The fungus P. brasilianum WZXY-m122-9 was isolated from an unidentified marine sponge, which was collected in July 2016 from Weizhou Island in the South China Sea, and was identified by microscopic examination and 18S rDNA ITS sequence's BLAST in GenBank (GenBank accession number HM469396). A voucher specimen (WZXY-m122-9) was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China.

## Genome Sequencing and Analysis

Genome sequencing of P. brasilianum WZXY-m122-9 was performed by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) with an Illumina HiSeq 2000 system. Sequence assembly was performed with SPAdes version 3.5.0 (http:// cab.spbu.ru/software/spades/) to yield 1,367 contigs covering approximately 34.7 Mb. Gene prediction was then performed with Prokka (https://github.com/tseemann/prokka). Anti-SMASH (antibiotics and secondary metabolite analysis shell) analysis of genome sequences was performed to detect secondary metabolite gene clusters, and accurate gene cluster alignment was performed manually by comparisons with homologous genes found in the NCBI database. Anti-SMASH of WZXYm122-9 genome analysis displayed that two gene clusters namely cluster A and cluster B, showed high identity compared with aus' clusters that had been reported in P. brasilianum MG11 (Matsuda et al., 2016).

#### Quantitative RT-PCR for Gene Expression

Degenerate primers were designed according to the conserved sequences of prenyltransferase (PM-122-9\_1376′ ) and terpene cyclase (PM-122-9\_1374) genes within clusters A and B, and the length of PCR products were 805 bp and 529 bp, respectively. The gene expression levels were detected by qRT-PCR. Total RNA of WZXY-m122-9 was obtained from the rice culture medium,

FIGURE 1 | Comparison of the gene clusters between P. brasilianum WZXY-m122-9 and P. brasilianum MG11. (A) Comparison of the genes in cluster A of P. brasilianum WZXY-m122-9 with the aus's genes (cluster A) from P. brasilianum MG11. The homologous genes between the gene clusters were linked with lines. (B) Comparison of the genes in cluster B of P. brasilianum WZXY-m122-9 with the aus's genes (cluster B) from P. brasilianum MG11. The homologous genes between the gene clusters were linked with lines. PM-122-9-5653 and others mean the genes in P. brasilianum WZXY-m122-9.

**41**

and cDNA was synthesized from 1 µg of total RNA in a volume of 20 µL using TransScriptIIAll-in-One first-strand cDNA synthesis super mix (Transgene) according to the manufacturer's instructions. 0.4 µL cDNA, forward primer (10µM), reverse primer (10µM) and 10µL 2× TransStart Top Green qPCRSuperMix (Transgene) were used in subsequent RT-PCR reactions with supplement ddH2O to 20 µL. The specific primers included actin-F (5′ -ACCTGCTCTGCGACTACAAC-3′ ), actin-R (5′ -ACACCGCCCTCATAATAAAG-3′ ), PM-122-9\_1376′ -F (5′ -CCACCAAAGGGGATTACCA-3′ ), PM-122-9\_1376′ -R (5′ -GAGCAGAAATGTCGCAGGAA-3′ ), PM-122-9\_1374-F (5′ -CGGTAGGATGGTCGGTCAAC-3′ ), PM-122-9\_1374-R (5′ -ACGGCGGAGTGTAGGAAGAA-3′ ). Each primer pair gave a single PCR product. The β-actin gene was treated as an internal reference gene, optimized PCR conditions were 94◦C for 30 s; 45 cycles of 94◦C for 5 s; 60◦C for 15 s; and 72◦C for 10 s; followed by dissociation stage. Three parallel tests for each reaction and recording their respective Ct values. According to amplification curves, every sample was different from no template control (NTC), which indicated that the genes were amplified from cDNA template. Under current culture conditions, the aus′ gene cluster was expressed in the plasmids of WZXY-m122-9, and the expression level of prenyltransferase (PM-122-9\_1376′ ) and terpene cyclase (PM-122-9\_1374) genes was higher than β-actin due to Ct1376 and Ct1374 less than Ctactin.

## Fermentation of the Fungus

The fermentation was carried out in 40 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H2O (100 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum (107 /mL) and incubated at 25◦C for 25 days.

#### Extraction and Isolation of Metabolites

The fermented material was extracted with EtOAc (3 × 1 L), successively. The EtOAc extract was evaporated to dryness under reduced pressure to afford a crude residue (20.5 g). The crude extract was suspended in 90% MeOH-H2O and then extracted with petroleum ether (PE) for three times, while the MeOH layer was concentrated under vacuum to obtain MeOH soluble extract (8.0 g). Part of the MeOH fraction (4 g) was then subjected to silica gel (200–300 mesh) vacuum liquid chromatography with gradient elution using PE-EtOAc (from 8:1 to 0:1, v/v) to obtain four fractions (FA-FD). FC (0.9 g) was detected by 1H NMR spectrum, showing the signals featured meroterpenoid analogues, and it was chromatographed over an RP-C18 column eluting with MeOH-H2O (65:35, v/v) to afford six subfractions (FCa–FCf). FCb (60 mg) was separated on a semipreparative RP-C18 HPLC column using MeOH-H2O (50:50, v/v, 2 mL/min) to yield compounds **7** (8 mg) and **1** (4 mg). FCc (110 mg) was purified by a semipreparative RP-C18 HPLC column eluting with MeCN-H2O (40:60, v/v, 2 mL/min) to afford compounds **2** (6 mg), **3** (6 mg) and **6** (2 mg). FCd (70 mg) was separated using a semipreparative RP-C18 HPLC column eluting with MeOH-H2O (55:45, v/v, 2 mL/min) to afford **4** (5.0 mg), **5** (5.0 mg), and **8** (45 mg).

Brasilianoid A (**1**) (methyl (1R, 2S, 5R, 5aS, 7aS, 8R, 10S, 12R, 13aR, 13bS)-1, 10-dihydroxy-5,7a,10,12,13bpentamethyl-14-methylene-3,9,11-trioxotetradecahydro-

2,5:8,12-dimethanocycloocta[3,4]benzo[1,2 -c]oxepine-8(5H) carboxylate). White amorphous powder; [α] 20 D −22 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 203 nm; IR vmax (KBr) 3,419, 2,985, 2,935, 1,754, 1,720, 1,708, 1,662, 1,227, and 1,025 cm−1 . 1H and 13C NMR data, see **Tables 1**, **2**; HRESIMS m/z 473.2176 [M–H]− (calcd for C26H33O8, 473.2175).

Brasilianoid B (**2**) ((5aS, 5bS, 7R, 8aR, 9S, 11aR, 11bS, 13aS)- 8a-hydroxy-1, 1, 5a, 7, 9, 11b-hexamethyl-14-methylene-5a, 5b, 6, 7, 8a, 9, 11b, 12, 13, 13a-decahydro-1H, 11H-7,11amethanofuro[3′′,4′′:3′ ,4′ ]cyclohepta[1′ ,2′ :3,4]benzo[1,2-

c]oxepine-3,8,11-trione). Colorless monoclinic crystals (MeOH-Acetone-H2O, 5:5:1); m.p. 170–172◦C; [α] 20 D −40 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 206 nm; IR vmax (KBr) 3,420, 2,931, 2,966, 1,754, 1,693, 1,604, 1,384, and 1,043 cm−1 . 1H and 13C NMR data, see **Tables 1**, **2**; HRESIMS m/z 473.2170 [M + HCOO]− (cald for C26H33O8, 47 3.2175).

Brasilianoid C (**3**) ((5aS, 5bS, 7R, 8aR, 9R, 11aR, 11bS, 13aS)-8a-hydroxy-1, 1, 5a, 7, 9,11b -hexamethyl-14 methylene-5a,5b,6,7,8a,9,11b,12,13,13a-decahydro-1H,11H-7, 11a-methanofuro[3′′,4′′:3′ ,4′ ]cyclohepta[1′ ,2′ :3,4]benzo[1,2-

c]oxepine-3,8,11-trione). Colorless monoclinic crystals (MeOH-H2O, 8:1); m.p. 171–173◦C; [α] 20 D −40 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 208 nm; IR vmax (KBr) 3,420, 2,930, 2,876, 1,760, 1,694, 1,604, 1,385, and 1,027 cm−1 . 1H and 13C NMR data, see **Tables 1**, **2**; HRESIMS m/z 473.2181 [M + HCOO]− (cald for C26H33O8, 473.2175).

Brasilianoid D (**4**) (methyl 3-((3R, 3aR, 5R, 6aS, 7S, 8S, 10aS, 10bR)-3a-hydroxy-8- (2-hydroxypropan-2-yl)-3,5,7,10atetramethyl-11-methylene-1,4-dioxodecahydro-1H,3H-5,10bmethanobenzo[3,4]cyclohepta[1,2-c]furan-7-yl)propanoate). Colorless monoclinic crystals; (MeOH-CHCl3, 5:1); m.p. 255– 258◦C; [α] 20 D −60 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 202 nm; IR vmax (KBr) 3,420, 2,965, 2,856, 1,750, 1,661, 1,634, 1,384, and 1,072 cm−1 . 1H and 13C NMR data, see **Tables 1**, **2**; HRESIMS m/z 507.2590 [M + HCOO]− (calcd for C27H39O9, 507.2594).

Brasilianoid E (**5**) (methyl 3-((3S, 3aR, 5R, 6aS, 7S, 8S, 10aS, 10bR)-3a-hydroxy-8- (2-hydroxypropan-2-yl)-3,5,7,10atetramethyl-11-methylene-1,4-dioxodecahydro-1H,3H-5,10bmethanobenzo[3,4]cyclohepta[1,2-c]furan-7-yl)propanoate). Colorless orthorhombic crystals (MeOH-CHCl3-H2O, 5:3:1); m.p. 225–228◦C; [α] 20 D −60 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 202 nm; IR vmax (KBr) 3,445, 2,966, 2,939, 1,750, 1,662,

1,607, 1,385, and 1,065 cm−1 . 1H and 13C NMR data, see **Tables 1**, **2**; HRESIMS m/z 507.2600 [M + HCOO]− (cald for C27H39O9, 507.2594).

Brasilianoid F (**6**) ((1R, 4S, 5R, 5aS, 5bR, 7R, 8aR, 9R, 11aR, 11bS, 13aS)-5, 8a-dihydroxy-1,5a,7,9,11bpentamethyl-14-methylenedodecahydro-1H,11H-1,4:7,11adimethanofuro[3′′,4′′:3′ ,4′ ]cyclohepta[1′ ,2′ :3,4]benzo[1,2-

c]oxepine-3,8,11-trione). White amorphous powder; [α] 20 D −48 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 202 nm; IR vmax (KBr) 3,419, 2,986, 2,949, 1,754, 1,720, 1,603, 1,383, and 1,088 cm−1 . 1H and 13C NMR data, see **Tables 1**, **2**; HRESIMS m/z 443.2077 [M – H]− (cald for C25H31O7, 443.2070).

## ECD Calculation

Conformational searches were carried out by random searching in the Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 5.0 kcal/mol. Due to the confirmed NOESY correlations and relatively rigid skeleton, the results showed the lowest energy conformers for (1R, 2S, 4R, 5S, 8S, 9R, 10S, 1 ′R, 3′R, 5′ S)-**1** ′ , 1R, 2S, 4R, 5S, 8S, 9R, 10S, 1′R, 3′R, 5′R, 6 ′R-**6** ′ and for 5S, 8S, 9S, 10S, 1′R, 3′R, 5′R, 6′R-**7** ′ within 5.0 kcal/mol. Subsequently, the conformers were re-optimized using DFT at the B3LYP/6-31+G(d) level in gas phase by the GAUSSIAN 09 program. The energies, oscillator strengths, and rotational strengths (velocity) of the first 60 electronic excitations were calculated using the TDDFT methodology at the b3lyp/6- 311++g(d, p) level in vacuum. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, σ = 0.35 for **1** and 0.25 for **6**). By comparison of the calculated ECD spectra with the experimental curves, the absolute configuration of **1** and **6** were resolved.

## Assay for in Vitro Anti-HBV Effects

HepAD38 cells were cultured with DMEM in 48-well plate at 1 × 105 cells/well for 24 h, then treated with 10 µM of each compound for 72 h. After 4 days, the cells were washed with precooled PBS for 2 times. HBV progeny DNA of HepAD38 cells were extracted using QIAamp DNA Blood Mini kit (Biomiga) according to the manufacturer's instruction. Total DNA was reversely transcribed using PrimeScript RT reagent Kit (Takara, Dalian, China). The primers were designed and synthesized by Takara, and the sequences of the primers are indicated in Table S9. PCR amplification was performed on an StepOne Plus real time PCR system (Applied Biosystems, Foster City, CA) using the SYBR Green Master Mix (Applied Biosystems, Foster City, CA). All experiments were performed in triplicate, and the relative levels of assayed HBV DNA were calculated with the delta– delta CT method using lamivudine as a positive control, and normalized to non-treated control.

## Assay for Protective Effects on Skin Barrier Functions in in Vitro

HaCaT cells were cultured with DMEM in 6-well plate at 1 × 105 cells/well for 24 h, then treated with 20, 10, 5 µM of compounds **1**–**7** for 72 h. Total RNA of HaCaT cells were extracted using RNA Miniprep kit (Biomiga) according to the manufacturer's instruction. Then total RNA was reverse transcribed using PrimeScript RT reagent Kit (Takara, Dalian, China). The primers were designed and synthesized by Takara, the sequences of the primers are indicated in Table S10. PCR amplification was performed on an StepOne Plus real time PCR system (Applied Biosystems, Foster City, CA) using the SYBR Green Master Mix (Applied Biosystems, Foster City, CA). All experiments were performed in triplicate, and the relative levels of assayed mRNAs were calculated with the delta–delta CT method using ACTIN expressions as endogenous control, and normalized to non-treated control.

HaCaT cells were seeded at 5 × 105 cells per well in 6-well plates and incubated under starvation conditions for 24 h using serum-free MEM. HaCaT cells were exposed to UVB irradiation (UVB lamp: SUV-1000, Sigma-China) at a dose of 30 mJ/cm2 . After UVB irradiation, DMEM medium containing **1** (20µM) or epigallocatechin gallate (20µM) was added and the cells were incubated for 24 h, and then the MTT assay was performed.

## Assay for the Inhibition of Nitric Oxide Production in RAW264.7 Macrophages

All compounds were assayed by measuring the inhibitory effects on NO production induced by LPS in mouse macrophage RAW 264.7 cells. The RAW 264.7 cells were seeded in each well of a 96-well plate at a concentration of 5 × 105 cells/ml in DMEM medium containing 10% fetal bovine serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 100µg/ml streptomycin. After incubation at 37◦C in a humidified atmosphere of 5% CO2 air for 12 h, cells were treated with LPS (1µg/ml) and compounds (50µg/ml for each compound, and compound was dissolved in DMSO to dilute until the final DMSO concentration < 0.1%, v/v), then incubation for 24 h at 37◦C. NO production was determined by measuring the quantity of nitrite in the culture medium by the Griess reagent. Aminoguanidine was selected as a positive control. A 50 µl of the supernatant of culture medium was mixed with 100 µl of the Griess reagent. Using nitrite to generate a standard curve, nitrite production was measured by a microplate reader at 540 nm. Cell viability was examined by the MTT method (Wu et al., 2016). The compounds with inhibitory rate more than 50% at a dose of 50µg/ml were further detected in the gradient concentrations to calculate IC50 values.

## Statistical Analysis

All quantitative values are given as means ± SEMs. Significant differences among the experimental groups were assessed by one-way ANOVA. Statistical significances were considered at the ∗P < 0.05 and ∗∗P < 0.01 levels.

