Eutypellaolides A–J, Sesquiterpene diversity expansion of the polar fungus Eutypella sp. D-1

Eight new 12,8-eudesmanolide sesquiterpenes, eutypellaolides A–H (1–8), and two new eudesmane-type sesquiterpenes, eutypellaolides I–J (9–10), along with four known 12,8-eudesmanolide compounds 11–14, were isolated from the culture extract of the polar fungus Eutypella sp. D-1 by one strain many compounds (OSMAC) approach. The structures of these compounds were determined through comprehensive spectroscopic data and experimental and calculated ECD analysis. Antibacterial, immunosuppressive, and PTP1B inhibition activities of these compounds were evaluated. Compounds 1 and 11 exhibited strong inhibitory activities against Bacillus subtilis and Staphylococcus aureus, with each showing an MIC value of 2 μg/mL. Compound 9 displayed weak immunosuppressive activity against ConA-induced T-cell proliferation with an inhibitory rate of 61.7% at a concentration of 19.8 μM. Compounds 5, 11, and 14 exhibited weak PTP1B inhibition activities with IC50 values of 44.8, 43.2, and 49.5 μM, respectively.


Introduction
The polar regions are renowned for their harsh environmental conditions, characterized by extremely low temperatures, hurricanes, and intense ultraviolet radiation.These extreme conditions contribute to the development of unique physiological adaptations in microorganisms that inhabit the polar regions, leading to a diversity of microbial secondary metabolites (Santiago et al., 2015).Due to the exceptional conditions and abundant microbiological resources, polar regions have been an increasing interest in human activities and scientific research (Lu et al., 2014;Tian et al., 2017).However, compared with the vast number of natural products reported in tropical regions, compounds from the polar regions have been relatively limited, with only over one hundred new compounds being reported in recent years (Dos Santos et al., 2021).Consequently, polar microbiology has gained recognition as a crucial source of bioactive natural products.
The fungi of Eutypella genus have been extensively investigated for decades due to the respected biological and pharmacological activities of their secondary metabolites Ning et al. 10.3389/fmicb.2024.1349151Frontiers in Microbiology 02 frontiersin.org(Pongcharoen et al., 2006;Ning et al., 2023).The polar fungus Eutypella sp.D-1 has been discovered to have the ability to produce a variety of structurally distinct and biologically active secondary metabolites, such as cytochalasins, pimarane diterpenes, cytosporins, and sesquiterpenes, with significant antimicrobial and cytotoxic activities (Liu et al., 2014;Zhou et al., 2017;Wang et al., 2018;Yu et al., 2018a,b;Zhang et al., 2019).Previous research on Eutypella sp.D-1 has discovered one sesquiterpene, eut-Guaiane (11), with significant antibacterial activity (Zhou et al., 2017).The OSMAC (One Strain Many Compounds) approach has been extensively employed for the identification of novel metabolites from microorganisms.To enhance the sesquiterpene chemical diversity of Eutypella sp.D-1, drawing inspiration from the OSMAC strategy, we utilized different culture conditions.Subsequently, the EtOAc extracts of the different fermentation broths were subjected to HPLC analysis, and a large number of sesquiterpene analogs were found in solid defined medium compared with PDB medium (Supplementary Figure S110).A follow-up chemical investigation led to the isolation of ten new sesquiterpenes, eutypellaolides A-J (1-10), and four known related compounds 11-14 (Figure 1).Herein, we present the isolation, structure elucidation, and bioactive evaluation of these compounds.
2 Materials and methods

