Lachnuoic Acids A−F: Ambuic Acid Congeners from a Saprotrophic Lachnum Species

Chemical prospection of an extract derived from a saprotrophic fungus Lachnum sp. IW157 resulted in the isolation and characterization of six unprecedentedly reported ambuic acid analogues named lachnuoic acids A−F (1–6). Chemical structures of 1–6 were determined based on comprehensive 1D and 2D NMR spectroscopic analyses together with HR‐ESI‐MS spectrometry. The relative configurations of 1–3 were defined by ROESY spectroscopic analyses while their absolute configurations were unambiguously determined by Mosher's esters method. All isolated compounds were subjected to cytotoxic, antimicrobial, antibiofilm and nematicidal activity assays where only lachnuoic acid A (1) revealed potent antimicrobial activity against Staphylococcus aureus and Bacillus subtilis at MIC values of 16.6 and 8.3 μg/mL, respectively.


Introduction
The fungal kingdom currently comprises ca, 150,000 described species, which have already yielded numerous unique secondary metabolites with unprecedented chemical scaffolds.Some chemical scaffolds have become the basic structures for various developmental candidates that resulted in marketed drugs or agrochemicals. [1]However, most of the fungal taxa have been neither cultured nor explored for secondary metabolites.In our ongoing study, we are focusing on rare, inoperculate Discomycetes (class Leotiomycetes, order Helotiales) from Germany and have been able to culture the respective species from ascospores for the first time.We have recently reported the isolation and the characterization of two polyketides from the cultures of a Lachnum sp. that was derived from apothecia collected off the common reed grass, Phragmites communis in Germany. [2]Morphologically, it revealed characteristic features to be closely related to Lachnum carneolum.This fungus was selected because of strong activities of its crude extract in a screening of over twenty Leotiomycetes, and because HPLC-DAD/MS profiling revealed that it had an extraordinarily rich secondary metabolite profile.In the current paper, we wish to report the isolation, structure elucidation and biological activities of further metabolites that were produced by the same strain.

