Coumarins, dihydroisocoumarins, a dibenzo-α-pyrone, a meroterpenoid, and a merodrimane from Talaromyces amestolkiae

Abstract

Chemical investigation of an organic extract of a fungus isolated from submerged wood collected from fresh water (strain G173), identified as a Talaromyces amestolkiae (Eurotiales; Trichocomaceae), led to the isolation of three coumarins, three dihydroisocoumarins, a dibenzo-α-pyrone, a meroterpenoid, and a merodrimane. Three of the isolated compounds, namely 7-chloropestalasin A (3), 4-hydroxyaspergillumarin (6), and ent-thailandolide B (9) were new. The structures were elucidated using a combination of spectroscopic and spectrometric techniques. The absolute configurations of 2, 3, 5, and 6 were established via a modified Mosher’s ester method, whereas for 9 a combination of TDDFT ECD and ORD calculations were employed. Compounds 1–9 were evaluated for antimicrobial activity against a group of bacteria and fungi.

As part of an ongoing project to uncover new chemistry from nature,^1–5 our group has been investigating freshwater fungi.^6–11 Lignicolous freshwater fungi represent a viable resource for discovering new secondary metabolites with a broad range of biological activities.^12–14

A fungal strain accessioned as G173 and identified as Talaromyces amestolkiae (Eurotiales; Trichocomaceae) was isolated from submerged wood in a small pond near Bur-Mil Park, Guilford County, North Carolina. From an ecological point of view, strain G173 is not a true indweller of freshwater but can be defined as an immigrant species.^{15, 16} Fractionation of the organic extract of G173 using flash chromatography, followed by preparative RP-HPLC, resulted in the isolation of three coumarins (1–3), three dihydroisocoumarins (4–6), a dibenzo-α-pyrone (7), a meroterpenoid (8), and a merodrimane (9), with >97% purity according to UPLC-PDA (Fig. S1).

Compounds 1 (12.2 mg) and 2 (1.0 mg) were isolated as colorless amorphous solids with molecular formulas of C₁₂H₁₂O₅ and C₁₄H₁₆O₅, respectively, as determined by HRESIMS. The NMR (Fig. S2) and HRMS data identified 1 as the known compound, 3-hydroxymethyl-6,8-dimethoxycoumarin (Fig. 1), which was previously isolated from the soil fungus Talaromyces flavus.¹⁷ In addition, 2 was identified as pestalasin A (Fig. S3), a coumarin that was reported from the endophytic fungus Pestalotiopsis sp., which was isolated from the leaves of the Chinese mangrove Rhizophora mucronata.¹⁸ The absolute configuration of 2 was not previously reported; therefore it was assigned via a modified Mosher’s ester method,¹⁹ establishing the configuration as 2′S (Fig. 2 and S4).

Fig. 1.

Structures of compounds 1-9.

Fig. 2.

Δδ_H values [Δδ (in ppm) = δ_S − δ_R] obtained for (S)- and (R)-MTPA esters 2a and 2b for pestalasin A (2), 3a and 3b for 7-chloro-pestalasin A (3), and 6a and 6b for 4-hydroxy-aspergillumarin A (6), in pyridine-d₅.

Compound 3 (0.5 mg) was obtained as a white solid.²⁰ The molecular formula was determined as C₁₄H₁₅ClO₅ by HRESIMS and analysis of ¹H, HMBC, and edited-HSQC NMR data (Table 1 and Fig. S5–S7). The HRMS and NMR data indicated 3 as a chlorinated analogue of 2, which was supported by both the presence of the characteristic isotopic pattern of chlorine in the HRMS data of 3, and the replacement of the meta-coupled aromatic protons (δ_H 6.45 and 6.65 for H-5 and H-6, respectively, J_H-5/H-7 = 2.65 Hz) in 2 (Fig. S3), by a singlet aromatic proton (δ_H 6.70 for H-5) in 3 (Fig. S5). Analyses of the 2D NMR data (Fig. 3) gave the structure of 3, which was ascribed the trivial name 7-chloropestalasin A. The absolute configuration of 3 was assigned via a modified Mosher’s ester method,¹⁹ establishing the configuration as 2′S (Fig. 2 and S8).

Table 1.

¹H and ¹³C NMR data of 3 (400 MHz for ¹H; 100 MHz for ¹³C, CDCl₃) and 6 (700 MHz for ¹H; 175 MHz for ¹³C, CDCl₃)

Position	3		6
	δ C *	δH mult (J in Hz)	δ C	δH mult (J in Hz)
1			168.8
2	161.4
3	128.4		83.3	4.43, ddd (8.6, 3.4, 2.9)
4	137.6	7.95, s	67.4	4.78, dd (8.6, 2.3)
4a	109.6		141.9
5	100.0	6.70, s	116.1	7.07, d (7.5)
6	151.8		137.1	7.53, dd (8.0, 7.5)
7	118.1		117.8	6.98, d (8.0)
8	146.1		162.2
8a	137.9		106.8
9	57.4	3.92, s
10	56.9	3.95, s
1’	41.1	2.64, dd (13.7, 8.2)	30.7	1.76, m
		2.83, dd (13.7, 3.7)		1.92, m
2’	66.7	4.16, m	18.4	1.75, m
				1.90, m
3’	23.7	1.28, d (6.4)	42.9	2.56, ddd (9.2, 6.3, 2.9)
4’			209.2
5’			30.3	2.16, s
4-OH				2.76, br. s.
8-OH				10.91, s

^*13C NMR data for 3 were obtained from HMBC and edited-HSQC spectra.

