larvae.^11,12 Interestingly, the spruce beetle (Dendroctonus rufipennis) also has this symbiotic relationship with the actinomycete Micrococcus luteus. M. luteus has strong inhibitory activity against fungi, protecting beetles from pathogenic fungi, thereby increasing the survival rate of spruce beetles. ¹³ However, research on insect symbiotic actinomycetes is still in its infancy, and only the functions of a few insect symbiotic actinomycetes and secondary metabolites have been reported. Therefore, further isolation and analysis of symbiotic actinomycetes from insects and identification of related metabolites of actinomycetes will help in the discovery of new natural products with biological activity.

Cornifronone (Figure 1), a new cadinane-type sesquiterpene, was isolated from the body surface of a mason bee (Osmia cornifrons)–derived Streptomyces sp. OC1611-8A. Sesquiterpenoids have a variety of biological activities, such as antitumor, neurotoxicity, antibacterial, and anti-inflammatory, ¹⁴ and most are derived from plants. Up to now, only very few sesquiterpenoids have been isolated from actinomycetes, such as 15-hydroxy-T-muurolol, ¹⁵ strepsequitriol, ¹⁶ and elgonenes.^17,18 In the detection of sesquiterpenoids by thin-layer chromatography (TLC) analyses, terpene derivatives can be dyed blue or purple by anisaldehyde/sulfuric acid. However, because unsaturated fatty acid derivatives with similar color reactions masked them, their lipid-like nuclear magnetic resonance (NMR) spectra are usually not obvious; this is the reason for their low isolated yields. ¹⁵ Thus, cornifronone enriches the diversity of sesquiterpenoids isolated from actinomycetes.

Figure 1.

The structure of cornifronone (1).

Results and discussion

Structure elucidation

Compound 1 was obtained as a colorless oil. Its molecular formula (C₁₅H₂₄O₄) was determined by mass spectrometry: m/z 251.1641 ([M + H − H₂O]⁺, calcd for C₁₅H₂₃O₃, 251.1642) from high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The ¹H NMR spectrum Figure S3 in Supplemental material showed signals of three methyl groups at δ_H 2.04 (3H, s), 1.29 (3H, s), and 1.04 (3H, d, J = 6.5 Hz); an olefinic proton at δ_H 5.68 (1H, s); and three methylene groups at δ_H 1.73 (2H, m), δ_H 1.74 (2H, m), δ_H 3.58 (1H, d, J = 11.2 Hz), and a methine proton at δ_H 3.76 (1H, d, J = 11.3 Hz). There are 15 carbon signals according to the ¹³C NMR and DEPT-135 spectra Figure S4,S5 in Supplemental material, including three methyl carbons, three methylene carbons, five methine carbons, and three quaternary carbons (one carbonyl at δ_C 200.2, one olefinic carbon at δ_C 162.6, and one oxygenated carbon at δ_C 85.9). The carbonyl and double-bond carbons accounted for two out of the four degrees of unsaturation required by the molecular formula, and the remaining two were suggestive of two rings in 1. These spectroscopic data indicated 1 to be a cadinane-type sesquiterpene with an α,β-unsaturated carbonyl group. ¹⁹

These facts and four degrees of unsaturation indicate that the compound has a bicyclic ketone structure with a double bond. The planar structure of 1 was confirmed through the overall analysis of one-dimensional (1D) and two-dimensional (2D) NMR spectra. Two four-carbon fragments H-4/H-5/H-6/H-7 and H-4/H-5/H-10/H-9 can be found from the ¹H-¹H COSY spectrum Figure S8 in Supplemental material. The heteronuclear multiple-band coherence (HMBC) correlations Figure S7 in Supplemental material between H-14 and C-8, C-9, and C-10 indicate that CH₃-14 is connected to C-9. Similarly, the correlations between H-12 and C-6, C-13, and C-11 revealed that CH₃-12 is connected to C-11. What is more, CH₃-15 was shown to be connected to C-3 by the correlations in the HMBC spectrum between H-15 and C-4, C-2, and C-3 (Figure 2). The correlations from H-9 to C-10 and C-8, and H-7 to C-8 indicated that C-9 is connected to C-10 and C-8, and that C-7 is connected to C-8. The HMBC correlations from Me-12 and H-13 to C-6 indicate that C-11 is linked to C-6. The carbonyl group was positioned at C-1 due to its chemical shift, heteronuclear multiple-quantum coherence (HMQC) correlations Figure S6 in Supplemental material from H-2 to C-1, and weak HMBC correlations from H-5 to C-1. The above evidence indicated that 1 was a 9,10 saturated and 11,13-diol derivative of tatarinowii A. ¹⁹ Therefore, the planar structure of 1 was established.

Figure 2.

The key HMBC, ¹H-¹H COSY and NOESY correlations for 1.

