A glyoxylate-containing benzene derivative and butenolides from a marine algicolous fungus Aspergillus sp. SCSIO 41304

Abstract A new glyoxylate-containing benzene derivative, methyl 2-(4-hydroxy-3-(3′-methyl-2′-butenyl)phenyl)-2-oxoacetate (1), together with ten known compounds (2–11), were isolated from the marine algicolous fungus, Aspergillus sp. SCSIO 41304. Their planar structures and absolute configurations were elucidated by detailed NMR, MS spectroscopic analysis and comparing with literature data. Compound 1 was isolated as a new fungal secondary metabolite, possessing a methyl glyoxylate moiety R-CO-CO-OCH3, which is rare in natural sources. All the isolated compounds (1–11) were tested for their antibacterial and enzyme inhibitory activities against acetylcholinesterase (AChE) and pancreatic lipase (PL). Among these compounds, aspulvinone H (4) showed moderate inhibition against AChE and PL with IC50 values of 25.95 and 47.06 μM, respectively. Further molecular docking simulation exhibited that compound 4 could well bind to the catalytic pockets of the AChE and PL. Graphical Abstract


Introduction
Marine algicolous endophytic fungi parasitize on the surface and inside of alga. Up to now, over 300 new metabolites have been found and identified from marine algicolous fungi (Ji and Wang 2016). The fungi of genus Aspergillus from alga, have been proven to be one of the richest sources of biologically active secondary metabolites with pharmaceutical potential (Aparicio-Cuevas et al. 2017). Previous investigations on the chemical constitutes of the genus Aspergillus have led to the isolation of polyketides (Chen et al. 2014;Guo et al. 2016), alkaloids (Kong et al. 2019;Yang et al. 2021), terpenoids (Qi et al. 2016), coumarins (Shimada et al. 2004), and aromatic derivatives . A great deal of attention has been attracted by these metabolites because of their wide range of biological activities, including antimicrobial , antioxidant (Chen et al. 2020), antiviral , cytotoxic (Wang et al. 2017), and enzyme inhibitory activities (Cai et al. 2019).
In our current research for novel bioactive compounds from marine algicolous fungi, the fungal strain Aspergillus sp. SCSIO 41304 isolated from the brown alga Sargassum sp. collected at Yongxing Island (Xisha Islands, China). Chemical research on the fermentation products of the strain resulted in a new glyoxylate-containing benzene derivative (1), together with aromatic derivatives, butenolide derivatives, and cyclopentane derivatives. Herein, we reported the isolation, structure elucidation, and biological activities of these compounds.

Results and discussion
Compound 1 was obtained as yellow oil. Its molecular formula C 14 H 16 O 4 (seven unsaturations) was determined by a quasimolecular ion peak at m/z 247.0978 (calcd for C 14 H 15 O 4 -, 247.0976) in the HRESIMS spectrum. The 1 H and 13 C NMR data measured in chloroform ( ]. Based on the above 1 D NMR data, there may be a benzene ring and an olefin in compound 1. The HMBC correlations between H-5 and C-1/C-3, and between H-6 and C-2/C-4, suggested the presence of a 1,2,4-trisubstituted benzene ring. In addition, the 1 H-1 H COSY correlation between H 2 -1 0 and H-2 0 , the HMBC correlations between H 3 -4 0 and C-2 0 /C-3 0 /C-5 0 , and between H 2 -1 0 and C-3, implied that an isopentenyl group was attached to C-3. Moreover, the methyl ester group was identified based on HMBC correlation from H 3 -4 00 to C-2 00 . Based on the above findings, the structure of 1 was found to be similar to the reported methyl 4-hydroxy-3-(3 0 -methyl-2 0 -butenyl) benzoate (Rogelio et al. 1997), and the difference between them was an additional carbonyl group (d C 184.9), according to its HRESIMS data. The carbonyl group was located between the C-1 and C-2 00 , confirmed by the HMBC correlations between H-2 and C-1 00 , and between H-6 and C-1 00 . Hence, the planar structure of compound 1 was determined as methyl 2-(4-hydroxy-3-(3 0 -methyl-2 0 -butenyl)phenyl)-2-oxoacetate. Compound 1 was isolated as a new fungal secondary metabolite, possessing a methyl glyoxylate moiety R-CO-CO-OCH 3 , which is rare in natural sources.
All isolated compounds were assessed for their enzyme inhibitory activities against AChE and PL. Only compound 4 showed moderate inhibition against AChE and PL with IC 50 values of 25.95 ± 1.21 and 47.06 ± 1.75 lM, respectively. Subsequently, molecular docking research was conducted to investigate the binding modes between compound 4 and the two enzymes, respectively. As shown in Figure S2, compound 4 could tightly bind inside of the active site of AChE with the calculated binding affinity of À12.1 kcal/mol. Detailed analysis of molecular docking results showed that 4 could form strong interactions with AChE via multiple hydrophobic interactions and four hydrogen bonds with Arg-289, Tyr-121, Ser-122, and Trp-84 with the lengths of 3.1, 3.0, 3.1, and 3.8 Å, respectively. In addition, one of the benzene rings of 4 could generate p-p stacking interaction with Trp-279. The interaction modes characterized by multiple hydrophobic interactions, six hydrogen bonds, and two p-p stackings between 4 and PL were similar to that of AChE (binding affinity, À10.59 kcal/mol). Moreover, a salt bridge interaction between 4 and His-264 was observed in the molecular docking results ( Figure S3). The above docking results well supported the enzyme inhibition of 4 against AChE and PL.

