Cycloaspeptide H, a cyclopentapeptide from the endophytic fungus Penicillium virgatum

Abstract One new cyclopentapeptide, cycloaspeptide H (1), featuring a serine residue, along with seven known compounds (2–8), was isolated from the endophytic fungus Penicillium virgatum GDGJ-227. The planar structure of 1 was elucidated by a comprehensive analysis of NMR and MS spectroscopic spectra, and the absolute configuration was determined by single-crystal X-ray diffraction (Cu Kα) analysis. Compounds 7 and 8 displayed antibacterial activities against Bacillus subtilis and Enterobacter aerogenes with MIC values ranging from 12.5 to 50 μg/mL. Graphical Abstract


Introduction
Fungi are a promising source of beneficial drug leads due to the peculiar structural features and significant biological activities (Guo et al. 2015).Among them, the genus Penicillium is well-known to produce a variety of bioactive and structure novel compounds including the clinically used antibiotic penicillin and the antifungal agent griseofulvin (Ali et al. 2017;Orfali et al. 2020).Cyclic peptides isolated from natural resources show a vast range of biological activities, such as antifungal (Ekanayakeet al. 2021), antibacterial (Ma et al. 2016), cytotoxic (Liu et al. 2020), and antiviral activities (Lu et al. 2012).Given their special structural or conformational nature and ability to accommodate various amino acids, the cyclic peptides are good drug candidates and tools to study biochemical interactions (Bajaj 2019;Apostolopoulos et al. 2021).
Sophora tonkinensis is a traditional Chinese medicinal plant, which is used to treat throat inflammation and fever (Wei et al. 2020;Qin et al. 2022).As part of our continuing search for novel and bioactive metabolites from associated endophytic fungi (Xu et al. 2017;Zheng et al. 2018;Liang et al. 2019;Mo et al. 2021;Qin et al. 2022), the fungus Penicillium virgatum GDGJ-227 attracted our attention.Chemical investigation of the metabolites led to the isolation and structure elucidation of a new cyclopentapeptide, cycloaspeptide H (1), and seven known metabolites (2-8).Herein, the isolation, structural elucidation, and antibacterial activities were described.

Results and discussion
Compound 1 was obtained as a white amorphous powder, and its molecular formula was determined as C 36 H 43 N 5 O 7 on the basis of the HRESIMS at m/z 658.3234, (calcd for 658.3235, [M þ H] þ ), indicating eighteen degrees of unsaturation.Detailed analysis of the 1 H, 13 C, and multiplicity-edited heteronuclear single quantum coherence spectroscopy (HSQC) NMR spectra (Table S1) showed characteristics of a typical peptide, including three amide (-NH) proton signals at d H 11.94, 8.80, and 8.26, four a-amino protons at d H 5. 31, 4.78, 4.52, and 4.15, two N-methyl groups at d H 2.64, and 2.59, and three mono-substituted/di-substituted benzenes at d H 8. 74, 8.02, 7.51, 7.07, 7.28, 7.26, 6.93, and 6.69.In addition, five amide type carbonyls at d C 170.7, 169.5, 169.2, 168.6, and 168.2, and eighteen sp 2 hybridised carbons at d C 115.3-156.0,four methine carbons at d C 68.3, 62.5, 51.5, and 47.8, one oxygen-bearing methylene carbon at d C 59.9 were observed.The above NMR features accounted for 17 degrees of unsaturation.Further analysis of 1 D and 2 D NMR spectrum allowed five subunits to be established, including an anthranilic acid (ABA), a serine (Ser), an N-Me-phenylalanine (N-Me-Phe), a leucine (Leu), and an N-Me-tyrosine (N-Me-Tyr) residues, indicating that the structure of 1 was similar to that of cycloaspeptide A (Kobayashi et al. 1987).
The complete amino acid sequence of 1 was assigned by extensive analysis of inter-residue HMBC correlations.The HMBC correlations (Figure S1 were observed.All these data confirmed the cyclic structure of 1.Thus, the planar structure of 1 was established as shown in Figure 1.Furthermore, the absolute configuration of 1 was unambiguously determined as 2S, 12S, 18S, and 28S by single-crystal X-ray (Cu Ka) analysis (Figure S3), with Flack's parameter of 0.029 (13).
Up to now, only 10 natural cyclopentapeptides comprising ABA residue motif have been reported, including cycloaspeptides A-G (Kobayashi et al. 1987;Dalsgaard et al. 2004;Zhang et al. 2009) and avellanins A-C ( Yamazaki et al. 1987;Igarashi et al. 2014).Cycloaspeptides are structurally characterised by containing anthranilic acid residue (position p1), aromatic amino acid residues (positions p3 and p5), and leucine or alanine residue (position p 4) (de Mattos-Shipley et al. 2018).All reported cycloaspeptides possess fatty amino acids, alanine, valine or leucine residues, in positions p2 and p4.To the best of our knowledge, 1 is the first cycloaspeptide contained a nonaromatic hydrophilic amino acid serine residue.The simplest and minimal modification of a single amino acid or peptide bonds is represented by N-methylation, which can improve the pharmacokinetic properties of biologically active peptides as well as resulting in analogues (Di Gioia et al. 2016).Interestingly, compound 1 possess two Nmethylation amino acid residues.