Characteristics of the Producer Strain
The investigated strain, Lachnum sp.IW157 (DSM 116717), was derived from a specimen collected in Germany and tentatively identified as Lachnum (cf.) carneolum.The characteristics of the apothecia are depicted on the Internet.(https://asco-sonneberg.de/pages/gallery/lachnum-carneolum-220425-iw157-stma22045-nur-lsu-01xsmjj41478.php).A preliminary molecular characterization of the strains was reported in our previous paper [2] where it was also discussed that the clarification of the identity of this discomycete will afford additional work, including type studies, which are outside the scope of the current study.
The secondary metabolites that are described here were obtained from the same extract that yielded the diaporphasines E and F (Figure 1) as described previously. [2]solation and Structure Elucidation of Secondary Metabolites 1-6 Compound 1 was isolated as a white amorphous solid.Its molecular formula was determined to be C 9 H 12 O 6 based on HR-ESI-MS that revealed a sodium adduct at m/z 239.0526 [M + Na] + (calculated 239.0526) indicating four degrees of unsaturation.The 13 C NMR spectral data of 1 (Table 1) revealed the presence of two unprotonated carbon atoms identified as a sp 2 carbonyl carbon at δ C 167.4 and an oxygenated sp 3 carbon (δ C 79.4).In addition, the 13 C NMR spectrum of 1 also revealed the presence of seven methines distinguished into four sp 2 (δ C 124.3, 129.5, 130.5, 147.0) and three sp 3 (δ C 70.9, 74.1, 78.4) carbon atoms.The 1 H NMR and HSQC spectra of 1 revealed the presence of two doublet olefinic protons at δ H 6.05 and 6.72 ppm with a coupling constant of 15.5 Hz and they were directly correlated to two olefinic carbons at δ C 124.3 and 147.0, respectively.This notion suggested that compound 1 incorporates in its structure an α,β-unsaturated carbonyl moiety and to be of trans configuration.A literature search of 1 revealed that it combines structural features of ambuic acid analogues [3][4][5][6] and conduritols. [7,8]Further confirmation to the depicted structure of 1 (Figure 1) was obtained by 2D NMR spectroscopic analyses including 1 H-1 H COSY, HMBC and ROESY (Figure 2).The 1 H-1 H COSY spectrum revealed the first spin system between the two olefinic protons forming the α,β-unsaturated carbonyl moiety while the second spin system extends over three oxygenated methine protons at δ H 3.39 (d, J = 8.3 Hz), 3.74 (dd, J = 8.3, 3.7 Hz) and 4.14 (dt, J = 3.7, 2.1 Hz) ascribed to H-5, H-6 and H-9 in addition to two olefinic protons at δ H 5.52 (dt, J = 10.3, Figure 1.Structures of compounds lachnuoic acids AÀ F (1-6), diaporphasines E, F [2] and ambuic acid. [3]ble 1. 1 H and 13 C NMR data of 1-3.The HMBC spectrum of 1 (Figure 2) revealed key correlations from H-5 and H-9 to a carbon at δ C 147.0 (C-3) whose directly connected proton at δ H 6.72 (d, J = 15.5 Hz) revealed key correlation to a carbonyl carbon suggesting its existence as a terminal free carboxylic acid moiety.Further HMBC correlations were noticed from H-5 to C-7 (δ C 129.5) whereas H-6 and H-9 revealed a key correlation to C-8 (δ C 130.5).The relative configuration of 1 was deduced by ROESY spectrum that unveiled a key ROE correlation between H-5 and H-9 suggesting to be cofacial while H-6 revealed a key ROE correlation to H-3 and hence suggested to be facing the opposite side of the molecule.The absolute configuration of 1 was determined by analyzing the coupling constant values and performing Mosher's esters method (Figure 3, Table S3) and that revealed Δδ SR values diagnostic for (4R,5R,6S,9S) configuration.Based on the obtained results, compound 1 was identified as a previously undescribed natural product that was named lachnuoic acid A.
Compound 2 was obtained as a white amorphous solid.HR-ESI-MS determined its molecular formula to be C 9 H 10 O 5 by revealing a sodium adduct at m/z 221.0420 [M + Na] + (calculated 221.0420) indicating the presence of five degrees of unsaturation instead of four degrees in 1.The 1 H and 13 C NMR spectra of 1 and 2 (Table 1), revealed an equal number of olefinic and oxygenated methine protons suggesting that the additional degree of unsaturation might be due to an epoxide ring formation.This suggestion was further confirmed by 13    NMR and HSQC spectral data of 2 (Table 1) that revealed relatively shielded carbon resonances at δ C 60.0, 64.2, 63.5 and 63.1 assigned to C-4, C-5, C-6 and C-9, respectively, compared to their counterparts in 1.The obtained results and by comparison with those reported of ambuic acid analogues [3][4][5][6] and conduritols, [7,8] compound 2 was suggested to have an epoxide ring formed over C-4 and C-5 accounting for the additional degree of unsaturation.Further confirmation of the suggested structure of 2 (Figure 1) was provided by 1 H-1 H COSY and HMBC spectra (Figure 2).Similar to 1, the 1 H-1 H COSY spectrum of 2 revealed a spin system between two doublet olefinic protons at δ H 5.95 and 7.23 ppm with a coupling constant of 15.5 Hz indicating their presence as an α,βunsaturated carbonyl moiety in trans configuration.The 1 H-1 H COSY spectrum of 2 exhibited a second spin system extending from three aliphatic methine protons at δ H 3.29 (dt, J = 2.3, 1.0 Hz, H-5), 4.43 (p, J = 2.2 Hz, H-6) and 4.11 (dd, J = 4.8, 1.2 Hz, H-9) over two olefinic protons at δ H 5.43 (dtd, J = 10.5, 2.1, 0.8 Hz, H-7) and 5.60 (ddd, J = 10.5, 5.0, 2.3 Hz, H-8).The relative configuration of 2 was determined by ROESY spectrum (Figure 2) that revealed key ROE correlations from H-5, H-6 and H-9 to H-3 indicating that they are all on a cofacial orientation.The absolute configuration of 2 was determined by analyzing the coupling constant values of H-5, H-6 and H-9 in addition to performing Mosher's esters method that revealed Δδ SR values between its (S)-and (R)-MTPA esters (Figure 3, Table S4) diagnostic for (4S,5R,6R,9S) configuration.Based on the aforementioned results, compound 2 was identified as a previously undescribed ambuic acid derivative that was trivially named as lachnuoic acid B.
