*2.6. X-ray Crystallographic Analysis of Compound 1 and 10* 2.6.1. Penicicellarusin A (**1**)

Colorless needles of compound **1** were obtained from ethyl ether. Data collection was performed on a Eos CCD (Bruker, Bremen, Germany) using graphite-monochromated Cu K<sup>α</sup> radiation, *λ* = 1.54184 Å at 100.00(10) K. Crystal data: C24H32O6, *M* = 416.49, space group orthorhombic, *P212121*; unit cell dimensions were determined to be a = 5.70420(10) Å, b = 11.7760(2) Å, c = 33.3228(6) Å, *α* = *β* = *γ* = 90.00◦ , *V* = 2238.38(7) Å<sup>3</sup> , Z = 4, ρcalc =1.236 mg/mm<sup>3</sup> , *F* (000) = 896.0, *µ* (Cu Kα) = 0.715 mm−<sup>1</sup> . 16083 unique reflections were collected to 2*θmax* = 144.15◦ , in which 4334 reflections were observed [F<sup>2</sup> > 4*σ* (F<sup>2</sup> )]. The structure refinements were conducted by a previously reported method [19]. The final refinement gave R<sup>1</sup> = 0.0357, *w*R<sup>2</sup> = 0.0852 (*w* = 1/*σ*|F|<sup>2</sup> ), and S = 1.047. CCDC 2039557 contains the supplementary crystallographic data for **1**. These data can be obtained from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data\_request/cif (accessed on 20 October 2020).

#### 2.6.2. Penicyrone A (**10**)

Colorless needles of compound **10** from methanol were obtained. Data collection was performed on a Eos CCD using graphite-monochromated Cu K<sup>α</sup> radiation, *λ* = 1.54184 Å at 100.00(10) K. Crystal data: C24H34O7, *M* = 434.519, space group monoclinic, *P21*; unit cell dimensions were determined to be a = 5.96230(10) Å, b = 14.1879(3) Å, c = 14.0680(6) Å, *α* = *γ* = 90.00◦ , *β* = 95.885◦ *V* = 1183.78(4) Å<sup>3</sup> , Z = 2, ρcalc =1.219 mg/mm<sup>3</sup> , *F* (000) = 468.0, *µ* (Cu Kα) = 0.728 mm−<sup>1</sup> . 12357 unique reflections were collected to 2*θmax* = 140.124◦ , in which 4418 reflections were observed [F<sup>2</sup> > 4*σ* (F<sup>2</sup> )]. The structure refinements were conducted by the same method as described for compound **1**. The final refinement gave R<sup>1</sup> = 0.0341, *w*R<sup>2</sup> = 0.0842 (*w* = 1/*σ*|F|<sup>2</sup> ), and S = 1.048. CCDC 2039558 contains the supplementary crystallographic data for **10**. These data can be obtained from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data\_request/cif (accessed on 20 October 2020).

### *2.7. Alkaline Hydrolysis of Compound* **8** *and* **9**

Alkaline hydrolysis reaction was carried out following a previously described method [20]. Each compound (2.0 mg) was dissolved and hydrolyzed with 2 M NaOH/MeOH at 25 ◦C for 3 h. Then neutralized with 1 N HCl/MeOH and extracted with chloroform for two times (10 mL × 2). Methyl esters of the fatty acids were identified by GC-MS. The GC-MS was operated in EI mode (70 eV) scanning from 40 to 500 amu.

#### *2.8. Bioinformatic Analyses*

To identify Biosynthetic Gene Clusters (BGCs) in the genomes of *P. cellarum* YM1, antiSMASH 6.2 was used and only clusters containing a putative PKS similar to both VerA and CtvA protein were further considered [21,22]. The proteins in these clusters were additionally blasted against *P. polonicum* and *Aspergillus terreus* var*. aureus* to verify their presence. To find functional domains and predict a putative function, we resort to NCBI BLAST using Non-Redundant database and Interproscan.

### *2.9. Computation Section*

Systematic conformational analyses for **5a**, **5b**, **5c** and **5d** were performed using the CONFLEX softwre (version 7 Rev. A; CONFLEX Corporation, Tokyo, Japan) via the MMFF94 molecular mechanics force field. Using TDDFT at B3LYP/6-31+G(d,p) basis set level, the MMFF94 conformers were further optimized in methanol with PCM model. The stationary points have been checked as the true minima of the potential energy surface by verifying that they do not exhibit vibrational imaginary frequencies. ECD spectra were calculated by TD-DFT using a Gaussian function at the PBE1PBE/6-311G\* level. Using Boltzmann statistics, equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (∆G). According to Boltzmann weighting of main conformers, the overall ECD spectra were then generated [23].

#### *2.10. Evaluation of Biological Activities*

#### 2.10.1. Antimicrobial Bioassay

Assay for antibacterial activities including *Staphylococcus aureus* (ATCC 6538 and CGMCC 1.2465), meticillin-resistant *S. aureus* (MRSA, clinical isolates, Beijing Chao-yang Hospital, Beijing, China), *Enterococcus faecalis* (clinical isolates, Beijing Chao-yang Hospital), *Bacillus subtilis* (ATCC 6633), and antifungal activities including *Candida albicans* (ATCC 18804), and *Aspergilus fumigatus* (CGMCC 3.5835) were carried out as previously described method [24]. The inhibition rate was calculated and plotted versus test concentrations to afford the MIC. MIC values were defined as the minimum concentration of compounds that inhibited visible microbial growth. All the experiments were performed in triplicate.

#### 2.10.2. Cytotoxicity Assay

Cytotoxicity test against A549, HepG2, and K562 cell lines was carried out as previously described method [25]. Taxol, 5-flourouracil, and cisplatin were used as the positive controls.