General experimental procedures
NMR spectra were recorded on a Bruker AC 500 and 700 NMR (Broker, Fallanden, Switzerland) spectrometer with TMS as internal standard. High resolution mass spectra (HR-ESI-MS) were measured on a Bruker micro TOF-QII mass spectrometer (Bruker). Optical rotations were tested using Anton Par MCP-500 polarimeter (Hertford, UK). UV spectra were recorded on a Shimadzu UV-2600 PC spectrometer. TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10 À 40 lm) and over silica gel (200 À 300 mesh) (Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Biosciences), respectively. Spots were detected on TLC (Qingdao Marine Chemical Factory) under 254 nm UV light. All solvents employed were of analytical grade (Tianjin Damao Chemical and Industry Factory). Semi-preparative HPLC was performed by using an Agilent system with a YMC-Pack ODS-A column (YMC-pack ODS-A, YMC Co. Ltd., 250 Â 10 mml, 5 lm, 3.0 mL/min). The artificial sea salt was a commercial product (Guangzhou Guangyan Technology Company).

Fungal strain
The Aspergillus sp. SCSIO 41304 was isolated from the brown alga Sargassum sp. collected at Yongxing Island (Xisha Islands, China). This strain was stored on MB agar slants at 4 C and then deposited at the Marine Microbial Collection Center of CAS Key Laboratory of Tropical Marine Bio-resources and Ecology. The ITS sequence region of the strain SCSIO 41304 was amplified by PCR, and rDNA sequencing showed that it shared significant homology to several species of Aspergillus. The rDNA sequence has 99.82% sequence identity to that of Aspergillus terreus (GenBank accession no. NR_131276), so it was designated as Aspergillus sp. and named as Aspergillus sp. SCSIO 41304.

Molecular docking simulation
The crystal structures of AChE and PL (PDB ID: 6G1W and 1ETH) were retrieved from the Protein Data Bank (http://www.rcsb.org/pdb) for molecular docking research, which conducted by AutoDock (version 1.5.6). The structure of 4 was generated in ChemBio Office (version 17.0), followed by an MM2 calculation to minimize the conformation energy. The original ligand and crystal water were removed before the docking calculation. The hydrogens were added to the structures of AChE and PL, and Kollman united partial charges were assigned. A Lamarckian genetic algorithm was applied as a default search algorithm and allowed full flexibility of the active pockets of the ligand structure within the grid boxes size of 54 Å Â 56 Å Â 54 Å and 106 Å Â 108 Å Â 100 Å, with the spacing of 0.375 Å. During the docking, the default parameters were used if it was not mentioned. The docking pose that had the lowest binding energy was represented as the most favorable binding conformation.

Conclusion
A new glyoxylate-containing benzene derivative, methyl 2-(4-hydroxy-3-(3 0 -methyl-2 0butenyl)phenyl)-2-oxoacetate (1), together with ten known compounds (2-11), were isolated from the marine algicolous fungus, Aspergillus sp. SCSIO 41304. Their planar structures and absolute configurations were elucidated by detailed spectroscopic analysis and comparing with literature data. Compound 1 was isolated as a new fungal secondary metabolite, possessing a methyl glyoxylate moiety R-CO-CO-OCH 3 , which is rare in natural sources. All the isolated compounds (1-11) were tested for their antibacterial and enzyme inhibitory activities against AChE and PL. Among these compounds, compound 4 showed moderate inhibition against AChE and PL with IC 50 values of 25.95 and 47.06 lM, respectively. The simulation of the molecular docking also confirmed that compound 4 had corresponding inhibitory effects on AChE and PL. Since these isolated compounds showed no significant antibacterial activity, further investigation on the bioactivity of these compounds need to be undertaken in the future. This work not only enriched secondary metabolites in the genus Aspergillus, but also revealed that butenolide derivatives showed significant enzyme inhibitory activities (Figure 1).