Antibacterial Polyketides from the Deep-Sea Cold-Seep-Derived Fungus Talaromyces sp. CS-258

Abstract

Thirty-two fungal polyketide derivatives, including eleven new compounds, namely (3R,5′R)-5-hydroxytalaroflavone (1), talaroisochromenols A–C (3, 5, and 11), (8R,9R,10aR)-5-hydroxyaltenuene (13), (8R,9R,10aS)-5-hydroxyaltenuene (14), (8R,9S,10aR)-5-hydroxyaltenuene (15), nemanecins D and E (25 and 26), 2,5-dimethyl-8-iodochromone (27), and talarofurolactone A (29), together with one new naturally occurring but previously synthesized metabolite, 6-hydroxy-4-methoxycoumarin (28), were isolated and identified from the deep-sea cold-seep-derived fungus Talaromyces sp. CS-258. Among them, racemic ((±)-11) or epimeric (13–15, 25, and 26) mixtures were successfully separated by chiral or gradient elution HPLC. Meanwhile, compound 27 represents a rarely reported naturally occurring iodinated compound. Their planar structures as well as absolute configurations were determined by extensive analysis via NMR, MS, single-crystal X-ray diffraction, Mosher’s method, and ECD or NMR calculation (with DP4⁺ probability analysis). Possible biosynthetic routes of some isolated compounds, which are related to chromone or isochromone biosynthetic pathways, were put forward. The biological analysis results revealed that compounds 7, 9, 10, 18–22, 24, 30, and 31 showed broad-spectrum antibacterial activities against several human and aquatic pathogens with MIC ranges of 0.5–64 μg/mL.

1. Introduction

Polyketides are a big family of secondary metabolites generally produced from a common origin of acetyl-CoA polymerization by connecting acetic acid units via condensation reactions and intermediated by a non-reducing group of iterative polyketide synthases (PKSs) [1,2]. Fungal polyketides have attracted considerable attention due to their unique molecular scaffolds and diverse pharmacological activities [3,4,5]. Among them, a series of fungal polyketides, such as griseofulvin, brefeldin, and hypomycetin, have been selected as promising candidates for new antibiotic lead compounds and possess great commercial values [1,3,5].

Deep-sea cold-seep-derived fungi have formed a unique physiological process and metabolic mechanism during their adaptation to the typical chemosynthetic-driven ecosystems with hydrocarbon-rich fluid, strikingly emerging as a promising source for their prolific production of structurally diverse secondary metabolites with various biological properties in very recent years [6,7,8]. Increasing efforts have been made on deep-sea cold-seep fungi to seek and develop biologically active pharmaceutical molecules with antibiotic [7,9,10], antioxidant [11], phytotoxic [12], anti-inflammatory [13], cytotoxic [14], proangiogenic [15,16], and enzyme inhibitory [17] properties.

In the course of forwarding our program on discovering bioactive metabolites from deep-sea cold seep-derived fungi [6,15,16], a fungal strain Talaromyces sp. CS-258, which was isolated from a mussel sample collected in a cold seep area in the northeast of the South China Sea at a depth of −1200 m, displayed diverse classes of polyketide derivatives and exhibited antimicrobial activity in a preliminary screening. Intensive chemical investigations on the fermentation broth of the fungus resulted in the isolation and identification of 32 polyketides (Figure 1), including 11 new chromone- or isochromone-derived analogs (compounds 1, 3, 5, 11, 13–15, 25–27, and 29) and 1 new naturally occurring coumarin (28) that was previously obtained by chemical synthesis [18]. The isolated compounds were assayed for their antimicrobial activities against pathogenic microbes, which demonstrated that eleven polyketides, including 7, 9, 10, 18–22, 24, 30, and 31, possessed broad-spectrum antibiotic activities against human and aquatic pathogenic bacteria with MIC values ranging from 0.5 to 64 μg/mL. Herein, the details of isolation, structural elucidation, plausible biosynthetic pathways, and bioactivity assays of these compounds are described. The structure–activity relationships (SARs) for these antibacterial polyketides have been briefly discussed in this paper.

2. Results and Discussion

2.1. Structure Elucidation

The molecular formula of compound 1 was given as C₁₃H₁₀O₆ from HRESIMS data with nine degrees of unsaturation. A comprehensive analysis of its ¹H, ¹³C, and DEPT NMR spectra (

Table 1. ¹H and ¹³C NMR spectroscopic data of 1, 3, and 5 in DMSO-d₆ (δ in ppm, J in Hz).