Similar to 2, compound 3 was obtained as a white amorphous solid and HR-ESI-MS established its molecular formula to be C 9 H 10 O 5 by revealing a sodium adduct at m/z 221.0415 [M + Na] + (calculated 221.0420) indicating five degrees of unsaturation.The 1 H and 13 C NMR spectral data of 3 (Table 1) revealed close dataset values to those reported for 2 suggesting being of a comparable relative configuration.A major difference was noticed in the chemical shift values assigned to H-5 at δ H 3.10 (q, J = 1.6 Hz; δ C 63.5) and H-6 at δ H 4.20 (dt, J = 4.3, 1.7 Hz; δ C 61.8) suggesting an opposite configuration at H-6.The relative configuration of 3 was determined by ROESY spectrum (Figure 2) that revealed key ROE correlations from H-5 and H-9 to H-3 and H-2 indicating their cofacial existence while H-6 to be on the opposite side.The absolute configuration of 3 was determined by Mosher's esters method (Figure 3, Table S5) together with analyzing the coupling constant values of H-5, H-6 and H-9 that were diagnostic to (4S,5R,6S,9S) configuration.Therefore, compound 3 was identified as a previously undescribed natural product and was given a trivial name lachnuoic acid C.
Compound 4 was isolated as a yellowish-white amorphous solid.Its molecular formula was determined to be C 9 H 10 O 5 based on HR-ESI-MS that revealed a sodium adduct at m/z 221.0422 [M + Na] + (calculated 221.0420) indicating five degrees of unsaturation.Despite its identical molecular formula and degrees of unsaturation to those in 2 and 3, the 1 H and 13 C NMR spectral data of 4 (Table 2) revealed a second α,βunsaturated carbonyl moiety appearing as a ketocarbonyl carbon at δ C 197.7 (C-9) followed by two olefinic sp 2 carbon atoms at δ C 126.9 (C-8) and 148.6 (C-7).The latter carbons were directly correlated to two proton resonances at δ H 6.01 (ddd, J = 10.1, 2.6, 1.3 Hz, H-8) and 7.01 (ddd, J = 10.1, 5.1, 2.9 Hz, H-7), respectively, indicating their presence in cis configuration.In addition, the 1 H spectrum of 4 revealed the presence of one disastereotopic methylene group at δ H 2.33 (ddt, J = 19.4,8.4, 2.8 Hz) and 2.69 (dtd, J = 19.4,5.2, 1.4 Hz) that were directly correlated to a secondary sp 3 carbon at δ C 32.9 (C-6).The 1 H-1 H COSY spectrum of 4 (Figure 4) revealed a spin system extending over H-5 at δ H 3.86 (dd, J = 8.4, 5.2 Hz) to a diasteroptopic methylene group (H 2 -6) followed by H-7 and H-8.Based on the obtained results, compound 4 was deduced to incorporate a cyclohexenone moiety in its structure.A literature search of 4 revealed its structural resemblance to strobiloscyphones [4] and pestallic acids. [5]Further confirmation to the suggested structure of 4 was provided by HMBC spectrum (Figure 4 2, Figure S41) revealed two unprotonated carbon atoms, a carbonyl carbon at δ C 167.9 and an olefinic carbon at δ C 134.2 in addition to five protonated olefinic carbon atoms at δ C 118.7, 122.9, 133.9, 137.5 and 139.7 that account altogether for four degrees of unsaturation.Thus, compound 5 was deduced to have an acyclic structure.In addition, the 13 C NMR spectral data of 5 revealed two oxygenated methylene sp 3 carbon atoms (δ C 61.1 and 61.5).The 1 H NMR and 1 H-1 H COSY spectra of 5 (Table 2, Figure 4) revealed two distinct spin systems: one between two olefinic trans configured protons at δ H 5.92 (H-2) and 7.65 (H-3) with a characteristic coupling constant of 15.8 Hz while the second spin system originated from an olefinic proton at δ H 6.54 (d, J = 11.7 Hz, H-5) extending to two olefinic protons at δ H 6.78 (ddt, J = 15.0,11.7, 1.8 Hz, H-6) and 6.08 (dt, J = 15.0,4.7 Hz, H-7) then ending by a doublet hydroxymethylene group at δ H 4.10 (d, J = 4.7 Hz, H 2 -8).These two substructures recognized by the 1 H-1 H COSY spectrum were correlated by the HMBC spectrum of 5 (Figure 4) that revealed key correlations from a hydroxymethylene group at δ H 4.15 (br s, H 2 -9) to three olefinic carbon atoms at δ C 137.5 (C-3), 134.2 (C-4) and 133.9 (C-5) indicating that both spin systems and H 2 -9 are all connected at C-4 to form the depicted open chain structure of 5 (Figure 1).By analyzing coupling constant values and ROE correlations illustrated by 5 (Figure 4), the two double bonds at C-2/C-3 and C-6/C-7 were determined to be of trans configuration while the one at C-4/C-5 was found to be of cis configuration.Based on the obtained results, compound 5 was identified as a previously undescribed natural product that was given the trivial name lachnuoic acid E. Compound 6 was purified as a yellowish-white amorphous solid with its molecular formula established as C 9 H 8 O 5 based on HR-ESI-MS that revealed a sodium adduct at m/z 219.0260 [M + Na] + (calculated 219.0264) and thus indicating six degrees of unsaturation.The 1 H, 13 C NMR and HSQC spectra of 6 (Table 2, Figure S49) revealed, similar to 1-5, the presence of an α,βunsaturated carbonyl moiety in trans configuration that account for two degrees of unsaturation.In addition, the 13 C NMR spectral data of 6 (Table 2) exhibited the presence of six sp 3 carbon atoms distinguished into five shielded oxygenated methines (δ C 46.8, 47.3, 50.60, 50.63, 55.3) and one unprotonated oxygenated carbon (δ C 54.2).Based on the obtained results, compound 6 was deduced to have a cyclohexane ring fused with three epoxide rings over C-4/C-5, C-6/C7 and C-8/C-9.The 1 H NMR spectral data of 6 (Table 2) revealed the presence of five methine protons appearing over three signals.A proton resonance at δ H 3.60 (dt, J = 4.3, 1.5 Hz; δ C 50.63) was assigned to H-8 based on 1 H-1 H COSY spectrum (Figure 4) that revealed key correlations from H-8 to H-9 at δ H 3.60 (m, overlapped).The latter together with H-5 at δ H 3.64 (m, overlapped) revealed in the HMBC spectrum (Figure 4) key correlations to an olefinic carbon at δ C 143.3 assigned to C-3.Only the relative configuration of 6 could be determined by its ROESY spectrum that revealed key ROE correlations from H-5 and H-9 to H-2 at δ H 6.19 (d, J = 16.0Hz) and H-3 at δ H 6.54 (d, J = 16.0Hz) indicating their direction toward the same face of the molecule while H-6 and H-7 directing toward the opposite face.Thus, the relative configuration of 6 was concluded to be (4R*,5R*,6S*,7R*,8S*,9S*). Based on the aforementioned results, compound 6 was identified as a previously undescribed natural product and was named lachnuoic acid F.
All the isolated compounds were assessed for their cytotoxic, antimicrobial, biofilm inhibitory and nematicidal activities.The results obtained (Tables S1 and S2) revealed that apart from lachnuoic acid A (1) featuring significant antimicrobial activities against Staphylococcus aureus (MIC = 16.6 μg/mL) and Bacillus subtilis (MIC = 8.3 μg/mL), none of the tested compounds unveiled potential activity.

