Antimicrobial bisabolane-type sesquiterpenoids from thedeep-sea sediment-derived fungus Aspergillus versicolorSD-330

Xiao-Dong Lia,b, Xin Lia,c,d , Xiao-Ming Lia,c,d, Xiu-Li Yinb andBin-Gui Wanga,c,d

aKey Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy ofSciences, Qingdao, China; bYantai Institute of Coastal Zone Research, Chinese Academy of Sciences,Yantai, China; cLaboratory of Marine Biology and Biotechnology, Qingdao National Laboratory forMarine Science and Technology, Qingdao, China; dCenter for Ocean Mega-Science, Chinese Academyof Sciences, Qingdao, China

ABSTRACTOne new aromatic bisabolene-type sesquiterpenoid (1), alongwith four known analogues (2–5) were isolated and identifiedfrom the deep-sea sediment-derived fungus Aspergillus versicolorSD-330. Their structures were elucidated by NMR, HRESIMS, X-raycrystallographic analysis, and quantum chemical ECD calculationsas well as comparison of those data with literature data.Compounds 1–5 were evaluated for antimicrobial activitiesagainst human and aquatic pathogenic bacteria. Compounds 1, 2and 5 exhibited selective inhibitory activities against zoonoticpathogenic bacteria such as Aeromonas hydrophilia, Escherichiacoli, Edwardsiella tarda and Vibrio harveyi, with the MIC valuesranging from 1.0 to 8.0lg/mL.

ARTICLE HISTORYReceived 9 September 2019Accepted 14 November 2019

KEYWORDSAspergillus versicolor;deep-sea sediment-derivedfungus; bisabolene-typesesquiterpenoids; antimicro-bial activity

1. Introduction

Aspergillus versicolor is a biosynthetically talented fungal species with great potentialto produce a wide range of structurally diversified secondary metabolites, suchas alkaloids (Ji et al. 2013; Zhou et al. 2014; Cheng et al. 2016), polyketides (Fu et al.2015; Wang et al. 2015, 2016; Li et al. 2018; Wang et al. 2018; Yu et al. 2018;Ding, Ding et al. 2019; Song et al. 2019; Zhu et al. 2019), sterols (Ding, Xu et al. 2019),

CONTACT Bin-Gui Wang wangbg@ms.qdio.ac.cnSupplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1696792.

This article has been republished with minor changes. These changes do not impact the academic content of the article.� 2019 Informa UK Limited, trading as Taylor & Francis Group

NATURAL PRODUCT RESEARCHhttps://doi.org/10.1080/14786419.2019.1696792

and terpenoids (Cui et al. 2018). Some of these metabolites exhibited intriguing bio-logical properties including antimicrobial (Zhou et al. 2014; Wang et al. 2016; Ding,Ding et al. 2019), and cytotoxicity (Yu et al. 2018) as well as enzyme inhibiting activ-ities (Wang et al. 2015; Cheng et al. 2016).

With the purpose of searching for new bioactive metabolites from A. versicolor, wecontinued our studies, five compounds (Figure 1), including one previously unde-scribed bisabolene sesquiterpenoid (1) along with four analogues (2–5), were obtainedand identified from the culture extract of Aspergillus versicolor SD-330, a deep-sea sedi-ment-derived fungus. Their structures were identified by the detailed interpretation ofthe nuclear magnetic resonance (NMR), high-resolution electrospray ionization massspectrometry (HRESIMS) spectra data, the absolute configurations of the new com-pound 1 was determined by the combination of NOESY and quantum chemical ECDcalculations. All of these compounds were examined for antimicrobial activities againsthuman and aquatic pathogenic bacteria. The details of isolation, structural elucidationand bioactivity evaluation of these compounds are discussed below.

