Natural Products Research Update

This article covers the latest developments in natural product research, primarily including ribosomally synthesized peptides, sesquiterpenes, diterpenes, and triterpenes.

1. Discovery of two novel terpenoid skeletons rapidly from Daphne altaica Pall. Using GNPS in combination with MolNetEnhancer and NAP annotation tools.

Recently, the team led by Professor Shaojiang Song at Shenyang Pharmaceutical University employed a combination of molecular networking techniques (GNPS), MolNetEnhancer, and NAP annotation tools to isolate two terpenoid compounds (1-2) with two distinct novel skeletons from Daphne altaica Pall. Compound 1 has a 9-oxy-tetracyclic [6.6.1.0^2,6.0^8,13] pentadecane skeleton, and compound 2 features a tetracyclic [5.3.0.1^2,5.2^4,11] tridecane skeleton. The authors deduced the biosynthetic pathways of these two new terpenoid compounds using known compounds wikstronone A and wikstronone C as biosynthetic precursors. Bioactivity tests revealed that compound 2 exhibited superior neuroprotective activity to the positive control (water-soluble vitamin E) at concentrations of 12.5 and 25 µmol/L, while compounds 1 and 2 both displayed acetylcholinesterase inhibition activity better than the positive control (donepezil). Molecular docking results suggested that the acetylcholinesterase activity of compounds 1 and 2 might arise from hydrogen bonds and π-π interactions.

2. Discovery of novel pentacyclic triterpenoid compounds by overexpressing eupC in mutant fungi.

The team from Peking Union Medical College overexpressed eupC in fungi (Phoma sp.) and identified two new tropolone-derived sesquiterpenes, phomaketals A (1) and B (2), along with a biosynthetically related secondary metabolite, pughiinin B (3), and the known compound noreupenifeldin B (4). They determined their structures using nuclear magnetic resonance (NMR), computational electronic circular dichroism (ECD), computational NMR, and copper target single crystal diffraction. Compounds 1 and 2 are likely derived through different reaction cascades from the presumed tropolone-derived sesquiterpene intermediates neosetophomone B (6) and 9-R-neosetophomone B (6′). Activity tests demonstrated that compound 1 exhibited anti-proliferative activity against SUPB15 cells with an IC50 value of 4.85 µmol/L, while compound 4 displayed strong inhibitory activity against three types of tumor cells (SUPB15, EL4, H9) with IC50 values ranging from 0.36 to 27.08 µmol/L.

3. Discovery of 14 Verticillane-Type Diterpenoids with Anti-Inflammatory and Hepatoprotective Activities from Soft Coral.

A research team from China Ocean University has discovered 14 new verticillane-type diterpenoid compounds from the soft coral Heteroxenia ghardaqensis. These compounds all feature a rare 6/12 bicyclic carbon skeleton, which is uncommon in marine organisms. The authors determined their planar structures and absolute configurations through extensive spectroscopic analysis, single-crystal X-ray diffraction analysis, computational electronic circular dichroism (ECD) spectra, and computational NMR. Notably, the authors obtained single-crystal structures for seven compounds through crystal cultivation, which greatly contributed to the identification of the absolute configurations, particularly for the 12-membered macrocyclic structures. In zebrafish experiments, compounds 5, 8, and 9 exhibited some anti-inflammatory activity, while compounds 2, 8, and 12 showed hepatoprotective activity.

4. Discovery of Novel Shikimic Acid Trimer with Anti-Colorectal Cancer Activity from Endophytic Fungi in Coptis Chinensis.

Natural products derived from fungi are crucial sources of lead compounds for drug development, making significant contributions to human health. Examples include penicillin, an antibiotic derived from Penicillium sp., and cyclosporin A, an immunosuppressant from Fusarium sp. Professor Hu Changhua’s team at the School of Pharmacy, Southwest University, is dedicated to discovering structurally unique and highly active lead compounds from traditional Chinese medicine and microorganisms. Through solid-state fermentation of rice and non-repetitive separation analysis using HPLC/HRESIMS, they discovered a previously unreported molecule. Subsequent isolation and identification revealed it to be Citrinsorbicillin A (1), a shikimic acid trimer with a novel highly oxidized carbon skeleton containing a core structure formed by the linkage of two bicyclo[2.2.2]octanedione rings via an enol connection, and it possesses ten chiral centers. Compound 1 is the first shikimic acid trimer compound discovered from a terrestrial fungus.

Additionally, the research uncovered two shikimic acid monomers, Citrinsorbicillin B (2) and Citrinsorbicillin C (3). The team determined the planar structures of compounds 1-3 through methods like 1D/2D NMR and HRESIMS and established the absolute configuration of the novel skeleton compound 1 using X-Ray single-crystal diffraction. Activity studies indicated that compound 1 exhibits significant inhibition of colorectal cancer cells (HT-29), with preliminary mechanistic investigations revealing its anti-colorectal cancer activity by blocking the G0/G1 phase of the HT-29 cell cycle. Furthermore, the researchers deduced the biosynthetic pathway of compound 1, which is derived from the shikimic acid monomers through two [4+2] cycloadditions. These findings provide a new molecular template for chemists and pharmacologists to design and discover novel anticancer drugs.