Conclusions
The current study revealed several compounds that bear structural resemblance to a previously reported fungal metabolite ambuic acid (Figure 1), a cyclohexenone-based polyketide first reported from Pestalotiopsis and Monochaetia associated with rainforest plants and revealed potential activity against several phytopathogens of the genera Fusarium, Diplodia and Pythium. [3]In addition to the suggested phytoprotective role of ambuic acid, several related metabolites were reported such as strobiloscyphones, [4] pestallic acids [5] and pestalotic acids [6] that revealed potential antimicrobial or cytotoxic activities.Interestingly, these compound classes have hitherto only been obtained from members of the Sordariomycetes order Amphisphaeriales to which Monochaetia and Pestalotiopsis belong [9] and are the first metabolites of this type that were ever isolated from a species of Leotiomycetes.The Lachnum strain we studied was thus revealed to have a rather diverse secondary metabolism.12] However, the fact that the compounds reported here and in the preceding study [2] did not have any prominent antibiotic effects, while the total extract has shown very potent activities, suggests that we may have missed some unstable active principle/s during the isolation procedures.To exploit this will be the subject of future studies.

Fungal Material
A sample strain was collected off Sonneberg at location 50°21' 39'' N, 11°7' 33'' E, direction Meilschnitz, Thüringen (Germany) on an altitude of 359 m above the sea level.If was found saprotrophically growing on the reed grass Phragmites communis (Poaceae).The collection date was on 22 April 2022 by Ingo Wagner and a voucher strain was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) coded as IW157 (DSM 116717).The sequenced genes were deposited in the GenBank with the accession numbers, OR335757 and OR335766, for ITS and LSU single gene datasets, respectively.