The relative configuration of 1 was based on nuclear Overhauser effect spectroscopy (NOESY) correlations Figure S9 in Supplemental material. The correlations between H-5 and H-10 showed that H-5 and H-10 were on the same side of the compound. Moreover, the correlations between H-5 and H-6 and H-4 demonstrated that H-4, H-5, and H-6 were also on the same side of this compound, which was confirmed by the observed small coupling constant between H-5 and H-6 and H-4. The correlations between H-10 and H-14 revealed that H-10 and H-14 were spatially vicinal, and this also means that H-10 and H-14 are on the same side of the compound. Due to H-5 and H-10 being on the same side, this permits the assignment of the relative configuration as 5S, 10R, 4S, 6S, 9S or 5R, 10S, 4R, 6R, 9R. The absolute configuration of compound (1) was determined by comparing the electronic circular dichroism (ECD) data obtained from experimental tests with the ECD data obtained by calculating the quantum chemical time-dependent density functional theory (TDDFT). Each isomer was optimized using density functional theory (DFT) at the B3LYP/6-311+G(d,p) level in the Gaussian 09 program. The 4R, 5R, 10S, 6R, 9R configuration for 1 was determined by a combination of ECD and ¹³C NMR shift calculations. Therefore, two model molecules (4R, 5R, 10S, 6R, 9R and 4S, 5S, 10R, 6S, 9S) were selected for ECD calculations. The results (Figure 3) showed that the experimental ECD spectrum of 1 was close to the calculated ECD curves of the model molecules 4R, 5R, 10S, 6R, and 9R. Therefore, the absolute configuration of 1 was elucidated as 4R, 5R, 6R, 9R, and 10S and named cornifronone (Table 1).

Figure 3.

Experimental and calculated ECD spectra of compound 1.

Table 1.

¹H and ¹³C NMR data for compound 1 ^a .

Position	δC	δH, m (J in Hz)
1	200.2	—
2	125.6	5.68 (1H, s)
3	162.6	—
4	75.5	4.69 (1H, d, J = 11.2)
5	46.4	2.59 (1H, m)
6	45.0	2.01 (1H, m)
7	22.5	1.73 (2H, m)
8	34.2	1.74 (2H, m)
9	19.2	1.58 (1H, m)
10	52.5	2.24 (1H, dd, J = 11.6, 5.6)
11	85.9
12	26.1	1.29 (3H, s)
13	66.7	3.76 (1H, d, J = 11.3)3.58 (1H, d, J = 11.2),
14	19.8	1.04 (3H, d, J = 6.4)
15	18.8	2.04 (3H, s)

a

Measured in CDCl₃ (¹H NMR at 500 MHz and ¹³C NMR at 125 MHz).

Cytotoxicity and antimicrobial activity

The disk diffusion method was used for testing the antibacterial and antifungal activities of compound 1. The results showed that compound 1 was inactive against all the bacteria and fungi tested. The 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of compound 1 and the NO inhibitory activity of compound 1 was tested using an NO assay kit. The results showed that compound 1 was inactive. The hexokinase (HK) enzyme-linked immunosorbent assay (ELISA) kit measured the IC₅₀ of 1 as 124.3 μM.

Analysis of the crude extract of the mason bee (O. cornifrons)–derived Streptomyces sp. OC1611-8A led to the isolation and structural characterization of a new cadinane-type sesquiterpene, namely, cornifronone. The compound was characterized based on the NMR, high-resolution mass spectrometry (HRMS), and ECD data. The cornifronone showed weak HK inhibitory activity with a mean IC₅₀ of 124.3 μM.

Materials and methods

Microorganism material

Aiming to discover new antibiotics from insect symbiotic actinomycetes, the strain OC1611-8A was isolated from the body surface of O. cornifrons, which was collected in Weihai, Shandong Province, China. Its 16S ribosomal RNA (rRNA) nucleotide sequence (GenBank number: MK358137) was compared with the data in GenBank, and it was found to be similar to Streptomyces lavendulae (AY745498) and Streptomyces toxytricini (HQ844491), with similarities of 99.27% and 99.26%, respectively. It can be confirmed from the neighbor-joining analysis that the strain OC1611-8A and the members of the genus Streptomyces are closely related in phylogeny and clustered with Streptomyces toxytricini (Figure 4). Subsequently, strain OC1611-8A was fermented and the fermentation broth was extracted with ethyl acetate to obtain a crude extract. A new cadinane-type sesquiterpene was isolated from the crude extract and named cornifronone (1). This report describes in detail the method for the separation of cornifronone and its structural characterization.

Figure 4.

Neighbor-joining tree based on 16S rRNA gene sequences showing the relationships between strains OC1611-8A and related members of the Streptomyces family. Numbers at nodes indicate percentages of 1000 bootstrap re-samplings; only values above 50% are shown. The tree is rooted with Streptacidiphilus albus NBRC 100918^T (NR024930). Bar, 0.005 nucleotide substitution per position.