## Crystal Structure Analysis

X-ray crystal data of **2**–**5** and **8** were acquired on an Eos CCD with a graphite monochromated Cu Kα radiation (λ = 1.5418 Å) (Supporting Information). These structures was solved with ShelXT-97 using direct methods and refined with ShelXL using full matrix least-squares on F2. Crystallographic data for **2**–**5** and **8** have been deposited at the Cambridge Crystallographic Data Center with the numbers of CCDC 1572280 for **2**, CCDC 1572282 for **3**, CCDC 1576933 for **4**, CCDC 1576930 for **5** and CCDC 1583230 for **8**. Crystallographic data are deposited in the Cambridge Crystallographic Data Centre. and are available free of charge via the Internet at www.ccdc.cam.ac.uk/products/csd/ request.

## RESULTS

## Identification of New Compounds

The molecular formula of brasilianoid A (**1**) was determined as C26H34O8 according to the HRESIMS ion at m/z 473.2176 [M – H]− and the NMR data, requiring ten degrees of unsaturation. The 1H NMR data presented five tertiary methyl singlet at δH 0.76 (s), 1.17 (s), 1.29 (s), 1.33 (s), and 1.34 (s), and a methoxy group at δH 3.57 (s), one oxygenated methine at δH 3.47 (t, J = 4.2 Hz), as well as the terminal olefinic methylene protons at δH 4.67 (s) and 5.21 (s) (**Table 1**). The 13C NMR and DEPT spectra exhibited a total of 26 carbon resonances (**Table 2**), which were classified into six methyl, five methylene (one olefinic carbon), four methine, and eleven quaternary carbons involving two ketones and two ester carbonyl carbons. Analyses of the 1D and 2D NMR data revealed that **1** is a preaustinoid-related meroterpenoid, while the partial structure regarding rings C/D/E of **1** was corresponded to rings B/C/D of preaustinoid A2 (**8**) (dos Santos and Rodrigues-Fo, 2003) with the exception of ring A. The COSY correlation coupled H-2 (δH 2.50, dd, J = 4.2, 4.9 Hz) to H-1 (δH 3.47) and H2-15 (δH 1.81, 2.24), along with the extension of COSY coupling between H-1 and a D2O exchangeable proton at δH 5.16 (d, J = 4.2 Hz), ascertained the location of a hydroxy group at C-1 (δC 70.0). A methylene bridge across C-2 (δC 48.0) and C-4 (δC 86.8) was evident from the HMBC correlations from H3-14 (δH 1.29, s) to C-4, C-5 (δC 48.3), and C-15 (δC 40.7), and from H2- 15 to C-1, C-2, C-4, and C-5. Additional HMBC correlation from the carbonyl carbon C-3 (δC 177.5) to H-1, H-2, and H2-15 in association with the degrees of molecular unsaturation confirmed the formation of a γ-lactone for ring A.

The relative configuration of **1** was established by the NOE data. As shown in **Figure 3**, the NOE correlations from H-15a (δH 2.24) to OH-1 and H-5, and from H-9 (δH 1.50) to OH-1, H-5, and H3-9′ , in association with the correlation between H3- 12 (δH 1.17) and H3-13 (δH 0.76), clarified trans fusion between B/C and C/D rings. When H-1 was arbitrarily assigned to αorientation, the methylene bridge H2-15 was ascertained to be in β-orientation. The relative configuration in rings C to E was the same as that of preaustinoid A2, based on the similar NOE relationship. The spatial closure between H3-9′ and H-9 was recognized by their NOE interaction, while the NOE correlation between H3-12 and H2-7′ indicated that the exo-vinyl group across C-1′ and C-3′ was α-oriented. In biogenetic consideration, compound **1** was assumed to be derived from preaustinoid A2 by the formation of C-2/C-15 bond. Thus, both compounds were suggested to share the same absolute configuration in rings C-E. Since the absolute configuration of preaustinoid A2 was determined by the single crystal X-ray diffraction analysis using Cu Kα radiation in present work, the stereogenic centers in rings C-E of **1** were supposed to be 8S, 9S, 10R, 3′R, 5′ S, and 7 ′R configurations (Supporting Information). Thus, the absolute configuration in rings A and B was in agreement with 1R, 2R, 4S, and 5S, based on the NOE relationships. This assignment was further supported by the ECD data (Stephens and Harada, 2010), of which the Cotton effects of experimental and calculated ECD

for the model molecule of **1** with 1R, 2R, 4S, 5S, 8S, 9S, 10R, 3′R, 5 ′ S, and 7′R were comparable (**Figure 4**).

Brasilianoid B (**2**) was isolated as colorless monoclinic crystals. Its molecular formula was determined as C25H32O6 based on the HRESIMS (m/z 473.2170 [M + HCOO]−) and NMR data, containing ten degrees of unsaturation. The 1H and 13C NMR data of **2** (**Tables 1**, **2**) regarding rings A-C resembled those of preaustinoid A2, while the distinction was attributed to rings D and E. An exocyclic olefinic group at C-2′ (δC 147.6) in ring C was recognized by the HMBC correlations from the olefinic methylene protons H2-7′ (δH 4.86, 5.09) to C-1′ (δC 66.5) and C-3′ (δC 55.3). Additional HMBC correlations from H3-8′ (δH 1.20, s) to C-2′ , C-3′ , and a ketone carbon C-4′ (δC 213.4), and from OH-6′ (δH 6.95, s) to C-4′ , C-1′ , and C-6′ (δC 90.8) established a cyclopentanone unit for ring D. Moreover, the HMBC correlations from H-5′ (δH 4.30, q, J = 6.4 Hz) to C-1′ , C-4′ , C-6′ , and a carbonyl carbon C-10′ (δC 172.6), in addition to the COSY coupling between H-5′ and H3-9′ (δH 1.14, d, J = 6.4 Hz), deduced a γ-lactone unit for ring E, while a hydroxy group and a methyl group were located at C-6′ and C-5′ , respectively.

The relative configuration of **2** in rings A-C was the same as that of preaustinoid A2, based on the similar NOE relationships. The NOE correlations from OH-6′ to H-7a (δH 2.23) and H-9 (δH 1.99) demonstrated the spatial closure of these protons, suggesting OH-6′ to be in β-orientation. Additional NOE correlations between OH-6′ /H3-9′ , H-5′ /H2-7′ , H-5′ /H3-8′ , and H2-7′ /H3-12 (**Figure 2**) clarified the relative configuration in rings D and E. Based on the Flack parameters [−0.04(13)] measured by the Cu-Kα X-ray diffraction of the single crystal, the absolute configurations of **2** were finally determined as 5S, 8S, 9S, 10S, 1′R, 3′R, 5′R, and 6′R (**Figure 5**).

The planar structure of brasilianoid C (**3**) was the same as that of **2**, based on the 1D and 2D NMR data in association with the same molecular formula as determined by the HRESIMS data. The distinction was ascribed to the carbons C-5′ (δC 83.5) and C-9′ (δC 18.2), which showed more deshielded resonances than the corresponding carbons of **2** (**Table 2**). The NOE data raised from rings A-D were in accordance with those of **2**, indicating that this partial structure in **3** was the same as that of **2**. However, the NOE correlations between OH-6′ (δH 7.09, s) and H-5′ (δH 4.46, q) and from H3-9′ (δH 1.03, d) to H2-7′ (δH 4.93, 5.14) and H3-8′ (δH 1.16, s) suggested **3** to be an C-5′ epimer of **2**. This assignment was confirmed by the Cu-Kα X-ray diffraction data, that clarified 5′ S configuration in **3** (**Figure 5**).

Brasilianoid D (**4**) has a molecular formula of C26H38O7 as determined by the HRESIMS and NMR data, bearing nine degrees of unsaturation. Analyses of the 2D NMR (COSY, HMQC, HMBC) data established the partial structure in rings B**-**E of **4** to be the same as that of **3**. However, the NMR data of **4** presented a methoxy group (δH 3.57, δC 51.6), and the methoxy protons correlated to a carbonyl carbon C-3 (δC 174.3) in the HMBC spectrum to form a methyl ester. The HMBC correlations from H2-1 (δH 1.32, 2.43) and H2-2 (δH 1.72, 2.42) to C-3 and C-10 (δC 41.1) demonstrated a methyl propionate unit to be positioned at C-10. In addition, a D2O exchangeable proton at δH 4.09 (s) showed the HMBC correlation with C-4 (δC 74.3), C-5 (δC 51.1), and two methyl carbons C-14 (δC 28.0) and C-15 (δC 33.9), and therefore pointed the substitution of a 2-hydroxyisopropane unit at C-5. Thus, compound **4** was likely derived from preaustinoid A1 (Geris dos Santos and Rodrigues-Fo, 2002) by acyclized ring A and the cyclic rearrangement in ring D. The NOE relationship of OH-6′ (δH 7.04, s) with H-7a (δH 2.23), H-9 (δH 2.07), and H-5′ (δH 4.42), and from H3-9′ (δH 1.03, d) to H2-7′ (δH 4.89, 5.08), and H3-8′ (δH 1.12, s), deduced the same relative configuration of both **4** and **3**. The absolute configuration of **4** was in agreement with that of **3** according to the Flack parameters of the Cu-Kα X-ray diffraction for the single crystals (**Figure 5**).

The planar structure of brasilianoid E (**5**) was the same as that of **4**, as determined by the HRESIMS data (m/z 507.2600 [M + HCOO]−, cald for C27H39O9, 507.2594) and the comparable 1D and 2D NMR data. The distinction was attributed to the NMR resonances at C-5′ (δC 76.1) and C-9′ (δC 13.4) which shifted 7 and 5 ppm, respectively, to upfield in comparison with those of **4** (**Tables 1**, **2**). As the case of **2**, the NOE correlation between OH-6′ (δH 6.76, s) and H-5′ (δH 4.29) and between H3-9′ (δH 1.15) and H2-7′ (δH 4.82, 5.04) clarified **5** to be an C-5′ epimer of **4**. The Cu-Kα X-ray crystallographic diffraction of the single crystal using Flack parameters (-0.006(9)) further confirmed the absolute configurations of **5** to be 5S, 8S, 9S, 10S, 3′R, 5′R, 6′R, and 7′R (**Figure 5**).

Brasilianoid F (**6**) has a molecular formula of C25H32O6 as determined by the HRESIMS (m/z 443.2077 [M-H]−, cald for C25H31O7, 443.2070) and NMR data, requiring ten degrees of unsaturation. Analyses of 1D and 2D NMR data indicated that rings A to C are in accordance with those of **1**, as evident from the proton spin systems from OH-1 (δH 5.16, d, J = 4.5 Hz) to H2-15 (δH 1.87, 2.54) and from H-5 (δH 1.67) to H2-7 (δH 1.63, 2.16) in the COSY spectrum, in addition to the key HMBC correlations observed from H2-15 to C-1, C-2, C-3, C-4 and C-5; H3-14 to C-4, C-5 and C-15; H3-12 to C-7, C-8, C-9, and C-1′ ; H3-13 to C-1, C-5, C-9, and C-10, and OH-1 to C-1, C-2 and C-10. Moreover, the HMBC and COSY correlations assigned the partial structure of rings D to E to be the same as that of **2**. The similar NOE data, such as the key NOE correlations from OH-1 to H-15b (δH 2.54), H-5 and H-9 and between H3-12 and H3-13 as observed in the NOESY spectrum of **1** suggested the same relative configuration in rings A-C of both **6** and **1**. The NOE correlations from H-5 to H-15b and H-7b (δH 2.16), H-7b to H-9 and OH-6′ , and between OH-6′ and H3-9′ (**Figure 2**) clarified these protons to be cofacial, indicating the same relative configuration in rings C-E of both **6** and **2**. Additional NOE correlations from H-5′ to H2-7′ and H3-8′ supported the exo-vinyl bridge C-2′ to be in the same orientation as H-5′ . In order to assign the absolute configuration, the ECD data of **6** and its enantiomer were calculated at the B31YP/6- 311++G(2d, 2p) level in the gas phase using the B3LYP/6- 31G(d) optimized geometries after conformational searches via the MMFF94S force field (Ding et al., 2007). Comparison of the experimental ECD data with those calculated for the enantiomers indicated that **6** (**Figure 4**) led to the identification of 1R, 2S, 4R, 5S, 8S, 9S, 10R, 3′R, 5′R, 6′R, and 7′R configurations.

Analyses of 1D and 2D NMR data established the planar structure of **7** to be identical to preaustinoid D (Duan et al., 2016), while the similar NOE correlation and ECD data clarified both compounds to be identical. However, comparison of the experimental ECD data with that calculated for a model molecule of (5S, 8S, 9S, 10S, 3′R, 5′ S, 6′R, 7′R)**-7** (**Figure 4**) suggested that the structure of preaustinoid D should be revised to its enantiomer. Compound **8** was identical to preaustinoid A2 based on the comparison of its spectroscopic data with these reported in the literature (dos Santos and Rodrigues-Fo, 2003), while the absolute configuration was determined by the Flack parameters of the X-ray diffraction of the single crystals.

The NMR data of the epimers **2**/**3** and **4**/**5** revealed that the chemical shifts for C-5′ and C-9′ in **2** and **5** with 5′ R exhibited

the resonances at δC 76 (C-5′ ) and δC 13 (C-9′ ), whereas those for 5′ S isomers (**3**, **4**) showed more deshielded resonances at δC 83 (C-5′ ) and δC 18 (C-9′ ). The similar cases were also found in other analogues reported in literature. These NMR distinction provided the evidence to identify the configuration of C-5′ in austin-related meroterpenoids (Duan et al., 2016; Park et al., 2018). For instance, the chemical shifts of C-5′ (δC 76.2) and C-9′ (δC 13.3) in **6** and C-5′ (δC 78.5) and C-9′ (δC 12.2) in **7** implied the same orientation of OH-6′ and H3-9′ .

#### Biological Activity

Compounds **1–8** showed no cytotoxic activities toward tumor cell lines A549, U937 and SMMC7721 with IC50 > 10µM. However, compound **1** significantly stimulated the expression of filaggrin and caspase-14 in HaCaT cells with a dose dependent manner (**Figure 6**). Filaggrin is a key natural moisturizing factor that maintains the ability to regulate the skin moisture barrier (Rawlings and Harding, 2004), while the mutation of the filaggrin gene resulted in skin losing water to allow the entrance of bacteria, leading to allergies, irritation and infection (Nomura et al., 2007). Caspase-14 deficiency was associated with the accumulation of incompletely degraded filaggrin fragments within the stratum corneum, decreased stratum corneum hydration, increased transepidermal water loss, and sensitivity to UVB photodamage (Eckhart and Tschachler, 2011). To test the skin protective activity against UVB irradiation, the cytotoxicity of **1** against HaCaT cells was measured by the MTT assay. The cell viability was decreased to 70% compared to the normal group after exposure to UVB 30 mJ/cm2 . Treatment of the damaged cells with compound **1** (20µM) resulted in the cell viability increasing to 77%, while the positive control epigallocatechin gallate increased the cell viability to 75% at the same dose. This finding indicated that compound **1** is able reduce the UVB-induced cell damage.

Compounds **2** and **3** showed moderate inhibition toward NO production in LPS-induced RAW 264.7 macrophages (**Table 3**). In addition, compounds **3–5** (10µM) inhibited the DNA expression of the HBV virus in HepG2.2.15 cells with the inhibitory rates of 25, 15, and 10%, respectively, whereas lamivudine as a positive control exerted the inhibitory rate of 75% in the same dose (Supporting Information, Figure S8).

#### DISCUSSION

In present work, we reported P. brasilianum WZXY-m122- 9 to be obtained from a marine sponge for the first time. It provides a new source to produce austin-related

meroterpenenes with the uniquely modified structures, implying the same fungal species from different origins activating distinct biosynthetic pathway or enzyme function. Since austin-related meroterpenenes have been reported to possess insecticide and antibiotic activities, the sponge-associated fungus may generate the relevant metabolites to play chemoecological role in the association with host for the defense of micro- or macroorganism invasion.

