**1. Introduction**

Marine-derived fungi can thrive in the extreme environments including salinity, high pressure, low temperature and oligotrophic conditions compared to their terrestrial counterparts, which makes them able to produce structurally diverse bioactive compounds more easily [1–3]. Meanwhile, these compounds usually have unique structures that also provide the possibility for structural design and modification of the leading compounds [4]. As one special marine-derived fungus, *Acremonium chrysogenum* has made irreplaceable contributions to controlling the bacterial infections and saving countless patients for production of the *β*-lactam antibiotic cephalosporin C (CPC) and its derivatives [5]. The genomic sequences and annotation of *A. chrysogenum* was first completed in 2014, and a total of

**Citation:** Duan, C.; Wang, S.; Huo, R.; Li, E.; Wang, M.; Ren, J.; Pan, Y.; Liu, L.; Liu, G. Sorbicillinoid Derivatives with the Radical Scavenging Activities from the Marine-Derived Fungus *Acremonium chrysogenum* C10. *J. Fungi* **2022**, *8*, 530. https:// doi.org/10.3390/jof8050530

Academic Editors: Tao Feng and Frank Surup

Received: 26 April 2022 Accepted: 16 May 2022 Published: 20 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

42 secondary metabolite biosynthetic gene clusters, including 14 polyketide synthetase (PKS) clusters, 10 terpene synthase clusters, 8 hybrid clusters, 7 nonribosomal peptide synthetase clusters and 3 non-identified secondary metabolite clusters, were predicted [6].

Sorbicillinoids are a class of structurally diverse hexaketide metabolites with a characteristic sorbyl side chain residue [7–9]. They were first isolated from *Penicillium notatum* in 1948 and structurally elucidated by Cram and Tishler [10,11]. Up until now, more than 159 naturally occurring sorbicillinoids have been isolated and have displayed good biological activities in cytotoxic, antimicrobial and phytotoxic activities [7–9]. Because free radicals play an important role in the development of aging and many diseases, including cancer, arthritis and atherosclerosis, exploring the novel radical scavengers is crucial for developing new drugs to slow down the aging process and treat these diseases. Some sorbicillinoid derivatives have shown great antioxidative application prospects, such as bisorbicillinol (ED<sup>50</sup> = 31.4 µM) and bisorbibetanone (ED<sup>50</sup> = 62.5 µM), etc. [8]. Additionally, there is an urgent need to find more novel compounds for the emergence of microbial resistance. Some sorbicillinoids showed significant antimicrobial activity, indicating their potential as candidates [7]. Meanwhile, the sorbicillinoid biosynthetic gene clusters from *Penicillium chrysogenum* and *Trichoderma reesei* have been identified and their biosynthetic pathway has been partially revealed [12–14]. Generally, two PKSs SorA and SorB are responsible for the formation of sorbicillin and dihydrosorbicillin, which are then oxidative dearomatized to give sorbicillinol and dihydrosorbicillinol by the FAD-dependent monooxygenase SorC, respectively [15]. Sorbicillinol is regarded as the precursor of most sorbicillinoids since it is condensed with its derivatives or other compounds to form the dimeric and hybrid sorbicillinoids by Diels-Alder or Michael-addition-like reactions [16,17]. The sorbicillinoid biosynthetic gene cluster in *A. chrysogenum* has been regarded as the most ancient, based on evolutionary origin, and carries more modifier than other species [13], and disruption of these two PKS encoding genes results in the abolishment of sorbicillinoids [18]. However, there is lack of a systematic investigation about sorbicillinoids produced by *A. chrysogenum*.

Based on the chemical investigations in this study, the resulting crude extracts of *A. chrysogenum* C10 from the rice solid fermentation, which has a higher accumulation of compounds and reproducibility than submerged fermentation [19], had afforded three structurally unique compounds: acresorbicillinols A–C (**1**–**3**) and five known sorbicillinoids including trichotetronine (**4**) [20], trichodimerol (**5**) [21], demethyltrichodimerol (**6**) [21], trichopyrone (**7**) [22] and oxosorbicillinol (**8**) [20] (Figure 1). Compounds **1**–**8** were evaluated for their DPPH radical scavenging abilities and antimicrobial activities. In addition, the boundary of sorbicillinoid biosynthetic gene cluster (*Acsor*) was confirmed and its biosynthetic pathway was proposed. This study reported the isolation, structural elucidation and bioactivities of the isolated compounds from *A. chrysogenum* C10. *J. Fungi* **2022**, *8*, x FOR PEER REVIEW 3 of 15