5. Structural Revision of Nine Triterpenoid Compounds Using NAPROC-13 and Computational NMR.

It is estimated that approximately 3-9% of natural product structures are incorrectly elucidated. Therefore, the accurate determination and correction of natural product structures have become especially important. Recently, a research team from Panama employed a combination of NAPROC-13 and computational nuclear magnetic resonance (NMR) to revise the structures of nine triterpenoid compounds, which had previously been assigned the same carbon spectra in prior reports. Furthermore, the computational NMR spectra indicated that some chemical shifts needed reassignment.

By improving the fit between experimental and computational data, the authors corrected erroneous structures and correctly assigned each signal, which is crucial for elucidating compounds related to new structures. Additionally, the structural uncertainty of several compounds found in the literature and the Sci-Finder database was eliminated. By conducting chemical shift searches in NAPROC-13, incorrectly labeled structures were identified, and identical 13C NMR data revealed two or more different compounds. Through density functional theory (DFT) calculations, the structures with the highest probability and the best agreement with experimental 13C NMR data were determined.

6. Discovery of Cage-like [6,6,6,6,6] Novel Polyketides from Lichen-Endophytic Fungus.

A South Korean research team has discovered three unprecedented cage-like oxygenated anthraquinone [6,6,6,6,6] polyketides, known as Daldipyrenones A-C (1-3), from lichen-endophytic fungus Daldinia pyrenaica 047188. These compounds feature a spiro-azaphilone unit. The structures of 1-3 were determined through spectral analysis and chemical derivatization reactions. It is speculated that these compounds are formed from the chromane biosynthetic intermediate 1-(2,6-dihydroxyphenyl)but-2-en-2-one and a spiro-azaphilone known as pestafolide A, which combine to form a bicyclic [2.2.2]octane ring through radical coupling or Michael addition reactions. Genomic sequencing of the strain revealed two independent biosynthetic gene clusters responsible for the formation of these biosynthetic intermediates, further confirming the validity of the proposed biosynthetic pathway. Biological activity experiments showed that compound 1 significantly inhibits melanin production, with an EC50 value lower than the positive control. Additionally, compound 1 also exhibits moderate activity in promoting adiponectin secretion.

7. Discovery of Fungal Ribosomally Synthesized and Post-translationally Modified Peptides with Potential for Treating Parkinson’s Disease.

Peptides are a unique class of compounds that have been found to possess characteristics of both small-molecule drugs and large-molecule biologics. Recently, the research team led by Tan Renxiang and Hu Gang at Nanjing University of Chinese Medicine discovered a disulfide-bonded peptide compound called acalitide from the endophytic fungus Acaulium album of Cordyceps militaris. Using metabolomics and mass spectrometry analysis, they determined the structure of this compound through NMR and single-crystal diffraction. Acalitide consists of a 30-amino acid macrocyclic peptide with a three-ring scaffold, featuring two pairs of disulfide bonds formed by four cysteine residues.

Researchers used genomic sequencing and protein sequence analysis to propose the biosynthetic gene cluster of acalitide. They confirmed the biosynthetic pathway and cyclization method through heterologous expression in Aspergillus oryzae and point mutation experiments. In vitro activity studies revealed that acalitide possesses significant neuroprotective activity. Subsequent investigations, including brain-targeted lipid nanoparticle formulation and in vivo models in nematodes and mice, further validated the safety and potential for treating Parkinson’s disease with acalitide.

This research expands the category of fungal Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs), delves into the complexity of fungal RiPP biosynthesis, and offers a valuable lead compound for potential Parkinson’s disease treatment. It also serves as an example for characterizing low-abundance but high-value compounds found in nature.

References

1. W, Y, Zhou.; et al. Rapid Discovery of Two Unprecedented Meroterpenoids from Daphne altaica Pall. Using Molecular Networking Integrated with MolNetEnhancer and Network Annotation Propagation. Chinese Chemical Letters. 2023, 2: 109030.

2. C, Li.; et al. Phomaketals A and B, Pentacyclic Meroterpenoids from a eupC Overexpressed Mutant Strain of Phoma sp.. Chinese Chemical Letters. 2023, 2: 109019.

3. X, Han.; et al. Heterolactone and Heterolactams A-M, Verticillane Diterpenoids with Anti-Inflammatory and Hepatoprotective Activities from the Soft Coral Heteroxenia ghardaqensis. Journal of Natural Products. 2023, 86(9): 2131-2138.

4. G, P, Yin.; et al. Citrinsorbicillin A, a Novel Homotrimeric Sorbicillinoid Isolated by LC-MS-guided with Cytotoxic Activity from the fungus Trichoderma Citrinoviride HT-9. Chinese Chemical Letters. 2023, 3: 109035.

5. H, A. Sánchez-Martínez.; et al. Synergistic Combination of NAPROC-13 and NMR 13C DFT Calculations: A Powerful Approach for Revising the Structure of Natural Products. Journal of Natural Products. 2023.

6. S, J, Lee.; et al. Daldipyrenones A-C: Caged [6,6,6,6,6] Polyketides Derived from an Endolichenic Fungus Daldinia pyrenaica 047188. Organic Letters. 2023, 25(36): 6725-6729.

7. Z, W, Tong.; et al. Disulfide Bridge-targeted Metabolome Mining Unravels an Antiparkinsonian Peptide. Acta Pharmaceutica Sinica B. 2023, 19.