Fungal material
The strain Eutypella sp.D-1 was collected near the Ny-Ålesund District in the London Island of Kongsfjorden of the Arctic.It was purified at 20°C by using potato dextrose agar (PDA) medium and identified as Eutypella sp.through 18S rDNA gene sequence analysis (GenBank Accession number FJ430580).At present, the strain of Eutypella sp.D-1 was deposited at the Naval Medical University, Shanghai, China.
After subjecting the solid defined medium to 1 hour of ultrasonic treatment with a mixture of CH 2 Cl 2 /CH 3 OH (v/v, 1:1), it was subsequently extracted three times using the same mixture.The CH 2 Cl 2 /CH 3 OH solution was evaporated under reduced pressure to obtain an aqueous solution and then extracted with EtOAc three times under reduced pressure at 40°C to yield a dark brown gum (6.86 g).
Eutypellaolide D (4) was obtained as a yellowish oil, with a molecular formula of C 15 H 22 O 4 as determined by HRESIMS (m/z 289.1401 [M + Na] + ).Comparison of the 1 H and 13 C NMR spectra of 4 with those of 4β-hydroxy-5α,8β(H)-eudesm-7(11)-en-8,12olide (Zhang et al., 2012), with a difference in the presence of a hydroxyl group substituted at C-13 (δ C 54.7) in 4, was supported by the HMBC correlations from  (Wang et al., 2017).The agreement between the calculated ECD spectrum and the experimental CD spectrum also supported the absolute configuration of 4 (Figure 4).Eutypellaolide E (5) was isolated as a yellowish oil and had a molecular formula of C 16 H 20 O 5 based on HRESIMS (m/z 315.1204 [M + Na] + ).The NMR data of 5 closely resembled those of 12 (Wang et al., 2017), except for the presence of a hydroxy group at C-3 (δ C 75.2) and methoxy group at C-4 (δ C 80.1) in 5 and the absence of two olefinic carbons (δ C 131.2 and δ C 131.4) in 12.The C-3 was attached to C-16 (δ C 49.4) via the C-4, which was confirmed by the HMBC correlation from H 3 -16 (δ H , 3.13, s) to C-4 (δ C 80.1).The NOESY correlations from H 3 -14 to H-1α and from H-3 to H-1α and H 3 -15 suggested that H-3/ H 3 -14/H 3 -15 was located at the same orientation.Furthermore, a comparison between the calculated and the experimental ECD spectra confirmed the absolute configurations of 5 as 3S,4R,10S (Figure 4).
Eutypellaolide G (7) was obtained as a yellowish oil, and its molecular formula was deduced as C 15 H 16 O 4 due to its HRESIMS (m/z 283.0947 [M + Na] + ), indicating eight degrees of unsaturation.
Notably, the NMR data of 7 closely resembled that of chlorantholide A (Wang et al., 2012), except for the discernible hydroxy group at C-13 in 7, which was verified by the HRESIMS data and the HMBC correlations from H 2 -13 (δ H 4.56) to C-7 (δ C 148.3), C-11 (δ C 124.0), and C-12 (δ C 169.6).The NOESY correlations from H-6α (δ H 2.64) to H 3 -14 and H-6β (δ H 3.37) to H-5 confirmed that H 3 -14 and H-5 were in opposite orientations.The absolute configuration of 7 was determined by comparing its specific rotation with chlorantholide A. A comparison of the specific rotation between  (Wang et al., 2012).
The similarity between the calculated ECD spectrum and the experiment further supported the absolute configuration of 8 (Figure 5).et al., 1995), following the method as the cases of compounds 1, 3, 4, 7, and 8.
Eutypellaolide J (10) was a colorless oil and exhibited a planar structure identical to that of thomimarine E (Afiyatullov et al., 2017).However, the chemical shift of C-11 (δ C 41.3)  (δ H 0.93), indicating that these protons were on the same side.

Conclusion
In summary, the OSMAC approach effectively induced chemical diversities of the polar fungus Eutypella sp.D-1, using a modified solid nutrient medium, to produce fourteen sesquiterpene compounds.Among them, there were ten new sesquiterpenes eutypellaolides A − J (1-10) and four known 12,8-eudesmanolide compounds 11-14.Fortunately, the production of compound 11, which exhibits excellent antibacterial activity, increased sharply.Interestingly, these new metabolites were only detected in the solid nutrient medium and were not produced when the fungus was cultivated in potato dextrose broth (PDB) or other liquid media (Lu et al., 2014;Zhou et al., 2017;Wang et al., 2018).Therefore, it could be concluded that the OSMAC approach should be a feasible and effective strategy to trigger the production of bioactive secondary metabolites from the polar fungi.Compounds 5, 11, and 14 possess an α,β-unsaturated γ-lactone structure, which serves as a crucial pharmacophore for significant PTP1B inhibitory activity, and this characteristic was shared with sesterterpene phyllofolactones A and phyllofolactones F, as reported in the literature (Abdjul et al., 2015).The findings from this study contribute to the expanding knowledge of natural products from polar fungi and their potential for discovery as new drug leads.& editing.