Identification of fungal fossils and novel azaphilone pigments in ancient carbonised specimens of Hypoxylon fragiforme from forest soils of Châtillon-sur-Seine (Burgundy)

Abstract

Fungal stromata were recently discovered in association with charcoal and burnt soil aggregates during an archaeological survey in the Châtillon-sur-Seine forest massif. The wood and soil in the samples were dated to the medieval period (between 738 and 1411 AD). Light microscopy and scanning electron microscopy revealed that a few of the stromatal fragments still contained ascospores. Their macromorphological characters were described and secondary metabolite profiles were generated using high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS). The combination of these two data lines then allowed species identification. Most of the fragments were assigned to Hypoxylon fragiforme, the type species of the Hypoxylaceae (Xylariales). Two further species whose stromata grew on the fossil charcoal could be tentatively identified as Jackrogersella cohaerens and (more tentatively) as Hypoxylon vogesiacum. These three species are still commonly encountered in the forests of Central Europe today. Furthermore, the HPLC-HRMS data of H. fragiforme suggested the presence of unknown azaphilone dimers and of further new pigments. These archaeological compounds were compared to fresh stromata of H. fragiforme collected in Germany and subjected to the same analytical protocol. While the major components in both samples were identified as the known mitorubrin type azaphilones and orsellinic acid, the chemical structures of seven novel complex azaphilone pigments, for which we propose the trivial names rutilins C-D and fragirubrins A-E, were elucidated using spectral methods (NMR and CD spectroscopy, high resolution mass spectrometry). It appears that these pigments had indeed persisted for millennia in the fossil stromata.

Recent advances in the chemistry and biology of azaphilones

“…CRI7 33 Cytotoxic (A549: 46.5 mg mL À1 , HuCCA-1: 48.1 mg mL À1 , MOLT-3: 17.4 mg mL À1 ) Dothideomynones B (39), D-F (41)(42)(43) Dothideomycete sp. CRI7 34 Mycoleptone A (44) Mycoleptodiscus indicus 15 Cytotoxic (PC3: 10.0 mM); antileishmanial (Leishmania major: 28.5 mM) Mycoleptone B (45) Mycoleptodiscus indicus 15 Cytotoxic (PC3: 7.1 mM); antileishmanial (Leishmania major: 21.7 mM) Mycoleptone C (46) Mycoleptodiscus indicus 15 Deectin C1 (47) Aspergillus deectus NCC0415 36 Inhibit enzyme (SHP2: 29.3 mM, PTP1B: 40.4 mM) Deectin C2 (48) Aspergillus deectus NCC0415 36 Inhibit enzyme (SHP2: 21.1 mM, PTP1B: 19.8 mM) Deectin C3 (49) Aspergillus deectus NCC0415 36 Inhibit enzyme (SHP2: 16.2 mM, PTP1B: 16.5 mM) (51) Aspergillus deectus NCC0415 36 Inhibit enzyme (SHP2: 7.0 mM, PTP1B: 6.1 mM) Deectin E (52) Aspergillus deectus NCC0415 36 Inhibit enzyme (SHP2: 16.0 mM, PTP1B: 24.0 mM) 8,11-Didehydrochermesinone B (53) Nigrospora sp. YE3033 26 Colletotrichone A (54) Colletotrichum sp.…”

“…Azaphilones (96-106) were characterized with an aliphatic chain at C-7 lacking the benzoyl moiety, e.g., peyronellone F (96), 6 lenormandins F-G (97-98), 50 as well as fragirubrins A-E (102-106). 51 Phialomustins A, C, and D (99-101), three azaphilones sharing with an unprecedented skeleton, were discovered from an endophytic fungus Phialophora mustea. Interestingly, they showed moderate activities toward several human cancer cells, meanwhile 94 and 95 also displayed antifungal activities with IC 50 values of 14.3 and 73.6 mM against Candida albicans, respectively.…”

“…52 2.8.2 Mitorubrin derivatives with a 7-benzoyl. The azaphilones with a benzoyl group substituted at C-7 included 12 mitorubrin (107-118), such as 6 00 -hydroxy-(R)-mitorubrinic acid (109), 53 purpurquinone D (110), 53 talarophilones A-B (111-112), 54 pinophilins D-F (113-115), 55 rutilins C-D (116-117) 51 and pinazaphilone A (118). 12 Apart from these, (+)-6 00 -hydroxymitorubrinol acetate (107) and (+)-6 00 -hydroxymitorubrinol (108), were encountered in Hypoxylon rubiginosum, a novel species from northern Thailand.…”

“…Chemical structures of deflectin-type azaphilones(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65).…”

Collaborative Biosynthesis of a Class of Bioactive Azaphilones by Two Separate Gene Clusters Containing Four PKS/NRPSs with Transcriptional Crosstalk in Fungi

“…Other natural products containing both two scaffolds have also been isolated in other fungal samples, but the covalent bonds formed in these compounds were significantly different from that of compound 1 ( Figure S1). [5] Undoubtedly, heterologous reconstitution of the biosynthetic pathway is an effective strategy to explore silent BGCs in native microorganisms, and the previous characterizations of these two BGCs were both performed in this way. [4,6] However, it may not always be possible to reveal the original biosynthetic pathway in the natural host.…”