2. Results and discussion

Compound 1 was obtained as a colorless oil. HRESIMS ion at m/z 281.1380 [MþH]þ

indicated its molecular formula should be C15H20O5 (calculated for C15H21O5,281.1384), indicating six degrees of unsaturation. The 13C NMR and DEPT spectroscopicdata (Supplemental material, Table S1) along with HSQC spectra revealed the presenceof 15 carbon atoms, which were clarified into six non-protonated carbons, three aro-matic methines, four methylenes (with one oxygenated), and two methyls. The 1HNMR spectrum displayed three aromatic proton signals at d 7.37 (1H, d, J¼ 8.1 Hz, H-3), 7.31 (1H, d, J¼ 8.1 Hz, H-4) and 7.26 (1H, s, H-6); four methylene signals at d 3.05(1H, dd, J¼ 10.7, 10.6 Hz, H-12), d 1.87 (1H, t, J¼ 10.8 Hz, Ha-8) and d 1.64 (1H, t,J¼ 10.8 Hz, Hb-8), d 1.28 (1H, m, Ha-9) and d 1.02 (1H, m, Hb-9), d 1.21 (2H, m, H-10);and two methyl signals at d 0.89 (3H, s, H-13) and 1.48 (3H, s, H-14). Detailed analysisof the 1D and 2D NMR spectra displayed signals similar to those of sydowic acid, aphenolic bisabolane sesquiterpenoid originally isolated from Aspergillus sydowi(Hamasaki et al. 1975), revealing that 1 also belongs to the family of phenolic

Figure 1. The structures of compounds 1–5 isolated from A. versicolor.

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bisabolanes. The main differences between 1 and sydowic acid were the presence ofthe oxygenated methylene signals at dH/C 3.05/69.3 and the absence of one methylsignals, and some variations for the chemical shifts of C-7, C-10, C-11 and C-13. Thekey HMBC correlations (Supplemental material, Figure S7) H-12/C-10, C-13; H-13/C-10,C-12 and H-10/C-12, C-13 indicated that compound 1 was C-12 methyl hydroxylatedderivative of sydowic acid (Hamasaki et al. 1975). Based on the above spectroscopicevidence, the planar structure of 1 was determined.

The relative configuration of 1 was assigned by analysis of NOESY data (Supplementalmaterial, Figure S9). The key NOE correlation between Me-13 and Me-14 indicated thatthey were on the same side of the molecule, while the NOE correlation between H-8 andH-12 suggested that they were on the other face. The absolute configuration of 1 wasstudied by the TDDFT-ECD calculation. The experimental ECD spectrum of 1 was matchedwell with that of calculated for (7R, 11S)-1 (Supplemental material, Figure S11), leading tothe absolute configuration of 1 as 7R, 11S.

The planar structure of compound 2 was identified through the detailed analysis ofthe 1H, 13C and DEPT NMR data (Supplemental material, Table S1) and was comparedwith those of aspergoterpenin C reported in the literature (Guo et al. 2018). Combinedwith the close experimental ECD spectrum (Supplemental material, Figure S16) betweencompound 2 and aspergoterpenin C, we identified compound 2 as the same as aspergo-terpenin C, the absolute configuration of which was determined by comparing the ECDspectra with that of related known analog in the literature (Guo et al. 2018). Consideringits crystallographic data never being reported before, we crystallized it for an X-ray singlecrystallographic analysis and reported its crystallographic data here for the first time(Supplemental material, X-ray crystallographic analysis of compound 2 and Figure S15).

In addition to the compounds 1 and 2, three other bisabolane-type sesquiterpe-noids (3–5) were also isolated. By detailed spectroscopic analysis as well as compari-son with those data from the literatures, the structures of compounds 3–5 wereidentified as (7S,11S)-(þ)-12-hydroxysydonic acid (3) (Chung et al. 2013), (S)-(þ)-11-dehydrosydonic acid (4) (Lu et al. 2010), and engyodontiumone I (5) (Yao et al. 2014).

