A new furanosteroid from Talaromyces sp. lgt-4, a fungal endophyte isolated from Tripterygium wilfordii

Abstract Wortmannolol (1), a new furanosteroid, along with five known compounds, wortmannolone (2), ergosterol (3), p-hydroxyphenyl ethanol (4), trans-6-dodecene (5), (2Z, 4E) -5-(8-hydroxy-1,5-dimethyl-3-oxo-6-oxabicyclo [3.2.1] octan-8-yl) -3-methylpenta-2,4-dienoic acid (6) were isolated from a fungal endophyte Talaromyces sp. lgt-4. Their structures were elucidated by IR, MS, 1D and 2D NMR spectra. Compound 1 show weak monoamine oxidase inhibitory activity. Graphical abstract


Introduction
Endophytic fungi are microorganisms that inhabit the intercellular spaces of plant stems, petioles, roots and leaves without causing visible disease symptoms (Petrini 1991;Wilson 1995) and growth in this habitat involves continual metabolic interaction between fungus and host (Schulz et al. 2002). Many endophytic fungi have produced a good number of secondary metabolites in the process of growth. Some of them were directly or indirectly used as therapeutic agents against numerous diseases (Kharwar et al. 2011;Xu et al. 2014;Zhang et al. 2014). In the course of searching for new bioactive natural products, we found a new furanosteroid that we named wortmannolol, together with five known compounds 2-6 (Figure 1), from a fungal endophyte Talaromyces sp. lgt-4 which was isolated from Tripterygium wilfordii.

Results and discussion
Wortmannolol (1) was isolated as an amorphous powder. The molecular formula C 20 h 22 O 5 was determined from the pseudomolecular ion peak at m/z 343.1552 [M + h] + (Calcd for C 20 h 23 O 5 , 343.1540) in the hRESIMS spectrum. The IR spectrum showed absoprtions at 3405, 1656 cm −1 , suggestive of hydroxyl group and carbonyl group, respectively. analysis of the 1 h and 13 C NMR spectra of 1 revealed the presence of two methyl, four sp 3 methylenes, five sp 3 methines, one sp 2 methine, two sp 3 quaternary carbons and six sp 2 quaternary carbons. The 1 h NMR signals at δ h 3.70 (d, J = 3.81 hz), 3.45 (dd, J = 3.7, 2.7 hz), 5.21 (d, J = 2.7 hz), 3.74 (dd, J = 9.1, 7.8 hz) and 13 C NMR signals at δ C 55.94, 54.88, 61.10, 80.30) were characteristic of oxygenated methines. The 1 h NMR signals at δ h 3.70 (d, J = 3.81 hz), 3.45 (dd, J = 3.7, 2.7 hz) and carbon shifts at δ C 55.94, 54.88 showed the presence of an epoxide moiety. Comparison to the known compound of similar structure wortmannolone 2 (it's structure was unambiguously elucidated by single-crystal X-ray crystallography (CCDC1021903) (Ding et al. 2015), compound 1 showed a resonances for C-17 oxygenated methine signal at δ h 3.74 (dd, J = 9.1, 7.8 hz, h-17) (δ C 80.03, C-17) instead of carbonyl signal, which suggested that 1 was the 17-carbonyl reduction product of 2. The presence of hydroxyl group at C-17 was further confirmed by the observed hMBC correlations from h-18, h-16 and h-12 to C-17 and h-17 to C-12 ( Figure S1). The NMR coupling patterns shown for proton signals h-1, h-2 and h-3 in 1 were nearly identical with those of 2. The coupling constants of h-1, h-2, h-3 of compound 1 were also highly similar to those of 2 which suggested relative stereochemistry from C-1 to C-3 of 2 was identical with that of 1. In addition to coupling constant analysis, the relative stereochemistry of 1 was supported by NOESY correlations ( Figure S1). Correlations in the NOESY spectrum were observed from h-19 to h-1 and h-1 to h-2, which implied that h-1/h-2 are β oriented. Several additional NOESY correlations,in particular, is in the β orientation and h-17, h-14 are α orientation. Computational methods were used to clarify the absolute configuration of 1 by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. We found that the calculated ECD spectrum of the conformers of 1 with 1R, 2R, 3R, 10R, 13S, 14R, 17S but not the other isomers (1S, 2S, 3S, 10S, 13R, 14S, 17R) is in good accordance with the experimental spectrum ( Figure S2). Thus, the absolute configuration of 1 was unambiguously assigned as 1R, 2R, 3R, 10R, 13S, 14R, 17S.
The five known compounds were identified as wortmannolone (2)  The monoamine oxidase inhibitory activity of compounds 1-6 was tested. Only compound 1 showed weak anti-monoamine oxidase activity at the final concentration of 25 μg/ mL (the inhibitory rate was 30%).

General
Optical rotations were obtained on a JaSCO P-1020 polarimeter. IR spectra were recorded on a Perkin Elmer 1600 FT IR spectrophotometer as KBr pellets and the absorption frequencies are expressed in reciprocal centimeters (cm −1 ). Mass spectral studies were carried out on a hewlett-Packard 5989B MS. 1D and 2D NMR experiments were performed on a Bruker avance-3 spectrometer at 600 Mhz. Silica gel (200-400 mesh, Qingdao Marine Chemical Co, Ltd China), Sephadex Lh-20 (Pharmacia), MCI gel and macroporous resin (Mitsubshi Chemical Co, Ltd, Japan) were used for open column chromatography. hPLC (LC-2000) (JaSCO International Co, Ltd, Tokyo, Japan); C18 column (250 mm × 10 mm, 5 mm).

Plant materials and endophytic fungi
The T. wilfordii were collected from GanSu province, China which were identified by associate Professor Lin Yang who majored in plant classification, School of Life Science and Engineering, Lanzhou university of Technology, Lanzhou, China. a voucher specimen (No. 2011071715) is deposited at the School of Life Science and Engineering, Lanzhou university of Technology, China.
The strain lgt-4 was isolated from Chinese herb medicine T. wilfordii and was identified as Talaromyces sp. based on both morphology on PDa and analysis of the DNa sequences of the ITS1-5.8S-ITS2 ribosomal DNa gene region (GenBank accession No. KF850714).

ECD calculation
The geometry of the molecules was first optimised by Gaussian 09 package (Frisch et al. Gaussian 09, Revision a.1. Gaussian, Inc. Wallingford CT) at B3LYP/6-31G (d,p) computational level. The output structures were then saved as input structures to conduct ECD calculations using time-dependent density functional theory with B3LYP functional and DGDZVP basis set. all circular dichroism calculations were performed by considering the solvation effect using polarisable continuum model and the methanol was specified as the solvent, which was in agreement with the experiment condition.

MAO assays
The procedure of testing MaO-inhibiting activity was same with that reported in our previous paper (Zhi et al. 2014).

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