General experimental procedures
The NMR spectral data were recorded on Bruker AV 400 or 600 spectrometers (400 MHz for 1 H NMR and 100 MHz for 13 C NMR; 600 MHz for 1 H NMR and 150 MHz for 13 C NMR).Optical rotations were measured on a Bellingham-Stanley ADP 440þ polarimeter at 20 C. The HRESI-MS data were measured on a Micro Mass Q-TOF spectrometer (Waters Corporation, Milford, MA, USA).Column chromatography was performed using silica gel Qingdao Haiyang Chemical Co. Ltd.,China), ODS (50 lm, YMC, Japan) and Sephadex LH-20 (GE) were used for column chromatography.Semi-preparative high performance liquid chromatography (HPLC) was performed on a Shimadzu LC-20A system (Shimadzu, Japan) using an ODS column (250 Â 10 mm, 5 lm, 2.0 mL/min, YMC).

Fungal material
The strain GDGJ-227 was isolated from the roots of Sophorae tonkinensis, which was collected from Baise city, Guangxi province, People's Republic of China in 2017.It was further identified as Penicillium virgatum based on morphological characteristics and molecular analyses of ITS, and the sequence of its rDNA ITS region had been submitted to GenBank (the GenBank accession number ON891942).The strain is preserved in the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University.

Antibacterial assay
The antibacterial activities against Gram-positive bacteria, B. anthracis, B. megaterium, B. subtilis, B. paratyphosus B, and S. aureus, and Gram-negative bacteria E. coli and S. dysenteriae were evaluated by using the broth micro-dilution method in 96-well plates (Wiegand et al. 2008;Hoque et al. 2016;Qin et al. 2022).The bacteria were grown in liquid LB medium (37 Cfor 18-24 h), and the diluted bacterial suspension (10 6 CFU per milliliter) were used for assay.The respective compounds were dissolved in DMSO and diluted to the following final concentrations: 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 lg/ mL.Ciprofloxacin was used as a positive control.Finally, the plates were incubated at 37 C for 24 h.The MIC was determined as the lowest concentration at which no growth was observed.All MICs were determined in triplicates.

Conclusions
In summary, six cyclopentapeptides (1-6), containing an anthranilic acid residue, together with two coumarin derivatives (7 and 8), were obtained from the endophytic fungus P. virgatum GDGJ-227.Compound 1 was a new cycloaspeptide containing a nonaromatic hydrophilic amino acid serine and two N-methylation amino acid residues.Compounds 7 and 8 exhibited moderate antibacterial activities.

Disclosure statement
No potential conflict of interest was reported by the authors.
) from ABA-NH (d H 11.94) to N-Me-Tyr-CO (d C 168.6), N-Me-Tyr-H 3 (d H 2.59) and Leu-a-H (d H 4.44) to Leu-CO (d C 169.2), Leu-NH (d H 8.26) and N-Me-Phe-a-H (d H 5.31) to N-Me-Phe-CO (d C 169.5), N-Me-Phe-H 3 (d H 2.64) to Ser-CO (d C 170.7), and Ser-a-H (d H 4.52) and ABA-b-H (d H 8.02) to ABA-CO (d C 168.2) allowed the definition of the residue sequence of 1 as cyclo-(N-Me-Tyr!Leu!N-Me-Phe!Ser!ABA).Moreover, the complete amino acid sequence of 1 was also confirmed by the ESI MS 2 experiments (Figure S2).The key fragment ions at m/z 481.2434 [