The obtained compounds 1–5 were tested for antimicrobial activities against sevenzoonotic pathogenic bacteria (Supplemental material, Table S2). The new compound 1and the known compound 2 exhibited obviously inhibitory activities against A. hydrophilia,E. coli, E. tarda and V. harvey, each with MIC values of 2.0 to 8.0lg/mL. While the knowncompound 5 exhibited significant inhibitory activity on E. coli, with an MIC value of1.0lg/mL, which is more potent than that of the positive control chloramphenicol (MIC2.0lg/mL). Moreover, compound 5 also exhibited potent activities against A. hydrophilia,E. tarda, V. harveyi and V. parahaemolyticus each with an MIC value less than or equal to8.0lg/mL, which is comparable to that of chloramphenicol. Compounds 3 and 4 alsoexhibited some selective activities against the zoonotic pathogenic bacteria.

3. Experimental

3.1. General experimental procedures

Optical rotations were acquired on an Optical Activity AA-55 polarimeter at room tem-perature. UV spectra were recorded on a PuXi TU-1810 UV� visible

NATURAL PRODUCT RESEARCH 3

spectrophotometer. ECD spectra were measured on a Chirascan spectropolarimeter.1D and 2D NMR spectra were obtained at 500 and 125MHz for 1H and 13C, respect-ively, on a Bruker Avance 500MHz spectrometer with TMS as the internal standard.Mass spectra were generated on a VG Autospec 3000 or an API QSTAR Pulsar 1 massspectrometer. Analytical and semi-preparative HPLC were performed using a DionexHPLC system equipped with a P680 pump, an ASI-100 automated sample injector, anda UVD340U multiple wavelength detector controlled by Chromeleon software (version6.80). Commercially available Si gel (200–300 mesh, Qingdao Haiyang Chemical Co.),Lobar LiChroprep RP-18 (40–63lm, Merck), and Sephadex LH-20 (Pharmacia) wereused for open column chromatography. All solvents were distilled prior to use.

3.2. Fungal material

The fungus Aspergillus versicolor SD-330 was isolated from a marine sediment samplecollected in May, 2012, from the South China Sea at a depth of 1487m. The funguswas identified using a molecular biological protocol by DNA amplification and thesequencing of the ITS region, as described in our previous report (Wang et al. 2006).The sequenced data derived from the fungal strain have been deposited in GenBank(accession no. MN176407). A BLAST search result showed that the sequence was mostsimilar (99%) to the sequence of Aspergillus versicolor (compared to accession no.MH911415.1). The strain is preserved at the Key Laboratory of Experimental MarineBiology, Institute of Oceanology, Chinese Academy of Sciences, with accession numberSD-330.

3.3. Fermentation and extraction

For chemical investigations, the fungal strain was statically fermented for 35 days atroom temperature in a liquid medium containing 20% potato juice, 2% glucose, 0.5%peptone, and 0.3% yeast extract.

The entire fermented cultures were filtered to separate the broth from the mycelia.The broth was extracted three times with EtOAc, while the mycelia was extractedthree times with a mixture of ethyl alcohol and H2O (95:5, v/v). The ethyl alcohol solu-tion was evaporated under reduced pressure to afford an aqueous solution, which wasthen extracted with EtOAc for three times. As the TLC and HPLC profiles of the twoEtOAc solutions from the broth and mycelia were almost identical, they werecombined and concentrated under reduced pressure to give an extract (25.1 g) forfurther separation.

3.4. Isolation

The organic extract was fractionated by vacuum liquid chromatography (VLC) on silicagel eluting with different solvents of increasing polarity from petroleum ether (PE) toMeOH to yield 9 fractions (Frs. 1–9) that were pooled based on TLC analysis. Fr. 4(4.5 g), eluted with PE–EtOAc (2:1), was further purified by column chromatography(CC) on Sephadex LH-20 (MeOH) to afford 4 (17.0mg) and 5 (12.9mg). Fr. 6 (2.1 g),