The early-stage in the biosynthesis of **1**–**6** was considered to follow the similar pathway of austin (Scott et al., 1986; Ahmed et al., 1989). The PKS synthesizes DMOA, which followed by farnesylation, methylesterification, epoxidation, terpene cyclization, Baeyer-Villiger oxidation to yield preaustinoid A1 (a product coexisted with **1**–**6** in the fungal strain) (Itoh et al., 2012). The latter compound can be converted to preaustinoid A2 by dehydrogenation. Compounds **1** and **6** presented as unique scaffold with γ-lactone unit in ring A. Both compounds were biogenetically presumed to be converted from preaustinoid A2 by the radical-based pathway, similar to the case for the biotransformation of preandiloid C to andiconin (Matsuda et al., 2014). During the formation of γ-lactone, Me-15 in preaustinoid A2 was catalyzed by a Fe(II)/α-KG-dependent dioxygenase to yield a radical intermediate, which subsequently formed a C-15/C-2 bond with the electron-rich olefin at C-2 to generate an intermediate with a free radical at C-1. The latter carbon

TABLE 3 | Inhibitory effects against LPS induced NO production in RAW264.7 macrophages.

IC50, 50% inhibitory concentration; CC50, 50% cytotoxic concentration.

was hydroxylated to yield compound **1**. The formation of γlactone in **6** was suggested to follow the similar manner as for **1**. Conversion of preaustinoid A2 to **2** and **3** was presumably mediated by the enzymes as the case of the biotransformation from preaustinoid A3 to austinol. Coexistence of C-5′ epimers (**2**, **3**) with equal amount suggested that the stereogenic selection for the reduction of ketone at C-5′ to hydroxy group is unspecific. Compounds **4** and **5** are regarded to be derived from preaustinoid A1, which followed the similar rearrangement as that for the conversion of preaustinoid A2 to **2** and **3**. We isolated a dioxygenase from the fungus, which was identical to AusE (Matsuda et al., 2013; Valiante et al., 2017). The dioxygenase can convert preaustinoid A2 to preaustinoid A3 in vitro experiment. However, The transformation of preaustinoid A2 to **1** or **6** was unsuccessful. This data suggested that the other Fe(II)/α-KG-dependent dioxygenase is required for the generation of the methylene bridge in **1** and **6**. The methyl ester of **4** and **5** is derived by the hydrolysis of the lactone in ring-A to yield a carboxylic acid at the side chain, and then methyl esterification (**Scheme 1**). Although we found **4** and **5** in the HPLC chromatographic profile of EtOAc extract, whether both compounds to be derived by enzymatic step are uncertain.

## CONCLUSION

In conclusion, compounds **1** and **6** possess unprecedented skeleton featuring the formation of C-2/C-15/C-4 bridge in ring A, while **2** and **3** with 7/6/6/5/5 pentacyclic skeletons of meroterpenoids are found from nature for the first time. The bioassay results indicated that the structural variation directly induced the bioactive spectrum. Compound **1** is the first example of natural products used to promote filaggrin and caspase-14

expression for the protection of the UVB-induced cell damage, suggesting that it is a promising lead for the treatment of dermatological diseases.

#### AUTHOR CONTRIBUTIONS

JZ responses for the isolation of compounds. BY performed the biogenetic approach for synthetic gene screening. DL took the antiviral bioassay. SG assayed for the regulation of filaggrin and caspase-14. PP helped to revise the manuscript. WL elucidated the structures and edited the manuscript.

#### REFERENCES

#### FUNDING

Financial supports from the MOST-973 program (2015CB755906) and the National Natural Science Foundation of China (81630089, 41376127, U1606403) are highly appreciated.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2018.00314/full#supplementary-material

from marine mangrove rhizospheric soil. J. Nat. Prod. 75, 1888–1895. doi: 10.1021/np300377b

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhang, Yuan, Liu, Gao, Proksch and Lin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Polyketides From the Endophytic Fungus Cladosporium sp. Isolated From the Mangrove Plant Excoecaria agallocha

Liping Wang1,2†, Xiuli Han2,3†, Guoliang Zhu2 , Yi Wang2 , Arthit Chairoungdua4 , Pawinee Piyachaturawat 4 and Weiming Zhu1,2,5 \*

1 State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang, China, 2 Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China, 3 College of Life Sciences, Shandong University of Technology, Zibo, China, 4 Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand, 5 Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao, China

#### Edited by:

Xian-Wen Yang, Third Institute of Oceanography, State Oceanic Administration, China

#### Reviewed by:

Massuo Jorge Kato, Universidade de São Paulo, Brazil Roberta Teta, Università degli Studi di Napoli Federico II, Italy

> \*Correspondence: Weiming Zhu<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Medicinal and Pharmaceutical Chemistry, a section of the journal Frontiers in Chemistry

Received: 08 June 2018 Accepted: 23 July 2018 Published: 14 August 2018

#### Citation:

Wang L, Han X, Zhu G, Wang Y, Chairoungdua A, Piyachaturawat P and Zhu W (2018) Polyketides From the Endophytic Fungus Cladosporium sp. Isolated From the Mangrove Plant Excoecaria agallocha. Front. Chem. 6:344. doi: 10.3389/fchem.2018.00344 Five new polyketides (2–6) and ten known compounds (1 and 7–15) were obtained from the fermentation products of the endophytic fungus Cladosporium sp. OUCMDZ-302 with the mangrove plant, Excoecaria agallocha (Euphorbiaceae). The new structures of 2–6 were established on the basis of ECD, specific rotation and spectroscopic data as well as the chemical calculation. Compound 7 showed cytotoxicity against H1975 cell line with an IC50 value of 10.0µM. Compounds 4 and 8–10 showed radical scavenging activity against DPPH with the IC50 values of 2.65, 0.24, 5.66, and 6.67µM, respectively. In addition, the absolute configuration of compound 1 was solidly determined by Xray and sugar analysis of the acidic hydrolysates for the first time as well as those of compounds 8–10 in this paper.

Keywords: Cladosporium sp., mangrove fungus, Excoecaria agallocha, polyketides, anti-oxidation

## INTRODUCTION

Mangrove plants and endophytic fungi are two principal sources of new and bioactive natural products (Zhang et al., 2006; Wu et al., 2008). Excoecaria agallocha (Euphorbiaceae), also known as blind-your-eye, is mainly used to treat sores and stings.

In our ongoing investigations of new and bioactive compounds from endophytes associated with mangrove plants (Lin et al., 2008; Lu et al., 2009, 2010; Wang et al., 2012; Kong et al., 2014; Zhu et al., 2018), an endogenous fungal strain OUCMDZ-302 identified as Cladosporium sp., was isolated from the surface-sterilized stems of E. agallocha. The secondary metabolites of the genus Cladosporium were mainly reported as polyketides derivatives, such as macrolides (Jadulco et al., 2001; Zhang et al., 2001; Shigemori et al., 2004), α-pyrones (Jadulco et al., 2002), α-pyridone (Ye et al., 2005), and binaphthyl derivatives (Sakagami et al., 1995).

**52**

Herein we report five new polyketides (**2**–**6**) (**Figure 1**) isolated from the EtOAc extract of Cladosporium sp. OUCMDZ-302, along with the ten known structures (**Figure S36** and **Table S1**), (2R)-7-O-α-D-ribofuranosyl-5-hydroxy**-**2-methylchroman-4-one (**1**) (Hu et al., 2017), 7-O-α-D-ribosyl-5-hydroxy-2-propylchromone (**7**) (Zhao et al., 2015), 3-(2,3-dihydroxy phenoxy)butanoic acid (**8**) (Dai et al., 2009), (2S,4S)-4-methoxy-2-methylchroman-5-ol (**9**) (Wu et al., 2010), (2S,4S)-2-methylchroman-4,5-diol (**10**) (Teles et al., 2005), (±)-5,7-dihydroxy-2-methyl chroman-4-one (**11**) (Rao et al., 1994), (±)-5-hydroxy-2-methylchroman-4-one (**12**) (Dai et al., 2006), 1-(2,6-dihydroxyphenyl) ethanone (**13**) (Dhami and Stothers, 1965), 1-(2,6-dihydroxyphenyl)-1-butanone (**14**) (Huang et al., 2005), and 2-butyryl-3,5-dihydroxycyclohex-2-enone (**15**) (Igarashi et al., 1993). Compound **7** showed inhibitory activity against H1975 cell line with an IC50 value of 10.0µM. Compounds **4** and **8**–**10** exhibited radical scavenging activity against DPPH with IC50 values of 2.65, 0.24, 5.66, and 6.67µM, respectively. In addition, the absolute configurations of compounds **8**–**10** were resolved and that **1** was solidified in this paper.

## MATERIALS AND METHODS

#### General Experimental Procedures

The NMR, ECD, [α]D, UV and IR spectra were recorded on JEOL JNM-ECP 600, JASCO J-810, JASCO P-1020 digital, Beckman DU R 640 and Nicolet NEXUS 470 spectrophotometers, respectively. ESI-MS, EI-MS and GC-MS were measured on Q-TOF ULTIMA GLOBAL GAA076 LC, VG Autospec-3000 and Agilent 6890/5973 spectrometers, respectively. Semipreparative HPLC and chiral separation was performed on a YMC-pack ODS-A column [10 × 250 mm, 5µm, 4 mL/min] and a CHIRALPAK IA column [20 × 250 mm, 5µm, 10 mL/min]. TLC was performed on plates precoated with silica gel GF254 (10– 40µm). The column chromatography (CC) was performed over silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Sweden), respectively. The seawater for the cultural medium of Cladosporium sp. OUCMDZ-302 was collected from Yellow Sea near Qingdao.

#### Fungal Material

The strain Cladosporium sp. OUCMDZ-302 was isolated from the surface sterilized stems of the mangrove plant E. agallocha grown in Wenchang, Hainan, China. Briefly, the stems were washed with tap water and sterile distilled water in sequence. The stems with clean surface were further sterilized in a sequence of 75% ethanol for 2 min, 0.1% of HgCl2 for 3 min, and sterile distilled water. The outer bark was removed, and the inner bark was cut into small pieces that were then placed on a potato dextrose agar (PDA) plate and cultured at 28◦C for 3 days. A single colony was transferred to PDA media and was identified according to its morphological characteristics (**Figure S35**) by Prof. Kui Hong, Wuhan University. A voucher specimen is deposited in our laboratory at −80◦C. The working strain was prepared on PDA slants and stored at 4◦C.

#### Fermentation and Extraction

The producing fungal strain Cladosporium sp. OUCMDZ-302 was inoculated into a 500 mL cylindrical flask containing 100 mL of seawater consisting of 2% maltose, 2% mannitol, 1% glucose, 1% monosodium glutamate, 0.3% yeast extract, 0.1% corn flour, 0.05% KH2PO4, 0.03% MgSO4· 7H2O (pH 6.5) and cultured at 28◦C for 48 h on a rotary shaker at 120 rpm. The seed culture was transferred into three hundred and fifty 500 mL conical flasks (200 mL/flask) containing the same medium, and performed at 28◦C for 7 days on rotary shakers at 160 rpm. The whole fermentation broth (70 L) was filtered through cheese cloth to separate the mycelia from filtrate. The filtrate was concentrated to about one-quarter of the original volume under reduced pressure and then extracted three times with equal volumes of ethyl acetate (EtOAc) and concentrated to dryness. The mycelia were extracted three times with acetone and concentrated to an aqueous solution. The aqueous solution was subsequently extracted three times with equal volumes of EtOAc and concentrated. Both EtOAc extractions were combined to give 45 g of the extract.

#### Isolation

The extract (45 g) was separated into eight fractions (Fr.1–Fr.8) on a silica gel column (8.5 × 15 cm, 200–300 mesh) using a step gradient elution with CHCl3-petroleum ether (V/V 0:100–100:0, 4 L) and then MeOH–CHCl3 (V/V 0:100–100:0, 16 L). Fr.1 (5.4 g) was separated on a silica gel column (4.5×10 cm, 200–300 mesh) eluted with CHCl3-petroleum ether (V/V, 1: 1, 3L) to give **12** (1g). Fr.3 (0.3 g) was further purified by semipreparative HPLC (60% MeOH/H2O) to give **10** (7 mg, tR 4.97 min). Fr.4 (4.3 g) was separated into two subfractions by column chromatography over silica gel (RP-18) eluting with gradient H2O-MeOH (50– 100%). Fr.4-1 (1.4 g) was separated by Sephadex LH-20 (3 × 75 cm, MeOH, 300 mL) to obtain three fractions (130 mL, Fr.4- 1-1; 90 mL, Fr.4-1-2; 80 mL, Fr.4-1-3). Fr.4-1-2 (140 mg) was purified by semipreparative HPLC (30% MeOH/H2O) to yield **5** (1 mg, tR 8.24 min), **13** (30 mg, tR 20.20 min), and **15** (5 mg, tR 18.15 min). Fr.4-1-3 (190 mg) was purified by semipreparative HPLC (30% MeOH/H2O, 0.15% CF3CO2H) to give **4** (15 mg, tR 16.76 min) and **8** (30 mg, tR 14.41 min). Fr.4-2 (360 mg) was purified by semipreparative HPLC (50% MeOH/H2O) to give **6** (10 mg, tR 12.28 min), and **14** (24 mg, tR 18.12 min). Fr.5 (1.1 g) was separated into two subfractions by a silica gel column (2.6 × 10 cm, 200–300 mesh) eluted with MeOH–CHCl3 (V/V 1:40, 1L). Fr.5-1 (40 mg) was purified by semipreparative HPLC (25% MeOH/H2O) to give **3** (3 mg, tR 6.76 min), and Fr.5-2 (80 mg) was purified by semipreparative HPLC (50% MeOH/H2O, 0.15% CF3CO2H) to give compounds **11** (5 mg, tR 6.78 min). Fr.6 (2.8 g) was separated into two subfractions by a silica gel column (4.5 × 10 cm, 200–300 mesh) eluted with CHCl3-petroleum ether MeOH–CHCl3 (V/V 1:25, 2L). Fr.6-1 (110 mg) was purified by semipreparative HPLC (60% MeOH/H2O) to give **9** (10 mg, tR 10.28 min). Fr.6-2 (340 mg) was purified by semipreparative HPLC (50% MeOH/H2O) to give **7** (18 mg, tR 13.55 min) and the mixture of **1** and **2** (70 mg, tR 5.56 min). The mixture of **1** and **2** were further purified by a chiral column (Chiralpak IA,

MeOH–MeCN–EtOH 40:40:20) to yield compound **1** (35.4 mg, tR 8.22 min) and **2** (23.7 mg, tR 4.69 min).

## ECD, [α]D and Coupling Constant Calculation

Calculations for ECD and [α]D were performed in HyperChem 7.5 and Gaussian 03 (Frisch et al., 2004; Chen et al., 2016; Jin et al., 2018). Karplus formula was used to compute the coupling constant (3 J) from the proton-proton torsion angle (Haasnoot et al., 1980).

## Cytotoxic Assays

Cytotoxicities of compounds **1**–**14** against HL-60 and K562 cell lines were assayed by the MTT method (Mosmann, 1983), while those for BEL-7402, A549, HeLa, and H1975 cell lines were tested by SRB (Skehan et al., 1990) methods. Adriamycin was used as the positive control with the IC50 values of 0.02, 0.21, 0.48, 1.32, 0.32, and 0.38, respectively.

## Anti-oxidant Activities

The anti-oxidant activities of compounds **1**–**14** were evaluated by DPPH assay in vitro (Wang et al., 2007). Vitamin C was used as the positive control with an IC50 value of 3.29µM.

## Antimicrobial Assays

The antimicrobial activities of compounds **1**–**14** against E. coli, E. aerogenes, P. aeruginosa, B. subtilis, and C. albicans were evaluated by an agar dilution method (Zaika, 1988). Ciprofloxacin lactate and ketoconazole was used as the positive controls for bacteria and fungi with MIC values of 4.0, 0.5, 32.0, 16.0, 4.1µg/mL, respectively.

