Comparative Study of Toxic Terpenoidal Aldehydes and Lactone Derivatives from the European Polypore Bondarzewia mesenterica

Two unprecedented isomeric secondary metabolites named vibralactones Z5 (1a) and Z6 (1b), in addition to eleven known compounds (2–12), were isolated from solid-state rice culture medium of Bondarzewia mesenterica (Bondarzewiaceae). Chemical structures of the isolated compounds were established via spectral analyses. The new lactone derivatives were weakly active against Staphylococcus aureus without any significant cytotoxicity, while the molecules containing an aldehyde functionality showed significant antimicrobial and cytotoxic effects. For instance, erinacine P (7) and (+)-isovelleral (8) and erinacine P (7) were cytotoxic against all tested cell lines at IC50 values in the ranges of 3.5–14.2 and 2.8–30.2 μM, respectively. In addition, they revealed moderate antimicrobial activity with the lowest minimum inhibitory concentration (MIC) values recorded against Mucor hiemalis (8.3 μg/mL), Pichia anomala, and Rhodotorula glutinis at 16.6 μg/mL.


INTRODUCTION
Basidiomycota continue to be a bountiful source of secondary metabolites that can be used as templates for development of drugs and agrochemicals. 1 The genus Bondarzewia is a remarkable poroid basidiomycete genus whose species are characterized by large imbricate basidiomes.Some of the species are saprotrophs regarded as edible and/or medicinal especially in Asia, whereas others are tree pathogens. 2,3The genus was first coined in 1940 by Singer and typified by Bondarzewia montana, which is now regarded as a synonym of Bondarzewia mesenterica.This species shows apparent host specificity for Abies and mainly occurs in old forests. 3urrently, there are eleven species accepted in the genus, recorded from Asia, Europe, America, and Oceania, but with no records from Africa thus far. 2 Phylogenetically, it is closely related to Heterobasidion. 2 Both genera belong to the order Russulales, and an own family Bondarzewiaceae has eventually been created to accommodate them and some other genera. 4he genus Bondarzewia remains understudied concerning the chemistry of its secondary metabolites, except for a report of the weakly cytotoxic fruiting body constituent montadial and dihydrobenzofuran derivatives from cultures of a Chinese collection of Bondarzewia berkeleyi. 5,6In the latter study, no biological activity for the isolated metabolites has been reported and no proof was given as to whether the study culture was authentic, even though the specimen was identified by a specialist.In our ongoing attempts to study rare and hitherto untapped members of Basidiomycota, we recently came across a culture of B. mesenterica and decided to conduct an in-depth study on its secondary metabolism.The results are the subject of the current paper.

