Allelopathic Polyketides from an Endolichenic Fungus Myxotrichum SP. by Using OSMAC Strategy

Three new polyketides myxotritones A-C (2–4), together with a new natural product 7,8-dihydro-7R,8S-dihydroxy-3,7-dimethyl-2-benzopyran-6-one (1) were obtained from the endolichenic fungus Myxotrichum sp. by using OMSAC (One Strain, Many Compounds) method. The planar structures of these new compounds were determined by NMR experiment and HRESIMS data, and the absolute configuration of 1 was established by X-ray diffraction, and the stereochemistry of the new compounds 2-4 were determined by same biosynthesis origin, and similar CD spectra with 1. Allelopathic test showed that compound 4 significantly retarded root elongation of Arabidopsis thaliana seed, indicating that this fungus might contribute to the defense of its host lichen. From the view of biosynthetic pathway, all four compounds 1-4 might be originated from Non-Reduced Polyketide synthase (NR-PKS).

Snatzke' rule was also used to determine the diol of C-7 and C-8 [11][12][13] . The positive cotton effect at 327 nm observed in situ dimolybdenum CD spectra permitted the assignment of absolute configuration as 7R, 8S (Fig. S4).  Myxotritone A (2) was isolated as a yellow powder, [α ] D 22 = + 45.6 (c = 0.125, MeOH). Its molecular formula was determined as C 22 H 22 O 8 (12 degrees of unsaturation) by TOF-ESI-MS spectral data, which showed a pseudomolecular ion at m/z 437.1209 [M + Na] + (Fig. S10). The UV spectrum of 2 displayed the maximum absorptions at 217 nm (log ε 4.21), 256 nm (log ε 3.86) and 364 nm (log ε 3.97) (Fig. S12), revealing the presence of an extended conjugated system as the characteristic of azaphilones. The 1 H, 13 C NMR and HMQC spectra revealed that 1 contained four methyls (one methoxyl group), two methylenes with one oxygenated, an oxymethine unit, an oxygenated quaternary carbon, 12 olefinic carbons, an ester carbonyl carbon, and a keto carbonyl group, which explained all carbon signals of 2. Analysis of the 1 H and 13 C-NMR data of 2 revealed the same structural fragment (subunit A) as 1, except the H-5 in 1 was replaced by other moiety in 2, and this conclusion was supported by HMBC correlations (Fig. 4). The remaining connectivity was solved by detailed analysis of HMBC spectrum. The correlations from 10′ -CH 2 -to C-3′ a, C-4′ and C-5′ , from 3′ -CH 2 -to C-3′ a, C-4′ and C-7′ a, 8′ -CH 3 to C-5′ , C-6′ and C-7′ together with correlations of the 9′ -methoxyl with C-7′ established a hexa substituted phenyl ring. The key correlation from 3′ -CH 2 -to the ester carbonyl and considering the chemical shift value of C-7′ a (δ C 107.8) led to construct an isobenzofuran-1(3H)-one fragment (subunit B). The correlations from 10′ -CH 2 -to C-4a, C-5 and C-6 connected the subunit A with subunit B (Fig. 4). Considering the chemical shift values of C-7, C-8, and C-5′ and molecular formula, these three carbons must be anchored a free hydroxyl group, respectively. Thus the planar structure of 2 was determined. Compound 2 showed positive (371 nm, 311 nm and 229 nm) and negative (259 nm) cotton effects in the CD spectrum (Fig. 5). Based on the similar CD data and same biosynthetic pathway with 1, the relative and absolute configurations of 2 were postulated to be 7R, 8S.
Myxotritone B (3) was obtained as yellow powder, [α ] D 22 = + 6.0 (c = 0.067, MeOH). The molecular formula of 3 was deduced as C 23 H 24 O 9 on the basis of its TOF-ESI-MS spectrum, in which a pseudomolecular ion was observed at m/z 467.1313 [M + Na] + (Fig. S17). The 1 H and 13 C NMR spectra for 3 were similar with those of 2 except that one more methoxyl signal was observed, and the methyl anchored at the phenyl ring was disappeared in 3, which implied that the methyl group on the phenyl ring in 3 was methoxylation. Yet, careful analysis of the 1 H NMR of 2 and 3 revealed that the peak shape of 10′ -CH 2 in 2 and in 3 was completely different: singlet in 2, whereas two doublets in 3. This phenomenon implied that the substitutes around C-10′ in 3 were different from those in 2, leading to the chemical environment change, which produced anisotropic characteristics of 10′ -CH 2 in  3. Thus further HMBC spectrum was done to explain the phenomenon. The HMBC correlations clearly revealed that the connection of lactone ring was changed in 3 ( Fig. 4), which finally established the planar structure of 3. The relative and absolute configurations of 3 were postulated to be 7R, 8S, due to its similar CD data and same biosynthetic origin with 1.   (Fig. S24). The 1 H and 13 C NMR spectra of 4 suggested the presence of similar subunit found in 1 except for 9-methyl signal (δ C 17.9/δ H 2.18) replaced by a methylol signal (δ C 59.9/δ H 4.29). This hypothesis was further confirmed by HMBC correlations (Fig. S23) (Fig. 4). The HMBC correlations from 9-CH 2 OH to C-3, C-4 and C-4a confirmed that the additional methylol group was connected with C-3. Similarly, the relative and absolute configurations of 4 were also deduced as 7S and 8S.
From the structural features of compounds 1-4, all these four compounds might come from same Non-Reduced Polyketide synthase (NR-PKS) origins. The putative biosynthetic pathway was suggested in the Fig. 6. To study the potential effects of these metabolites, allelopathic potential of these compounds was tested with the root elongation of A. thaliana as model. The root growth of A. thaliana was inhibited after treatment with the compounds in a dose dependent manner (Fig. 7). The inhibition of compounds 1-4 at different concentrations was shown in Fig. 7. Compound 4 were found to retard the Arabidopsis seeds root significantly, with the inhibition rate of 75.9% at 8 μg/mL, whereas compounds 1 and 2 showed moderate inhibition activities. The results implied that this fungus might contribute to the defense of its host lichen.