No.	1		3		5
1		166.9 C
2				168.2 C		166.9 C
2a				99.1 C 1		97.8 C
3		91.4 C		165.0 C		166.3 C
3a		149.9 C
4	5.76, d (1.6)	100.8 CH	6.44, s	103.2 CH	6.33, s	101.4 CH
5		164.6 C		163.9 C		163.8 C
6	6.34, s	103.3 CH	6.98, s	106.9 CH	6.37, s	102.1 CH
6a				133.1 C		137.2 C
7a		103.5 C		130.1 C		118.1 C
7		158.7 C		150.8 C 1	6.89, dd (6.9, 2.6)	42.0 CH
8	1.76, s	12.9 CH3		197.0 C	3.67, m	83.3 CH
9			3.12, d (18.0) 2.88, d (18.0)	46.8 CH2	4.47, d (2.4)	78.1 CH
9a				81.4 C		153.1 C
10			1.70, s	27.3 CH3	1.29, d (6.9)	18.5 CH3
2′		169.2 C
3′	6.36, s	129.8 CH
4′		200.1 C
5′	4.61, s	78.2 CH

¹ Observed in HMBC spectrum.

) revealed great similarity to talaroflavone (2), a previously described compound isolated from the sponge-derived fungus Alternaria sp. F49 [19]. However, the signals of a methoxy group at δ_C/H 55.8/3.75 at C-5 in the NMR spectra of 2 were absent in those of 1. In comparison to 2, obvious upfield shifts for C-5 in 1 were detected. These differences suggested that compound 1 was a 5-demethylation derivative of 2, which was further supported by the HMBC correlations of H-4/H-6 to C-5 (Figure 2). From a biosynthetic point of view, the stereoscopic configuration of 1 was assumed to be the same as that of 2. After slow evaporation of the MeOH solvent, single crystals of 2 were obtained. The relative configuration of 2 was thus assigned by the result of a single-crystal X-ray diffraction experiment using Cu Kα radiation as 3S*, 5′S* (Figure 3). Accordingly, the relative configuration of 1 was also established as 3S*, 5′S*, the same as 2. The absolute configuration of 1 was further studied by the time-dependent density functional (TDDFT) ECD calculation. The calculated ECD curve of (3R,5′R)-1 agreed well with the experimental ECD curve of 1 (Figure 4), which ascertained compound 1 as (3R,5′R)-5-hydroxytalaroflavone.

The protonated molecular ion peak at m/z 261.0398 [M − H]^– in HRESIMS revealed the molecular formula of C₁₃H₁₀O₆ for 3 with nine degrees of unsaturation. As shown in Table 1, the ¹H NMR spectrum exhibited signals for two singlet aromatic protons (δ_H 3.26 and 2.94), one doublet methylene (δ_H 3.12 and 2.88), and one singlet methyl (δ_H 1.70). The ¹³C and DEPT NMR spectra data of 3 indicated the presence of 13 carbon signals, which were sorted into one methyl, one methylene, two methines, and nine quaternary carbons. Detailed analysis of the NMR data revealed that the structure of 3 was similar to that of compound 4, an altenusin analog bearing a 6/6/5 tricyclic ring skeleton obtained from the mangrove endophytic fungus Alternaria sp. SK6YW3L [20]. However, resonances for a methoxy group of 4 were absent in the NMR spectra of 3, which suggested that 3 was a 5-demethylated derivative of 4. The above deduction was supported by HMBC correlation from H-4 to C-5 (Figure 2). The absolute configuration of 3 was established as 9aS based on the quantum chemical calculation of ECD (Figure 4) and nominated as talaroisochromenol A.

Compound 5 was afforded as a yellow oil, and the molecular formula of C₁₃H₁₂O₆ for 5 was assigned by its positive HRESIMS. The NMR spectra of 5 were identical to those of 6, a known altenusin derivative with a 6/6/5 tricyclic ring [20], except for the obvious differences in the chemical shifts of CH-8, CH-9, and CH₃-10. The above observation suggested that 5 was a new epimer of 6 (epimeric at the C-9 position), which was further proved by the NOESY correlations from H-7 to H-9 and from H₃-10 to H-8 (Figure 5). Hence, the absolute configuration of 5 was assigned as 7R, 8S, 9R, which was further confirmed by comparison of its calculated ECD for 7R, 8S, 9R-5 with the measured ECD spectra (Figure 4). Thus, compound 5 was acknowledged as a new compound, namely talaroisochromenol B.