2.10.3. 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) Glucose Uptake Assay

This experiment was consistent with those reported in our previous work [26]. The HepG2 hepatoma cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA), 100 U/mL penicillin/streptomycin. The cells reaching confluence were treated with 10−<sup>6</sup> M insulin for 24 h to generate insulin resistance. Compounds or positive drug (rosiglitazone) were mixed and incubated for 24 h, with the final concentration of 100, 50, 25, and 12.5 µM; then, 100 nM of insulin was added and incubated for 30 min at 37 ◦C followed by addition of 50 µM (2-NBDG). After that, cells were washed with ice-cold PBS and 100 µL FBS-free DMEM was added to each well. The level of 2-NBDG uptake was determined on microplate reader (Bio-Tek Instruments, VT, USA) at 485 nm excitation and 528 nm emission. All data were handled with GraphPad Prism 5 and reported as mean ± SD of three independent experiments.

#### **3. Results**

In this study, the MS/MS-based molecular networking strategy was applied for target isolation of new verrucosidins. First, *Penicillium* strains were cultured on rice substrates and the resulting EtOAc extracts was screened by HPLC-UV-DAD analysis (Figure S1). Then, the ethyl acetate extract of *P. cellarum* YM1 that produced secondary metabolites with similar retention time and UV characteristics to those of deoxyverrucosidin was further investigated by UPLC-HRMS/MS. The LC-MS/MS data were used to generate a visualized molecular networking that was further annotated by Cytoscape 3.8.2 (Figure S2).

In details, the HPLC-HRMS/MS analysis in the positive ion mode was conducted on the ethyl acetate extract from *P. cellarum* YM1 with deoxyverrucosidin as the phishing probe. The obtained fragmentation data were organized by molecular networking, yielding a metabolite-level view of the data. Individual MS/MS spectrum was organized into 106 clusters consisting of 899 connected nodes (Figure S2). Using the MS/MS data of deoxyverrucosidin as "seed" spectra, an initial focal point (a blue hexagon with *m/z* 401.232) was generated in the global molecular networking. A close examination of the molecular network indicated some nodes connected to deoxyverrucosidin (Figure 2), which predicted the presence of potential natural analogs. Under the guidance of MS/MS-based molecular networkings, seven new verrucosidins, namely penicicellarusins A-I (**3–9**), in addition to five known polyketides verrucosidin (**1**) [4,27], normethylverrucosidin (**2**) [27], penicyrone A-B (**10–11**) [28], and deoxyverrucosidin (**12**) [29] were obtained by the isolation workflow. The structures of known compounds were determined by comparing their NMR and MS data with literature data.

Compound **1** was obtained as white needle-like crystals and identified as verrucosidin by comparison of the NMR data reported in the literature [4,27] and the single-crystal X-ray crystallographic analysis (Figure 3).

Penicicellarusin A (**3**) was isolated as yellow oil with a molecular formula C24H32O<sup>7</sup> (indicating nine degrees of unsaturation) as deduced by HRESIMS data ([M+Na]<sup>+</sup> *m/z* 455.2041; calcd. 455.2040). The <sup>1</sup>H-, <sup>13</sup>C-NMR and HSQC spectra of **3** revealed the presence of eight methyls, including one oxygenated one [*δ*H*/δ*<sup>C</sup> 1.23 (3H, d, 6.8 Hz) /19.2, 1.40 (3H, s)/22.1, 1.44 (3H, s)/15.7, 1.48 (3H, s)/13.8, 1.98 (3H, s)/18.7, 2.04 (3H, s)/10.4, 2.12 (3H, s)/9.7, and 3.91(3H, s)/61.3], one hydroxymethyl [*δ*H/*δ*<sup>C</sup> 4.36 (d, *J* = 12.2 Hz), 4.41 (d, *J* = 12.2 Hz)/59.6], five methines including three oxygenated [*δ*H*/δ*<sup>C</sup> 3.60 (s)/68.7, 3.81 (s)/64.6, 4.09 (d, 6.8 Hz)/78.4] and two *sp*<sup>2</sup> methines [*δ*H*/δ*<sup>C</sup> 5.60 (brs) /133.9, 5.98 (brs)/134.9]. The NMR data of compound **3** were similar with those of verrucosidin (**1**) [27], indicating the presence of a 3,6-dioxabicyclic[3.1.0]hexane moiety, a 2*H*-pyran-2-one moiety, and a polyene chain in **3** (Figure 1).

The HMBC correlations were detected from H3-21 to C-11 and C-12, from H-13 to C-12 and C-14, from H3-22 to C-14, from H3-23 to C-14 and C-15, from H-15 to C-12, C-14 and C-23, which together with the <sup>1</sup>H-1H COSY correlations of H-15-H3-23 confirmed the presence of the 3,6-dioxabicyclic[3.1.0]hexane moiety. The HMBC correlations of H3-16 to

C-1, C-2, and C-3, H3-24 to C-3, H3-17 to C-3, C-4, and C-5, and as well as the chemical shifts of C-1 (*δ* 167.5), C-2 (*δ* 111.6), C-3 (*δ* 170.2), C-4 (*δ* 112.2), and C-5 (*δ* 157.3) completed the assignment of α-pyranone moiety. Furthermore, the <sup>1</sup>H-1H COSY correlations of H-7- H3-19-H-9-H3-20-H-11 together with the HMBC correlations from H3-18 to C-5 and C-6, from H-7 to C-5, C-6, and C-8, from H3-19 to C-7 and C-8, from H-9 to C-7, C-8, and C-10, from H3-20 to C-9, C-10, and C-11, and from H-11 to C-10 and C-12 supported a heptadiene moiety which was connected with α-pyranone moiety through C-5 and linked with 3,6-dioxabicyclic[3.1.0]hexane moiety through C-12 (Figure 4). *J. Fungi* **2022**, *8*, x FOR PEER REVIEW 9 of 17 *J. Fungi* **2022**, *8*, x FOR PEER REVIEW 9 of 17

