New citrinin derivatives from the deep-sea-derived fungus Cladosporium sp. SCSIO z015

Abstract During the course of our search for novel bioactive compounds from marine fungi, four new citrinin derivatives, cladosporins A–D (1–4) were isolated from a culture broth of the deep-sea-derived fungus Cladosporium sp. SCSIO z015. Their complete structural assignments were elucidated by the extensive spectroscopic investigation. The absolute configurations of 1–3 were established by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. Compounds 1–4 showed weak toxicity towards brine shrine naupalii with LC50 values of 72.0, 81.7, 49.9 and 81.4 μM, respectively. And 4 also showed significant antioxidant activity against ɑ,α-diphenyl-picrylhydrazyl (DPPH) radicals with an IC50 value of 16.4 μM. Graphical Abstract


Introduction
Marine fungi have been studied for their bioactive compounds and have proved to be a rich and promising source of novel compounds showing anticancer, antibacterial, antiplasmodial, antiviral and anti-filming activities (Hong et al. 2015;Mayer et al 2011;Saba et al. 2015). It is acknowledged that fungi have a potential of secondary metabolites far beyond the number of compounds known. It has been estimated that about 1 million natural products are isolated from biological sources, out of these, about 40% are from microbes and fungi stands out as the most prominent source of bioactive compounds, with 42% of the total (Demain 2014). The majority of fungi inhabiting in the marine environment has not yet been described. The recent study of the fungal genome has shown that many gene clusters are silent, proving that there are possibilities for any more new compounds (Marmann et al. 2014). The genus Cladosporium consists of approximately 800 species (Crous et al. 2007). Cladosporium genera are widespread, pathogenic and promising sources for new metabolites with pharmaceutical applications (Wu et al. 2018). Secondary metabolites isolated from the genus Cladosporium were mainly the derivatives of polyketides, pyrones, binapthyl, alkaloids and phenols Peng et al. 2013). Studies revealed that marine fungus Cladosporium produce structurally diverse and biologically active metabolites with antifouling, antiviral, antibacterial and cholesterol lowering activities (Bensch et al. 2012;Yu et al. 2018). Cladospolide E and F (Wu et al. 2018), sporiolides A and B (Yu et al. 2018), and cladosporin and napthopyranone derivatives are one of the common examples isolated from this genus .
The biodiversity, pathogenicity and the less documented work on marine microbes attracted us to carry out the research on their secondary metabolites and their bioactivities. The research was conducted on Cladosporium cladosporioides SCSIO z015 isolated from the deep-sea sediments collected from Okinawa Trough. This study was chiefly aimed at pursuing new biologically active secondary metabolites from marine-derived fungus. In our search for new interesting metabolites, we obtained four new citrinin derivatives cladosporins A-D (1-4) ( Fig. 1) from a fermentation product of C. cladosporioides SCSIO z015. Citrinin is a polyketide mycotoxin and a broad spectrum antibiotic isolated for the first time from Penicillium citrinum (Clark et al. 2006). Later on it has been reported from Aspergillus, Monascus and a number of Penicillium species (Bennett and Klich 2003). Citrinin has been known for several decades; most of its derivatives were only recently reported. Until now, a large number of citrinin derivatives have been reported from various natural resources. They exhibited diverse biological activities such as antibacterial , antifungal (Wakana et al. 2006) and cytotoxic activities (Hu et al. 2017). Bioactivities of antibacterial, anti-biofilm, antioxidant, and toxicity of the isolated compounds were evaluated. Herein, we report the isolation, structure elucidation and biological activities of these compounds.