Fig. 3.

Key HMBC, COSY, and NOESY correlations of 3, 6 and 9.

Compounds 4 (10.5 mg; colorless oil) and 5 (2.0 mg; colorless crystal) were isolated with molecular formulas of C₁₄H₁₆O₄ and C₁₄H₁₈O₄, respectively, as determined by HRESIMS. The NMR (Fig. S9 and S10) and HRMS data identified 4 and 5 as the known dihydroisocoumarins, aspergillumarins A and B, respectively (Fig. 1), which were previously reported from the culture broth of a marine-derived fungus Aspergillus sp. isolated from the fresh leaf of the mangrove tree Bruguiera gymnorrhiza collected from the South China Sea.²¹ The NMR data of 5 matched those reported by Li and co-workers, except for the chemical shift of the 5′-methyl group (δ_H 2.34, d, J = 6 Hz),²¹ which was observed at δ_H 1.22, d, J = 6 Hz (Fig. S10). The absolute configuration of 5 at C-4′ was not determined by Li and co-workers.²¹ Therefore, we attempted to assign the absolute configuration via a modified Mosher’s ester method;¹⁹ however, these results indicated that 5 was a racemic mixture. Indeed, four products were observed, a major and a minor product from each reaction in a 3:1 ratio (Fig. S11).

Compound 6 (0.6 mg) was obtained as a white solid,²² with a molecular formula of C₁₄H₁₆O₅ as determined by HRESIMS along with ¹H, ¹³C, and edited-HSQC NMR data (Table 1, Fig. S12 and S13), establishing an index of hydrogen deficiency of 7. The NMR data suggested 6 as a dihydroisocoumarin analogue of 4. A key difference was replacement of the allylic methylene moiety (δ_H/δ_C 2.93/34.1, m, for H₂-4/C-4) in 4 by an oxymethine in 6 (δ_H/δ_C 4.78/67.4, dd, J = 8.6, 2.3 for H-4/C-4). These data, along with a 16 amu difference in the HRMS data between 4 and 6, indicated hydroxylation at the C-4 position in 6. The coupling constant (J_H-4/H-3 = 8.6 Hz) implied a pseudoaxial/pseudoequatorial trans orientation in 6 (Table 1, Fig. S12). A NOESY correlation observed between 4-OH and H-3 indicated that these two protons were on the same face (Fig. 3 and S15). Analyses of the COSY and HMBC NMR data (Fig. 3 and S14), established the structure of 6, which was given the trivial name 4-hydroxyaspergillumarin A. The absolute configuration of 6 was assigned via a modified Mosher’s ester method¹⁹ as 4S (Fig. 2 and S16).

Compounds 7 (5.8 mg) and 8 (6.3 mg) were isolated as colorless crystalline solids and identified using HRMS and NMR data as graphislactone A (a dibenzo-α-pyrone)²³ and berkeleyacetal C (a meroterpenoid)²⁴ (Fig. S17 and S18), respectively. Graphislactone A was first isolated from the lichen Graphis scripta var. pulverulenta,²⁵ while berkeleyacetal C was isolated from extracts of a Penicillium sp.²⁴

Compound 9 (2.9 mg) was obtained as a white solid,²⁶ with a molecular formula of C₂₇H₃₂O₈ as determined by HRESIMS and NMR data (Table S3 and Fig. 3 and S19–S22), establishing an index of hydrogen deficiency of 12. The HRMS and NMR data of 9, including the NOESY spectrum, were identical to that of thailandolide B, a merodrimane isolated from Talaromyces thailandiasis.²⁷ However, the specific rotation of 9 ([α]_D²⁰ −47, CHCl₃, c 0.05) was found to be opposite to that of thailandolide B ([α]_D²⁴ +134, CHCl₃, c 0.1), suggesting that 9 could be an enantiomer of thailandolide B.²⁷ Thus, the absolute configuration of 9 was determined using electronic circular dichroism (ECD) and optical rotatory dispersion (ORD) spectroscopy combined with time-dependent density functional theory (TDDFT) and quantum chemical calculations. The calculated TDDFT-ECD spectrum of 9 matched the measured data, displaying two positive (~230 and ~310 nm) and two negative (~270 and ~350 nm) Cotton effects, respectively (Fig. 4). The calculated spectra for thailandolide B was, as expected, opposite to 9 (Fig. 4). Unfortunately, no experimental data were published for thailandolide B for comparison purposes. However, the calculated ORD value for 9 ([α]_D²⁰ −88.5 in CHCl₃) agreed with the experimental data. Thus, the absolute configuration of 9 was established as 5S,7R,8S,9S,10R,18S,19S and given the trivial name ent-thailandolide B.

Fig. 4.

Comparison of experimental and calculated ECD spectra of 9 and thailandolide B in MeOH.

Compounds 1–9 were tested for antimicrobial activity against a group of bacteria and fungi²⁸ and found to be inactive.