Molecular Study
DNA of the fungus was extracted from the mycelia grown on yeast malt agar (YM agar; malt extract 10 g/L, yeast extract 4 g/L, Dglucose 4 g/L, agar 20 g/L, pH 6.3 before autoclave).Extracted using the fungal gDNA Miniprep Kit EZ-10 Spin Column protocol (NBS Biologicals, Cambridgeshire, UK).The amplification of the ITS and LSU were performed as previously described. [13]

Fermentation, Extraction and Isolation of Metabolites
A pre-culture was initiated by inoculating five well-grown 14-dayold Lachnum sp.(DSM 116717) pieces from a YM agar plate into a 500 mL Erlenmeyer flask containing 200 mL of ZM 1 = 2 medium (5 g/ L molasses, 5 g/L oatmeal, 1.5 g/L D-glucose, 4 g/L sucrose, 4 g/mL mannitol, 0.5 g/L edamin, 0.5 g/L ammonium sulfate, 1.5 g/L calcium carbonate, pH = 7.2).The pre-culture underwent a ten-day incubation period at 23 °C with a shaker rotating at 140 rpm.Employing an Ultra-Turrax (T25 easy clean digital, IKA) at 8000 rpm for 10 seconds, the pre-culture was homogenized.Subsequently, a 2-mL aliquot of the homogenized material was added to 200 mL of ZM 1 = 2 liquid media in a 500-mL Erlenmeyer flask and repeated over 25 flasks to afford 5 L total liquid culture volume.Daily glucose levels were monitored using Medi-Test glucose strips (Macherey-Nagel, Düren, Germany).Three days post-glucose depletion, cultures were extracted.Mycelia and supernatant were separated by vacuum filtration.The supernatant underwent ethyl acetate extraction (2×5 L), discarding the aqueous phase, and filtering the organic phase through anhydrous sodium sulfate.Mycelia were immersed in acetone (2×3 L) and sonicated at 40 °C for 30 minutes.Organic phases were evaporated under vacuum at 40 °C resulting in total mycelial extract of 2.61 g and total broth extract of 1.86 g.