First, the body surface of the mason bee was washed with 2 mL of sterile water to obtain an actinomycete suspension; 200 µL of the suspension was pipetted, transferred to the petri dishes containing Gauze’s medium (20 g starch, 1 g KNO₃, 0.5 g NaCl, 0.5 g K₂HPO₄·3H₂O, 0.5 g MgSO₄· 7H₂O, and 0.01 g FeSO₄·7H₂O per liter of water) containing nystatin (10,000 units/mL) and cyclohexanimide (5%, w/v) as an antibacterial agent, and the petri dishes were cultured at 35 °C. The actinomycetes colonies were singled out and purified to obtain pure morphologies. Genomic DNA was extracted using a DNA isolation kit (Shanghai Sangon Biotech Co., China), and 16S rRNA was amplified by polymerase chain reaction (PCR) using primers Fd2 (5′-GAGTTTGATCATGGCTCAG-3′) and 16Sr (5′-TTGCGGGACTTAACCCAACAT-3′). Finally, sequencing was performed by Shanghai Sangon Biotech Co., China. The nucleotide sequence was stored in GenBank (Accession number MK358137), and BLASTN software was used to retrieve closely related taxa from the GenBank database. The strain (No. OC1611-8A) was stored in the Laboratory of Natural Products Chemistry, Department of Pharmacy, Shandong University at Weihai.

Fermentation and separation

Strain Streptomyces sp. OC1611-8A was initially cultured on Gauze’s medium (20 g/L starch, 1 g/L KNO₃, 0.5 g/L NaCl, 0.5 g/L K₂HPO₄·3H₂O, 0.5 g/L MgSO₄·7H₂O, and 0.01 g/L FeSO₄·7H₂O) for 7 days. Then, a selected single colony was inoculated into 100 mL of Gauze’s liquid medium in Erlenmeyer flasks (250 mL) on a rotary shaker (220 r/min) at 32 °C for 4 days to prepare the seed broth. Subsequently, 10 mL of seed broth was transferred into a sterile 500 mL Erlenmeyer flask containing 150 mL of Gauze’s medium. About 30 L of this fermentation broth was incubated for 14 days at 220 r/min and 32 °C. The fermentation broth was then extracted three times with 30 L of EtOAc. The combined EtOAc solution was evaporated to dryness using a rotary evaporator at 40 °C to give 4.0 g of EtOAc extract. The extract was separated by chromatography on a silica gel (200–300 mesh) column eluting with n-hexane-acetone (10:1, 5:1, 2:1, 1:1, acetone), finally eluting with MeOH. Five fractions (A–E) were collected according to the results of TLC analysis. Fraction B (286 mg) was separated by eluting with silica gel column chromatography with n-hexane-acetone (10: 1, 5: 1, 2: 1) to obtain four subfractions B1–B4. Finally, B4 (51 mg) was purified through a C-18 reverse column (H₂O-MeOH, 3:2) to give compound 1 (5 mg).

Cornifronone (1)

Colorless oil.

$${[\alpha ]}_D^{20}=-33.4\left(c0.100,\mathrm{MeOH}\right)$$

Infrared (IR; KBr): 3442, 2929, 1653, 1619, 1440, 1379, 1239, 1072, and 1046 cm⁻¹.

HRESIMS: m/z = 251.1641 ([M + H − H₂O]⁺, calcd for C₁₅H₂₃O₃: 251.1642).

¹H NMR (500 MHz, CDCl₃): δ_H = 5.68 (1H, s, H-2), 4.69 (1H, d, J = 11.2 Hz, H-4), 3.76 (1H, d, J = 11.3 Hz, H-13), 3.58 (1H, d, J = 11.25 Hz, H-13), 2.59 (1H, m, H-5), 2.24 (1H, dd, J = 11.6 Hz, 5.6 Hz, H-10), 2.04 (3H, s, Me-15), 2.01 (1H, m, H-6), 1.74 (2H, m, H-8), 1.73 (2H, m, H-7), 1.58 (1H, m, H-9), 1.29 (3H, s, Me-12), 1.04 (3H, d, J = 6.45 Hz, Me-14). ¹³C NMR (125 MHz, CDCl₃): δ_C = 200.2 (C-1), 162.6 (C-3), 125.6 (C-2), 85.9 (C-11), 75.5 (C-4), 66.7 (C-13), 52.5 (C-10), 46.4 (C-5), 45.0 (C-6), 34.2 (C-8), 26.1 (C-12), 22.5 (C-7), 19.8 (C-14), 29.2 (C-9), and 18.8 (C-15).

Supplemental Material

supporting_information_2 – Supplemental material for Cornifronone: A cadinane-type sesquiterpene from a mason bee (Osmia cornifrons)–derived Streptomyces sp

Supplemental material, supporting_information_2 for Cornifronone: A cadinane-type sesquiterpene from a mason bee (Osmia cornifrons)–derived Streptomyces sp by Guo-Li Li, Ze-Yu Xu, Nan Li, Zhe Wang, Tian Tian and Tong Shen in Journal of Chemical Research