Asperlones A and B, Dinaphthalenone Derivatives from a Mangrove Endophytic Fungus Aspergillus sp. 16-5C

Racemic dinaphthalenone derivatives, (±)-asperlone A (1) and (±)-asperlone B (2), and two new azaphilones, 6″-hydroxy-(R)-mitorubrinic acid (3) and purpurquinone D (4), along with four known compounds, (−)-mitorubrinic acid (5), (−)-mitorubrin (6), purpurquinone A (7) and orsellinic acid (8), were isolated from the cultures of Aspergillus sp. 16-5C. The structures were elucidated using comprehensive spectroscopic methods, including 1D and 2D NMR spectra and the structures of 1 further confirmed by single-crystal X-ray diffraction analysis, while the absolute configuration of 3 and 4 were determined by comparing their optical rotation and CD with those of the literature, respectively. Compounds 1, 2 and 6 exhibited potent inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) with IC50 values of 4.24 ± 0.41, 4.32 ± 0.60 and 3.99 ± 0.34 μM, respectively.


Introduction
Tuberculosis (TB) ranks as the second leading cause of death from an infectious disease worldwide; an estimated 9.0 million people developed TB and 1.5 million died from the disease in 2013, according to the WHO [1]. In recent years, extensively drug-resistant TB (DR-TB), multidrug-resistant TB (MDR-TB) and HIV-associated TB have made clinical treatment even more difficult and complex. New chemotherapeutic approaches and unusual anti-infective agents are in urgent need, especially those applying to new targets and based on different mechanisms. Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) is secreted by the microbe and manipulates host signal transduction pathways, which has proven to be an essential virulence factor when M. tuberculosis hosts macrophages [2][3][4][5][6]. Increased research reveals that it exhibits unique and multiple activities against immune responses [7][8][9][10][11][12][13]. Therefore, finding new inhibitors of MptpB could be a promising strategy against M. tuberculosis infection and conducive to the treatment of TB.
In light of our hypothesis that all these compounds are the product of a fungal polyketide synthase [26]. The unusual dinaphthalenone derivatives, named (±)-asperlones A (1) and B (2), are A B C presumed to be started from five acetate units through a series of polyketide biosynthesis, reduction, dehydration, oxidation and dimerization [27][28][29], as shown in Scheme 2. The initial condensations of tetraoxodecanoic acid and further reduction leading to compound A, followed by oxidation to generate compound B and subsequent dimerization, oxidation and rearrangement to generate (±)-asperlone B (2). On the other way, the reduction and dehydration of A leading to C and further oxidation to generate D. Subsequent dimerization of the rearrangement structure of B and D afford the dinaphthalenone skeleton and then further oxidize and rearrange to generate (±)-asperlone A (1).

General
Melting points were measured on an X-4 micromeltin-point apparatus (Cany Precision Instruments Co., Ltd., Shanghai, China) and are uncorrected. Optical rotations were determined with a RUDOLPH Autopol III polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) at 20 °C. UV data were measured on a PERSEE TU-1900 spectrophotometer (Purkinje General Instrument Co., Ltd., Beijing, China). IR spectra were measured on a Nicolet Nexus 670 spectrophotometer (Thermo Fisher Scientific, Inc., Hudson, NH, USA), in KBr discs. CD data were measured on a Chirascan™ CD spectrometer (Applied Photophysics, London, UK). 1

Fungal Material
The fungus used in this study was isolated from a mangrove endophytic fungus from the leaves of S. 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 strain was deposited in School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, China, with the access code, 16-5C.

Cloning, Expression and Purification of MptpB
The full-length PTPB gene was amplified from genomic DNA of the Mtb H37Rv strain (School of Life Sciences, Sun Yat-sen University: Guangzhou, China). PCR products were cloned in frame with an N-terminal His6 tag into the pET28a (+) vector (Novagen, Merck KGaA, Darmstadt, Germany). For protein expression, plasmids were transformed into Escherichia coli BL21(DH3) cells (Invitrogen, Thermo Fisher Scientific, Inc., Hudson, NH, USA) and grown in LB medium containing 50 μg/mL kanamycin at 37 °C till the OD600 of the solution was about 0.6. After the addition of 0.1 mM IPTG, the culture was grown for another 16 h at 20 °C. The cells were harvested by centrifugation at 5000 rpm for 5 min at 4 °C. The bacterial cell pellets were resuspended in the buffer containing 20 mM Tris, pH 7.9, 500 mM NaCl, 5 mM imidazole and were lysed by sonication on ice. Cellular debris was removed by centrifugation at 10,000 rpm for 30 min at 4 °C. The protein was purified from the supernatant using glutathione-Sepharose 4B (GE Healthcare, Buckinghamshire, UK), according to the manufacturer's instructions. Protein concentration was determined using the Bradford dye binding assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's recommendations, with bovine serum albumin as the standard. The purified MptpB was stored in 20% glycerol at −20 °C.

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. The IC50 value was determined at five different substrate concentrations by non-linear regression fitting of the inhibitor concentration versus inhibition rate. All measurements were done in triplicate from three independent experiments. The reported IC50 were the average value of three independent experiments.

Conclusions
Aspergillus sp. 16-5C, an endophytic fungus from the South China Sea, was proven as a prolific producer of bioactive metabolites. Two unusual pairs of enantiomers of dinaphthalenone derivatives and two new azaphilones, together with four known compounds, were isolated from this fungal strain, and the structures of 1-8 were elucidated primarily by NMR experiments. The structure of 1 confirmed by single-crystal X-ray diffraction analysis, while 3 was determined by the optical rotation and 4 was assigned on the basis of the CD and NOESY spectra. The biosynthetic pathways for all these compounds were proposed and suggested that the secondary metabolites are the product of a fungal polyketide synthase. In the bioassay, the isolated compounds were evaluated for their inhibitory activity against MptpB; compounds 1, 2 and 6 exhibited strong inhibitory activity that suggested they could represent a new type of lead compounds for the development of new anti-tuberculosis drugs.