A new cycloheptane derivative from the fungus Penicillium crustosum JT-8

Abstract A new highly oxygenated cycloheptane derivative crustane (1), along with fourteen known compounds (2–15) were isolated from Penicillium crustosum JT-8. The structure of compound 1 was determined by extensive spectroscopic data, DP4+ probability analyses and dimolybdenum CD method. Compound 1 exhibited moderate antibacterial activity against Staphylococcus aureus with MIC of 4.0 μg/mL. Graphical Abstract


Results and discussion
Compound 1 was a white powder. It is puzzling that there were obvious signals of glycerol (d C 75.7 and 64.4) in 13 C NMR spectra (Supplementary information Figure S3). However, only one light grey spot was observed using CHCl 3 -MeOH (4:1) as a developing solvent in TLC experiment. In addition, compound 1 and glycerol located at different places in TLC experiment (Supplementary information Figure S10). This phenomenon might be caused by the existence of intermolecular hydrogen bonding interactions between the two compounds (Cai et al. 2014), and the percentage composition of crustane and glycerol was nearly 1:1.2 (Davis and Wareham 1999;Bitta and Kubik 2001). The molecular formula of compound 1 was deduced to be C 7 H 12 O 4 by HR-ESI-MS (m/z 183.0660 [M þ Na] þ , calcd for C 7 H 12 O 4 Na: 183.0628). By analysing the 1 H and 13 C NMR, DEPT, COSY and HMBC spectra, compound 1 exhibited characteristic NMR features of C-1 (d C 105.4 s), C-3 (d C 70.1 d), C-4 (d C 75.7 d) and C-5 (d C 76.9 d), which revealed that C-3, C-4 and C-5 linked with one oxygen atom and C-1 connected with two oxygen atoms. The 1 H-1 H COSY correlations (Supplementary information Figure S4) Figure S5) from H-2 to C-1 and C-7 (d C 34.9 t) as well as HMBC correlation from H-6 to C-1 confirmed the existence of a cycloheptane ring. In addition, the carbon chemical shift of C-1 and the key HMBC correlation from H-5 to C-1 confirmed that C-1 and C-5 was linked by one oxygen bridge. In NOESY spectrum (Supplementary information Figure S7), the correlations of H-4/H-2b and H-5 and H-5/ H-6b confirmed these groups were b-oriented. The NOE correlations of and H-3/H-6a and H-2a revealed that these groups were a-oriented. In addition, accurate computations of the proton À proton spin À spin coupling constants 3 J 4,5 of 5S Ã -1 and 5R Ã -1 at APFD/6-311 þ g(2d,p) level of theory were performed to confirmed the relative configuration of C-5. As a result, calculated coupling constants (Supplementary information  Table S1) 3 J 4,5 of 5R Ã -1 and 5 S Ã -1 were 4.22 Hz and 2.44 Hz, respectively, these data compared with experimental value (H-4, d H 3.46 dd, 3 J ¼ 4.38 Hz) from 1 H spectrum determined the relative configuration of C-5 was R Ã . Moreover, 1 H and 13 C NMR chemical shift calculations (in chloroform) were performed to confirm the four possible The gauge independent atomic orbital (GIAO) method (Wolinski et al. 1990) at the PCM/mPW1PW91/6-311 þ G(d,p) level of theory was employed for chemical shift calculations and DP4þ probability analyses. The comparing of theoretical chemical shifts of (1S Ã ,3S Ã ,4S Ã ,5R Ã )-1 (a), with experimental data was by total absolute deviation (TAD) mean absolute error (MAE) and DP4þ method ). The result of 13 C data and all data ( 1 H þ 13 C) of DP4þ method (Supplementary information Figure S13) showed that 1 (d) was the most possible stereoisomer. Furthermore, the absolute configuration of the 3,4-diol in 1 was assigned by a dimolybdenum CD method (Frelek et al. 2003;G orecki et al. 2007;. Upon the addition of Mo 2 (OAc) 4 to a solution of compound 1 in DMSO, a chiral dimolybdenum complex was generated in situ as an auxiliary chromophore. The positive CD Cotton effects at approximate 300 nm (Supplementary information Figure S14) permitted us to assign the (3S,4R)-configurations on the basis of Snatzke's empirical rule, with the large group pointing away from the remaining portion of the complex. Therefore, compound 1 were determined to be (1S,3S,4R,5R)-8oxabicyclo[3.2.1]octane-1,3,4-triol and named crustane.

