Sesquiterpenes with new carbon skeletons from the basidiomycete Phlebia tremellosa

Three new sesquiterpenes, phlebidiol, phlebioic acid, and phlebiolide, as well as the known compound tremetriol, were isolated from cultures of the basidiomycete Phlebia tremellosa. The structures of all isolated compounds were established by extensive spectroscopic analyses, including those involving extensive two-dimensional nuclear magnetic resonance. The absolute configurations of phlebidiol, phlebioic acid, and phlebiolide were determined by comparisons of experimental and calculated electronic circular dichroism spectra. Phlebidiol and phlebioic acid have previously unreported carbon skeletons, for which we propose the skeletal names “seco-sterpurane” and “phlebiane,” respectively. Phlebiolide is also the second published example of a merulane sesquiterpene. Electronic supplementary material The online version of this article (10.1007/s11418-019-01286-8) contains supplementary material, which is available to authorized users.


Results and discussion
Phlebia tremellosa ECN184 was isolated from the healthy leaves of Senna alata and identified by sequencing the D1/ D2 26S rRNA gene and internal transcript spacers (ITS) of the ribosomal DNA. The whole mycelia of P. tremellosa, which were cultured on 180 plates of 2% malt extract agar (MEA) for 30 days, were extracted three times with MeOH at room temperature and concentrated under reduced pressure to afford the crude extract. The MeOH extract was then partitioned between ethyl acetate and water. The ethyl acetate extract was subjected to repeated silica gel and Sephadex LH-20 column chromatography (CC) to obtain three new compounds (1)(2)(3). In addition, the known compound tremetriol was also isolated and identified based on comparisons of spectroscopic data with spectra published in the literature [3].
To elucidate the absolute configuration of 1, the electronic circular dichroism (ECD) spectrum was calculated by quantum-chemical methods and compared with the experimental spectrum. The preliminary conformers, which were generated using the GMMX add-on module (energy window = 10 kcal/mol), were optimized using density functional theory (DFT) at the CAM-B3LYP/6-31+G(d,p) level. ECD spectra of the DFT-optimized conformers were calculated using time-dependent DFT at the CAM-B3LYP/6-31+G(d,p) level with the conductor-like polarizable continuum model (CPCM) solvation model in MeOH. The obtained ECD spectra were Boltzmann averaged on the basis of the calculated relative energies of the DFT-optimized conformers. The experimental and calculated ECD spectra of 1 were in good agreement for the (4R, 5S, 7R) absolute configuration (Fig. 3b).
Phlebioic acid (2), [ ] 20 D = + 173.8, was obtained as a colorless oil. HRESIMS revealed an [M+Na] + ion peak at 289.1396, indicating the quasi-molecular formula The IR spectrum exhibited absorptions for hydroxy groups (3435 cm −1 ) and carbonyl groups (1699 cm −1 ). The 13 C NMR and DEPT135 spectra (Table 1) showed the presence of four methyls, three methylenes, two methines, and six nonprotonated carbons, including four sp 2 carbons assigned to a tetra-substituted double bond [δ C 132.8 (C-1) and 155.8 (C-10)], a carboxy group [δ C 182.8 (C-14)], and a ketonic carbonyl group [δ C 208.2 (C-2)]. The DQF-COSY spectrum showed two solitary C-4/C-5/C-6 and C-8/C-9 sequences (Fig. 2). HMBC correlations [H 2 -8/C-10; H 2 -9/C-1, C-10, C-15; H 3 -15/C-1, C-10] indicated that both C-9 and C-15 were bound to an sp 2 carbon (C-10) within the double bond. HMBC correlations   -3), and a methylene carbon (C-4), suggestive of the C-2/C-3/C-4 sequence with two methyl groups at C-3. The HMBC correlation from H 2 -6 to C-1 confirmed the linkage between C-1 and C-5. The remaining indices of hydrogen deficiency and the chemical shifts of C-1 and C-10 implied a linkage between C-1 and C-2. Hence, the planar structure of 2 was determined to be a sesquiterpene with a previously unreported skeleton. The relative structure of 2 depicted in Fig. 4a (Table 1) indicated the presence of three methyls, three methylenes, two sp 3 methines, and an sp 2 methine. Additionally, the 13 C NMR spectrum displayed signals for 15 carbon atoms, which could be classified as three methyls, three methylenes, two sp 3 and one sp 2 methines, and two sp 3 and four sp 2 nonprotonated carbons, including a carbonyl carbon. The DQF-COSY spectrum revealed correlations indicative of the C-4/C-5/C-6 connection (Fig. 2). The HMBC spectrum showed correlations from both H 3 -13 and H 3 -14 to an olefinic methine (C-2), a quaternary carbon (C-3), and a methylene (C-4), as well as mutual correlations (C-14 or C-13). The HMBC correlations [H-2/C-1, C-5; H 2 -4/C-1, C-2] suggested a linkage between C-1 and C-5, revealing the presence of a 3,3-dimethylcyclopentene moiety. The HMBC spectrum also revealed correlations from the uncoupled methyl group [δ H 1.33 (3H, s, H 3 -15)] to a methine at δ C 66.0 (C-6), a nonprotonated carbon at δ C 61.0 (C-7), and a methylene at δ C 30.5 (C-8). Furthermore, H 2 -8 correlated with two sp 2 quaternary carbons at δ C 122.9 (C-9) and 153.8 (C-12) and a carbonyl carbon at δ C 174.3 (C-10). The carbon signal of C-12 also correlated with an olefinic proton (H-2) within a 3,3-dimethylcyclopentene moiety. These correlations allowed a seven-membered ring bearing a methyl group and a carbonyl group at C-7 and C-9, respectively, to be constructed. The remaining oxygenated methylene (H 2 -11) was proposed to be attached to C-12 based on HMBC correlations from H 2 -11 to C-9 and C-12. The location of C-2 was also corroborated by an NOE between H-2 and H 2 -11, as observed in the NOESY spectrum. Finally, the correlation from H 2 -11 to C-10 confirmed the formation of a γ-lactone ring. The oxygen functional groups at C-6 and C-7 were determined to be an epoxy group based on the remaining indices of hydrogen deficiency and the chemical shifts in the 1 H and 13 C NMR spectra. The presence of the epoxy group was also supported by the fact that the IR spectrum showed no absorption for hydroxy groups. The relative configurations of C-5, C-6, and C-7 were deduced to be as shown in Fig. 5a (Fig. 5b).
All isolated compounds exhibited no agonistic activities for peroxisome proliferator-activated receptor γ, retinoid X receptor α, and liver X receptor α in a luciferase reportor assay based on the method described in our previous paper Fig. 4 a Key NOESY correlations (arrows) in phlebioic acid (2). b Comparison of the experimental ECD spectrum of 2 with the calculated ECD spectrum of (4R, 5S, 7S)-2