RESULTS AND DISCUSSION
2.1.Strain Identification.The fungal isolate IHI 766 was collected by one of the authors (H.K.) at the Bavarian Forest National Park in Bavaria, Germany.Morphologically, it was identified as similar to another B. mesenterica isolate (IHI 668) collected at the same location in 2016 and deposited at the International Institute Zittau, Technical University of Dresden (Germany) as DSM 108281.A draft genome of the latter is assembled and available under NCBI Bioproject PRJNA521458.
Compound 1 was obtained as a yellow oil that revealed a single peak during both analytical and preparative chromatographic separations.The molecular formula of  S3) revealed the presence of two sets of comparable proton resonances in a ratio of 5:6 according to their integration indices indicating its possible existence as an inseparable mixture of two isomeric derivatives (1a and 1b).The same notion was also obvious in the 13 C NMR spectrum (Table 1, Figure S4) that revealed twined peaks of comparable resonances and can be recognized into four unprotonated sp 2 carbon pairs namely two carbonyl carbon pairs at δ C 177.69/177.73(C-6) and 176.6 (C-5), two olefinic carbon pairs at δ C 171.97/172.03(C-3) and 139.05/139.16(C-9).In addition, the 13 C NMR spectral data of 1a and 1b (Table 1) revealed the presence of two tertiary sp 2 carbon atoms each corresponding to an isomer at δ C 124.4 and 121.9 that were directly correlated via the heteronuclear single quantum coherence (HSQC) spectrum (Figure S7) to two olefinic protons at δ H 5.29 (td, J = 7.7, 1.5 Hz, 1H) and at δ H 5.43 (td, J = 7.3, 1.4 Hz, 1H).The 13 C NMR spectral data of 1 unveiled the presence of one pair of electromagnetically equivalent methine sp 2 carbon atoms at δ C 116.5 (C-4) directly correlated via the HSQC spectrum to two electromagnetically equivalent olefinic protons at δ H 5.91 (q, J = 1.7 Hz, 2H), one pair of methine sp 3 carbon atoms at δ C 45.2/45.0(C-1), four pairs of methylene sp 3 carbons at δ C 31.00/30.89(C-2), δ C 31.27/ 31.34 (C-7), an electromagnetically equivalent pair at δ C 75.0 (C-12), and an oxygenated methylene pair at δ C 61.2 (C-11 in 1a)/68.5 (C-10 in 1b).The interpreted spectral data accounted for four degrees of unsaturation and thus suggesting that 1a and 1b comprisemonocyclic structures.A literature search based on the obtained results revealed that 1a and 1b are closely related to vibralactone J (2) 7 with a clear difference of the absence of two allylic methyl groups and the emergence of two hydroxymethylene moieties instead.Apart from that difference, the 1 H and 13 C NMR data of both 1a/1b and 2 came in close accordance.
Further confirmation to the depicted structures of 1a and 1b was obtained by a 1 H− 1 H COrrelated SpectroscopY (COSY) spectrum (Figure 2) that revealed the presence of two comparable spin systems extending over H-8/H 2 -7/H-1/H 2 -2, whereas the heteronuclear multiple bond correlation (HMBC) spectrum (Figure 2) confirmed the presence of one lactone ring in each isomer by revealing key correlations from diastereotopic methylene protons at δ H 4.84/4.88(dd, J = 17.9, 1.7 Hz, H 2 -12) and an olefinic methine proton at δ H 5.91 (q, J = 1.7 Hz, H-4) to one carbonyl carbon at C-5 (δ C 176.6) and a deshielded quaternary carbon pair at δ C 171.97/172.03(C-3), thus confirming the presence of comparable α,βunsaturated lactone rings in 1a and 1b, respectively.To distinguish the depicted orientation of terminal hydroxymethylene moieties in 1a and 1b, the rotating-frame Overhauser spectroscopy (ROESY) spectrum (Figure 2) was recorded and revealed key ROE correlations from diastereotopic methylene protons at δ H 4.05/4.09(d, J = 12.2 Hz, H 2 -11) to diastereotopic methylene protons at δ H 2.39/2.47(H 2 -7) in 1a, whereas the hydroxymethylene protons in 1b at δ H 3.94 (d, J = 1.4 Hz, H 2 -10) revealed key ROE correlation to an olefinic proton at δ H 5.43 (tq, J = 7.3, 1.4 Hz, H-8), confirming the presence of hydroxymethylene groups at C-11 and C-10 in 1a and 1b, respectively.Both compounds 1a and 1b revealed a single secondary carboxylic acid chiral carbon (C-1) resembling vibralactone J (2) and sterepinic acid A (3). Being isolated as an inseparable mixture hindered determining their absolute configuration by the phenylglycine methyl ester (PGME) method that previously established (S) configuration for sterepinic acid A. 9 9 in addition to be of a common biosynthetic origin, then at least one of the compounds 1a/1b could presumably feature a similar (S) configuration at C-1.Based on the aforementioned results, compound 1 was unambiguously confirmed to be a mixture of two isomeric vibralactone derivatives and hence were trivially named as vibralactones Z 5 (1a) and Z 6 (1b).
To the best of our knowledge, (+)-isovelleral ( 8) was unprecedented in fungal cultures and this encouraged us to investigate whether it is a genuine product or evolved from an enzymatic conversion of stearyl-velutinal as previously reported in the literature. 13In order to define its source, two different mycelial portions of equal age were separately extracted by ethyl acetate.One portion was directly extracted without any mechanical treatment while the other was crushed and kept for 30 min before extraction.Both extracts were individually analyzed using HPLC-DAD-MS to screen for the molecular weight of 530 g/mol ascribed to stearyl-velutinal.The obtained results revealed the presence of (+)-isovelleral (8) in comparable abundances in both extracts as that obtained in the main extract of this study.Intriguingly, no traces of stearylvelutinal could be detected in all of the analyses supporting its probable production during an advanced growth phase of the fungus in its natural habitat as a defense mechanism against competing predators or parasites, rather than being induced by lab cultivation.However, upon trying the same extraction scheme on dry fruiting bodies of B. mesenterica, we could detect neither stearyl-velutinal nor isovelleral.

Biological Activities of Compounds 1−12.
The isolated compounds demonstrated various biological effects in the antimicrobial and cytotoxicity assays based on our  standardized methods. 19The obtained results (Table 2) unveiled that the new vibralactones Z 5 /Z 6 (1) together with Z 2 (5) and sterepinic acid A (3) had weak antibacterial effects against Staphylococcus aureus at minimum inhibitory concentration (MIC) values of 66.6 μg/mL.The compounds were non-cytotoxic to cell lines L929 and KB3.1; hence, they were not further tested.

