Pseudocercones A-C, three new polyketide derivatives from the endophytic fungus Pseudocercospora sp. TSS-1

Abstract Chemical investigation of an EtOAc extract of the endophytic fungus Pseudocercospora sp. TSS-1 led to the isolation of three new polyketide derivatives, including one benzophenon derivative (1), two spirocyclic polyketides (4 and 5), along with four known compounds (2, 3, 6 and 7). Their structures and the absolute configurations were characterized by means of NMR, HRESIMS, 13C NMR and theoretical electronic circular dichroism calculations. Furthermore, all compounds were evaluated for their antibacterial activity against four microbial pathogens (Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli), and compounds 1, 2, 3 and 5 displayed significant selective antibacterial activity against S. aureus with MIC values ranging from 3.9 to 7.8 µg/mL. Graphical Abstract


Introduction
Endophytic fungi are prolific producers of secondary metabolites with diverse structures and most of their metabolites have a wide range of biological activities (Tan and Zou 2001;Zhang et al. 2006;Nett et al. 2009;Berdy 2012;Liu and Liu 2018).Pseudocercospora, more than 400 species have been described worldwide, many of which are phytopathogenic fungi and can cause serious plant diseases (Kimunye et al. 2020).Generally, phytopathogenic fungi owned many virulence factors that were countered by plant defense mechanisms, and bioactive secondary metabolites produced by phytopathogenic fungi emerged to defend against the stress of ecological conditions (Kusari et al. 2012).However, to the best of our knowledge, the studies on secondary metabolites of Pseudocercospora remain very limited, and only two polyketide derivatives with antioxidant activity were reported so far (Prihantini and Tachibana 2017), which indicated that fungi of the genus Pseudocercospora was a promising reservoir for exploring structurally novel and bioactive natural products for drug discovery.
We are committed to discovering new secondary metabolites with diverse structures and bioactivities from endophytic fungi (Kang et al. 2021;Zhang et al. 2021;Zhang et al. 2021;Zhang et al. 2021;Zhang et al. 2021;Chen et al. 2022).In our previous research, it was found that the metabolites of a strain Pseudocercospora sp.TSS-1 was structurally diverse by TLC and HPLC-DAD analyses, and its EtOAc extract displayed certain inhibitory activity against Staphylococcus aureus with a MIC value of 0.5 mg/mL.To explore the antibacterial material basis of strain Pseudocercospora sp.TSS-1, we performed a further chemical investigation on the EtOAc extract to afford three new polyketide derivatives (1, 4 and 5), along with four known compounds (2, 3, 6 and 7).Herein, the isolation, structural elucidation, and antimicrobial activity against four microbial pathogens of all the isolates were described.
Subsequently, the structure of 1 was further confirmed by 13 C NMR calculation.The gauge-independent atomic orbital (GIAO) DFT 13 C NMR calculations (McWeeny 1962;Ditchfield 1972) towards 1 were performed at the xB97xD/6-31G Ã (Chai and Head-Gordon 2008) level, and the calculations data were then compared with their experimental 13 C NMR data following the reported sorted training set strategy (STS) protocol (Li et al. 2020).According to linear regression analysis of 13 C NMR chemical shifts, the values of the correlation coefficient (R 2 ) was 0.9963 for 1 (Figure S2).Moreover, the resulting P rel value (Table S2) of 1 is 100%, and the MAE, RMS, and P mean values of 1 further showed that the calculated 13 C NMR data matched well with the experimental one.Therefore, compound 1 was completely determined as shown in Figure 1, and identified as pseudocercone A.
Compound 4 was isolated as a pale yellow powder.Its molecular formula was determined as C 18 H 16 O 7 based on HRESIMS ion peak at m/z 345.0979 ing 11 degrees of unsaturation.Detailed comparison of the NMR data of 4 (Table S1) with the established structure (Samanthi et al. 2015) revealed that both compounds shared the same planar structure, and key HMBC correlation (Figure S1) proved the correctness of the structure.However, the absolute configuration of 4 has not been identified.Hence, we subsequently determined the absolute configuration by theoretical electronic circular dichroism (ECD) calculation (Figure S3), and the absolute configuration was determined to be 1S, Therefore, compound 4 was completely determined as shown in Figure 1, and identified as pseudocercone B, and it is absolute configuration was established for the first time.
Compound 5 was isolated as a pale yellow powder and had a molecular formula of C 18 H 16 O 7 with 11 degrees of unsaturation by the positive HRESIMS ion peak at m/z 367.0791 [M þ Na] þ (calcd for C 18 H 16 O 7 Na þ , 367.0788).Further analyses of NMR data (Table S1) for 5 demonstrated that 5 possessed a planar structure completely consistent with a reported Trypacidin (Lu et al. 2005;Song et al. 2021), and key HMBC correlation (Figure S1) can be observed, but the absolute configuration has not been determined, theoretical ECD calculation was applied to determine the absolute configuration of 5.As shown in Figure S4, the calculated ECD curve of 1S was well matched with the experimental one.Therefore, the absolute configuration of compound 5 was completely determined and reported for the first time, which was identified as pseudocercone C and shown in Figure 1.
Furthermore, the antibacterial activity of all compounds against four common clinical pathogens (S. aureus, E. faecalis, P. aeruginosa, and E. coli) was evaluated.As shown in Table S3, compounds 1, 2, 3, and 5 displayed significant selective antibacterial activity in S. aureus with MIC values ranging from 3.9 to 7.8 mg/mL.Compound 7 displayed moderate antibacterial activity against S. aureus with MIC values of 15.6 lg/ mL.Partial compounds also showed weak antibacterial activity against E. faecalis and E. coli.