**Figure 2.** The subnetwork of tandem MS/MS molecular working for crude extracts of the fungus *P. cellarum*. The entire network and subnetwork are presented in Figure S2. **Figure 2.** The subnetwork of tandem MS/MS molecular working for crude extracts of the fungus *P. cellarum*. The entire network and subnetwork are presented in Figure S2. sidin by comparison of the NMR data reported in the literature [4,27] and the single-crystal X-ray crystallographic analysis (Figure 3).

Penicicellarusin A (**3**) was isolated as yellow oil with a molecular formula C24H32O7 (indicating nine degrees of unsaturation) as deduced by HRESIMS data ([M+Na]+ *m/z*

Penicicellarusin A (**3**) was isolated as yellow oil with a molecular formula C24H32O7 (indicating nine degrees of unsaturation) as deduced by HRESIMS data ([M+Na]+ *m/z* 455.2041; calcd. 455.2040). The 1H-, 13C-NMR and HSQC spectra of **3** revealed the presence of eight methyls, including one oxygenated one [*δ*H*/δ*C 1.23 (3H, d, 6.8 Hz) /19.2, 1.40 (3H, s)/22.1, 1.44 (3H, s)/15.7, 1.48 (3H, s)/13.8, 1.98 (3H, s)/18.7, 2.04 (3H, s)/10.4, 2.12 (3H, s)/9.7,

of eight methyls, including one oxygenated one [*δ*H*/δ*C 1.23 (3H, d, 6.8 Hz) /19.2, 1.40 (3H, s)/22.1, 1.44 (3H, s)/15.7, 1.48 (3H, s)/13.8, 1.98 (3H, s)/18.7, 2.04 (3H, s)/10.4, 2.12 (3H, s)/9.7, and 3.91(3H, s)/61.3], one hydroxymethyl [*δ*H/*δ*C 4.36 (d, *J* = 12.2 Hz), 4.41 (d, *J* = 12.2 Hz)/59.6], five methines including three oxygenated [*δ*H*/δ*C 3.60 (s)/68.7, 3.81 (s)/64.6, 4.09 (d, 6.8 Hz)/78.4] and two *sp*2 methines [*δ*H*/δ*C 5.60 (brs) /133.9, 5.98 (brs)/134.9]. The NMR data of compound **3** were similar with those of verrucosidin (**1**) [27], indicating the

(d, 6.8 Hz)/78.4] and two *sp*2 methines [*δ*H*/δ*C 5.60 (brs) /133.9, 5.98 (brs)/134.9]. The NMR data of compound **3** were similar with those of verrucosidin (**1**) [27], indicating the

**Figure 3.** The X-ray crystallographic structure of **1** and **10**. **Figure 3.** The X-ray crystallographic structure of **1** and **10**.

**Figure 3.** The X-ray crystallographic structure of **1** and **10**.

presence of a 3,6-dioxabicyclic[3.1.0]hexane moiety, a 2*H*-pyran-2-one moiety, and a pol-

presence of a 3,6-dioxabicyclic[3.1.0]hexane moiety, a 2*H*-pyran-2-one moiety, and a pol-

The HMBC correlations were detected from H3-21 to C-11 and C-12, from H-13 to C-12 and C-14, from H3-22 to C-14, from H3-23 to C-14 and C-15, from H-15 to C-12, C-14 and C-23, which together with the 1H-1H COSY correlations of H-15-H3-23 confirmed the presence of the 3,6-dioxabicyclic[3.1.0]hexane moiety. The HMBC correlations of H3-16 to C-1, C-2, and C-3, H3-24 to C-3, H3-17 to C-3, C-4, and C-5, and as well as the chemical shifts of C-1 (*δ* 167.5), C-2 (*δ* 111.6), C-3 (*δ* 170.2), C-4 (*δ* 112.2), and C-5 (*δ* 157.3) completed the assignment of α-pyranone moiety. Furthermore, the 1H-1H COSY correlations of H-7- H3-19-H-9-H3-20-H-11 together with the HMBC correlations from H3-18 to C-5 and C-6, from H-7 to C-5, C-6, and C-8, from H3-19 to C-7 and C-8, from H-9 to C-7, C-8, and C-10, from H3-20 to C-9, C-10, and C-11, and from H-11 to C-10 and C-12 supported a heptadiene moiety which was connected with α-pyranone moiety through C-5 and linked with 3,6-

The HMBC correlations were detected from H3-21 to C-11 and C-12, from H-13 to C-12 and C-14, from H3-22 to C-14, from H3-23 to C-14 and C-15, from H-15 to C-12, C-14 and C-23, which together with the 1H-1H COSY correlations of H-15-H3-23 confirmed the presence of the 3,6-dioxabicyclic[3.1.0]hexane moiety. The HMBC correlations of H3-16 to C-1, C-2, and C-3, H3-24 to C-3, H3-17 to C-3, C-4, and C-5, and as well as the chemical shifts of C-1 (*δ* 167.5), C-2 (*δ* 111.6), C-3 (*δ* 170.2), C-4 (*δ* 112.2), and C-5 (*δ* 157.3) completed the assignment of α-pyranone moiety. Furthermore, the 1H-1H COSY correlations of H-7- H3-19-H-9-H3-20-H-11 together with the HMBC correlations from H3-18 to C-5 and C-6, from H-7 to C-5, C-6, and C-8, from H3-19 to C-7 and C-8, from H-9 to C-7, C-8, and C-10, from H3-20 to C-9, C-10, and C-11, and from H-11 to C-10 and C-12 supported a heptadiene moiety which was connected with α-pyranone moiety through C-5 and linked with 3,6-