CONCLUSIONS
To this end, we have studied secondary metabolites, mostly consisting of lactone moieties from B. mesenterica.It is noteworthy that similar derivatives have been widely reported from the basidiomycete Boreostereum vibrans (formerly Stereum vibrans), 20,21 a fungus considered as a "talented strain" due to its ability to produce a vast array of natural products. 8evertheless, isolation of similar compounds from different fungi belonging to the same class in the phylum Basidiomycota is a commonly encountered scenario.Notably, the aldehyde functionality has been shown to affect the antimicrobial and cytotoxic effects similar to the isolated cyathane-xyloside (7) 19,22 and sesquiterpenoid (8). 23The presence of two aldehyde groups in (+)-isovelleral (8) has been demonstrated to be responsible for its mutagenic, antimicrobial, cytotoxic, and antifeedant properties. 13,23

General Experimental Procedures. HPLC-DAD-
MS analyses were performed on an amaZon speed ETD ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) on positive and negative ionization modes.The HPLC system consisted of a Dionex UltiMate 3000 UHPLC (Thermo Fisher Scientific Inc., Waltham, MA) equipped with a C 18 Acquity UPLC BEH column (Waters, Milford).The solvent phase consisted of solvent A (deionized H 2 O + 0.1% formic acid) and solvent B (MeCN + 0.1% formic acid).The separation gradient was as follows: 5% solvent B for 0.5 min, 5−100% solvent B over 20 min, and holding the gradient isocratically at 100% solvent B for 10 min.The flow rate employed was 0.6 mL/min, and UV−Vis detections were recorded at 190−600 nm.HR-(+)ESI-MS data were recorded on a maXis ESI-TOF (Time-Of-Flight) mass spectrometer (Bruker Daltonics, Bremen, Germany) connected to an Agilent 1260 series HPLC-UV system (Agilent Technologies, Santa Clara, CA) equipped with a C 18 Acquity UPLC BEH column (Waters).The solvent system consisted of solvent A (deionized H 2 O + 0.1% formic acid) and solvent B (MeCN + 0.1% formic acid).The separation gradient was as follows: 5% solvent B for 0.5 min, 5−100% solvent B over a period of 19.5 min, and eventually holding solvent B at 100% for 5 min.The flow rate employed was 0.6 mL/min at 40 °C and the UV−Vis detection was recorded at 200−600 nm.Molecular formulas of the detected compounds were calculated using the Smart Formula algorithm of the Compass Data Analysis software (Bruker, version 6.1).An Avance III 500 ( 1 H 500 MHz, 13 C 125 MHz, Bruker Daltonics, Bremen, Germany) spectrometer was used to record 1D and 2D NMR spectra of compounds dissolved in methanol-d 4 .Chemical shifts were recorded in parts per million (ppm), and coupling constants were calculated in Hertz (Hz).UV−Vis spectra were obtained using a Shimadzu UV−Vis 2450 spectrophotometer (Kyoto, Japan) at a concentration of 0.02 mg/mL in methanol Uvasol (Merck, Darmstadt, Germany).Optical rotations were recorded on an Anton Paar MCP-150 Polarimeter (Graz, Austria) with a sodium D line at 589 nm and 100 mm path length at a concentration of 1.0 mg/mL in methanol.
4.2.Collection of the Fungal Specimen and Preparation of Cultures.Basidiocarps of B. mesenterica were collected at Bavarian Forest National Park (location: Mittelsteighuẗte) in Bavaria, Germany, in August 2021, on the base (putatively emerging from the buried root) of an old-growth Abies alba by one of the authors (H.K.).The mycelial cultures were established from the fresh basidiomes and maintained on YMG (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, and 20 g of agar in 1 L of deionized water) medium.

Fermentation of Cultures and Extraction of Metabolites.
YMG agar plates were prepared and used to inoculate fresh pieces of basidiomes to obtain axenic cultures as previously described. 19Subsequently, YMG agar plates with fully grown mycelia of B. mesenterica were used to inoculate 6 × 500 mL Erlenmeyer culture flasks containing sterile rice medium.After 55 days of static incubation at 24 °C in the dark, secondary metabolites were extracted as initially reported. 24In brief, the fermented cultures were initially soaked overnight using 3 × 500 mL of acetone per flask and sonicated at 40 °C.The filterate was evaporated until an aqueous phase was attained and then partitioned twice against EtOAc (1:1).The aqueous phase was discarded, and the EtOAc phase was dried under vacuum to yield the total extract (1.5 g).