Fungus and culturing condition. The endolichenic fungus Myxotrichum sp. was isolated from the lichen
Cetraria islandica (L.) Ach. collected from Laojun Mountain, Yunnan Province, People's Republic of China. The isolate was identified on the basis of the internal transcribed spacer region sequences of the rDNA (Genbank Accession No. HQ324780) (Fig. S1) and the fungus assigned the accession no.20081189 was deposited at lichen laboratory's culture collection in College of Life Sciences, Shandong Normal University, Jinan. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 15 days. Then, the proper fungus strain was inoculated in five Erlenmeyer flasks (500 mL) each containing 200 mL PDB (20% potato and 2% glucose) media. Flask cultures were incubated at 25 °C on a rotary shaker at 110 rpm for seven days as spore seeds. These spore seeds were used to inoculate in Fernbach flasks (500 mL), each containing 60 g of rice, and incubated at 25 °C for 40 days.
Extraction and isolation. The fermented material was extracted with ethyl acetate (6 L for four times).

H (600MHz) and 13 C NMR (150 MHz) spectroscopic data (MeOH-d 4 ) of myxotritone A-C (2-4).
Scientific RepoRts | 6:19350 | DOI: 10.1038/srep19350 t R = 13 min). Fraction (863 mg) eluted with CH 2 Cl 2 - Seedling growth test. Arabidopsis thaliana seeds were surface sterilized by 5% sodium hypochlorite for 5 min, followed by washing with sterile distilled water for five times. Compounds were dissolved with DMSO to final concentration of 40 mg/mL. Then 20 μL of them were added to 25 mL 1/2 MS medium supplemented with 0.8% (w/v) agar to get plates with different concentrations of compounds (8,16, 32 μg/mL). To eliminate the effect of DMSO on the growth of A. thaliana, plates with 20 μL DMSO were used as blank control. Fifteen seeds were distributed on each Petri dishes described before. Each concentration was conducted in triplicate. The Petri dishes were placed in a growth chamber at 23 ± 1 °C under light for 8 h and darkness for 6 h. The lengths of roots were measured after 9 days. The percentage of growth inhibition of root lengths was calculated as the following equation: where T stands for the average length of treatment (cm) and C stands for the average length of control (cm) 14,15 .