Fungal material
The endophytic fungal strain of Pseudocercospora sp.TSS-1 was isolated from Lycopodiastrum casuarinoides, from huangsang Nature Reserve in Suining County, Shaoyang City, Hunan Province, was collected in July 2019.This endophytic strain was further identified through the sequence analysis of the rDNA ITS region, and the sequence of its rDNA ITS region had been submitted to GenBank (Accession: OP106990).This endophytic fungus is preserved in Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central South University.

Fermentation, extraction and isolation
The fungus was cultured in a solid medium, which was prepared by autoclaving each 500 mL conical flask with 150 g of rice and 200 mL of ultrapure water in it.The fermentation was performed in 80 flasks under static conditions at a constant temperature of 28 C for 4 weeks.The fungal culture was extracted with EtOAc, and the extract was subsequently decompression concentration to afford 85.0 g.

13 C NMR calculation and ECD calculation methods
The conformational search was performed by Confab (O'Boyle et al. 2011).Optimization and frequency calculation of each conformer was performed on the B3LYP/SVP level of theory by using Gaussian09 software package (Frisch et al. 2016).DFT GIAO 13 C NMR calculation was calculated on the xB97xD/6-31G Ã (IEFPCM, DMSO) level, and the data processing followed the reported STS protocol.The calculated shielding tensors of compound 1 were Boltzmann averaged based on Gibbs free energy.The theoretic ECD spectra were calculated on xB97xD/jun-cc-pvdz level of theory and were Boltzmann averaged according to Gibbs free energy.SpecDis v1.71 was used to simulate the ECD curve of compounds 4 and 5, with sigma/gamma value of 0.30 eV.(Bruhn et al. 2013).The averaged calculated ECD curves of compounds 4 and 5 were adjusted by red shifting and blue shifting for 10 nm, respectively.

Antibacterial assay
Antibacterial activity of all compounds against four common clinical pathogens (S. aureus ATCC29213, E. faecalis ATCC29212, P. eruginosa ATCC27853 and E. coli ATCC25922) were tested by using the broth macrodilution method according to Clinical and Laboratory Standards Institute (CLSI) recommendation on 96-well plates (Zhao et al. 2018).Vancomycin and Amikacin were used as positive control.The MIC was determined as the lowest concentration that inhibited visible growth of bacteria.