Compound 11 possessed a molecular formula of C₁₂H₁₀O₆ as established from its HRESIMS spectrum, accounting for eight degrees of unsaturation. In the ¹H NMR spectra, resonances for two phenolic hydroxyl groups (δ_H 11.08 and 10.99), two meta-substituted aromatic protons (δ_H 6.62, d, J = 2.2 Hz; δ_H 6.46, d, J = 2.2 Hz), one singlet oxymethine (δ_H 5.49), a chemically nonequivalent methylene (δ_H 3.26 and 2.94), and one singlet methyl (δ_H 1.51) were observed. The investigation of ¹³C NMR and HSQC spectral data displayed attribution signals of two lactone carbonyl groups, six aromatic carbons (including two methines and four quaternary carbons), one oxygenated quaternary carbon, one oxygenated methine, one methylene, and one methyl. These data demonstrated great similarity to those of 12, a known polyketide yielded from an endolichenic fungus Ulocladium sp. [21], except for the absence of a methoxy group at C-8 (

Table 2. ¹H and ¹³C NMR spectroscopic data of 11 in DMSO-d₆ (δ in ppm, J in Hz).

No.	11
2		173.2 C
3	3.26, d (17.6) 2.94, d (17.6)	42.2 CH2
3a		85.9 C
5		166.0 C
5a		97.8 C
6		163.5 C
7	6.46, d (2.2)	104.3 CH
8		165.2 C
9	6.62, d (2.2)	110.9 CH
9a		133.3 C
9b	5.49, s	76.9 CH
10	1.51, s	19.9 CH3
6-OH	10.99, s
8-OH	11.08, s

). Compared to 12, the obvious upfield shift of C-8 in 11 was observed. Thus, 11 was determined as an 8-hydroxylated derivative of 12.

The relative configuration of 11 was determined by the NOESY correlation between H-9b and H₃-10 as 3aS*, 9aS* (Figure 5). The planar structure and relative configuration of 11 were further verified by an X-ray diffraction experiment (Figure 3). However, compound 11 was acquired as a racemate with an optical rotation value near zero, and no obvious Cotton effect was observed from the ECD spectrum. The chiral HPLC separation of 11 on a Chiralcel IG column (hexane/isopropanol = 80:20, flow rate of 1 mL/min) succeeded in separating two isomers (Figure S2). Finally, as shown in Figure 4, the ECD computation allowed the assignment of absolute configurations for two isomers as (+)-11-(3aR, 9aR) and (−)-11-(3aS, 9aS), respectively. Thus, 11 was named talaroisochromenol C.

Compounds 13–15 were originally isolated as yellow oils and owned the same molecular formula of C₁₄H₁₄O₆ based on their positive HRESIMS data. The UV spectra of 13–15 showed similar characteristic absorptions for an isocoumarin chromophore at λ_max 242, 282, and 323 nm [22]. Subsequent interpretation of their NMR data (

Table 3. ¹H and ¹³C NMR spectroscopic data of 13–15 in DMSO-d₆ (δ in ppm, J in Hz).

No.	13		14		15
2		168.3 C		167.8 C		167.7 C
2a		98.8 C		98.3 C		98.1 C
3		163.1 C		163.3 C		163.2 C
4	6.29, s	102.3 CH	6.26, d (2.1)	102.6 CH	6.28, overlap	102.7 CH
5		165.1 C		165.5 C		165.8 C
6	6.53, s	103.4 CH	6.47, d (2.1)	103.3 CH	6.53, d (2.2)	103.8 CH
6a		139.4 C		137.7 C		138.2 C
7a		132.2 C		131.4 C		133.5 C
7	6.14, d (3.1)	130.3 CH	6.05, d (2.5)	130.3 CH	6.28, overlap	127.8 CH
8	3.95, t (4.0)	69.7 CH	4.07, dd (7.9, 2.5)	72.5 CH	4.08, t (4.6)	64.9 CH
9	3.68, dt (7.9, 3.9)	68.8 CH	3.59, ddd (12.0, 7.9, 3.7)	70.2 CH	3.66, dt (12.3, 3.7)	65.4 CH
10	β 2.24, dd (14.0, 3.5) α 1.93, dd (14.5, 8.1)	38.7 CH2	β 2.14, dd (12.0, 3.7) α 2.01, t (12.3)	43.5 CH2	β 2.21, t (12.0) α 1.90, dd (11.6, 3.3)	38.4 CH2
10a		80.9 C		82.0 C		81.6 C
11	1.45, s	27.4 CH3	1.48, s	26.2 CH3	1.43, s	25.8 CH3
3-OH	11.22, s		11.25, s		11.28, s

