Application of Feature-Based Molecular Networking for Comparative Metabolomics and Targeted Isolation of Stereoisomers from Algicolous Fungi

Abstract

Seaweed endophytic (algicolous) fungi are talented producers of bioactive natural products. We have previously isolated two strains of the endophytic fungus, Pyrenochaetopsis sp. FVE-001 and FVE-087, from the thalli of the brown alga Fucus vesiculosus. Initial chemical studies yielded four new decalinoylspirotetramic acid derivatives with antimelanoma activity, namely pyrenosetins A–C (1–3) from Pyrenochaetopsis sp. strain FVE-001, and pyrenosetin D (4) from strain FVE-087. In this study, we applied a comparative metabolomics study employing HRMS/MS based feature-based molecular networking (FB MN) on both Pyrenochaetopsis strains. A higher chemical capacity in production of decalin derivatives was observed in Pyrenochaetopsis sp. FVE-087. Notably, several decalins showed different retention times despite the same MS data and MS/MS fragmentation pattern with the previously isolated pyrenosetins, indicating they may be their stereoisomers. FB MN-based targeted isolation studies coupled with antimelanoma activity testing on the strain FVE-087 afforded two new stereoisomers, pyrenosetins E (5) and F (6). Extensive NMR spectroscopy including DFT computational studies, HR-ESIMS, and Mosher’s ester method were used in the structure elucidation of compounds 5 and 6. The 3′R,5′R stereochemistry determined for compound 6 was identical to that previously reported for pyrenosetin C (3), whose stereochemistry was revised as 3′S,5′R in this study. Pyrenosetin E (5) inhibited the growth of human malignant melanoma cells (A-375) with an IC50 value of 40.9 μM, while 6 was inactive. This study points out significant variations in the chemical repertoire of two closely related fungal strains and the versatility of FB MN in identification and targeted isolation of stereoisomers. It also confirms that the little-known fungal genus Pyrenochaetopsis is a prolific source of complex decalinoylspirotetramic acid derivatives.

Progress and challenges in exploring aquatic microbial communities using non-targeted metabolomics

“…Especially in the realm of supervised multivariate statistics, machine learning classification tools such as random forest and XG-Boost are commonly used [42,158]. At the same time, some of the above mentioned in silico spectrum annotation tools make use of deep neural networks to learning MS/MS fragmentation, compound class prediction, or de novo structure elucidation and are rapidly improving [110,114,159]. However, high confidence compound annotation through the prediction of MS/MS spectra from structure libraries is still an unsolved problem, and the field is still waiting for an "AlphaFold Moment" in mass spectrometry-based metabolomics.…”

“…FBMN can also make use of ion mobility separation information in addition to its algorithm to characterize chromatographic features and separate isomers with similar MS/MS spectra. The FBMN concept and workflow allows for improved annotation of mass spectra in combination with the ability for semi-quantitative or relative quantitative analysis, enhancing the user's downstream research and potential for new compound discovery and quantification from understudied organisms such as from aquatic fungi and aquatic macroalgae [110].…”

Marine natural products

“…244) and a strain of Pyrenochaetopsis yielded pyrenosetin C (for which the configuration was revised to 761 ) and related compounds 762 and 763 . 245 Talaverrucin A 764 , a heterodimeric oxaphenalenone with a unique 6/6/6/5/5/5/6 fused ring system, was isolated from an Antarctic sponge-derived strain of Talaromyces . 246 Other Talaromyces strains yielded a diverse range of metabolites including thioester-containing benzoate derivatives 765 – 770 , 247 sulfur containing spiro alkaloid 771 , unusual diacid 772 and alkaloids 773 – 777 , 248 aromatic polyketides 778 – 782 , 249 tripeptides 783 and 784 , containing an N-trans -cinnamoyl group, 250 chlorinated unsaturated alcohol 785 and coumarin derivative 786 , 251 decalin derivatives 787 and 788 , 252 meroterpenoid 789 (ref.…”

A review on the chemical ecology of the Fucaceae holobionts: from fundamental knowledge to applications

“…Chemical exploration using a bioactivity-driven molecular networking approach on the endophytic fungi Pyrenochaetopsis sp. from F. vesiculosus led to the identification of six new hybrid polyketides, thought to be synthesized by polyketide synthases/non-ribosomal peptide synthetases (PKS-NRPS) [193][194][195], Pyrenosetins A-F (61-66). They have antineoplastic and cytotoxic activities, except for Pyrenosetin E (65) which shows only cytotoxic activity, and Pyrenosetin F (66) bearing no biological activity on the assayed targets [193][194][195].…”