## RESULTS AND DISCUSSION

## Identification of Compounds

Compounds **1** and **2** were first isolated as an isomeric mixture whose molecular formula was determined to be C15H18O8 by HRESIMS at m/z 327.1068 [M+H]+ (calcd 327.1080), indicating seven degrees of unsaturation. An interpretation of the 1D (**Table 1, Figures S1**–**S3**) and 2D NMR (**Figure 2** and **Figures S4**–**S6**) spectra established a pentose moiety and a benzopyrane moiety similar to those of 5,7-dihydroxy-2 methylchroman-4-one (**11**) (Rao et al., 1994). The upfield shift of C-7 (−1.5 ppm) and the key HMBC correlations between the anomeric proton (δH−1 ′ 5.66/5.68) and C-7 (δ 165.1/165.0) indicated that **1** and **2** were 7-O-pentosides of **11**. Acidic hydrolysis of the mixture of **1** and **2** with 2 M HCl yielded (±)-**11** and D-ribose that was identified by GC-MS analysis of the reaction products with L-cysteine methyl ester and Me3SiCl (**Figures S33**, **S34**) (Deyrup et al., 2007). These data indicated that **1** and **2** are a pair of epimers at C-2. Separation of 2-epimeric mixture of **1** and **2** was achieved on a chiral column using MeOH-MeCN-EtOH as eluent. And then, NMR data of optically-pure **1** (**Figures S7**, **S8**) and **2** (**Figures S9**, **S10**) were obtained. Xray single crystal diffraction of **1** revealed the α-glycosidic bond and 2R-configuration (**Figure 3**). The ECD Cotton effects of compounds **1** and **2** were opposite in sign (**Figure 4**), confirming the opposite configuration of C-2. Thus, the structures of **1** and **2** were unambiguously elucidated as (2R)- and (2S)-7-O-α-Dribofuranosyl-5-hydroxy**-**2-methylchroman-4-one, respectively. This is the first time that to solidify the absolute configuration of compound **1**, although it was reported last year (Hu et al., 2017).

The molecular formula of compound **3** was determined to be C10H12O4 based on the HRESIMS peak at m/z 195.0659 [M– H]− (calcd 195.0657), indicating five degrees of unsaturation. The NMR data (**Table 1**, **Figures S11**–**S14**) was similar to those of **10** (Teles et al., 2005), except for the upfield methylene signal at δH/C 1.50 & 1.88/38.0 that was replaced by the one of an oxygenated methine at δH/C 3.45/69.8. This was further supported by the 1H-1H COSY from H-9 (δ 1.31) to H-4 (δ 4.48) through H-2 (δ 4.14) and H-3 (δ 3.45) (**Figure 2** and **Figure S15**), and the key HMBC correlations of H-9 to C-3 (δ 69.8) and H-3 to C-4a (δ 111.2) (**Figure 2** and **Figure S16**). In order to confirm the relative configuration, we calculated the coupling constant of H2-H3 and H3-H4 for the four possible relative configurations **3A**–**3D** (**Figure 5**). The computational 3 J value of **3A** was most near to the measured result (**Table 3**).

aMeasured in CDCl3 and δC−1 was 174.0.

The absolute configuration was established by calculation of the specific rotation. The measured [α]D value of **3** (−53.6) is consistent with the calculated one for (2S,3S,4R)-**3** (−100) and opposite to the calculated one for (2R,3R,4S)-**3** (+102). Thus, the structure of **3** was identified as (2S,3S,4R)-2-methylchroman-3,4,5-triol.

Compound **4** showed the molecular formulae of C11H14O5 based on HRESIMS peaks at m/z 225.0767 [M–H]− (calcd 225.0763), indicating five degrees of unsaturation. The 1D (**Figures S17**–**S19**) and HMQC (**Figure S20**) NMR spectra of **4** displayed three sp2 methines and four sp2 quaternary carbon signals, one sp3 oxygenated methine signals, one sp3 methylene signals and two methyl group (including one methoxy). The 1D NMR data (**Table 1**) of **4** were almost identical to those of **8** [**Figure S1**; (Dai et al., 2009)] except for an additional methoxy (δH/C 3.78/52.5) and the upfield shift for carbonyl carbon (−3.5 ppm), indicating that **4** is the methyl ester of **8**. This was confirmed by analysis of 1H-1H COSY correlation (**Figure S21**) and the key HMBC between the methoxy protons at δH 3.78 and the carbonyl carbon at δC−1 174.0 (**Figure 2** and **Figure S22**). The specific rotations of both **4** ([α]D +14.4) and **8** ([α]D +8.2) were opposite to the synthetic analog, R-3-(3-methoxyphenyloxy)butanoic acid ([α]D −31.2) (Kawasaki et al., 2008), indicating both **4** and **8** as S-configuration. The S-configuration of **4** was also backed by the coincidence of experimental and calculated ECD curves (**Figure 6**). Thus, the structure of compounds **4** and **8** were established as methyl (3S)-3-(2,3-dihydroxyphenyloxy) butanoate and (3S)-3- (2,3-dihydroxyphenyloxy)butanoic acid, respectively.

TABLE 2 | 1H (600 MHz) and 13C (150 MHz) NMR Data of Compounds 5 and 6 in DMSO-d6.

aMeasured in CDCl3.

The molecular formula of compound **5** was determined as C7H12O2 based on the HREIMS peak at m/z 128.0845 [M]+ (calcd 128.0837), corresponding to two degrees of unsaturation. The IR spectrum showed hydroxy groups at 3442 cm−1 and double bonds at 3080 and 1646 cm−1 . The 1D NMR spectra

(**Table 2**, **Figures S23**–**S25**) of **5** showed two double bonds one of which is terminal, two oxygenated methines and one methyl group (**Table 1**). These groups were connected to the full structure of CH2 =CH–CH=CH–CH (OH)–CH (OH)– CH3 on the basis of 1H-1H COSY correlations from the methyl (δH 1.01) to the methylene (δH 5.03/5.17) through the two oxygenated methines (**Figure 2** and **Figure S26**). The large

TABLE 3 | The calculated 3JH−2,H−3 and 3JH−3,H−4 values of compound 3 for the four possible relative configurations.

value of 3 JH−4,H−5 (15.4 Hz) corresponded to E-14 double bond. The large 3 JH−2,H−3 value (6.0 Hz) and the downfield methyl carbon signal (δC−1 19.1) indicated an anti-conformation (Jarvis et al., 1996; Zhang and O'doherty, 2005; Nilewski et al., 2009), corresponding to threo-configuration of 2,3-diol (Zheng et al., 2010). In order to confirm the relative configuration of compound **5**, 3 JH−2,H−3 of threo-**5** and erythro-**5** were computed. The results showed that the predicted 3 JH−2,H−3 values of threo-**5** (5.5 Hz) matched with the measured one (6.0 Hz) while the calculated one of erythro-**5** (3.8 Hz) was inconsistent, indicating threo- configuration. The direction of the specific rotation of **5** ([α]D −6.8) were similar to the structurally related tbutyl (6S,7S)-6,7-dihydroxyocta-2,4-dienoate ([α]D −23) (Zhang and O'doherty, 2005), and opposite to t-butyl (6R,7R)-6,7 dihydroxyocta-2,4-dienoate ([α]D +22.9) (Zhang and O'doherty, 2005). The structure of **5** was thus deduced as (2S,3S,4E)-hepta-4,6-diene-2,3-diol.

The molecular formula of compound **6** was determined to be C11H18O2 based on the HREIMS peak at m/z 182.1305 [M]+ (calcd. 182.1307), indicating three degrees of unsaturation. The EIMS of **6** illustrated in **Figure 7** indicates the existence of

The relative configurations of compounds **9** and **10** were determined as (–)-trans-4-methoxy-2-methylchroman-5-ol (Wu et al., 2010) and (–)-trans-2-methyl chroman-4,5 -diol (Teles et al., 2005), respectively. The absolute configuration of compound **9** was determined by quantum chemical ECD calculation. The measured ECD of **9** was coincident with the calculated ECD of (2S,4S)-**9** and opposite to ECD of (2R,4R)-**9** (**Figure 8**). Thus, compound **9** was established to be (2S,4S)-4 methoxy-2-methyl chroman-5-ol. The similar sign of the specific rotations of **9** and **10** ([α] 22 D −2.0 vs. [α] 22 D −6.0, MeOH) suggests the same absolute configuration. Therefore, compound **10** was determined to be (2S,4S)-2-methylchroman-4,5-diol. The

absolute configurations of compounds **9** and **10** were determined for the first time in this study.

(2R)-7-O-α-D-Ribofuranosyl-5-hydroxy-2-methylchroman-4-one (**1**): White amorphous powder; [α] 23 D +198.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.62), 278 (3.55), 320 (2.77) nm; ECD (MeOH) λmax (1ε) 211 (+14.21), 284 (−3.51), 327 (+3.42); IR (KBr) νmax 3,416, 1,646, 1,573, 1,354, 1,295, 1,195, 1,155, 1,076, 1,029 cm−1 ; 1H and 13C NMR (**Table 1**); HRESIMS m/z 327.1068 [M+H]+ (calcd for C15H19O8 327.1080).

(2S)-7-O-α-D-Ribofuranosyl-5-hydroxy-2-methylchroman-4-one (**2**): White amorphous powder; [α] 23 D +118.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.62), 278 (3.55), 320 (2.77) nm; ECD (MeOH) λmax (1ε) 211 (−10.04), 284 (+9.40), 330 (−2.13); IR (KBr) νmax 3,416, 1,646, 1,573, 1,354, 1,295, 1,195, 1,155, 1,076, 1,029 cm−1 ; 1H and 13C NMR (**Table 1**); HRESIMS m/z 327.1068 [M+H]+ (calcd for C15H19O8 327.1080).

(2S,3S,4R)-2-Methylchroman-3,4,5-triol (**3**): Colorless oil; [α] 23 D −53.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.25), 270 (2.26) nm; IR (KBr) νmax 3,429, 2,356, 1,627, 1,4,01, 1,090 cm−1 ; 1H and 13C NMR (**Table 1**); HRESIMS m/z 195.0659 [M–H] − (calcd. for C10H11O4: 195.0657).

### X-ray Crystallographic Data of 1

Compound **1** was obtained as a colorless monoclinic crystal with molecular formula of C15H18O8 from MeOH and H2O. Space group P21, a = 7.0121(7) Å, b = 10.6659(11) Å, c = 9.8560(8) Å, α = 90.00◦ , β = 95.3230(10)◦ , γ = 90.00◦ , V = 733.95(12) Å3 , Z = 2, Dcalcd = 1.476 mg/m3 , µ = 0.121 mm−1 , F(000) = 344, crystal size 0.42 ×0.30 ×0.21 mm. A total of 3413 unique reflections (2θ<50◦= were collected on a CCD area detector diffractometer with graphite monochromated Mo-Ka radiation (λ = 0.71073 Å). The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of full-matrix least squares refinement was based on 2053 unique reflections (2θ <50◦ ) and 210 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0421, Rw = 0.0981 and R = 0.0374 for I>2sigma(I) data. Crystallographic data (excluding structure factors) for structure **1** in this paper have been deposited in the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 883328 [fax: +44 (0)-1223-336033 or e-mail<EMAIL_ADDRESS>
#### Biogenetic Origin

These compounds were postulated to be biosynthesized by the polyketide pathway from acetyl coenzyme A (**Figure 9**). The acetyl-CoA units underwent condensation, cyclization, dehydration and hydrogenation to produce compounds **11** and **12**. Compound **11** formed compounds **1** and **2** by glycosidation. (S)-**12** underwent oxidation and reduction to yield compound **3**. The reduction of (S)-**12** produced compound **10** that was transformed to compound **9** followed by methylation. (S)-**12** was subjected to Baeyer-Villiger oxidation followed by methanolysis and hydrolysis to yield compounds **4** and **8**, respectively. Compounds **5** and **6** were formed from different lengths of acetyl-CoA units by condensation, reduction, dehydration, and decarboxylation. The condensation of acetyl-CoA units followed by cyclization and reduction formed compound **15** that was transformed to compound **14** after enolization and dehydration.

#### Biological Activity

Compounds **1**–**14** were tested for cytotoxic effects on the HL-60, BEL-7402, K562, A549, HeLa, and H1975 cell lines, DPPH scavenging activity, and antimicrobial activities against E. coli, E. aerogenes, P. aeruginosa, B. subtilis, and C. albicans. As the results, compound **6** was cytotoxic to H1975 cell line with an IC50 values of 10.0µM, while compounds **4** and **8**–**10** showed DPPH radical scavenging activity with the IC50 values of 2.65, 0.24, 5.66, and 6.67µM, respectively. None of the compounds exhibit antimicrobial activities.

#### CONCLUSIONS

Five new polyketides were isolated and identified from the fermentation of the mangrove fungus Cladosporium sp. OUCMDZ-302 with Excoecaria agallocha. The new compound **4** showed DPPH radical scavenging activity with an IC50 value of 2.65µM.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

## REFERENCES

#### FUNDING

This research was financially supported by the National Natural Science Foundation of China (NSFC, Nos.<PHONE_NUMBER>2, U1501221 and 21502034), the 100 Leading Talents of Guizhou Province (fund for W Zhu), the science and technology project of Guizhou (Grant No. QKHT Z-2014-4007), the technology plan of Guizhou (Grant No. QKH YSZ-2015-4009), and by the Thailand Research Fund (TRF) through the International Research Network (IRN-58W0004). The fungus Cladosporium sp. OUCMDZ-302 was identified by Prof. Kui Hong, Wuhan University.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2018.00344/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Wang, Han, Zhu, Wang, Chairoungdua, Piyachaturawat and Zhu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Talaropeptides A-D: Structure and Biosynthesis of Extensively N-methylated Linear Peptides From an Australian Marine Tunicate-Derived Talaromyces sp.

Pradeep Dewapriya1 , Zeinab G. Khalil 1 , Pritesh Prasad1 , Angela A. Salim1 , Pablo Cruz-Morales 2,3, Esteban Marcellin2 and Robert J. Capon1 \*

1 Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia, 2 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia, 3 Joint BioEnergy Institute, Emeryville, CA, United States

#### Edited by:

Xian-Wen Yang, Third Institute of Oceanography, State Oceanic Administration, China

#### Reviewed by:

Yonghui Zhang, Huazhong University of Science and Technology, China Fernando Reyes, Fundación Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Spain Kirk R. Gustafson, National Cancer Institute (NCI), United States

#### \*Correspondence:

Robert J. Capon<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Medicinal and Pharmaceutical Chemistry, a section of the journal Frontiers in Chemistry

Received: 02 July 2018 Accepted: 14 August 2018 Published: 04 September 2018

#### Citation:

Dewapriya P, Khalil ZG, Prasad P, Salim AA, Cruz-Morales P, Marcellin E and Capon RJ (2018) Talaropeptides A-D: Structure and Biosynthesis of Extensively N-methylated Linear Peptides From an Australian Marine Tunicate-Derived Talaromyces sp. Front. Chem. 6:394. doi: 10.3389/fchem.2018.00394 An Australian marine tunicate-derived fungus, Talaromyces sp. CMB-TU011 was subjected to a program of analytical microbioreactor (MATRIX) cultivations, supported by UHPLC-QTOF profiling, to reveal conditions for producing a new class of extensively N-methylated 11-12 residue linear peptides, talaropeptides A-D (2-5). The structures for 2-5, inclusive of absolute configurations, were determined by a combination of detailed spectroscopic and chemical (e.g., C3 and C18 Marfey's) analyses. We report on the biological properties of 2-5, including plasma stability, as well as antibacterial, antifungal and cell cytotoxicity. The talaropeptide mega non-ribosomal peptide synthetase (NRPS) is described, as second only in size to that for the fungus-derived immunosuppressant cyclosporine (an 11-residue extensively N-methylated cyclic peptide).