**Figure 4.** Selected key HMBC and 1H-1H COSY correlations of **3–6**. **Figure 4.** Selected key HMBC and <sup>1</sup>H-1H COSY correlations of **3–6**. **Figure 4.** Selected key HMBC and 1H-1H COSY correlations of **3–6**.

dioxabicyclic[3.1.0]hexane moiety through C-12 (Figure 4).

dioxabicyclic[3.1.0]hexane moiety through C-12 (Figure 4).

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 10 of 17

yene chain in **3** (Figure 1).

yene chain in **3** (Figure 1).

The relative configuration of **3** was confirmed by NOESY experiment (Figure 5). The NOE correlations H-11 (*δ* 5.60) to H3-23 (*δ* 1.23) and H-13 (*δ* 3.60), H3-22 (*δ* 1.48) to H-13 and H3-23, and H-15 (*δ* 4.09) to H3-21 (*δ* 1.40), indicated that H3-22, H3-23, H-11, and H-13 were on the same face, while H3-21 and H-15 were on the opposite face. The geometry of C8=C9 and C10=C11 were confirmed to be *E* by the NOE correlations of H-7 (*δ* 3.81) with H-9 (*δ* 5.98) and H3-20 with H3-21. The relative configuration of **3** was confirmed by NOESY experiment (Figure 5). The NOE correlations H-11 (*δ* 5.60) to H3-23 (*δ* 1.23) and H-13 (*δ* 3.60), H3-22 (*δ* 1.48) to H-13 and H3-23, and H-15 (*δ* 4.09) to H3-21 (*δ* 1.40), indicated that H3-22, H3-23, H-11, and H-13 were on the same face, while H3-21 and H-15 were on the opposite face. The geometry of C8=C<sup>9</sup> and C10=C<sup>11</sup> were confirmed to be *E* by the NOE correlations of H-7 (*δ* 3.81) with H-9 (*δ* 5.98) and H3-20 with H3-21. The relative configuration of **3** was confirmed by NOESY experiment (Figure 5). The NOE correlations H-11 (*δ* 5.60) to H3-23 (*δ* 1.23) and H-13 (*δ* 3.60), H3-22 (*δ* 1.48) to H-13 and H3-23, and H-15 (*δ* 4.09) to H3-21 (*δ* 1.40), indicated that H3-22, H3-23, H-11, and H-13 were on the same face, while H3-21 and H-15 were on the opposite face. The geometry of C8=C9 and C10=C11 were confirmed to be *E* by the NOE correlations of H-7 (*δ* 3.81) with H-9 (*δ* 5.98) and H3-20 with H3-21.

**Figure 5. Figure 5.** Selected key NOE correlations of Selected key NOE correlations of **3–6 3–6**. .

**Figure 5.** Selected key NOE correlations of **3–6**. In the experimental ECD spectrum, compound **3** showed similar Cotton effects as verrucosidin (**1**) (Figure 6), supporting the same configuration at C-6 and C-7 between **1** and **3**. Thus, compound **3** was assigned a 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, 15*R* configuration and named penicicellarusin A.

Penicicellarusin B (**4**) was obtained as yellow oil with the molecular formula C23H30O<sup>7</sup> and nine degrees of unsaturation, as deduced from HRESIMS data. The 1D NMR spectroscopic data of **4** (Table 1) were similar with those of **3**, except for the lack of a singlet methyl group and the presence of one additional olefinic proton at *δ* 5.66 in **4**. HMBC correlations of H-2 (*δ* 5.66) to C-1, C-3 and C-4, H3-17 to C-3 and C-5, H3-24 to C-3 confirmed the structural changes on the 2*H*-pyran-2-one moiety in **4**. A further comprehensive analysis of its <sup>1</sup>H-1H COSY, HMQC, and HMBC spectra assigned the planar structure of **4** (Figure 4).

The NOESY correlations of H-11 with H-13 and H3-22, H3-23 with H-13 and H3-22, H-15 with H3-21, H-7 with H-9 and H3-17, H3-20 with H3-21 supported the same relative configurations for double bonds and 3,6-dioxabicyclic[3.1.0]hexane moiety between 4 and compound **3** (Figure 5). Compound **4** showed similar Cotton effects in the experimental electronic circular dichroism (ECD) spectrum with those of **3** (Figure 6), indicating it has the absolute configuration of 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*, as described in **3**.

**Figure 6.** Experimental CD spectra of **1**, **3**, and **4** in MeOH. **Figure 6.** Experimental CD spectra of **1**, **3**, and **4** in MeOH.

named penicicellarusin A.

Penicicellarusin B (**4**) was obtained as yellow oil with the molecular formula C23H30O7 and nine degrees of unsaturation, as deduced from HRESIMS data. The 1D NMR spectroscopic data of **4** (Table 1) were similar with those of **3**, except for the lack of a singlet methyl group and the presence of one additional olefinic proton at *δ* 5.66 in **4**. HMBC correlations of H-2 (*δ* 5.66) to C-1, C-3 and C-4, H3-17 to C-3 and C-5, H3-24 to C-3 confirmed the structural changes on the 2*H*-pyran-2-one moiety in **4**. A further comprehensive analysis of its Compound **5** was assigned the molecular formula of C24H34O<sup>7</sup> (eight degree of unsaturation) on the basis of its HRESIMS at *m/z* 457.2190 [M+Na]<sup>+</sup> and NMR data (Table 2). The <sup>1</sup>H-, <sup>13</sup>C-NMR, and UV spectra of **5** were similar with those of verrucisidinol [6], with the notable difference in the <sup>1</sup>H-NMR data of C-3, C-4, C-5, and H-7 (Table 2). A comprehensive analysis of its 2D NMR spectra including <sup>1</sup>H-1H COSY, HMQC, and HMBC experiments confirmed the planar structure of **5** (Figure 4).