) revealed that compounds 13–15 were characterized as three new stereoisomers of a known analog 5-hydroxyaltenuene, which were isolated from an endophytic fungus Penicillium sp.FJ-1 of Ceriops tagal [22].

The relative configurations of 13–15 were assigned by NOESY, X-ray single-crystal diffraction experiment, or NMR calculations with DP4+ probability analysis. As shown in Figure 5, NOESY correlations from Hα-10 to H-8 and H₃-11 implied the α-orientation of CH₃-11 and β-orientation of OH-8 in 13. Meanwhile, NOESY correlations from H₃-11 to H-9/Hβ-10 and from H-8 to Hα-10 suggested a syn orientation of CH₃-11 and H-9 and an opposite orientation of H-9 and H-8 in 14. The relative configurations of 13 and 14 were further confirmed by single-crystal X-ray diffraction experimental data as (8R*,9R*, 10aR*)-13 and (8S*,9S*,10aR*)-14 (Figure 3). Moreover, NOESY correlations of 15 from H₃-11 to H-9 and Hα-10 established its relative configuration of 9S*, 10aR*. The experimental ¹H and ¹³C NMR data of 15 were compared with the calculated NMR data of 15a and 15b (two possible isomers of 15, Figure 6) and matched well with those calculated for the isomer 15a (8R*,9S*,10aR*) with a DP4+ probability of 100% (Table S16). The absolute configurations of 13–15 were determined by comparing the experimental ECD data with the calculated ones (Figure 4), suggesting 13–15 to be (8R,9R,10aR)-, (8R,9R, 10aS)- and (8R,9S,10aR)-5-hydroxyaltenuenes, respectively. Notably, the orientation of the methyl group at C-11 significantly determined the ECD Cotton effects observed for 5-hydroxyaltenuene. This was supported by the observation that compounds 13 and 15, with 10aR absolute configurations, displayed positive Cotton effects at 236 nm and negative ones at 282 nm, in contrast to 14 with a 10aS absolute configuration. This was the first isolation of three new epimers of 5-hydroxyaltenuene from the fungus Talaromyces sp.

Compounds 25 and 26 were both obtained as yellow oily substances with similar chromatographic properties in the semipreparative HPLC separation [t_R (25) = 14 min, t_R (26) = 12 min]. The HRESIMS analysis displayed pseudomolecular ion peaks at m/z 255.1228 [M + H]⁺ (calcd for 255.1227) of 25 and at m/z 255.1226 [M + H]⁺ (calcd for 255.1227) of 26, assigning their molecular formula both as C₁₃H₁₈O₅ holding five degrees of unsaturation. Their UV spectra exhibited absorption maxima at 209 and 320 nm, revealing structural analogies between 25 and 26. A detailed inspection of ¹H and ¹³C NMR spectra of 25 and 26 (

Table 4. ¹H and ¹³C NMR spectroscopic data of 25–26 in DMSO-d₆ (δ in ppm, J in Hz).

No.	25		26
1	4.56, dd (10.7, 5.4) 3.69, dd (13.4, 10.7)	68.6 CH2	4.56, dd (10.7, 5.4) 3.67, m	68.6 CH2
3		165.0 C		164.9 C
4	5.58, s	101.8 CH	5.59, s	101.7 CH
4a		151.0 C		150.9 C
5	5.54, d (2.2)	114.4 CH	5.55, d (2.1)	114.3 CH
6		200.6 C		200.5 C
7		77.1 C		77.0 C
8	3.45, dd (10.1, 4.5)	73.7 CH	3.45, dd (10.1, 4.6)	73.6 CH
8a	2.58, m	37.7 CH	2.59, m	37.7 CH
9	2.25, m	44.2 CH2	2.32, dd (14.0, 6.8) 2.17, dd (14.0, 6.2)	44.1 CH2
10	3.86, dq (12.1, 6.2)	64.1 CH	3.88, m	64.0 CH
11	1.07, d (6.2)	18.6 CH3	1.07, d (6.2)	18.6 CH3
12	1.10, s	23.4 CH3	1.10, s	23.3 CH3
7-OH	4.93, s		4.92, s
8-OH	5.33, d (4.5)		5.31, d (4.6)
10-OH	4.69, d (5.0)		4.69, d (5.2)