Keywords: marine-derived, fungus, Talaromyces, talaropeptide, NRPS, linear peptide, secondary metabolite, natural product

#### INTRODUCTION

In an earlier study, we described the structure elucidation of a first-in-class cyclic hexapeptide containing a rare hydroxamate residue, talarolide A (**1**) (**Figure 1**), isolated from an Australian marine tunicate-derived fungus, Talaromyces sp. CMB-TU011 (Dewapriya et al., 2017). In an effort to optimize the production of **1**, we now report on a 24-well microbioreactor cultivation analysis (known in-lab as the MATRIX), using a combination of 11 different media and 3 phases (i.e., solid agar, as well as static and shaken broth). In situ solvent extraction on individual MATRIX culture wells yielded 33 extracts, which were subjected to UHPLC-DAD and UHPLC-QTOF-MS/MS analysis. While this study successfully revealed optimal conditions for the production of **1**, including new analogs (work-in-progress), it also revealed conditions where talarolide production was fully suppressed in favor of a new class of extensively N-methylated linear peptides. This report provides an account of the production, isolation and characterisation of these new peptides, talaropeptides A-D (**2**-**5**), where structure elucidation inclusive of absolute configurations was achieved by a combination of detailed spectroscopic and chemical analyses. We also take the opportunity to report on the biological properties of **2**-**5**, and document the mega non-ribosomal peptide synthetase (NRPS) responsible for the biosynthesis of talaropeptides.

**61**

#### Dewapriya et al. Talaropeptides From a Marine-Derived Fungus

## MATERIALS AND METHODS

#### General Experimental Details

Specific rotations ([α]D) were measured on a JASCO P-1010 polarimeter in a 100 × 2 mm cell at room temperature. NMR spectra were acquired on a Bruker Avance 600 MHz spectrometer with either a 5 mm PASEL 1H/D–13C Z-Gradient probe or 5 mm CPTCI 1H/19F-13C/15N/DZ-Gradient cryoprobe, controlled by TopSpin 2.1 software. In all cases, NMR spectra were acquired at 25◦C (unless otherwise specified) in hexadeuterated dimethylsulfoxide (DMSO-d6) and tetradeuterated methanol (methanol-d4) with referencing to residual 1H (δH 2.50 and δH 3.31, respectively) or 13C (δC 39.51 and δC 49.15, respectively) NMR resonances. HPLC-DAD-ESIMS data were acquired on an Agilent 1100 series separation module equipped with an Agilent 1100 series HPLC/MSD mass detector and Agilent diode array detector. Semi-preparative and preparative HPLCs were performed using Agilent 1100 series HPLC instruments with corresponding detectors, fraction collectors, and software. HRESI(+)MS spectra were obtained on a Bruker micrOTOF mass spectrometer by direct injection in MeCN at 3 µL/min using sodium formate clusters as an internal calibrant. UHPLC-QTOF analysis was performed on UHPLC-QTOF instrument comprising an Agilent 1290 Infinity II UHPLC equipped with a Zorbax C8 column (2.1 mm × 50 mm, 1.8µm particles), running with H2O/MeCN inclusive of 0.1% formic acid coupled to an Agilent 6545 Q-TOF. MS/MS analysis was performed on the same instrument for ions detected in the full scan at an intensity above 1,000 counts at 10 scans/s, with an isolation width of 4 ∼m/z using a fixed collision energy and a maximum of 3 selected precursors per cycle. Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (L-FDAA, synonym 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide) and Nα-(2,4-dinitro-5 fluorophenyl)-D-alanine amide (D-FDAA, synonym 1-fluoro-2-4-dinitrophenyl-5-D-alanine amide) were purchased from NovaBiochem. Amino acids and standards were purchased from NovaBiochem, BAChem Biosciences, Sigma, Fluka, or Merck.

#### Fungus Isolation and Taxonomy

Talaromyces sp. CMB-TU011 was isolated from an unidentified tunicate collected near Tweed Heads, NSW, Australia, and taxonomically identified as previously reported (Dewapriya et al., 2017).

### Analytical (MATRIX) Cultivation of Talaromyces sp. CMB-TU011

Talaromyces sp. CMB-TU011 was cultured in a 24-well microbioreactor plate (Khalil et al., 2014) using a combination of 11 culture media and 3 phases (i.e., solid agar, and liquid static and liquid shaken) known in-lab as the MATRIX (**Table S1**). Briefly, a sterile loop was used to transfer mycelia/spores from an agar plate cultivation to 24-well microbioreactor plate (2.5 mL agar for solid cultures, 1.5 mL of broth for liquid cultures). The microbioreactor plates were sealed with air permeable membranes and incubated at 26.5◦C for 10 days (190 rpm for shaken broth). The resulting 33 cultures were extracted in situ with EtOAc (2.0 mL/well), with the decanted solvent filtered and dried under N2. Secondary metabolite production was then analyzed by HPLC-DAD-ESIMS and UHPLC-QTOF.

### Scale-Up Cultivation of Talaromyces sp. CMB-TU011 and Isolation of 2-5

Agar cubes (∼1 cm2 ) recovered from 7 day CMB-TU011 agar plate cultures (3.3% artificial sea salt containing M1 medium) were used to inoculate 10 flasks (500 mL) charged with YES broth (160 mL). Individual flasks were covered with air permeable sterile cotton plugs, and incubated under static conditions for 10 days at 26.5◦C, after which the combined broths/mycelia were extracted with EtOAc (4 × 500 mL) to yield a crude extract (1.65 g) which was partitioned between hexane (200 mL) and 1% aqueous MeOH (50 mL) and dried in vacuo to yield hexane (495 mg) and MeOH (1.15 g) solubles. The MeOH solubles were further fractionated by gel chromatography (Sephadex R LH-20, MeOH) into 20 fractions, which were selectively combined on the basis of HPLC-DAD-ESIMS analysis (Zorbax SB-C8 column, analytical gradient 90% H2O/MeCN – 100% MeCN inclusive of an isocratic 0.05% formic acid) to yield a fraction of interest (49 mg) that was resolved by optimized semi-preparative HPLC (Zorbax SB C3 column (9.4 mm × 25 cm), 40% MeCN/H2O elution at 3.0 mL/min inclusive of an isocratic 0.01% TFA modifier) to yield talaropeptide A (**2**) (tR = 7.11 min, 1.3 mg), talaropeptide B (**3**) (tR = 8.78 min, 1.3 mg), talaropeptide C (**4**) (tR = 16.89 min, 1.8 mg), and talaropeptide D (**5**) (tR = 22.96 min, 2.8 mg) (**Supplementary Scheme S1**).

Talaropeptide A (2): white powder; [α]D 22 −135.8 (c 0.04, MeOH); 1D and 2D NMR (600 MHz, DMSO-d6) see **Table 1** and **Supplementary Material**; HRESI(+)MS m/z 1254.8435 [(M+H)+] (calcd for C65H112N11O13, 1254.8436).

Talaropeptide B (3): white powder; [α]D 22-110.0 (c 0.05, MeOH); 1D and 2D NMR (600 MHz, methanol-d4) see **Table 2** and **Supplementary Material**; HRESI(+)MS m/z 1375.8920 [M+Na]+ (calcd for C70H120N12O14Na, 1375.8939).

Talaropeptide C (4): white powder; [α]D 22 −151.6 (c 0.05), MeOH); 1D and 2D NMR (600 MHz, methanol-d4) see **Table 3** and **Supplementary Material**; HRESI(+)MS m/z 1318.8376 [(M+Na)+] (calcd for C67H113N11O14Na, 1318.8361).

Talaropeptide D (5): white powder; [α]D 22 −182.9 (c 0.05), MeOH); 1D and 2D NMR (600 MHz, methanol-d4) see **Table 4** and **Supplementary Material**; HRESI(+)MS m/z 1417.9052 [(M+Na)+] (calcd for C72H122N12O15Na, 1417.9045).

#### Marfey's Analyses

Analyses were carried out following the published method (Vijayasarathy et al., 2016). Briefly, an aliquot (50 µg) of each talaropeptide in 6 M HCl (100 µL) was heated to 100◦C in a sealed vial for 12 h, after which the hydrolysate was concentrated to dryness at 40◦C under a stream of dry N2. The hydrolysate was

**Abbreviations:** UHPLC, ultra-high-performance liquid chromatography; HPLC, high-performance liquid chromatography; QTOF, quadrupole time of flight; ESI, electrospray ionization; SIE, single ion extraction; DAD, diode array detector; NRPS, non-ribosomal peptide synthetase; LSI, Latent semantic indexing; ORFX, orphan protein/enzyme.

then treated with 1 M NaHCO3 (20 µL) and L-FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) as a 1% solution in acetone (40 µL) at 40 ◦C for 1 h, after which the reaction was neutralized with 1 M HCl (20 µL) and filtered (0.45µm PTFE) to generate an analyte.

C3 Marfey's analysis. An aliquot (10 µL) of each analyte was subjected to HPLC-DAD-MS analysis (Agilent Zorbax SB-C3 column, 5µm, 4.6 × 150 mm, 50◦C, with a 1 mL/min, 55 min linear gradient elution from 15–60% MeOH/H2O with a 5% isocratic modifier of 1% formic acid in MeCN) with amino acid content assessed by DAD (340 nm) and ESI(±)MS monitoring, supported by SIE (single ion extraction) methodology, with comparison to authentic standards.

C18 Marfey's analysis. An aliquot (10 µL) of each analyte was subjected to HPLC-DAD analysis for (Agilent Zorbax SB-C18 HPLC column, 5µm, 4.6 × 150 mm, 50 ◦C, with a 1 mL/min, 50 min isocratic elution of 21% MeOH/H2O for N-Me-Ala and 34 % MeOH/H2O for N-Me-Phe, with a 5% isocratic modifier of 1% formic acid in MeCN) with amino acid content assessed by DAD (340 nm), with comparison to authentic standards.

#### Genome Mining of Talaromyces sp. CMB-TU011

Genomic DNA from Talaromyces sp. CMB-TU011 was extracted using a standard chloroform protocol (Nikodinovic et al., 2003). The extracted DNA was fragmented using a Covaris focused ultrasonicator and the resulting fragments (∼ 1 KB) were used for library construction using a Thrulex DNA-Seq kit (Rubicon Genomics). The library was sequenced using a Next Seq platform in the paired-end (2 × 150) format to yield a total of 6,674,290 reads (1 GB). The raw reads were filtered and trimmed using Trimmomatic v0.36 (Bolger et al., 2014) to yield a total of 5,821,558 high quality reads (0.873 GB), which were assembled using Velvet 1.2.10 (Zerbino and Birney, 2008), Abyss v.2.0.3 (Simpson et al., 2009) and SPAdes v3.11.1 (Bankevich et al., 2012) assemblers with a window of Kmers between 41 and 121, with iterations every 10 units. The best assembly (Velvet with Kmer =51) was annotated for natural products biosynthetic gene clusters using the Fungal implementation of AntiSMASH 4.0 (Blin et al., 2017). The output was manually curated and domain annotation was improved using pFAM (Finn et al., 2016) and the NCBI Conserved Domain Search tool

#### TABLE 1 | Continued

a−eAssignments with the same superscript are overlapping. f−lAssignments with the same superscript may be interchanged. ndresonances not detected.

(Marchler-Bauer et al., 2017). Adenylation domain specificity was predicted using the LSI based A-domain functional predictor (Baranašic et al., 2014 ´ ). Manual sequence curation was done using the Artemis Genome Browser (Rutherford et al., 2000).

#### Antibacterial Assay

The bacterium to be tested was streaked onto a tryptic soy agar plate and was incubated at 37◦C for 24 h. One colony was then transferred to fresh tryptic soy broth (15 mL) and the cell density was adjusted to 104 -105 CFU/mL. The compounds to be tested

(Continued)

#### TABLE 2 | Continued

a−iAssignments with the same superscript are overlapping. j−rAssignments with the same superscript may be interchanged. ndresonances not detected.

were dissolved in DMSO and diluted with H2O to give 600µM stock solution (20% DMSO), which was serially diluted with 20% DMSO to give concentrations from 600µM to 0.2µM in 20% DMSO. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate and freshly prepared microbial broth (190 µL) was added to each well to give final concentrations of 30−0.01µM in 1% DMSO. The plates were incubated at 37◦C for 24 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate (BMG LABTECH, Offenburg, Germany). Each test compound was screened against the Gram-negative bacteria Escherichia coli ATCC 11775 and Pseudomonas aeruginosa ATCC 10145 and the Gram-positive bacteria Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6051. Rifampicin was used as a positive control (40µg/mL in 10% DMSO). The IC50 value was calculated

Dewapriya et al. Talaropeptides From a Marine-Derived Fungus

#### TABLE 3 | Continued

a−gAssignments with the same superscript are overlapping. h−rAssignments with the same superscript may be interchanged. ndresonances not detected.

as the concentration of the compound or antibiotic required for 50% inhibition of the bacterial cells using Prism 7.0 (GraphPad Software Inc., La Jolla, CA).

#### Antifungal Assay

The fungus Candida albicans ATCC 10231 was streaked onto a Sabouraud agar plate and was incubated at 37◦C for 48 h. One colony was then transferred to fresh Sabouraud broth (15 mL) and the cell density adjusted to 104 -105 CFU/mL. Test compounds were dissolved in DMSO and diluted with H2O to give a 600µM stock solution (20% DMSO), which was serially diluted with 20% DMSO to give concentrations from 600 to 0.2µM in 20% DMSO. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate and freshly prepared fungal broth (190 µL) was added to each well to give final concentrations of 30–0.01µM in 1% DMSO. The plates were incubated at 37◦C for 24 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate (BMG LABTECH, Offenburg, Germany). Amphotericin B was used as a positive control (30µg/ml in 10% DMSO). Where relevant, IC50 value were calculated as the concentration of the compound or antifungal

(Continued)

Dewapriya et al. Talaropeptides From a Marine-Derived Fungus

#### TABLE 4 | Continued

a−fAssignments with the same superscript are overlapping. g−qAssignments with the same superscript may be interchanged. ndresonances not detected.

drug required for 50% inhibition of the fungal cells using Prism 7.0 (GraphPad Software Inc., La Jolla, CA).

#### Cytotoxicity Assay

Adherent cell SW620 (human colorectal carcinoma) and NCI-H460 (human lung carcinoma) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. All cells were cultured as adherent mono-layers in flasks supplemented with 10% fetal bovine serum, L–glutamine (2 mM), penicillin (100 unit/mL), and streptomycin (100µg/mL), in a humidified 37◦C incubator supplied with 5% CO2. Briefly, cells were harvested with trypsin and dispensed into 96-well microtiter assay plates at 3,000 cells/well, after which they were incubated for 18 h at 37◦C with 5% CO2 (to allow cells to attach as adherent monolayers). Test compounds were dissolved in 20% DMSO in PBS (v/v) and aliquots (10 µL) applied to cells over a series of final concentrations ranging from 10 nM to 30µM. After 48 h incubation at 37◦C with 5% CO2 an aliquot (20 µL) of 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffered saline (PBS, 5 mg/mL) was added to each well (final concentration 0.5 mg/mL), and microtiter plates were incubated for a further 4 h at 37◦C with 5% CO2. After final incubation, the medium was aspirated and precipitated formazan crystals dissolved in DMSO (100 µL/well). The absorbance of each well was measured at 580 nm with a PowerWave XS Microplate Reader from Bio-Tek Instruments Inc. Where relevant, IC50 values were calculated using Prism 7.0, as the concentration of analyte required for 50% inhibition of cancer cell growth (compared to negative controls). Negative control was 1% aqueous DMSO, while positive control was doxorubicin (30µM). All experiments were performed in duplicate.

#### Plasma Stability Assay

An aliquot of talaropeptide D (**5**) (10 µL, 1 mM in DMSO) was added to rat plasma (190 µL) pooled from >3 different rats, and heated to 37◦C in a circulating water bath. Aliquots (20 µL) were taken at time points 0, 60, 120, and 180 min and added to MeCN (80 µL). Samples were centrifuged at 13,000 g for 3 min, and the supernatants concentrated to dryness under N2. After resuspending in MeOH (30 µL), and aliquot (1 µL) was analyzed by UHPLC-QTOF (MS), to detect and quantify residual talaropeptide D (**5**).