Results and discussions
Cladosporin A (1) was obtained as pale yellow oil having a molecular formula of C 13 H 15 NO 3 as determined by the HRESIMS at m/z 232.0984 [M þ H] À (calcd m/z 232.0979). The UV absorption bands were found at 207 and 283 nm, while the IR absorption bands for the hydroxyl and nitrile groups were observed at 3385 and 2259 cm À1 , respectively. The 1 H NMR spectrum (Table S1) displayed signals for two hydroxyl protons (d H 9.33, s and 9.76, s), one aromatic proton (d H 6.33, s), three methine protons (d H 5.58, s and 4.14, q, J ¼ 6.7 Hz, and 2.67, q, J ¼ 6.8 Hz), three methyl groups (d H 1.95, s, and 1.30, d, J ¼ 6.8 Hz, and 1.07, d, J ¼ 6.9 Hz). The 13 C NMR and DEPT spectra revealed 13 carbons including three methyls (d C 9.5, 18.9, and 20.3), four methines (d C 57.2, 74.1, 33.3, 100.0), and six olefinic quaternary carbons (d C 103.6, 112.3, 120.0, 136.7, 151.4, 156.0). These data showed similarity to that of penicitrinol F (Nong et al. 2013) and nigrospins B and C (Dong et al. 2014), which suggested that 1 was also a citrinin derivative. The planar structure of 1 was further revealed through HMBC and HSQC spectrum analyses ( Figure S37). The HMBC correlations from H-1 to C-3, C-9, C-10, from H-3 to C-1 and C-10, and from H-4 to C-9 and C-10 demonstrated the existence of a multi-substituted dihydropyrane. Furthermore, the HMBC correlations of H-7 with C-5, C-6, C-8 and C-9, H-13 with C-5, C-6 and C-10, 8-OH with C-7, C-8 and C-9, and 6-OH with C-5, C-6 and C-7 suggested the linking of the second benzene ring to the dihydropyrane through C-9 and C-10. And combining with the chemical shift of C-14 (d C 120.0) and the molecular formula of C 13 H 15 NO 3 , the HMBC correlation from H-1 to C-14 suggested one cyano group attached at C-1. The relative configuration of 1 was further determined by the NOESY spectrum showing correlations of H-4 with H-11, and H-3 with H-12 ( Figure S38) that suggested H-4 and CH 3 -11 at the same side, while H-3 and CH 3 -12 at the other side. However, no obvious NOE correlation for H-1 was observed.
Cladosporin B (2) had the same molecular formula C 13 H 15 NO 3 as 1. The 1 H and 13 C NMR spectral data of 2 (Table S1) were greatly similar to that of 1. The only obvious difference between them was the chemical shift of H-3 (d H 4.14 in 1, and d H 3.70 in 2). The analysis of 2D NMR spectra of 2 established the same planar structure as 1, while their NOESY spectra had an obvious difference. The NOESY spectrum of 2 showing correlations of H-11 with H-1 and H-4, and H-3 with H-12 ( Figure S38), suggested that H-1, H-4, and CH 3 -11 were at the same side, while H-3 and CH 3 -12 at the other side. Its relative configuration was identical to that of penicitrinol F (Nong et al. 2013), and nigrospins B and C (Dong et al. 2014) according to their identical NOE data. Although the specific optical rotation value of 2 ([a]25 D þ 0.1 (c 0.1, MeOH)) was small, it was similar to that of penicitrinol F ([a]20 D 24.8 (c 0.08, MeOH)), and nigrospins B and C ([a]20 D 62.0 (c 0.4, MeOH) and [a]20 D 59.0 (c 0.4, MeOH), respectively), and opposite to that of 1 ([a]25 D À42.6 (c 0.1, MeOH)). The above data indicated that 2 was an epimer of 1 at C-1.
The absolute configurations of 1 and 2 were further determined by comparison of the experimental electronic circular dichroism (ECD) curves with the calculated curves for the truncated model (1R, 3R, 4S)-1 and (1S, 3R, 4S)-2 ( Figure S39), respectively, using time dependent density functional theory (DFT). The DFT reoptimization of the initial Merck molecular force Field (MMFF) minima was performed at the B3LYP/6-3 þ g(d) level with a polarizable continuum model (PCM) solvent model for MeOH. The best agreement of the calculated ECD spectra of (1R, 3R, 4S)-1 and (1S, 3R, 4S)-2 with the experimental data suggested the absolute configuration of 1 moiety as (1R, 3R, 4S) and 2 moiety as (1S, 3R, 4S) ( Figure S39), respectively. Cladosporin C (3) was obtained as a colorless gum and established to have the molecular formula C 14 H 16 O 4 from HRESIMS at m/z 247.0983 [M þ H] À . The 1 H and 13 C NMR spectral data of 3 (Table S1) were similar to that of 1, penicitrinol F and nigrospins B and C. The most obvious difference between 3 and 1 was the additional presence of one methylene group and the presence of one carbonyl carbon (d C 167.3) in 3 instead of one nitrile carbon (d C 120.09 in 1). In the HMBC spectrum ( Figure S37), correlations of H-14 (d H 2.71, 2H) with C-1 (d C 67.3), C-9 (d C 118.8), C-15 (d C 167.3) suggested that the additional methylene attached on C-1 and connected with the carbonyl group to form the -CHCH 2 COO-fragment. Combining with the requirement of the molecular formula, the high-field shift of the chemical shift of C-8 (d C 147.1 in 3 and 151.4 in 1) suggested that the -CHCH 2 COO-fragment connected with C-8 to form a six-membered lactone. In addition, the NOESY spectrum showed correlations of H-3 with H-1 and H-12, and H-4 with H-11 ( Figure S38), which suggested that H-4 and CH 3 -11 were at the same side, while H-1, H-3 and CH 3 -12 were at the other side. So, the structure of 3 was determined as shown.
Cladosporin D (4) was isolated as orange brown oil. It had the molecular formula C 12 H 16 O 4 as determined by HRESIMS at m/z 225.1127 [M þ H] þ . The 1 H and 13 C NMR spectral data of 4 (Table S1) showed similarity to that of phenol A and phenol A acid (Zhang et al. 2007). The only obvious difference between 4 and phenol A acid was the presence of a formyl group (d H 10.46, d C 193.6) instead of a carboxyl group in 4. The analysis of 2D NMR spectra of 4 ( Figure S37) established the planar structure of 4 as 5-(3-hydroxybutan-2-yl)-2-formyl-4-methylbenzene-1,3-diol. The coupling constant J H-7, H-8 = 6.1 Hz and the chemical shifts of CH 3 -9 and CH 3 -10 (d C 20.4 and 16.2 in DMSO-d 6 ) of 4 were close to those of phenol A (J ¼ 6.0 Hz, d C 22.2 and 16.3 in actone-d 6 ) and nigrospine (J ¼ 6.0 Hz, d C 21.3 and 15.8 in DMSO-d 6 ) (Dong et al. 2014), which suggested that the relative configuration of C-7 and C-8 in 4 was the same as that in phenol A and nigrospine. So, the structure of 4 was determined as shown.
The bioactivities of antibacterial, anti-biofilm, antioxidant and toxicity towards brine shrimp of the isolated compounds 1-4 were evaluated. All of the compounds didn't show obvious antibacterial and anti-biofilm activities towards Staphylococcus aureus, Escherichia coli, and Bacillus subtilis. Compound 4 and positive control ascorbic acid exhibited significant potent antioxidant activity against DPPH radicals with IC 50 values of 16.4 and 4.9 lM, respectively, while the other compounds had no obvious activity with IC 50 >100 lM. Brine shrimp lethality bioassay showed that 1-4 showed medium toxicity towards brine shrine naupalii with LC 50 values of 72.0, 81.7, 49.9 and 81.4 lM, respectively, comparing with the positive control toosendanin (LC 50 21.2 lM).