) prominently indicated practically identical signals, suggesting that they shared the same planar structural scaffold. Both ¹H NMR spectral data of 25 and 26 exhibited characteristic resonances ascribed to two olefinic protons (represented for two trisubstituted double bands), three sp³-hybridized methines (including two oxygenated), two sp³-hybridized methylenes (including one chemically nonequivalent O-substituted), two methyl groups (as a singlet and a doublet), and three exchangeable protons. Analyses of their ¹³C, DEPT, and HSQC spectrum confirmed the presence of a carbonyl group, two pairs of olefin carbons, two oxygenated methines, one sp³-hybridized methine, two methylene groups, two methyls, and one quaternary carbon.

The ¹H-¹H COSY and HMBC correlations of 25 and 26 shown in Figure 2 established the same planar structure, which was similar to nemanecins A–C, three azaphilone analogs isolated from the culture broth of the fungus Nemania sp. BCC 30850 [23], indicating that 25 and 26 are a pair of stereoisomers. However, the signal of a singlet methyl in the ¹H and ¹³C NMR spectra of nemanecins A–C were absent in those of 25 and 26. Instead, signals for a 2-hydroxypropyl group were observed in the NMR spectra of 25 and 26. The above inference was further confirmed by the key ¹H-¹H COSY correlation from H-10 to H-9 and H-11 as well as the observed HMBC correlations from H-9 to C-3, C-4, and C-10 and from H-11 to C-9 and C-10 (Figure 2). Thus, the planar structures of 25 and 26 were assigned.

The relative configurations of compounds 25 and 26 of C-7, C-8, and C-8a were both assigned as 7S*,8R*, and 8aR* based on the NOESY analysis and scalar coupling constant data (Figure 5). Both of them displayed a NOESY correlation from H₃-10 to H-8a, which implied that H₃-10 and H-8a were co-facial and arbitrarily assigned in β-orientation. The large coupling constant between H-8a and H-8 (J = 10.1 Hz) confirmed the trans-relationship of these protons.

The modified Mosher’s method using an NMR tube [24,25] was used to determine the absolute configuration of C-10 in 25 and 26, leading to the assignments of S- for 25 and R-absolute configuration for 26 based on Δδ values (Figure 7). ECD calculations were performed to further solve the absolution configurations of C-7, C-8, and C-8a in 25 and 26. The experimental ECD spectra of 25 and 26 both exhibited a strong positive Cotton effect at 214 nm and a negative one at 345 nm, which was in good agreement with the calculated ECD curve of (7S,8R,8aR,10S)-25 and (7S,8R,8aR,10R)-26 (Figure 5). The results of ECD calculations indicated that the configuration of C-10 made no contribution to the ECD Cotton effects. Thus, the structures of compounds 25 and 26 were finally identified and named nemanecins D and E, respectively.

An analysis of the HRESIMS spectrum for m/z 316.9668 [M + H]⁺ (calcd for 316.9669) determined the molecular formula of compound 27 (yellow oil) as C₁₁H₁₀O₃I requiring seven degrees of unsaturation. Its ¹H and ¹³CNMR data (

Table 5. ¹H and ¹³C NMR spectroscopic data of 27–29 in DMSO-d₆ (δ in ppm, J in Hz).