In the experimental ECD spectrum, compound **3** showed similar Cotton effects as verrucosidin (**1**) (Figure 6), supporting the same configuration at C-6 and C-7 between **1** and **3**. Thus, compound **3** was assigned a 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, 15*R* configuration and

1H-1H COSY, HMQC, and HMBC spectra assigned the planar structure of **4** (Figure 4). The NOESY correlations of H-11 with H-13 and H3-22, H3-23 with H-13 and H3-22, H-15 with H3-21, H-7 with H-9 and H3-17, H3-20 with H3-21 supported the same relative configurations for double bonds and 3,6-dioxabicyclic[3.1.0]hexane moiety between 4 and compound **3** (Figure 5). Compound **4** showed similar Cotton effects in the experimental electronic circular dichroism (ECD) spectrum with those of **3** (Figure 6), indicating it has the absolute configuration of 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*, as described in **3**. Compound **5** was assigned the molecular formula of C24H34O7 (eight degree of unsaturation) on the basis of its HRESIMS at *m/z* 457.2190 [M+Na]+ and NMR data (Table 2). The 1H-, 13C-NMR, and UV spectra of **5** were similar with those of verrucisidinol [6], with the notable difference in the 1H-NMR data of C-3, C-4, C-5, and H-7 (Table 2). A comprehensive analysis of its 2D NMR spectra including 1H-1H COSY, HMQC, and HMBC experiments confirmed the planar structure of **5** (Figure 4). The partial relative configuration of **5** was confirmed by a NOESY experiment (Figure 5). The geometry of C8=C9 and C10=C11 were confirmed to be *E* by analysis of the NOESY The partial relative configuration of **5** was confirmed by a NOESY experiment (Figure 5). The geometry of C8=C<sup>9</sup> and C10=C<sup>11</sup> were confirmed to be *E* by analysis of the NOESY observations. The key NOESY correlations of H-11 with H-13 and H3-22, H3-23 with H-13 and H3-22, and H-15 with H3-21 supported the same relative configurations on furan ring as verrucisidinol [6]. Considering the same biosynthesis origin, compound **5** is deduced to share the same absolute configuration with those of **1**–**4** in the furan ring. In addtion, the optical rotation data of **5** ([α] 25 *D* = +45.0, *c* = 0.1, MeOH) were opposite to that of verrucisidinol ([α] 25 *D* = −10.0, *c* = 0.1, MeOH), implying the enantiomeric relationship between them. To determine the absolute configurations at C-6 and C-7, ECD calculation method was applied. The four configurations (**5a**, **5b**, **5c** and **5d**, Figure 7) were calculated using time-dependent density functional theory (TDDFT) at PBE1PBE/6-311 G\* level with PCM model in methanol, and 60 exciting states were calculated. By comparison of the experimental and simulated ECD curves (Figure 7), the experimental ECD was match better with **5a** (6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*). Thus, the compound **5** was assigned as 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*, and named as penicicellarusin C.

observations. The key NOESY correlations of H-11 with H-13 and H3-22, H3-23 with H-13 and H3-22, and H-15 with H3-21 supported the same relative configurations on furan ring as verrucisidinol [6]. Considering the same biosynthesis origin, compound **5** is deduced to share the same absolute configuration with those of **1**–**4** in the furan ring. In addtion, the optical rotation data of **5** ([α] 25 D = +45.0, *c* = 0.1, MeOH) were opposite to that of verrucisidinol ([α] 25 D = −10.0, *c* = 0.1, MeOH), implying the enantiomeric relationship between them. To determine the absolute configurations at C-6 and C-7, ECD calculation method was applied. The four configurations (**5a**, **5b**, **5c** and **5d**, Figure 7) were calculated using time-dependent density functional theory (TDDFT) at PBE1PBE/6-311 G\* level with The molecular formula of penicicellarusin D (**6**) was determined to be C23H32O<sup>7</sup> with the unsaturation degrees of eight on the basis of the HRESIMS data at *m*/*z* 443.2047 [M+Na]<sup>+</sup> (calcd. for C23H32O7Na *m*/*z* 443.2040) and NMR data (Table 2). The NMR data of **6** were similar to those of **5** except for the absence of one singlet methyl group. The key HMBC correlations from H-2 (*δ* 5.60) to C-1, C-3 and C-4, as well as the upfield shift of C-2 (*δ* 88.5) confirmed the disappearance of the methyl group on the C-2 position in **6**. Furthermore, Compound **6** showed similar Cotton effects in the experimental CD spectrum with those of **5** (Figure S5), which assigned the absolute configurations of **6** as 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*. It was designated as penicicellarusin D.