Fig. 5 a Key NOESY correlations (arrows) in phlebiolide (3). b
Comparison of the experimental ECD spectrum of 3 with the calculated ECD spectrum of (5R, 6S, 7R)-3 [20]. Further studies of the biological activities of isolated compounds are in progress.
In this study, we isolated three new sesquiterpenes with distinct carbon frameworks. Most notably, 1 and 2 have new skeletons that are named "seco-sterpurane" and "phlebiane", respectively. The biogenetic pathway of phlebidiol (1), the first described seco-sterpurane, is possibly explained by the oxidative aliphatic C-C bond cleavage of the α-hydroxy ketones within the cyclobutane ring of an oxidized tremetriol (Fig. 6). A similar oxidative cleavage of a C-C bond was previously proposed in the clavicoronane sesquiterpenes [7]. Cytochrome P450 17A1 (CYP17A1) is an example of an enzyme that can cleave α-hydroxy ketones devoid of α-C-H bonds. CYP17A1 is a heme enzyme that catalyzes the cleavage of the C-17 to C-20 bond in 17α-hydroxypregnenolone during the biosynthesis of dehydroepiandrosterone via the ferric peroxo-hemiacetal intermediate [21,22]. Metalloenzymes similar to CYP17A1 seem to be involved even in the biosynthesis of 1. Meanwhile, the first phlebiane sesquiterpene, phlebioic acid (2), is potentially biosynthesized by the secondary ring-opening rearrangement of the isolactarane sesquiterpene, as shown in Fig. 6, although no plausible precursor has been isolated from P. tremellosa. Furthermore, the biosynthetic pathway to phlebiolide (3) also seems to involve the secondary ring-opening rearrangement of an isolactarane sesquiterpene. Merulactone [3], a previously isolated isolactarane from P. tremellosa, is likely to be the precursor of 3; this biogenesis can be rationalized by rearrangement with ring-expansion initiated by the nucleophilic substitution of a hydroxyl group at the C-6 of merulactone. To the best of our knowledge, this is the first example of a proposed scaffold transformation from isolactarane to merulane involving a plausible precursor.