chroism spectrometer, the Thermo Scientific GENESYS 10S UV‐Vis and the Thermo Sci‐ entific Nicolet IS5 spectrophotometers, respectively. HRESIMS data and MS were ob‐ tained using an Agilent 6520B Q‐TOF Mass instrument equipped with an ESI source. All MS experiments were performed in positive ion mode. NMR data were acquired with the AVANCE‐500 spectrometer (Bruker, Bremen, Germany) using solvent signals (CD3OD, *δ*<sup>H</sup> 3.30/*δ*<sup>C</sup> 49.9, DMSO, *δ*<sup>H</sup> 2.50, 3.30/*δ*<sup>C</sup> 39.5, and CDCl3, *δ*<sup>H</sup> 7.26/*δ*<sup>C</sup> 77.16) as references. Octadecylsilyl (ODS, 50 μm, YMC Co., Ltd. Japan) and SephadexTM LH‐20 (Cytiva, Upp‐ sala, Sweden) were used for column chromatography. High performance liquid chroma‐ tography (HPLC) was performed on the SHIMADZU LC20AT system equipped with UV diode array detector using the Thermo Hypersil Gold‐C18 columns (5 μm, 250 mm × 4.6 mm) at a flow rate of 1 mL/min. For semi‐preparative HPLC, Waters 1525 system equipped with the UV/Visible detector and the Thermo Hypersil Gold‐C18 columns (5 μm, 250 mm × 10 mm) was used and performed at a flow rate of 2 mL/min. Solvents in‐ cluding methanol and ethyl acetate (EtOAc) for extraction and chromatographic separa‐ tion were analytical grade. HPLC‐grade solvents (acetonitrile and formic acid) were used

One high CPC‐producing strain of *A. chrysogenum* C10 (ATCC 48272) was released by PanLab. This fungus was inoculated on the rice solid medium in 500 mL Erlenmeyer flasks containing 80 g of rice and 120 mL of H2O, and cultivated at 28 °C for 7 days for the production of sorbicillinoids. A total of 10 kg fermentation sample was harvested.

The rice solid fermentation of *A. chrysogenum* was extracted with EtOAc (3 × 5 L) under the ultrasonication processing. The organic solvents were filtered and evaporated by the vaccum to get the crude extracts (25 g). Extracts were fractionated by ODS reverse silica gel using the gradient MeOH/H2O (*v*/*v*, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%) to afford 15 fractions (Fr.1–Fr.15). Fr.8 (MeOH/H2O (*v*/*v*, 65%)) (150 mg) was further subjected to the SephadexTM LH‐20 and eluted with MeOH to give 30 subfractions. Fr.8–24 (30 mg) was purified by semi‐preparative RP‐ HPLC using 50% acetonitrile in acidic water (0.1% formic acid) to give compounds **2** (5.0

**Figure 1.** Structures of compounds **1**–**8**. **Figure 1.**Structures of compounds **1**–**8**.

*2.1. General Experimental Procedure*

**2. Materials and Methods**

for the HPLC and semi‐preparative HPLC analysis.

*2.2. Fungal Materials and Fermentation*

*2.3. Extraction and Isolation*

#### **2. Materials and Methods**

#### *2.1. General Experimental Procedure*

Optical rotations, ECD spectra, UV and IR data were measured on the Austria Anton Paar MCP 200 Automatic Polarimeter, the Applied Photophysics Chirascan circular dichroism spectrometer, the Thermo Scientific GENESYS 10S UV-Vis and the Thermo Scientific Nicolet IS5 spectrophotometers, respectively. HRESIMS data and MS were obtained using an Agilent 6520B Q-TOF Mass instrument equipped with an ESI source. All MS experiments were performed in positive ion mode. NMR data were acquired with the AVANCE-500 spectrometer (Bruker, Bremen, Germany) using solvent signals (CD3OD, *δ*<sup>H</sup> 3.30/*δ*<sup>C</sup> 49.9, DMSO, *δ*<sup>H</sup> 2.50, 3.30/*δ*<sup>C</sup> 39.5, and CDCl3, *δ*<sup>H</sup> 7.26/*δ*<sup>C</sup> 77.16) as references. Octadecylsilyl (ODS, 50 µm, YMC Co., Ltd. Japan) and SephadexTM LH-20 (Cytiva, Uppsala, Sweden) were used for column chromatography. High performance liquid chromatography (HPLC) was performed on the SHIMADZU LC20AT system equipped with UV diode array detector using the Thermo Hypersil Gold-C18 columns (5 µm, 250 mm × 4.6 mm) at a flow rate of 1 mL/min. For semi-preparative HPLC, Waters 1525 system equipped with the UV/Visible detector and the Thermo Hypersil Gold-C18 columns (5 µm, 250 mm × 10 mm) was used and performed at a flow rate of 2 mL/min. Solvents including methanol and ethyl acetate (EtOAc) for extraction and chromatographic separation were analytical grade. HPLCgrade solvents (acetonitrile and formic acid) were used for the HPLC and semi-preparative HPLC analysis.