Preparation of (R)-and (S)-MTPA Ester Derivatives of 1-3
A 0.5-mg aliquot was dissolved in 50 μL of pyridine-d 5 and 10 μL of (R)-(À )-methoxy-α-(trifluoromethyl)phenylacetyl (MTPA) chloride were added for the preparation of the (S)-MTPA ester.The reaction mixture was incubated at room temperature for 60 minutes and monitored by LC-MS.After the reaction was completed, the mixture was diluted to 200 μL using pyridine-d 5 for NMR measurements.Similarly, 10 μL of (S)-MTPA chloride was added to an equal aliquot (0.5 mg) of the compound to synthesize the (R)-MTPA ester.Following these reactions, 1 H, 1 H-1 H COSY and HSQC spectra were obtained.This procedure was done the same way for compounds 2 and 3.

Biofilm Inhibitory Assay
Biofilm inhibitory activity against Staphylococcus aureus (DSM 1104) of the isolated compounds were examined following a previously reported protocol. [14,16]In brief, S. aureus was pre-cultured, and the biofilm assay involved 24-hour growth with serially diluted compounds (125-1.9μg/mL).Biofilm inhibition was examined and evaluated by crystal violet staining, washing, and subsequent biomass analysis using a microtiter plate reader (Synergy 2, BioTek).Microporenic acid (MAA) [14] was used as a positive control while methanol served as a solvent control, considered 100 % with no inhibition against S. aureus biofilms.

Nematicidal Activity
Nematicidal activity was assessed using Caenorhabditis elegans.C. elegans was kept on a nematode growth media (NGM) that was prepared as previously described. [17]In brief, an aliquot suspension with a concentration of approximately 1,000 nematodes/mL was prepared.Pure compounds were tested against the nematodes at concentrations of 100, 50 and 10 μg/mL.The assay was carried out in a 48-well microtiter plate.The compounds dissolved in MeOH were added to the well plate and subsequently dried under nitrogen.Once the solvent was completely evaporated, 300 μL of the nematode suspension was added to each well.Each treatment was replicated three times.Ivermectin at 1 μg/mL was used as a positive control, and MeOH was used as the negative control.Nematodes were monitored 15 minutes after inoculation.The plates were incubated at 150 rpm and 24 °C for 18 h.After incubation, both alive and dead nematodes were counted in three replicates under a stereomicroscope, and the mortality rate was calculated.Erect and non-moving nematodes were considered dead.A compound is deemed active if the lethal dose at 100 μg/ml is at least 50 %.
) that revealed key correlations from H-5 to C-3 (δ C 143.8), C-4 (δ C 80.6), C-6, C-7 and C-9 whereas H-2 at δ H 5.95 (d, J = 15.6 Hz) and H-3 at δ H 6.87 (d, J = 15.6 Hz) exhibited key correlations to a terminal carboxylic acid carbon at δ C 167.0 (C-1), C-4 and C-9 confirming the binding of the trans configured α,β-unsaturated carbonyl moiety at C-4.The relative configuration of 4 was deduced by ROESY spectrum (Figure4) that revealed a key ROE correlation from H-5 and H-3 suggesting their cofacial projection.The absolute configuration of 4 was determined as (4S,5R) based on the comparison of coupling constant at H-5 to its peers in 1-3 (Table1) that assigned (R) configuration to C-5 and consequently (S) configuration to C-4.Based on the obtained results, compound 4 was identified as a previously undescribed cyclohexenone derivative and it was named lachnuoic acid D. Compound 5 was obtained as a white amorphous solid.HR-ESI-MS revealed a sodium adduct at m/z 207.0621 [M + Na] + (calculated 207.0628) determining its molecular formula as C 9 H 12 O 4 thus indicating four degrees of unsaturation.The 13 C NMR and HSQC spectra of 5 (Table

-6.
a at 175 MHz / b at 700 MHz / c at 125 MHz / d at 500 MHz.e Assignment confirmed by HMBC and HSQC spectra.* Values may switch within the same column.