## RESULTS

#### Production and Isolation

UHPLC-QTOF analysis of MATRIX cultivations (i.e., microbioreactor well, **Figure S1**) revealed that YES static broth cultivation was optimum for the production of talaropeptides (**Figures S2**–**S4**). Reversed phase HPLC-DAD-ESIMS analysis of a 10 day YES static broth cultivation of CMB-TU011 revealed complete suppression of talarolide A (**1**) biosynthesis, in favor of four new higher molecular weight putative peptides, eluting in the order m/z 1254.8 (**2**), 1353.8 (**3**), 1318.8 (**4**), and 1417.9 (**5**) (**Figure S6**). Subsequent studies confirmed the production of **2**–**5** in YES static broth flask cultivations (80 mL broths in 250 mL flasks), with a >10-fold increase in production in flasks sealed with an air permeable cotton plug, as opposed to an air impermeable screw cap (**Figure S5**). Based on these results, scaled up production (160 mL broth in 10 × 500 mL flasks) successfully yielded a crude EtOAc extract (1.65 g), which was subjected to solvent trituration to yield hexane (495 mg) and MeOH (1.15 g) solubles. With analytical HPLC-DAD-ESIMS localizing **2-5** in the MeOH solubles, this material was subjected to gel chromatography (Sephadex LH-20, MeOH) followed by semi-preparative reversed phase HPLC chromatography, to yield talaropeptides A (**2**, 1.3 mg), B (**3**, 1.3 mg), C (**4**, 1.8 mg), and D (**5**, 2.8 mg) (**Figure 1**).

## Talaropeptide A (2)

HRESI(+)MS analysis of **2** returned a protonated molecular ion attributed to a molecular formula (C65H111N11O13, 1mmu−0.1) requiring 16 double bond equivalent (DBEs). Consistent with its putative peptide status, C3 and C18 Marfey's analyses (**Figure 2**), together with careful consideration of 1D and 2D NMR (DMSOd6) data (**Table 1**, **Figures S8**–**S13**), confirmed the presence of 11 amino acid residues [L-Thr, L-Pro, L-Val, L-Leu, N-Me-L-Ala, N-Me-L-Val (×4), N-Me-L-Phe and N-Me-L-Ile]. Whereas the C3 Marfey's method proved very effective at discriminating most amino acids, and in particular L vs D N-Me-Ile and N-Me-allo-Ile (**Figure 2H**), the C18 Marfey's method was needed to discriminate L vs D N-Me-Ala (**Figure 2D,** inset). The presence of multiple (×4) N-Me-L-Val residues was apparent from the complex array of isopropyl methyl resonances in the 1H NMR data for **2** (**Table 1**). This assessment of the amino acid content in **2** accounted for all DBE and was indicative of a linear undecapeptide. Although overlapping 1D NMR resonances prevented assignment of the complete amino acid sequence, diagnostic 2D NMR HMBC and ROESY correlations did identify a number of partial sequences; (i) N-Me-L-Ala1 - N-Me-L-Val2 -N-H (e.g., an HMBC correlation from N-Me-L-Val2 to C-1 in N-Me-L-Ala1 ); (ii) L-Thr4 -N-Me-L-Val5 (e.g., a ROESY correlation between H-2 in L-Thr4 and N-Me-L-Val5 ); (iii) L-Pro6 -N-Me-L-Val7 -N-Me-L-Val8 -N-Me-L-Phe9 (e.g., HMBC correlations from H-2 in N-Me-L-Val7 to C-1 in L-Pro6 , from N-Me-L-Val8 to C-1 in N-Me-L-Val7 , and from H-2 in N-Me-L-Phe9 to C-1 in N-Me-L-Val8 ); (iv) N-Me-L-Ile10-L-Leu11 (e.g., an HMBC correlation from the N-H in L-Leu11 to C-1 in N-Me-L-Ile10) (see **Figure 3**). While the HMBC and ROESY data failed to link fragments (i–iv), or locate L-Val3 , these issues were ultimately resolved by diagnostic UHPLC-QTOF (MS/MS) fragmentation patterns, which identified two consolidated partial sequences; (v) N-Me-L-Ala1 -N-Me-L-Val2 - L-Val3 -L-Thr4 -N-Me-L-Val5 and (vi) N-Me-L-Val8 -N-Me-L-Phe9 -N-Me-L-Ile10-L-Leu11 (**Figure 3**, **Figure S14**). Assembly of the partial sequences i-vi returned the complete structure for talaropeptide A (**2**) as shown (**Figure 1**).

## Talaropeptide B (3)

HRESI(+)MS analysis of **3** returned a sodiated molecular ion attributed to a molecular formula (C70H120N12O14, 1mmu−1.9) requiring 17 DBEs, suggestive of a Val homolog of **2**. The 1H NMR (DMSO-d6) spectrum of **3** (**Figure S16**) revealed resonances closely resembling **2**, however, the extra Val residue was not observed in HSQC (DMSO-d6) spectrum. Therefore, we re-acquired the NMR data in methanol-d4, which revealed resonances attributed to the additional Val residue (δH 3.50, δC 60.3) (**Figure S19**). This hypothesis was confirmed by C3 and C18 Marfey's analyses (**Figure S23**) and 1D and 2D NMR (methanol-d4) data (**Table 2**, **Figures S17**–**S21**), which confirmed the presence of 12 amino acid residues [L-Thr, L-Pro, L-Val (×2), L-Leu, N-Me-L-Ala, N-Me-L-Val (×4), N-Me-L-Phe and N-Me-L-Ile], accounting for all DBE and requiring that **3** be a linear dodecapeptide. Diagnostic 2D NMR HMBC and ROESY correlations identified key partial sequences; (i) N-Me-L-Ala1 -N-Me-L-Val2 (e.g., an HMBC

correlation from H-2 in N-Me-L-Val2 to C-1 in N-Me-L-Ala1 ); (ii) L-Pro6 -N-Me-L-Val7 -N-Me-L-Val8 -N-Me-L-Phe9 -N-Me-L-Ile10 (e.g., HMBC correlations from H-2 in N-Me-L-Val7 to C-1 in L-Pro6 , from N-Me-L-Val8 to C-1 in N-Me-L-Val7 , from N-Me-L-Phe9 to C-1 in N-Me-L-Val8 , and from N-Me-L-Ile10 to C-1 in N-Me-L-Phe9 ) (**Figure 4**). Similarly, diagnostic UHPLC-QTOF (MS/MS) fragmentation patterns identified consolidated partial sequence; (iii) N-Me-L-Ala1 -N-Me-L-Val2 - L-Val3 -L-Thr4 -L-Val∗ -N-Me-Val5 , and (iv) N-Me-L-Val8 -N-Me-L-Phe9 -N-Me-L-Ile10-L-Leu11 (**Figure 4**, **Figure S22**). Assembly of the partial sequences i-iv returned the complete structure for talaropeptide B (**3**) as shown (**Figure 1**), with the following caveat. As N-Me-Val5 and L-Leu11 are isomeric, their relative position cannot be determined by MS/MS fragmentation (or by way of overlapping 1D NMR resonances). To establish the regiochemistry of these amino acid residues, we draw on biosynthetic comparisons to the co-metabolite **2**, as well as knowledge of the talaropeptide biosynthetic gene cluster (see below).

#### Talaropeptide C (4)

HRESI(+)MS analysis of **4** returned a sodiated molecular ion attributed to a molecular formula (C67H113N11O14, 1mmu +1.5) requiring 17 DBEs, suggestive of an acetylated homolog of **2** (i.e., +42 Da). Comparison of the 1H NMR (methanol-d6) data for **4** with **2** supported the latter hypothesis, with the only significant difference being the appearance of resonances attributed to an acetyl moiety (δH 1.99, δC 22.3), with an HMBC correlation from

N-Me-L-Ala1 to N-COCH3 being diagnostic for an N-terminal acetamide moiety. C3 and C18 Marfey's analyses (**Figure S31**) together with 1D and 2D NMR (methanol-d4) data (**Table 3**, **Figures S24**–**S29**), confirmed the presence of 11 amino acid residues (L-Thr, L-Pro, L-Val, L-Leu, N-Me-L-Ala, N-Me-L-Val (×4), N-Me-L-Phe and N-Me-L-Ile), accounting for all DBE and requiring that **4** be a linear undecapeptide. Diagnostic 2D NMR HMBC and ROESY correlations identified key partial sequences; (i) N-Me-N-Ac-L-Ala1 -N-Me-L-Val2 (e.g., an HMBC correlation from H-2 in N-Me-L-Val2 to C-1 in N-Me-N-Ac-L-Ala1 ); (ii) L-Thr4 -N-Me-L-Val5 (e.g., an HMBC correlation from an N-Me-L-Val5 to C-1 in L-Thr4 ), and (iii) L-Pro6 -N-Me-L-Val7 -N-Me-L-Val8 -N-Me-L-Phe9 -N-Me-L-Ile10 (e.g., HMBC correlations from H-2 in N-Me-L-Val7 to C-1 in L-Pro6 , and from N-Me-L-Val8 to C-1 in N-Me-L-Val7 , and from N-Me-L-Phe9 to C-1 in N-Me-L-Val8 , and from N-Me-L-Ile10 to C-1 in N-Me-L-Phe9 ) (**Figure 5**). Similarly, diagnostic UHPLC-QTOF (MS/MS) fragmentation patterns identified the consolidated partial sequence (iv) N-Me-L-Val8 -N-Me-L-Phe9 - N-Me-L-Ile10-L-Leu11 (**Figure 5**, **Figure S30**). Assembly of the partial sequences i–iv returned the complete structure for talaropeptide C (**4**) as shown (**Figure 1**).

#### Talaropeptide D (5)

HRESI(+)MS analysis of **5** returned a sodiated molecular ion attributed to a molecular formula (C72H122N12O15, 1mmu +0.7) requiring 18 DBEs, suggestive of an acetylated homolog of **3** (i.e., +42 Da). Comparison of the 1H NMR (methanol-d4) data for **5** with **3** supported the latter hypothesis, with the only significant difference being the appearance of resonances attributed to an acetyl moiety (δH 1.99, δC 22.5), with HMBC correlations to the N-Me-L-Ala1 position it on the N-terminus. C3 and C18 Marfey's analyses (**Figure S38**) and 1D and 2D NMR (methanol-d6) data (**Table 4**, **Figures S32**–**S36**), confirmed the presence of 12 amino acid residues [L-Thr, L-Pro, L-Val (×2), L-Leu, N-Me-L-Ala, N-Me-L-Val (×4), N-Me-L-Phe and N-Me-L-Ile], accounting for all DBE and requiring that **5** be a linear dodecapeptide. Diagnostic 2D NMR HMBC and ROESY correlations identified key partial sequences; (i) N-Me-N-Ac-L-Ala1 -N-Me-L-Val2 (e.g., HMBC correlations from N-Me-L-Ala1 to N-COCH3, and from H-2 in N-Me-L-Val2 to C-1 in N-Me-L-Ala1 ); (ii) L-Val∗ -N-Me-L-Val5 (e.g., an HMBC correlation from an N-Me-L-Val5 to C-1 in L-Val∗ ), and (iii) L-Pro6 -N-Me-L-Val7 -N-Me-L-Val8 -N-Me-L-Phe9 -N-Me-L-Ile10 (e.g., HMBC correlations from H-2 in N-Me-L-Val7 to C-1 in L-Pro6 , and from N-Me-L-Val8 to C-1 in N-Me-L-Val7 , and from N-Me-L-Phe9 to C-1 in N-Me-L-Val8 , and from an N-Me to C-1 in N-Me-L-Phe9 ) (see **Figure 6**). Similarly, diagnostic UHPLC-QTOF (MS/MS) fragmentation patterns identified the partial sequence (iv) N-Me-L-Val8 -N-Me-L-Phe9 -N-Me-L-Ile10-L-Leu11 (**Figure 6**, **Figure S37**). Assembly of the partial sequences i–iv returned the complete structure for talaropeptide D (**5**) as shown (**Figure 1**), with the following caveat. As the NMR and MS/MS data for **5** could not provide an experimental assignment of relative regiochemistry for the dipeptide fragment comprised of L-Val3 and L-Thr4 , we draw on biosynthetic comparisons to the co-metabolite **3**, as well as TABLE 5 | Draft genome sequence of Talaromyces sp. CMB-TU011.

knowledge of the talaropeptide biosynthetic gene cluster (see below).

#### Talaropeptide Biosynthesis

A genome sequence of Talaromyces sp. CMB-TU011 was obtained, with coverage of 31X length of 27.5 MB, and a GC content of 47 %, consistent with related species (**Table 5**). Natural product genome mining of this sequence identified 17 biosynthetic gene clusters (BGCs, **Table S7**) including three nonribosomal peptide synthetases (NRPS). A very large intron-less mega synthase that includes 12 modules and 44 domains encoded in a single gene (45,892 bases, 15,297 amino acids) was identified as a plausible talaropeptide NRPS. Of note, this NRPS is only 3.2 kb smaller than the largest NRPS ever reported (49,104 bases, plu2670, 16,367 amino acids), being that documented for the extensively N-methylated and commercially important fungal cyclic undecapeptide cyclosporine from Tolypocladium inflatum (GenBank accesion: CAA82227, 15281 amino acids) (Weber et al., 1994).

The putative talaropeptide NRPS (**Figure 7**) exhibits an N-terminus condensation domain with a similar configuration to that of previously reported C domains associated with peptides incorporating N-terminal acyl moieties, consistent with the N-terminal N-acylation observed in talaropeptides C (**4**) and D (**5**). This domain might have been skipped during the biosynthesis of talaropeptide A (**2**) and B (**3**), or alternatively the N-Ac moiety may have been deleted after the biosynthesis (i.e. hydrolysed). A total of 12 adenylation domains were detected, in agreement with the number of amino acid residues found in talaropeptides B (**3**) and D (**5**). Predicted amino acid specificities for these domains are largely in agreement with those observed for **3** and **5**, except for modules 1 and 4 (**Table 6**). Seven methyl transferase domains were consistent with Nmethylation sites in **2**-**5**, with exceptions for N-methylation of residues 1 and 2, which may be installed post NRPS assembly. Alternatively, the methyl transferase in module 3 appears to be inactive on its corresponding extension step (i.e., L-Val3 ), and may be responsible for methylation of the first two residues (i.e., N-Me-N-Ac-L-Ala1 and N-Me-L-Val2 ). The methylation domain at module 5 (L-Val∗ ) appears to be inactive during the biosynthesis of talaropeptides B (**3**) and D (**5**), while the entire module 5 inactive in the biosynthesis of talaropeptides A (**2**) and C (**4**). Finally, a thioesterase domain was detected at the C-terminus of the talaropeptide NRPS, accounting for the release of the peptide product with a C-terminus carboxylic acid.

TABLE 6 | Comparison of a predicted product for ORFX (talaropeptide synthase) with structure for talaropeptide D (5). Adenylation (A) domain specificity was calculated using the LSI based A-domain functional predictor.

domains marked with an asterisk are skipped during biosynthesis, while module 5 (highlight light green) is skipped during talaropeptides A (2) and C (4) biosynthesis.

#### Talaropeptide Biological Activity

Talaropeptides A-D (**2**-**5**) exhibited no growth inhibitory activity when tested (up to 30µM) against human lung (NCI-H460) and colon (SW620) carcinoma cells, or when tested against the Gram-negative bacteria Escherichia coli ATCC 11775 and Pseudomonas aeruginosa ATCC 10145, the Gram-positive bacteria Staphylococcus aureus ATCC 25923 and S. aureus ATCC 9144, or the fungus Candida albicans ATCC 10231 (**Supplementary Material**). By contrast, talaropeptides A (**2**) and B (**3**) alone exhibited promising growth inhibitory activity (IC50 1.5 and 3.7µM) against the Gram-positive bacteria Bacillus subtilis ATCC 6633 (**Figure S41**). As might be predicted for an extensively N-methylated linear peptide, talaropeptide A (**2**) proved stable to rat plasma (i.e., proteases) (**Figure S39**).