PCM model in methanol, and 60 exciting states were calculated. By comparison of the experimental and simulated ECD curves (Figure 7), the experimental ECD was match Penicicellarusins E-G (compounds **7–9**) were determined to be fatty acid esters of **5** by interpretation of the HRESIMS, 1D and 2D NMR data (Table 3, Figures S6 and S7), and ECD spectra (Figure S8). The MS/MS data of **7–9** confirmed the presence of the fatty acid moiety in their structures. The pseudo molecular ion peaks [M+H]<sup>+</sup> at *m*/*z* 673.4669 in **7**, *m/z* 699.4819 in **8**, and *m/z* 697.4668 in **9** together with the fragment ion peaks [M+H-C16H32O2] + at *m*/*z* 435.2381 in **7**, [M+H-C18H32O2] <sup>+</sup> at *m*/*z* 435.2375 in **8**, [M+H-C18H30O2] <sup>+</sup> at *m*/*z* 435.2374 in **9** due to the loss of the corresponding fatty acid moiety. To assign the structure of fatty acid moieties, compounds **7–9** was hydrolyzed with alkaline solution followed by methyl esterification. The fatty acid chain in **7–9** was determined to be the palmitic acid, the oleic acid, and the linoleic acid, respectively, by comparison of the retention time and

MS spectrum with those of standards by GC-MS analysis (Figure S9). Compounds **7–9** showed similar Cotton effects in the experimental CD spectrum with those of **5** (Figure S8), which assigned their absolute configurations as 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*. better with **5a** (6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*). Thus, the compound **5** was assigned as 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*, and named as penicicellarusin C.

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 12 of 17

**Figure 7.** Experimental CD spectra of **5**, the calculated ECD spectra and the structure of **5a**, **5b**, **5c**  and **5d** (bandwidth σ = 0.30 eV). **Figure 7.** Experimental CD spectra of **5**, the calculated ECD spectra and the structure of **5a**, **5b**, **5c** and **5d** (bandwidth σ = 0.30 eV).

The molecular formula of penicicellarusin D (**6**) was determined to be C23H32O7 with the unsaturation degrees of eight on the basis of the HRESIMS data at *m*/*z* 443.2047 [M+Na]+ (calcd. for C23H32O7Na *m*/*z* 443.2040) and NMR data (Table 2). The NMR data of **6** were similar to those of **5** except for the absence of one singlet methyl group. The key HMBC correlations from H-2 (*δ* 5.60) to C-1, C-3 and C-4, as well as the upfield shift of C-2 (*δ* 88.5) confirmed the disappearance of the methyl group on the C-2 position in **6**. Furthermore, Compound **6** showed similar Cotton effects in the experimental CD spectrum With the help of single-crystal X-ray crystallographic analysis, the 6*R*, 9*R*, 12*S*, 13*S*, 14*R*, and 15*R* absolute configuration of penicyrone A (**10**) was determined. The value of the Flack absolute structure parameter 0.03 (8) was obtained, and a perspective ORTEP plot was shown in Figure 3 (CCDC 2039558). According to X-ray diffraction analysis, the configuration at the C-6 positions in **10** was 6*R*, instead of 6*S* reported in the literature [28]. The structures of other known compounds were determined by comparing spectroscopic data with those in the literature. *J. Fungi* **2022**, *8*, x FOR PEER REVIEW 13 of 17 To explore the bioactivities of verrucosidins, compounds **1–12** were evaluated for the

> with those of **5** (Figure S5), which assigned the absolute configurations of **6** as 6*R*, 7*S*, 12*S*, 13*S*, 14*R*, and 15*R*. It was designated as penicicellarusin D. Penicicellarusins E-G (compounds **7–9**) were determined to be fatty acid esters of **5** by interpretation of the HRESIMS, 1D and 2D NMR data (Table 3, Figure S6 and S7), and ECD spectra (Figure S8). The MS/MS data of **7–9** confirmed the presence of the fatty acid moiety in their structures. The pseudo molecular ion peaks [M+H]+ at *m*/*z* 673.4669 in **7**, *m/z* 699.4819 in **8**, and *m/z* 697.4668 in **9** together with the fragment ion peaks [M+H-C16H32O2]+ at *m*/*z* 435.2381 in **7**, [M+H-C18H32O2]+ at *m*/*z* 435.2375 in **8**, [M+H-C18H30O2] + at *m*/*z* 435.2374 in **9** due to the loss of the corresponding fatty acid moiety. To assign the To explore the bioactivities of verrucosidins, compounds **1–12** were evaluated for the antimicrobial effect, cytotoxic activity, and hypoglycemic activity. As a result, **1–12** showed no significant bioactivity in the antimicrobial assays and cytotoxicity assays at the dose of 100 µM. However, Compounds **1–4** were found to enhance the insulin-stimulated uptake of 2-NBDG in insulin-resistant HepG2 cells with the EC<sup>50</sup> values at 47.2 ± 1.2, 9.9 ± 2.5, 93.2 ± 1.2 and 40.2 ± 1.3 µM, respectively, while the other compounds showed no significant activity (Figure 8). In particular, compounds **1**, **2**, and **4** showed much stronger activity than the positive drug (rosiglitazone) in the range of 25–100 µM. antimicrobial effect, cytotoxic activity, and hypoglycemic activity. As a result, **1–12** showed no significant bioactivity in the antimicrobial assays and cytotoxicity assays at the dose of 100 µM. However, Compounds **1–4** were found to enhance the insulin-stimulated uptake of 2-NBDG in insulin-resistant HepG2 cells with the EC50 values at 47.2 ± 1.2, 9.9 ± 2.5, 93.2 ± 1.2 and 40.2 ± 1.3 µM, respectively, while the other compounds showed no significant activity (Figure 8). In particular, compounds **1**, **2**, and **4** showed much stronger activity than the positive drug (rosiglitazone) in the range of 25–100 µM.