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eluted with CHCl3–MeOH (10:1), was purified by CC on silica gel eluting with aCHCl3–MeOH gradient (40:1 to 10:1) to afford two subfrations (Fr. 6-1 and Fr. 6-2). Fr.6-1 was further purified by CC over RP-18 eluting with a MeOH–H2O gradient (1:9 to1:0) and by semi-preparative HPLC (MeOH/H2O; 75% to 80%; 8mL/min; UV detection:210 nm; running time 30.0min; column: Thermo Syncronic C18; type: 250mm �4.60mm, RP18, 5 lm) to obtain 1 (15.4mg, tR 21.0min). Fr. 6-2 was further purified bySephadex LH-20 (MeOH) and by semi-preparative HPLC (75% MeOH/H2O; 8mL/min;UV detection: 210 nm; running time 30.0min; column: Thermo Syncronic C18; type:250mm � 4.60mm, RP18, 5lm) to yield 3 (15.7mg, tR 20.2min). Fr. 7 (3.7 g), elutedwith CHCl3–MeOH (5:1), was further purified by CC on Sephadex LH-20 (MeOH) andthen purified by CC over RP-18 eluting with a MeOH–H2O gradient (1:9 to 1:0) and byrecrystallized to obtain 2 (8.5mg).

12-Hydroxysydowic acid (1): Colorless oily liquid; [a]20 D¼ þ5.1 (c 0.38, MeOH); UV(MeOH) kmax (log e) 211 (2.15), 246 (0.61), 299 (0.21) nm; ECD (0.35mg/mL, MeOH) kmax

(De) 206 (–5.38), 297 (þ1.19) nm; 1H and 13C NMR data (Supplemental material, Table S1);HRESIMS m/z 281.1380 [MþH]þ (calcd for C15H21O5, 281.1384, D –1.2440ppm).

3.5. Antimicrobial assay

Antimicrobial evaluation against seven zoonotic pathogenic bacteria between humanand aquatic animals (E. coli QDIO-1, Aeromonas hydrophilia QDIO-3, E. tarda QDIO-4,Pseudomonas aeruginosa QDIO-6, V. anguillarum QDIO-8, V. harveyi QDIO-9, and V. par-ahaemolyticus QDIO-10) was carried out by the microplate assay with three repetitions(Pierce et al. 2008). The pathogenic bacteria and aquatic pathogen strains were pro-vided by the Institute of Oceanology, Chinese Academy of Sciences. Chloramphenicolwas used as a positive control.

3.6. X-ray crystallographic analysis

(Supplemental material, Experimental section).

3.7. Computational section

Conformational searches were performed via molecular mechanics using theMMþmethod in HyperChem 8.0 software, and the geometries were further optimizedat B3LYP/6-31G(d) PCM/MeCN level via Gaussian 09 software (Frisch et al. 2013) togive the energy-minimized conformers. After this, the optimized conformers were sub-jected to the calculations of ECD spectra using TDDFT at BH and HLYP/TZVP; solventeffects of the MeCN solution were evaluated at the same DFT level using the SCRF/PCM method.

4. Conclusion

One new bisabolene sesquiterpenoid along with four known analogues had been iso-lated from the culture extract of Aspergillus versicolor SD-330, a deep-sea sediment-

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derived fungus. The structures of these compounds were elucidated using NMR andHRESIMS as well as quantum chemical ECD calculations and X-ray crystallographic ana-lysis. Compounds 1, 2 and 5 exhibited obviously inhibitory activities against zoonoticpathogenic bacteria between human and aquatic animals with the MIC values rangingfrom 1.0 to 8.0lg/mL.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This research work was financially supported by the Aoshan Scientific and TechnologicalInnovation Project of Qingdao National Laboratory for Marine Science and Technology(2016ASKJ14), and the National Natural Science Foundation of China (31700043 and 81600672),and the Key Research and Development Program of Shandong Province (2019GSF107091). X.-D.Li and X. Li appreciate the China Postdoctoral Science Foundation (2016LH00033, 2017M612358,and 2017M612360) for project funding. B.-G. Wang acknowledges the support of the ResearchVessel KEXUE of the National Major Science and Technology Infrastructure from the ChineseAcademy of Sciences (KEXUE2018G28) and the Taishan Scholar Project from Shandong Province.X. Li appreciate the Shandong Provincial Natural Science Foundation (ZR2017BB073).

ORCID

Xin Li http://orcid.org/0000-0001-5622-7333Bin-Gui Wang https://orcid.org/0000-0003-0116-6195

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