A new polyketide, penicillolide from the marine-derived fungus Penicillium sacculum

Abstract A new polyketide, penicillolide (1) was isolated from the fermentation broth of the marine-derived fungus Penicillium sacculum GT-308. Compound 1 is a polyketide with a unique carbon skeleton. The structure of this compound was established via extensive spectroscopic analyses including 1D-, 2D-NMR, and HRESI-MS. Graphical abstract


Introduction
Nowadays, the marine environment has become an increasingly important source of natural products (Tohme et al. 2011;Gerwick & Fenner 2013;Sawadogo et al. 2013;Blunt et al. 2014). Marine-derived micro-organisms, particularly marine-derived fungi, are prolific producers of new structural compounds, and are recognized as an important source of structurally novel and bioactive secondary metabolites for drug discovery (Bhatnagar & Kim 2010;Debbab et al. 2010;Liu et al. 2011;Yan et al. 2012). Fungi belonging to the genus Pencillium have been studied as an major producer of interesting compounds, especially of which derived from marine environment (da Silva et al. 2013;Zhan et al. 2013;Sun et al. 2014;Afshari et al. 2015;Diblasi et al. 2015). Our previous chemical investigation of a marine-derived fungus Penicillium sacculum GT-308 has resulted in the isolation of over 10 compounds, including a new sulfo-xanthone . As part of our continuing studies, the chemical constituents from other chromatographic fractions of fungus P. sacculum GT-308 were investigated, and a polyketide with a new skeleton, penicillolide (1), has now been isolated from the EtOAc extract of fermented broth, and its structure was elucidated on the basis of extensive spectroscopic experiments including 1D-, 2D-NMR and HRESI-MS. In this article, we report the isolation and structure elucidation of this compound.

Results and discussion
The EtOAc extract of fermented broth of P. sacculum GT-308 was fractionated by repeated column chromatography to yield compound 1 (Figure 1). The structure was elucidated based on spectroscopic methods.
Above structural units with sp 2 carbons accounted for 7 of the required 12 unsaturation degrees, requiring penicillolide to be pentacyclic (A-E) ( Figure S9). The structure of aromatic ring A could be assigned by HMBC correlations of H-4 (δ H 6.37) to C-5 (δ C 148.0), C-6 (δ C 131.6) and C-8 (δ C 118.5) and NOESY correlation of H-4 to 5-OCH 3 (δ H 3.88). The structure of ring B was indicated by HMBC correlations of H-2 (δ H 5.01, 5.08) to C-3 (δ C 131.9), C-4 (δ C 96.0), C-8 Figure 1. the structure of compound 1. and C-11 (δ C 88.8) and of H-9 (δ H 4.99) to C-3, C-7 (δ C 139.7), C-8 and C-11. Additionally, 1 H-1 H COSY correlation of H-9 to H-10b (δ H 2.77) indicated that C-9 connected with C-11 through C-10. The ether linkage between C-2 and C-11 could be assigned by the down-field shifts of C-2 (δ C 72.0) and C-11 (δ C 88.8), as well as the long range correlation from H-2 to C-11. The lactone ring C could be established by the cross-peaks from H-12 (δ H 3.90) to C-10, C-11, and C-13 (δ C 168.8) and from H-9 to C-13 in the HMBC spectrum. The structure of rings D and E were indicated by HMBC correlations of H-12 to C-16 (δ C 117.2), C-19 (δ C 138.3), of H-19 to C-16, C-17 (δ C 161.9), C-20 (δ C 117.4) and of H-10 to C-15 (δ C 189.9). The chemical shift of C-15 (δ C 189.9) indicated the presence of an α, β-unsaturated carbonyl group. In addition, furan ring E could be formed by C-17 connected with C-19 through an oxygen atom because of their down-field shifts (δ C 161.9 and 138.3). The connectivities of C-15 and C-16 were determined by the molecular formula and 12 unsaturation degrees. The 1 H-and 13 C-NMR data (Table S1) were assigned based on the HSQC, 1 H-1 H COSY and HMBC experiments.

Fungal material and culture
The marine-derived fungus P. sacculum GT-308 was obtained from the halophyte Atriplex sp. in the mesolittoral zone of Dongying City, Shandong Province, China. The fungus was identified by Prof. Li Tian of the First Institute of Oceanography SOA and Qingdao university of Science & Technology, China. The fungus was cultivated by shaking at 150 rpm and at 24 °C in 500-mL flasks, each containing the liquid medium (150-mL per flask) composed of 200 mL of potato extract, 2 g of peptone, 1 g of yeast extract, 15 g of glucose and 1000 mL of aged seawater. After 17 days, the fermented broth (75 L) was filtered through cheesecloth and separated into the supernatant and the mycelia.

Conclusion
Our investigation on the metabolites of the marine-derived fungus P. sacculum GT-308 has resulted in the isolation of one new polyketide, penicillolide (1), which represents a novel skeleton of polyketide.