General experimental procedures
See Supporting Information.

Fungal strain
The fungal strain Cladosporium cladosporioides SCSIO z015 was collected and from the deep sea sediments at a depth of 1330 m from Okinawa Trough (26 30 0 N, 128 00 0 E). It was identified as C. cladosporioides. According to a molecular biological protocol calling for DNA amplification and ITS region sequence comparison with the Gene Bank database and shared 99% similarity with C. cladosporioides (KX258800). The fungus was stored at À80 C in RNAM Center, South China Institute of Oceanology, Chinese Academy of Science.

Fermentation and extraction
C. cladosporioides SCSIO z015 spores were inoculated into 500 mL Erlenmeyer flasks each containing 200 mL liquid medium (D-sorbitol 2%, Maltose 2%, monosodium glutamate (MSG) 1%, Yeast extract 0.3%, Sea Salt 3%, KH 2 PO 4 0.05%, L-Tryptophan 0.05% pH = 6.5) and kept for static fermentation at 30 C for 30 days. The fermented whole broths (80 L) were filtered through cheesecloth. Sterilized XAD-16 resins were added to the liquor and stirrer for an hour to absorb the organic products. The resins were washed with distilled water to remove medium residue and then eluted with ethanol. The ethanol solvent was removed under vacuum to give a brown residue (35 g). The mycelium portion was crushed and extracted thrice with acetone. The acetone soluble fraction was concentrated in vacuum under reduced pressure to afford an aqueous solution. The aqueous solution was extracted with EtOAc thrice (v/v, 50/50) to give an EtOAc crude fraction (60 g). The residue liquor and mycelium extracts were combined together after analyzing by HPLC and thin layer chromatography (TLC).

Biological assays
Antibacterial and anti-biofilm activities against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis were performed by using the micro dilution method (Wang et al. 2017).
3.5.1. Antioxidant assay DPPH % radical and scavenging activity. See Supporting Information about the details.
3.5.2. Brine shrimp bioassay  See Supporting Information about the details.

Computational methods
Conformational searches were performed by employing a systematic procedure implemented in Spartan'14 software package using Molecular Merck force field (MMFF) (Wavefunction Inc., Irvine, CA, 2013). DFT/TDDFT calculations were conducted with the Guassian09 program (Gaussian, Inc., Pittsburgh PA, 2011). See Supporting Information about the details.