## DISCUSSION

Although fungi are well-known to produce cyclic peptides, linear peptides > 7 amino acid residues are comparatively rare (Komatsu et al., 2001; Boot et al., 2006). For example, excluding peptaibols such as the recently described trichodermides (Jiao et al., 2018), which are dominated by non-proteinogenic amino acids [e.g., α-aminoisobutyric acid (Aib) and D–isovaline (D– Iva)], only a handful linear peptides of > 7 amino acid residues have been reported from fungi. Interestingly, these reports feature peptides from marine-derived fungi, including the dodecapeptide dictyonamides A and B from a marine red alga-derived fungus (Komatsu et al., 2001), and N-methylated octapeptides RHM 1 and RHM 2 from a marine sponge-derived Acremonium (Boot et al., 2006). Also of note, no linear peptides have been reported from the genus Talaromyces.

The talaropeptides A-D (**2**-**5**) represent a new class of extensively N-methylated linear peptide natural product, and at the same time feature peptide amino acid sequences that are unprecedented in the scientific literature. That the talaropeptide pharmacophore lacks mammalian cell cytotoxicity, and exhibits highly selective antibacterial properties (albeit with modest potency), with a clear structure activity relationship requirement built around N-terminal acetylation, is intriguing.

From an ecological perspective, the link between antibacterial activity and acetylation suggests that control of N-acetylation, perhaps as a post-NRPS modification by hydrolysis of the acetyl group or by an unknown biosynthetic mechanism that lead to domain skipping, may bias production in favor of **2** and **3** as an antibacterial defense, or **4** and **5** as putative antibacterial prodrugs. In an ecological setting rich in microbial competitors, this putative biosynthetic mechanism of control may be mediated by inter-species or even inter-kingdom chemical communication.

The discovery that talaropeptide production was highly culture media and phase dependent (i.e., YES broth, static flask with an air permeable seal) raises the possibility that, the paucity of published fungal linear peptides may be due to a bias for cultivation conditions that disfavor linear peptides. Our application of systematic miniaturized microbioreactor approach

#### REFERENCES

to trialing cultivation conditions (i.e., MATRIX) provides a low cost, practical means to access this silent potential.

## DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

The GeneBank accession; Bankit212474, talarolide\_synthase – MH479449.

#### AUTHOR CONTRIBUTIONS

RC initiated and oversaw all research. PD performed all fungal cultivations, and isolated and characterized talaropeptides. PD, PP, AS, ZK, and RC performed data analysis and talaropeptide structure elucidations. ZK isolated fungal DNA, and together with PD carried out bioassays. PC-M and EM performed all genomic analyses, and identified the talaropeptide NRPS. RC and PD co-drafted the manuscript.

#### FUNDING

This work was supported in part by The University of Queensland, the Institute for Molecular Bioscience and the Australian Institute for Bioengineering and Nanotechnology.

#### ACKNOWLEDGMENTS

We thank R Damodar for the original isolation of Talaromyces sp. CMB-TU011.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2018.00394/full#supplementary-material

Supplementary Material | The includes fungal cultivation trial details, as well as tabulated and annotated 1D and 2D NMR data and spectra, C3 and C18 Marfey's and MS/MS analyses, and biological assay data for 2–5.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Dewapriya, Khalil, Prasad, Salim, Cruz-Morales, Marcellin and Capon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# α-Glucosidase Inhibitors From the Coral-Associated Fungus Aspergillus terreus

Mengting Liu1†, Changxing Qi 1†, Weiguang Sun1†, Ling Shen1 , Jianping Wang1 , Junjun Liu1 , Yongji Lai 2 , Yongbo Xue1 , Zhengxi Hu1 \* and Yonghui Zhang1 \*

1 Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 2 Department of Pharmacy, the Central Hospital of Wuhan, Wuhan, China

#### Edited by:

Xian-Wen Yang, Third Institute of Oceanography, State Oceanic Administration, China

#### Reviewed by:

Junfeng Wang, South China Sea Institute of Oceanology (CAS), China Valeria Costantino, Università degli Studi di Napoli Federico II, Italy

#### \*Correspondence:

Zhengxi Hu<EMAIL_ADDRESS>Yonghui Zhang<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Medicinal and Pharmaceutical Chemistry, a section of the journal Frontiers in Chemistry

Received: 24 July 2018 Accepted: 24 August 2018 Published: 13 September 2018

#### Citation:

Liu M, Qi C, Sun W, Shen L, Wang J, Liu J, Lai Y, Xue Y, Hu Z and Zhang Y (2018) α-Glucosidase Inhibitors From the Coral-Associated Fungus Aspergillus terreus. Front. Chem. 6:422. doi: 10.3389/fchem.2018.00422 Nine novel butenolide derivatives, including four pairs of enantiomers, named (±)-asperteretones A–D (1a/1b–4a/4b), and a racemate, named asperteretone E (5), were isolated and identified from the coral-associated fungus Aspergillus terreus. All the structures were established based on extensive spectroscopic analyses, including HRESIMS and NMR data. The chiral chromatography analyses allowed the separation of (±)-asperteretones A–D, whose absolute configurations were further confirmed by experimental and calculated electronic circular dichroism (ECD) analysis. Structurally, compounds 2–5 represented the first examples of prenylated γ -butenolides bearing 2-phenyl-3-benzyl-4H-furan-1-one motifs, and their crucial biogenetically related metabolite, compound 1, was uniquely defined by an unexpected cleavage of oxygen bridge between C-1 and C-4. Importantly, (±)-asperteretal D and (4S)-4-decarboxylflavipesolide C were revised to (±)-asperteretones B (2a/2b) and D (4), respectively. Additionally, compounds 1a/1b–4a/4b and 5 were evaluated for the α-glucosidase inhibitory activity, and all these compounds exhibited potent inhibitory potency against α-glucosidase, with IC50 values ranging from 15.7 ± 1.1 to 53.1 ± 1.4 µM, which was much lower than that of the positive control acarbose (IC50 = 154.7 ± 8.1 µM), endowing them as promising leading molecules for the discovery of new α-glucosidase inhibitors for type-2 diabetes mellitus treatment.

Keywords: coral-associated fungus, Aspergillus terreus, butenolide derivatives, structure reassignments, α-glucosidase inhibitors

#### INTRODUCTION

Diabetes mellitus (DM) is one of the most serious chronic diseases with the ever-increasing incidence rates of obesity and aging of the general population throughout the world (Kopelman, 2000) In 2013, it was estimated that over 382 million people all over the world have DM and this number is predicted to increase up to 500 million in 2030, when this disease will be excepted to be the 7th leading cause of death (Lauritano and Ianora, 2016). Globally, type-2 diabetes (non-insulin-dependent DM) covered 90–95% of all the diabetes cases (Lauritano and Ianora, 2016). Postprandial hyperglycemia is an important factor for the induction of type-2 diabetes and complications related to the diseases, such as micro- and macro-vascular diseases (Baron, 1998). A good strategy to maintain the normal level of postprandial plasma glucose is to medicate in

**76**

combination with dietary restriction and an exercise plan (Kim et al., 2008). In type-2 diabetes, delaying glucose absorption after meals by inhibition of α-glucosidase is known to help the therapy (Kim et al., 2008). For diabetic patients, α-glucosidase inhibitors (AGIs) are widely applied either as monotherapy or in combination with other oral hypoglycemic agents or insulin (Hung et al., 2012). However, AGIs-induced serious liver injuries and gastrointestinal side effects restricted the clinical practice (Yin et al., 2014; Kao et al., 2016). In view of the limited number of safe anti-diabetic drugs with low toxicity and everincreasing number of diabetic patients, the exploration for new α-glucosidase inhibitors, attracted, and still attract great interests from scientific community.

In our continuous search for chemically novel and bioactive secondary metabolites from marine fungi (Hu et al., 2014; Liu et al., 2018a,b; Yang et al., 2018), we focused our attention on a coral-associated fungus Aspergillus terreus. A systematic chemical investigation on the ethyl acetate extracts of rice medium of this fungal strain facilitated the isolation and identification of nine novel butenolide derivatives, including four pairs of enantiomers, named (±)-asperteretones A–D **(1a/1b–4a/4b)**, and a racemate, named asperteretone E **(5)**. Structurally, compounds **2**–**5** represented the first examples of prenylated γ -butenolides bearing 2-phenyl-3-benzyl-4H-furan-1-one motifs, and their crucial biogenetically related metabolite, compound **1**, was uniquely defined by an unexpected cleavage of oxygen bridge between C-1 and C-4. Importantly, by an experimental validation method, (±)-asperteretal D and (4S)-4 decarboxylflavipesolide C were revised to (±)-asperteretones B **(2a/2b)** and D **(4)**, respectively. Herein, the detailed isolation, structure elucidation, and α-glucosidase inhibitory activity of these compounds (**Figure 1**) are described.

#### MATERIALS AND METHODS

#### General Experimental Procedures

Optical rotations and IR data were collected from a PerkinElmer PE-341 instrument (PerkinElmer, Waltham, MA, USA) and a Bruker Vertex 70 FT-IR spectrophotometer (Bruker, Karlsruhe, Germany) with KBr pellets, respectively. UV and ECD spectra were collected from a Varian Cary 50 UV/vis spectrophotometer (Varian, Salt Lake City, UT, USA) and a JASCO-810 spectrometer, respectively. 1D and 2D NMR spectra were measured by using a Bruker AM-400 NMR spectrometer (Bruker, Karlsruhe, Germany). The solvent or solvent impurity peaks for methanold4 (δH 3.31 and δC 49.0) and CDCl3 (δH 7.24 and δC 77.23) were referenced to the 1H and 13C NMR chemical shifts. High-resolution electrospray ionization mass spectrometry (HRESIMS) were measured by using a Thermo Fisher LTQ XL LC/MS (Thermo Fisher, Palo Alto, CA, USA). Column chromatography (CC) was carried out by using silica gel (200– 300 mesh; Qingdao Marine Chemical Inc., China), Sephadex LH-20 (40–70 µm, Amersham Pharmacia Biotech AB, Uppsala, Sweden, Sweden), and octadecylsilyl (ODS, 50 µm, YMC Co. Ltd., Japan). Semi-preparative HPLC separations were carried out on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18 (9.4 × 250 mm) and a Daicel IC column (5 µm, 4.6 ×

250 mm, Daicel Chiral Technologies Co., Ltd., China) column. Thin-layer chromatography (TLC) was carried out by using silica gel 60 F254 (Yantai Chemical Industry Research Institute) and RP-C18 F254 plates (Merck, Germany).

#### Fungus Material

The fungus Aspergillus terreus was isolated from the soft coral Sarcophyton subviride, which was collected from the Xisha Island in the South China Sea. This strain was cultivated on potato dextrose agar (PDA) medium and identified by one of the authors (JW), based on its morphological properties and ITS sequence analysis (GenBank access no. MF972904). The fungal strain was reserved in the culture collection of Tongji Medical College, Huazhong University of Science and Technology.

#### Cultivation, Extraction, and Isolation

The strain Aspergillus terreus was cultivated on PDA (Potato Dextrose Agar) medium at 28◦C for 1 week to prepare the seed cultures. Agar plugs were cut into small pieces (approximately 0.5 × 0.5 × 0.5 cm3 ) and then was inoculated in 300 × 500 mL Erlenmeyer flasks which were previously sterilized by autoclaving, each containing 200 g rice and 200 mL distilled water. All flasks were incubated at 28◦C for 28 days. Then, the whole rice solid medium was extracted seven times in 95% aqueous EtOH at room temperature, and the solvent was removed under reduced pressure to afford a crude extract, which was partitioned with ethyl acetate against water to obtain the ethyl acetate soluble part (1.5 kg). The organic extract was separated by silica gel CC (100–200 mesh) with a stepwise gradient elution of petroleum ether–ethyl acetate–MeOH (10:1:0, 7:1:0, 5:1:0, 3:1:0, 1:1:0, 2:2:1, 1:1:1) to afford seven fractions (A–G).

Fraction C (75 g) was separated by an RP-C18 column using MeOH–H2O (from 20:80 to 100:0, v/v) to give five main fractions (C1–C5). Fraction C3 (2.3 g) was further purified by Sephadex LH-20 using CH2Cl2-MeOH (1:1, v/v) to yield two fractions (C3.1–C3.2). Fraction C3.2 was applied to silica gel CC eluted with petroleum ether–ethyl acetate (stepwise 4:1–1:1) to furnish four additional fractions (C3.2.1–C3.2.4). Repeated purification of fraction C3.2.3 (92 mg) by semi-preparative HPLC with CH3CN–H2O (50:50, v/v, 3.0 mL/min) to yield compounds **2** (4.8 mg; tR 12.5 min) and **4** (4.0 mg; tR 28.2 min). Afterwards, compound **2** was further purified by chiral HPLC using a Daicel IC column (isopropanol–n-hexane, 8:92, v/v, 2.0 mL/min) to give **2a** (2.2 mg; tR 60.7 min) and **2b** (2.3 mg; tR 55.2 min). The enantiomers **4a** (1.8 mg; tR 50.5 min) and **4b** (2.0 mg; tR 41.8 min) were obtained by chiral HPLC separation of compound **4** using a Daicel IC column eluted with isopropanol–n-hexane (8:92, v/v, 2.0 mL/min).

Fraction D (198 g) was subjected to an RP-C18 column eluted with MeOH–H2O (from 20:80 to 100:0, v/v) to yield five fractions (D1–D5). Fraction D4 (2.4 g) was applied to Sephadex LH-20 eluted with CH2Cl2-MeOH (1:1, v/v) and further purified by semi-preparative HPLC using CH3CN–H2O (60:40, v/v, 3.0 mL/min) to yield a racemic mixture **3** (9.6 mg; tR 38.2 min). The enantiomers **3a** (1.1 mg; tR 12.5 min) and **3b** (1.8 mg; tR 14.8 min) were further purified by chiral HPLC using a Daicel IC column eluted with isopropanol–n-hexane (8:92, v/v, 2.0 mL/min).

Fraction E (186 g) was chromatographed on silica gel CC (CH2Cl2-MeOH, 1:0–50:1, v/v) to yield five main fractions (E1– E5). Repeated purification of fraction E5 (1.3 g) using Sephadex LH-20 with CH3OH as eluent and RP-C18 column (MeOH–H2O, from 30:70 to 100:0, v/v) to give three mixtures (E5.1–E5.3). Fractions E5.1 (210 mg) was subjected to semi-preparative HPLC (MeOH–H2O, 60:40, v/v, 3.0 mL/min) to afford compound **1** (6.7 mg; tR 43 min). Subsequently, compound **1** was subjected to chiral HPLC using a Daicel IC column (isopropanol–n-hexane,

aRecorded at 400 MHz.

b "m" means overlapped or multiplet with other signals.

cRecorded at 100 MHz.

7:93, v/v, 2.0 mL/min), resulting in the separation of **1a** (2.7 mg; tR 12.4 min) and **1b** (2.5 mg; tR 16.2 min). A racemic mixture **5** (3.2 mg) was isolated by semi-preparative HPLC (MeOH–H2O, 70:30, v/v, 3.0 mL/min) from fraction E5.2 (54.5 mg).

(±)-Asperteretone A (**1**). White, amorphous powder; [α] 25 D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 202 (4.63), 229 (4.06), 279 (3.43) nm; IR νmax = 3435, 2925, 1718, 1623, 1511, 1446, 1381, 1257, 1179, 1047, 829, 573 cm−1 ; HRESIMS m/z 421.1622 [M + Na]+ (calcd for C23H26O6Na, 421.1627); For 1H NMR and 13C NMR data, see **Table 1**.

(−)-Asperteretone A (**1a**). White, amorphous powder; [α] 25 D −34 (c 0.1, MeOH); ECD (c 0.1, MeOH) = 1ε210 −0.85, 1ε227 −1.52.

(+)-Asperteretone A (**1b**). White, amorphous powder; [α] 25 D +37 (c 0.1, MeOH); ECD (c 0.1, MeOH) = 1ε209 +6.18, 1ε226 +5.80.