General procedure
Melting points was measured on a XRC-1 Melting Point Apparatus (Sichuan University Science Instrument Co., Chengdu, China). Optical rotations were conducted on a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). A Nicolet Magna-IR 550 spectrometer (Thermo Nicolet Co., Madison, WI, USA) was used for scanning IR spectroscopy with KBr pellets. NMR spectra were recorded on Bruker Avance 600 MHz spectrometer (Bruker, Karlsruhe, Germany). HRESIMS analyses were recorded with Agilent G3250AA (Agilent, Santa Clara, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, USA). Silica gels (200-300 mesh or 300-400 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Xciences AB) were used for column chromatography. Fractions were monitored by TLC and visualized by dipping into vitriolated chromogenic agent or under UV light. All the solvent used in the experiment were redistilled before use.

Fungal material and fermentation
Penicillium crustosum JT-8 was isolated from the ripe root of Paris polyphylla var. yunnanensis collected from Kunming, Yunnan Province, of China in November 2018. The ITS-rRNA sequences of this fungus was compared with those available in the GeneBank data bases to obtain the closest related species by BLAST analysis with the accession number of MH345907.1. It was preserved in the Key Laboratory of Medicinal Chemistry for Natural Resources at Yunnan University. Potato dextrose agar (PDA) slant culture medium was used to cultivate the strain in a constant-temperature incubator at 28 C for 6 days. Then, the mature P. crustosum JT-8 fungus was added to the solid-state fermentation culture medium (100 g potato as Culture substrate in each bottle) that had been sterilized at 121 C for 30 min before, and then incubated at 28 C for 30 days.

Antibacterial activity assay
Antibacterial activity assays were performed in sterilized 96-well microplates using amicrodilution method described previously (Dong et al. 2015). The 18-h-old bacterial cultures from Staphylococcus aureus (ATCC 25917), Bacillus subtilis (ATCC 6633), and Escherichia coli (ATCC 25922) was added to LB broth medium (1 L of water, 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl) to reach 1 Â 10 5 colony forming units/ mL. All compounds and the positive control were dissolved in dimethylsulfoxide (DMSO), and their final concentrations were ranged from 0.125 to 512 lg/mL as determined by using a 2-fold serial dilution method. The wells containing strains and diluted samples were incubated at 37 C (24 h). Kanamycin that has prominent antibacterial activity against B. subtilis, E. coli and S. aureus was introduced in the experiments as a positive control. All experiments were repeated two times. CX21BIM-set 5 microscope was used to monitor the growth of test strains. The minimal inhibitory concentration (MIC) was defined as the lowest antibiotic concentration that produced complete growth inhibition of the microorganisms. Test result display that Compound 1 exhibited moderate antibacterial activity against S. aureus with MIC of 4.0 lg/mL. Compound 4 exhibited antibacterial activity against B. subtilis, E. coli and S. aureus with MICs of 2.0 lg/mL, 2.0 lg/mL, 1.0 lg/mL. The remaining compounds were inactive against all strains tested.

Mo2(OAc) 4 -induced CD experiment
Absolute configuration of the 1,2-diol moiety in compound 1. HPLC grade DMSO was dried with 4 Å molecular sieves. According to a published procedure (Frelek et al. 2003;G orecki et al. 2007), mixture of the 1:2 diol À Mo 2 (OAc) 4 of compound 1 was subjected to CD measurements at a concentration of 0.1 mg/mL. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary (about 10 min after mixing). The inherent CD spectrum was subtracted. The observed signs of the diagnostic bands in the region of 300-400 nm in the induced CD spectrum were correlated to the absolute configuration of the 1,2-diol moiety.

Conclusion
A new cycloheptane derivative crustane (1) and fourteen known compounds (2-15) were isolated from fermentation products of P. crustosum JT-8. Their structures were established on the basis of extensive spectroscopic, DP4þ probability analyses and Mo 2 (OAc) 4 -induced CD experiment. Crustane (1) exhibited moderate antibacterial activity against S. aureus.