**Figure 8.** Stimulation on 2-NBDG glucose uptake in insulin-resistant HepG2 cells. (*celD*), the acyl-acyltransferase gene (*celF*), and the lyase gene (*celH*) potentially involved **Figure 8.** Stimulation on 2-NBDG glucose uptake in insulin-resistant HepG2 cells.

zymes involved in the biosynthesis of verrucosidin are largely uncharacterized.

In our work, with the genome of *P. cellarum* YM1 sequenced in our group, we searched for the gene cluster for penicicellarusins. By similarity analysis with the polyketide synthase gene *ctvA* and *verA*, the putative gene cluster *celA* was found in the genome of *P. cellarum* (Table 4 and Figure S11). Further bioinformatic analysis revealed seven genes, the polyketide synthase gene (*celA*), the SAM-dependent methyltransferase gene (*celB*), the flavin-dependent monooxygenase (*celC1 and celC2*), the cytochrome P450 gene

found in fungi. In this study, seven new verrucosidin derivatives (compounds **3–9**), were isolated together with five previously identified compounds from the fermentation products of fungus *P. cellarum*, suggesting that this fungus is an important producer of verru-

Verrucosidins share similar structural features with the citreoviridins, including a methylated α-pyrone, a polyene linker, and a tetrahydrofuran ring. The citreoviridin biosynthetic gene cluster containing a polyketide synthase (CtvA), a SAM-dependent methyltransferase (CtvB), a flavin-dependent monooxygenase (CtvC), and a hydrolase (CtvD) has been identified in *Aspergillus terreus var. aureus* [21]. CtvC is the only monooxygenase in the cluster, which can iteratively oxidize the terminal triene portion of the precursor into a bisepoxide moiety. As a regioselective hydrolase, CtvD can transform the bisepoxide moiety into a tetrahydrofuran ring moiety [21,30]. In addition, the verrucosidin bosynthetic gene cluster was confirmed by constructing deletion mutants for *verA* gene coding for HR-PKS known to be the key enzyme of the biosynthesis. Different from citreoviridin, the bosynthetic gene cluster for verrucosidin in the genome of *P. polonicum* contains two flavin-dependent monooxygenases, VerC1 and VerC2, which means that the cluster can synthesize compounds with higher oxidation degree [22]. However, the en-

**4. Discussion** 

cosidins.

#### **4. Discussion**

Up to now, less than 20 verrucosidins and structurally-related compounds have been found in fungi. In this study, seven new verrucosidin derivatives (compounds **3–9**), were isolated together with five previously identified compounds from the fermentation products of fungus *P. cellarum*, suggesting that this fungus is an important producer of verrucosidins.

Verrucosidins share similar structural features with the citreoviridins, including a methylated α-pyrone, a polyene linker, and a tetrahydrofuran ring. The citreoviridin biosynthetic gene cluster containing a polyketide synthase (CtvA), a SAM-dependent methyltransferase (CtvB), a flavin-dependent monooxygenase (CtvC), and a hydrolase (CtvD) has been identified in *Aspergillus terreus var. aureus* [21]. CtvC is the only monooxygenase in the cluster, which can iteratively oxidize the terminal triene portion of the precursor into a bisepoxide moiety. As a regioselective hydrolase, CtvD can transform the bisepoxide moiety into a tetrahydrofuran ring moiety [21,30]. In addition, the verrucosidin bosynthetic gene cluster was confirmed by constructing deletion mutants for *verA* gene coding for HR-PKS known to be the key enzyme of the biosynthesis. Different from citreoviridin, the bosynthetic gene cluster for verrucosidin in the genome of *P. polonicum* contains two flavin-dependent monooxygenases, VerC1 and VerC2, which means that the cluster can synthesize compounds with higher oxidation degree [22]. However, the enzymes involved in the biosynthesis of verrucosidin are largely uncharacterized.

In our work, with the genome of *P. cellarum* YM1 sequenced in our group, we searched for the gene cluster for penicicellarusins. By similarity analysis with the polyketide synthase gene *ctvA* and *verA*, the putative gene cluster *celA* was found in the genome of *P. cellarum* (Table 4 and Figure S11). Further bioinformatic analysis revealed seven genes, the polyketide synthase gene (*celA*), the SAM-dependent methyltransferase gene (*celB*), the flavin-dependent monooxygenase (*celC1 and celC2*), the cytochrome P450 gene (*celD*), the acyl-acyltransferase gene (*celF*), and the lyase gene (*celH*) potentially involved in the biosynthesis of penicicellarusins in *P. cellarum*. Based on above evidence, we propose the biosynthetic pathway of **1–12** (Figures 9 and S10). Verrucosidin (**1**) could be formed from **12** by oxidation of the olefinic bond, and further oxidation produces **3–6**. Compounds **7–9** were biosynthesized by esterification of **5** with different fatty acids. Compounds **10** and **11** can be transformed from **5** or **6** by dehydration reaction.


**Table 4.** Penicicellarusins biosynthetic genes and gene function prediction in *P. cellarum* and their homologs in other fungal species.

**Figure 9.** The biosynthetic gene clusters and postulated biogenetic pathway of **1**–**12**. (**a**) The penicicellarusin biosynthesis gene cluster in *P. cellarum*. *celA*: polyketide synthase gene, *celB*: the SAMdependent methyltransferase gene, *celC1*/*celC2*: the flavin-dependent monooxygenase gene, *celD*: the cytochrome P450 gene, *celF*: the acyl-acyltransferase gene, *celE*: transcriptional factor gene, and *celH*: lyase gene. (**b**) postulated biogenetic pathway of **1**–**12**. PKS domain abbreviations: KS ketosynthase, AT acyltransferase, DH dehydratase, MT methyltransferase, KR keroreductase, ACP acyl car-**Figure 9.** The biosynthetic gene clusters and postulated biogenetic pathway of **1**–**12**. (**a**) The penicicellarusin biosynthesis gene cluster in *P. cellarum*. *celA*: polyketide synthase gene, *celB*: the SAMdependent methyltransferase gene, *celC1*/*celC2*: the flavin-dependent monooxygenase gene, *celD*: the cytochrome P450 gene, *celF*: the acyl-acyltransferase gene, *celE*: transcriptional factor gene, and *celH*: lyase gene. (**b**) postulated biogenetic pathway of **1**–**12**. PKS domain abbreviations: KS ketosynthase, AT acyltransferase, DH dehydratase, MT methyltransferase, KR keroreductase, ACP acyl carrier protein.