(±)-Asperteretone B (**2**). White, amorphous powder; [α] 25 D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 202 (4.21), 215 (3.85), 285 (3.50) nm; IR νmax = 3425, 2927, 2853, 1747, 1611,

TABLE 2 | Comparison of the 1H and 13C NMR data for reported (±)-asperteretal D and 2 in CDCl3 (δ in ppm and J in Hz).

aRecorded at 600 MHz.

bRecorded at 400 MHz.

c "m" means overlapped or multiplet with other signals.

dRecorded at 150 MHz.

eRecorded at 100 MHz.

1514, 1440, 1372, 1342, 1269, 1204, 1144, 1113, 1020, 987, 946, 839, 789, 557, 534 cm−1 ; HRESIMS m/z 403.1526 [M + Na]+ (calcd for C23H24O5Na, 403.1521); For 1H NMR and 13C NMR data, see **Table 2**.

(−)-Asperteretone B (**2a**). White, amorphous powder; [α] 25 D −140 (c 0.1, MeOH); ECD (c 0.1, MeOH) = 1ε207−16.99, 1ε232−2.90, 1ε282−10.57.

(+)-Asperteretone B (**2b**). White, amorphous powder; [α] 25 D +136 (c 0.1, MeOH); ECD (c 0.1, MeOH) = 1ε207 +10.92, 1ε232 +3.56, 1ε282 +8.98.

(±)-Asperteretone C (**3**). White, amorphous powder; [α] 25 D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 202 (4.23), 223 (3.77), 285 (3.67) nm; IR νmax = 3483, 2991, 2952, 2878, 1712, 1637, 1453, 1434, 1396, 1351, 1267, 1170, 1133, 1008, 988, 967, 942, 900, 838, 736, 702, 605, 564 cm−1 ; HRESIMS m/z 411.1802 [M + H]+ (calcd for C24H27O6, 411.1808) and m/z 433.1635 [M + Na]+ (calcd for C24H26O6Na, 433.1627); For 1H NMR and 13C NMR data, see **Table 1**.

(−)-Asperteretone C (**3a**). White, amorphous powder; [α] 25 D −135 (c 0.1, MeOH); ECD (c 0.1, MeOH) = 1ε207 −9.13, 1ε232 −3.29, 1ε282 −5.67.

(+)-Asperteretone C (**3b**). White, amorphous powder; [α] 25 D +137 (c 0.1, MeOH); ECD (c 0.1, MeOH) = 1ε208 +9.79, 1ε236 +1.78, 1ε284 +8.67.

(±)-Asperteretone D (**4**). White, amorphous powder; [α] 25 D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 202 (4.62), 221 (4.32), 283 (4.05) nm; IR νmax = 3432, 2974, 2930, 2853, 1756, 1616, 1503, 1444, 1374, 1263, 1208, 1158, 1116, 1028, 994, 837, 670 cm−1 ; HRESIMS m/z 381.1692 [M + H]+ (calcd for C23H25O5, 381.1702) and m/z 403.1513 [M + Na]+ (calcd for C23H24O5Na, 403.1521); For 1H NMR and 13C NMR data, see **Table 1**.

(−)-Asperteretone D (**4a**). White, amorphous powder; [α] 25 D −120 (c 0.1, CH2Cl2); ECD (c 0.1, MeOH) = 1ε207−13.10, 1ε232−3.81, 1ε283−7.07.

(+)-Asperteretone D (**4b**). White, amorphous powder; [α] 25 D +116 (c 0.1, CH2Cl2); ECD (c 0.1, MeOH) = 1ε207 +9.95, 1ε232 +4.60, 1ε282 +9.14.

Asperteretone E (**5**). White, amorphous powder; [α] 25 D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) = 202 (4.55), 218 (4.29), 285 (4.06) nm; IR νmax = 3428, 2965, 2926, 2853, 1750, 1615, 1512, 1441, 1373, 1266, 1207, 1109, 1032, 946, 837, 565 cm−1 ; HRESIMS m/z 421.1626 [M + Na]+ (calcd for C23H26O6Na, 421.1627); For 1H NMR and 13C NMR data, see **Table 1**.

#### ECD Calculations

The theoretical calculations of compounds **1a**/**1b** and **2a**/**2b** were performed using Gaussian 09 and figured using GaussView 5.0 (He et al., 2017a,b,c; Hu et al., 2017). Conformation search using molecular mechanics calculations was performed in the Discovery Studio 3.5 Client with MMFF force field with 20 kcal mol−1 upper energy limit (Smith and Goodman, 2010). The optimized conformation geometries and thermodynamic parameters of all selected conformations were provided. The predominant conformers were optimized at B3LYP/6-31G(d,p) level. The theoretical calculation of ECD was performed using time dependent Density Functional Theory (TDDFT) at the B3LYP/6-31G(d,p) level in MeOH with PCM model (Miertus et al., 1981). The ECD spectra of compounds **1a**/**1b** and **2a**/**2b** were obtained by weighing the Boltzmann distribution rate of each geometric conformation (Tähtinen et al., 2003).

The ECD spectra were simulated by overlapping Gaussian functions for each transition according to:

$$\wedge \varepsilon(E) = \frac{1}{2.297 \times 10^{-39}} \times \frac{1}{\sqrt{2\pi\sigma}} \sum\_{i}^{A} \wedge E\_i R\_i e^{-\left[\left(E - E\_i\right)/\left(2\sigma\right)\right]^2}$$

The σ represented the width of the band at 1/e height, and 1Ei and Ri were the excitation energies and rotational strengths for transition i, respectively. Rvel had been used in this work.

## α-Glucosidase Inhibitory Assay

The α-glucosidase enzyme from Saccharomyces cerevisiae (Sigma Aldrich, USA) solution (1.5 U/mL) was prepared by dissolving the α-glucosidase in 200 M phosphate buffer (pH 6.8). The α-glucosidase enzyme solution (20 µL), test compounds (10 µL) and buffer (40 µL) were pipetted and mixed in a 96 well microtiter plate. The mixture was incubated at 37◦C for 10 min. After incubation, p-nitrophenyl-α-D-glucopyranoside (PNP-G) substrate solution (10 µL, in 20 mM phosphate buffer) was added. The increment of absorbance due to the hydrolysis of PNP-G by α-glucosidase was measured at the wavelength of 410 nm with a microplate reader (Thermo Scientific, Waltham, MA). Acarbose was used as a positive control and averages of three replicates were calculated. The α-glucosidase inhibitory activity was expressed as percentage inhibition and was calculated using the following formula: inhibition (%) = [1**–**(ODsample/ODblank)] × 100. The halfmaximal inhibitory concentration (IC50) was calculated as the compound concentration that is required for 50% inhibition, and the IC50 value of the acarbose was 154.7 ± 8.1 µM.

#### Molecular Docking Simulation

The virtual docking was implemented in the Surflex-Dock module of the FlexX/Sybyl software, which is a fast docking method that allows sufficient flexibility of ligands and keeps the target protein rigid. Molecules were built with Chemdraw and optimized at molecular mechanical and semiempirical level by using Open Babel GUI. The crystallographic ligands were extracted from the active site and the designed ligands were modeled. All the hydrogen atoms were added to define the correct ionization and tautomeric states, and the carboxylate, phosphonate and sulphonate groups were considered in their charged form. In the docking calculation, the default FlexX scoring function was used for exhaustive searching, solid body optimizing and interaction scoring. Finally, the ligands with the lowest-energy and the most favorable orientation were selected.

## RESULTS AND DISCUSSION

(±)-Asperteretone A (**1a**/**1b**) were obtained as white, amorphous powders. The molecular formula C23H26O6 was deduced from the HRESIMS data at m/z 421.1622 [M + Na]+ (calcd for C23H26O6Na, 421.1627), requiring 11 degrees of unsaturation. Its IR spectrum displayed intense absorption bands for hydroxyl (3,435 cm−1 ), carbonyl (1,718 cm−1 ), and aromatic ring (1,623 and 1,511 cm−1 ). The 1H NMR spectrum (**Table 1**) of **1** showed characterized signals for a para-disubstituted phenyl group at δH 7.21 (d, J = 8.4 Hz, H-2', 6') and 6.79 (d, J = 8.4 Hz, H-3', 5'), a 1,3,4-trisubstituted phenyl group at δH 6.60 (d, J = 2.0 Hz, H-2"), 6.59 (d, J = 8.2 Hz, H-5"), and 6.55 (dd, J = 2.0, 8.2 Hz, H-6"), a prenyl group at δH 3.21 (m, H2-7"), 5.27 (m, H-8"), 1.74 (s, H3-10"), and 1.70 (s, H3-11"), a methoxy group at δH 3.52 (s, OMe-4), two methines at δH 3.61 (d, J = 11.5 Hz, H-2) and 3.26 (m, H-3), and a methylene at δH 2.38 (dd, J = 8.9, 13.7 Hz, H-5) and 2.58 (dd, J = 4.0, 13.7 Hz, H-5). The 13C NMR and DEPT spectra (**Table 1**) of **1** showed 23 carbon resonances categorized as two methyls, two methylenes, ten methines (including eight olefinic), eight nonprotonated carbons (including six olefinic, one carboxyl, and one ester carbonyl), and one methoxyl. The diagnostic data above indicated that compound **1** was a butenolide derivative.

The protons and protonated carbon resonances in the NMR spectra were unambiguously assigned through the HSQC experiment. In the HMBC experiment (**Figure 2**), the correlations from H3-10" to C-8", C-9", and C-11" and from H2-7" to C-2", C-3", and C-4", as well as the 1H–1H COZY correlations (**Figure 2**) of H2-7"/H-8" and H-5"/H-6", confirmed the presence of the 1,3,4-trisubstituted phenyl group (subunit A)

with a hydroxyl and a prenyl motif attached at C-4" and C-3", respectively. Besides, the 1H–1H COZY correlations of H-2'/H-3' and H-5'/H-6' and HMBC correlations from both H-2' and H-3' to C-4' (δC 157.8) confirmed the presence of the paradisubstituted phenyl group (subunit B) with a hydroxyl motif attached at C-4'. Subunits A and B were connected through a "-CH2(5)-CH(3)-CH(2)-" group, as confirmed by the 1H–1H COZY correlations of H-2/H-3/H2-5 and HMBC correlations from H2-5 to C-1", C-2", and C-6" and from H-2 to C-1', C-2', and C-6'. In addition, the key HMBC correlations from H-3,

B (2a/2b) and D (4), respectively.

H2-5, and OMe-4 to C-4 (δC 177.4) and H-2 to C-1 (δC 178.3) indicated that a methyl ester and a carboxyl group were attached at C-3 and C-2, respectively. Thus, the planar structure of **1** was determined.

The experimental electronic circular dichroic (ECD) spectrum was measured in MeOH. Surprisingly, no apparent ECD Cotton effects were observed, suggesting that compound **1** was racemic, which adhered well to its lack of optical rotation. Subsequently, compound **1** was separated into two optically pure enantiomers, (−)-asperteretone A (**1a**, [α] 25 D −34) and (+)-asperteretone A (**1b**, [α] 25 D +37) using high performance liquid chromatography (HPLC) on a Daicel IC column (**Figure 3**).

The large coupling constant (J = 11.5 Hz) suggested the trans-relationship of H-2 and H-3, thus two possible optimal conformations existed for its conformation analysis (**Figure 4**). When the relative configuration of **1** was 2S ∗ ,3R ∗ , despite there were obvious NOE correlations of H-2/H2-5 and H-3/H-6' (or H-2'), the diagnostic NOE correlation of H2-5/H-2' (or H-6') was not observed in the NOESY spectrum, indicating that the assumption for 2S ∗ ,3R ∗ -configuration should be wrong. Thus, the relative configuration of **1** was deduced to be 2R ∗ ,3R ∗ , which was completely supported by the NOE correlations of H-2/H2-5 and H-3/H-2' (or H-6'). Accordingly, the relative configuration of **1** was determined to be 2R ∗ ,3R ∗ .

To further confirm the above conclusion and determine the absolute configurations of **1a** and **1b**, a time-dependent density functional theory (TDDFT) method at the at B3LYP/6-31G(d,p) level in MeOH with PCM model was performed for (2S,3R)**- 1**, (2R,3S)**-1**, (2S,3S)**-1**, and (2R,3R)**-1** (**Figure 5**), of which the DFT-calculated ECD spectra of (2S,3S)**-1** and (2R,3R)**-1** showed close similarities to the experimental ECD spectra of **1a** and **1b**, suggesting that the absolute structures of **1a** and **1b** should be assigned as (2S,3S)- and (2R,3R)-configuration, respectively.

(±)-Asperteretone B (**2a/2b**) were also obtained as white, amorphous powders and assigned the molecular formula C23H24O5, as determined from the HRESIMS analysis at m/z 403.1526 [M + Na]+ (calcd for C23H24O5Na, 403.1521) and 13C NMR data. The 1D (**Table 2**) and 2D NMR spectra of **2** were completely identical to that of the reported (±)-asperteretal D (Sun Y. et al., 2018), which drove us to believe that they shared the same structures. The 1,3,4-trisubstituted phenyl group with a C-4" hydroxyl and a C-3" prenyl motif and para-disubstituted phenyl group with a C-4' hydroxyl motif were explicitly confirmed by detailed analysis of the 2D NMR data (**Figure 2**) of **2**. However, a strong four-bond HMBC correlation from H2-5 to C-4 made us confused about the correctness of (±) asperteretal D. After careful examination of the HMBC spectrum (**Figure 2**) of (±)-asperteretal D, key correlations from H-2' to C-3 and from H-4 to C-1' were not observed in the HMBC spectrum; on the contrary, two four-bond HMBC correlations from H-2' to C-2 and from H2-5 to C-4 were observed, which were also found in the HMBC spectrum of **2**. These data above suggested that (±)-asperteretal D should be structurally revised from 2-benzyl-3-phenyl-type to 2-phenyl-3-benzyl-type.

The chiral resolution (**Figure 3**) of **2** afforded a pair of enantiomers, (±)-asperteretone B (**2a/2b**). To determine their absolute configurations, the ECD spectra of (4S)**-2** and (4R)**-2** were calculated by the TDDFT methodology, which matched well with those of (−)-asperteretone B (**2a**) and (+)-asperteretone B (**2b**), respectively, indicating that the absolute stereochemistry of **2a** and **2b** should be 4S- and 4R-configuration, respectively. Furthermore, by comparison of their experimental ECD spectra (**Figure 5**) and similar specific rotation values, the structures of (−)-asperteretal D and (+)-asperteretal D were revised to **2a** and **2b** (**Figure 6**), respectively.

(±)-Asperteretone C (**3a/3b**), obtained as white, amorphous powders, were determined to have the molecular formula

C24H26O6, as deduced from the HRESIMS data at m/z 411.1802 [M + H]+ (calcd for C24H27O6, 411.1808) and m/z 433.1635 [M + Na]+ (calcd for C24H26O6Na, 433.1627), which was indicative of twelve degrees of unsaturation. Its high similarities of NMR data (**Table 1**) with those of **2** suggested that **3** was also a butenolide derivative, with the differences that the paradisubstituted phenyl group was attached with a C-4' methoxy motif in **3** rather than a C-4' hydroxyl motif in **2**, and the 1,3,4-trisubstituted phenyl group attached with a C-4" hydroxyl and a C-3" prenyl motif in **2** was replaced by a 2-(2,3 dihydrobenzofuran-2-yl)propan-2-ol motif in **3**, as supported by the 2D NMR spectra (**Figure 2**), including HMBC and 1H–1H COZY correlations. Thus, the structure of **3** was determined.

Considering the similar structural features of **2** and **3**, we deduced that compound **3** was likely a racemic mixture. As expected, by chiral HPLC resolution (**Figure 3**), two isolates were obtained. Since no apparent Cotton effects were decisive for the absolute stereochemistry of C-8", the experimental ECD spectra (**Figure 5**) of **3a** and **3b** were closely similar to those of (−) asperteretone B (**2a**) and (+)-asperteretone B (**2b**), respectively, indicating that compounds **3a** and **3b** possessed the 4S- and 4Rconfiguration, respectively. Regrettably, the configuration of C-8" was difficult to be determined (Liu et al., 2018b).