A new anthraquinone from mangrove endophytic fungus Aspergillus sp. 16-5C

Abstract A new anthraquinone, asperquinone A (1), and four known anthraquinone derivatives 2–5 were isolated from the mangrove endophytic fungus Aspergillus sp. 16-5C. These structures were elucidated by spectroscopic analysis and the absolute configuration of 1 was unambiguously determined by ECD calculation. Compounds 1–5 showed no significant inhibitory effect against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB). Graphical Abstract


Introduction
Tuberculosis (TB) remains the world's most deadly infectious disease, which is caused by the bacillus Mycobacterium tuberculosis. The 2020 World Health Organization (WHO) global TB report (World Health Organization 2020) showcases an estimated 10 million people fell ill with TB and 1.2 million TB deaths in 2019. In recent years, extensively drug-resistant TB (DR-TB), multidrug-resistant TB (MDR-TB), HIV-associated TB and impacts of the COVID-19 pandemic have made clinical treatment even more difficult and complex. M. tuberculosis protein tyrosine phosphatase B (MptpB) is an essential virulence factor when M. tuberculosis hosts macrophages and exhibits unique and multiple activities against immune responses (Singh et al. 2003;Beresford et al. 2007;N€ oren-M€ uller et al. 2008;Vintonyak et al. 2010). Putting the spotlight on new inhibitors of MptpB could be a promising strategy against M. tuberculosis infection and conducive to the treatment of TB (Zhou et al. 2010;Huang et al. 2013;Xiao et al. 2015).
In the process of our screening program for novel bioactive metabolites from mangrove endophytic fungi from the South China Sea Xiao et al. 2013;Cai et al. 2019), a mangrove endophytic fungus, named Aspergillus sp. 16-5C, attracted our attention. During the course of our investigation on the chemical constituents from this fungus, a new anthraquinone, asperquinone A (1), together with four known compounds, 6,8-di-O-methylnidurufin (2), 6,8-di-O-methylaverufin (3), aversin (4) and averythrin (5) (Figure 1), were isolated. Compounds 1-5 were preliminarily screened for the ability to against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB), none of the compounds showed significant inhibitory activity (IC 50 > 60 lg/ mL). In this report, we describe the isolation, structural identification, and biological activity of the metabolites.
The activity of compounds 1-5 against MptpB was evaluated and none of them showed significant inhibitory activity (IC 50 > 60 lg/mL, Table S8).

Fungal material
The fungus used in this study was isolated from a mangrove endophytic fungus from the leaves of Sonneratia apetala, which were collected in Hainan Island, China. The fungus was identified as Aspergillus sp. according to a molecular biological protocol by DNA amplification and sequencing of the ITS region (deposited in GenBank, accession No. JX993829). A voucher specimen has been deposited in School of Chemistry, Sun Yat-sen University, Guangzhou, China, with the access code, 16-5C.

Calculation of ECD spectra
Molecular mechanics calculations and DFT/TD-DFT calculations were carried out with Spartan'14 software (Wavefunction Inc., Irvine, CA, USA) and Gaussian 09 program (Gaussian, Wallingford, CT, USA), respectively. Conformers with Boltzmann distribution over 1% were chosen for ECD calculations in methanol at B3LYP/6-311 þ G (2d, p) level. The IEF-PCM solvent model for MeOH was used. ECD spectra were generated using the program SpecDis 3.0 (University of W€ urzburg, W€ urzburg, Germany) and OriginPro 8 (OriginLab, Ltd., Northampton, MA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma ¼ 0.30 eV and UV shift ¼ þ15 nm. All calculations were performed with High-Performance Grid Computing Platform of Sun Yat-Sen University.

MptpB Inhibition assay
The inhibition assays were performed using the RediPlate 96 EnzChek Tyrosine Phosphatase Assay kit (Invitrogen, Thermo Fisher Scientific, Inc., Hudson, NH, USA) by monitoring the hydrolysis of the fluorogenic phosphatase substrate, 6,8-difluoro-methylumbelliferyl phosphate (DiFMUP), according to the manufacturer's instruction. All measurements were done in triplicate from three independent experiments.

Conclusion
A new anthraquinone, asperquinone A (1), together with four known anthraquinone derivatives 2-5 were isolated from Aspergillus sp. 16-5C, an endophytic fungus from the South China Sea. The structures of 1-5 were elucidated primarily by NMR experiments and the absolute configuration of 1 was confirmed by ECD calculation. In the bioassay, the isolated compounds were evaluated for their inhibitory activity against MptpB and none of them showed significant inhibitory (IC 50 > 60 lg/mL).

Disclosure statement
No potential conflict of interest was reported by the authors.