**5. Conclusions**  In summary, a MS/MS-based molecular networking for the target discovery of verrucosidin-like polyketides was established in this study. The stereochemistry of the new compounds was determined by electronic circular dichroism (ECD) methods or comparison of experimental ECD spectra. The absolute configuration of penicyrone A (**10**) was corrected based on X-ray diffraction analyses. Bioactivity screening indicated that compounds **1**, **2**, and **4** showed much stronger promising hypoglycemic activity than the positive drug (rosiglitazone) in the range of 25–100 µM. The promising hypoglycemic activity Early studies have demonstrated that verrucosidins and structurally-related compounds are endowed with several interesting bioactivities, such as antibacterial activities [3,10], antitumor [7,8], antiviral [9], and neurological activities [11]. In this work, it was found that compounds **1**–**4** show promising hypoglycemic activity, especially compounds **2** and **4**. Preliminary structure-activity relationship showed that the formation of epoxy three-membered ring on C6-C<sup>7</sup> in the structures contributes greatly for the glucose uptakeenhancing activity in insulin-resistant HepG2 cells. The promising hypoglycemic activity is an interesting new bioactivity for this class of compounds.

#### the efficacy of the molecular networking in discovering natural products with unique **5. Conclusions**

ridin, Figures S11–S40: NMR spectra for compounds **3–9**.

rier protein.

structural features. **Supplementary Materials:** The following are available online at www.mdpi.com/xxx/s1, Figure S1: The HPLC profiles of metabolites extracted from the culture medium of *Penicillium* strains, Figure S2: The molecular network obtained by combining the LC-MS/MS analyses of extracts from *P. cellarum* YM1, Figure S3: Phylogenetic analysis and morphological characters of *P. cellarum* YM1, Figure S4: Most stable conformers of **5** in solvated model calculations at the B3LYP/6-31+G(d,p) level (d), Figure S5: Experimental CD spectra of **5** and **6** in MeOH, Figure S6: Selected key HMBC and 1H-1H COSY correlations of **7–9**, Figure S7: Selected key NOE correlations of **7–9**, Figure S8: Experimental CD spectra of **5** and **7–9** in MeOH, Figure S9: GC-MS analysis of methyl linoleate, methyl oleate and products of alkaline hydrolysis-methyl esterification of compounds **8** and **9**, Figure S10: Gene cluster In summary, a MS/MS-based molecular networking for the target discovery of verrucosidin-like polyketides was established in this study. The stereochemistry of the new compounds was determined by electronic circular dichroism (ECD) methods or comparison of experimental ECD spectra. The absolute configuration of penicyrone A (**10**) was corrected based on X-ray diffraction analyses. Bioactivity screening indicated that compounds **1**, **2**, and **4** showed much stronger promising hypoglycemic activity than the positive drug (rosiglitazone) in the range of 25–100 µM. The promising hypoglycemic activity is an interesting new bioactivity for this class of compounds. This work further proved the efficacy of the molecular networking in discovering natural products with unique structural features.

schematic illustrating comparative organization of the penicicellarusin, verrucosidin, and citreovi-

is an interesting new bioactivity for this class of compounds. This work further proved

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jof8020143/s1, Figure S1: The HPLC profiles of metabolites extracted from the culture medium of *Penicillium* strains, Figure S2: The molecular network obtained by combining the LC-MS/MS analyses of extracts from *P. cellarum* YM1, Figure S3: Phylogenetic analysis and morphological characters of *P. cellarum* YM1, Figure S4: Most stable conformers of **5** in solvated model calculations at the B3LYP/6-31+G(d,p) level (d), Figure S5: Experimental CD spectra of **5** and **6** in MeOH, Figure S6: Selected key HMBC and <sup>1</sup>H-1H COSY correlations of **7–9**, Figure S7: Selected key NOE correlations of **7–9**, Figure S8: Experimental CD spectra of **5** and **7–9** in MeOH, Figure S9: GC-MS analysis of methyl linoleate, methyl oleate and products of alkaline hydrolysis-methyl esterification of compounds **8** and **9**, Figure S10: Gene cluster schematic illustrating comparative organization of the penicicellarusin, verrucosidin, and citreoviridin, Figures S11–S40: NMR spectra for compounds **3–9**.

**Author Contributions:** Conceptualization, J.H.; validation, B.C.; software, R.Z., G.Z., data curation, J.H., B.C., R.Z., J.Z. and H.D.; formal analysis, W.L., X.L. and W.Y.; investigation, J.H. and H.L.; resources, T.W. and J.S.; writing—original draft preparation, J.H. and B.C.; writing—review and editing, H.L.; project administration, H.L.; funding acquisition, J.H., E.L., H.L. and H.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was supported by the National Special Project for Key Science and Technology of Food Safety (grant No. 2017YFC1601302), the National Key R&D Program of China (grant No. 2017YFE0108200), and the National Natural Science Foundation (Grant Nos. 22177131, and 82073723).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank the National Special Project and the National Natural Science Foundation for funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