Conclusion
Three novel sesquiterpenes (1)(2)(3) were isolated from the cultures of Phlebia tremellosa. The absolute configurations of 1-3 were confirmed by CD spectroscopy and DFT calculations. All new compounds are likely to have originated from sterpurane and/or isolactarane sesquiterpenes. Despite the fact that sterpurane and isolactarane sesquiterpenes have been isolated from several fungal species as mentioned previously, with the exception of merulactone, seco-sterpurane, phlebiane, and merulane sesquiterpenes have never been detected previously [23]. Therefore, our present study revealed the existence of distinctive biosynthetic pathways for the production of rare sesquiterpenes in the basidiomycete P. tremellosa.

Fungal material and identification
Phlebia tremellosa ECN184 was isolated from the healthy leaves of Senna alata cultivated in the Herbal Garden of Gifu Pharmaceutical University (Gifu, Japan) in November 2016. The surfaces of the leaves were sterilized by sequential soaking in 95% EtOH for 30 s, 0.5% NaClO for 2 min, and 70% EtOH for 2 min. The surface-sterilized leaves were cut into 1-cm 2 pieces and cultured on MEA containing 2% malt extract, 0.1% bacto peptone, 2% d-glucose, and 1.5% agar supplemented with 0.005% chloramphenicol in 9-cm petri dishes. The dishes were then incubated at 27 °C. Emergent organisms were isolated on new MEA. On the basis of the DNA sequencing of the ITS of rDNA and the D1/D2 domain of the 26S rDNA, the isolate belonged to genus Phlebia. The sequence data of P. tremellosa have been deposited at the DNA Data Bank of Japan (DDBJ) under the access numbers LC424440 (26S rRNA) and LC424443 (ITS). The strain was deposited at Department of Microbiology, School of Pharmacy, Aichi Gakuin University (ECN-184).

Fermentation, extraction, and isolation
The fungus P. tremellosa was inoculated onto 150 MEA plates without chloramphenicol. After incubation at 27 °C for 30 days, the fermented materials were extracted with MeOH (2 × 4 L, each 24 h) at room temperature, and the solution was evaporated in vacuo to obtain the MeOH extract (71.8 g). The MeOH extract was partitioned three times with equal amounts of ethyl acetate and water, and the ethyl acetate solution was concentrated under vacuum to yield the ethyl acetate soluble fraction (7.2 g). The ethyl acetate fraction was separated on a silica gel column with CHCl 3 /MeOH (gradient 50:1 to 8:1, v/v) as the eluent, to give fractions (Frs.) 1-12. Fr. 8 was purified with silica gel CC (n-hexane/ethyl acetate 5:1 to 3:1, v/v) to obtain phlebiolide (3; 2.1 mg). Fr. 9 was subjected to silica gel column purification (n-hexane/ethyl acetate 5:1 to 3:1, v/v) to obtain tremetriol (4.5 mg). Phlebioic acid (2; 2.1 mg) was isolated from Fr. 10 with the aid of a silica gel column (n-hexane/ acetone, 1:1). Fr. 11 was purified with a Sephadex LH-20 column (MeOH) to yield phlebidiol (1; 41.4 mg).

Computational methods
Conformers of 1-3 were generated using the GMMX add-on module of GaussView 6 with an energy window of 10 kcal/ mol. Optimization of suggested conformers followed by TDDFT calculations were performed using Gaussian 16 with various combinations of functionals (B3LYP, CAM-B3LYP, APFD, ωB97X-D) and basis sets [6-311+G(d,p), 6-31+G(d,p)] with the CPCM solvent model. ECD spectra were generated by the SpecDis program using a Gaussian band shape with 0.3-0.35 eV [24,25]. The overall CD spectra of the obtained conformers were Boltzmann weighted at 298 K after UV correction.