No.	27		28		29
2		160.9 C		161.9 C		170.5 C
3	5.75, s	109.8 CH	5.85, s	90.3 CH		126.5 C
4		176.9 C		165.7 C	3.09, dd (17.5, 8.6) 2.55, dd (17.0, 3.0)	31.5 CH2
4a		108.7 C 1		115.6 C
5		138.4 C	7.10, d (2.8)	106.8 CH	5.03, q (7.1)	77.4 CH
6	6.19, s	120.0 CH		153.7 C
7		159.1 C	7.06, dd (8.8, 2.9)	120.7 CH
7a
8		75.3 C	7.25, d (8.8)	117.5 CH
8a		- 2		146.0 C
9	2.22, s	19.1 CH3
10	2.48, s	20.4 CH3
1′					5.73, m	128.7 CH
2′					5.83, m	135.4 CH
3′					3.98, brs	71.6 CH
4′					3.30, m	66.2 CH2
5′
1″					6.54, tt (7.6, 3.0)	141.4 CH
2″					2.16, m	23.3 CH2
3″					1.02, t (7.5)	12.9 CH3
4-OMe			3.99, s	57.0 CH3
6-OH			9.90, brs
3′-OH					4.92, brs
4′-OH					4.64, brs

¹ Observed in HMBC spectrum, ² missed signal.

) were in good accord with the NMR spectral information of the known chromone derivative 2,5-dimethychromone [26], except that the signal of the methine of C-8 resonating at δ_C 100.4 in the NMR spectra of 2,5-dimethychromone was replaced by a deprotonated carbon resonating at δ_C 75.3 in those of 27. The chemical shift of C-8 at δ_C 75.3 suggested the substitution by an iodine atom according to the previously reported compounds 2-iodo-5-methoxyphenol (δ_C 74.4) [27] and (aS)-6-iodofonsecinone A (δ_C 79.3) [28]. Thus, the structural assignment of 27 was assigned as 2,5-dimethy-8-iodochromone as shown in Figure 1, which was further confirmed by the HMBC correlations from H₃-9 to C-2/C-3, H-3 to C-2/C-4a, and H-10 to C-4a/C-5/C-6 (Figure 2).

Compound 28 was obtained as a yellow oil, and its ¹H and ¹³C NMR spectra data (Table 5) exactly matched those of the known compound 6-hydroxy-4-methoxycoumarin synthesized in the previous literature [18]. It is now isolated for the first time from a natural source. We supplemented its relevant NMR, MS, and UV data here.

The HRESIMS data established the molecular formula of compound 29 as C₁₁H₁₆O₄ with four degrees of unsaturation. As shown in Table 5, the ¹H and ¹³C NMR spectrum displayed signals corresponding to two quaternary carbons (one lactone and one olefinic), five methines (three olefinic and one oxygenated), three methylenes (one oxygenated), and one methyl. The ¹H-¹H COSY correlations from H-4 to H-5 and HMBC correlations from H₂-4 to C-2 and C-3 as well as H-5 to C-4 established a dihydrofuran-2(3H)-one moiety. The assignment for a spin coupling system of [=CHCH₂CH₃] was identified by ¹H-¹H COSY correlations from H-2” to H-1” and H-3”. Furthermore, a 3,4-dihydroxybut-1-en-1-yl group was assigned by ¹H-¹H COSY correlations from H-2′ to H-1 and H-3′, from H-3′ to H-4′ and 3′-OH, and from H-4′ to 4′-OH. The moiety of [=CHCH₂CH₃] spin system was attached to C-3 outside the lactone ring with regard to the HMBC correlations from H-1″ to C-2 and C-4 and from H-2″ to C-3. In addition, the 3,4-dihydroxybut-1-en-1-yl group was bonded to C-5, as proven by the ¹H-¹H COSY correlation from H-1′ to H-5 and HMBC correlations from H-1′ to C-4 and C-5 and from H-2′ to C-5. The E-configuration of double bonds Δ^{1′, 2′} was assigned based on the large coupling constants of J = 15.5 Hz, while the cis relation of Δ^{3, 1″} was ascertained by NOESY correlation between H₂-4 and H-1″ (Figure 8A).

The relative configuration of 29 was determined by NMR calculation with DP4⁺ analysis. As a result, the experimental NMR data of 29 corresponded to the computed NMR data for (5R*,3′S*)-isomer 29b (99.12% probability, Table S19 in the supplementary material). The absolute configuration of 29 was assigned by ECD calculation. The calculated ECD curves of (5R,3′S)-29 coincided with the measured curve, which demonstrated the absolute configuration of 29 as 5R, 3′S (Figure 8B). Thus, the structure of 29 was established, and it was named talarofuranone A trivially_.

In addition, based on the comparison of their NMR and optical rotation data with those reported in the literature, the structures of the other sixteen polyketides were identified as phialophoriol (7) [29], (1S,3S)-2,3-dihydro-3,6,8-trihydroxy-1-methylcyclopenta[c][2]-benzopyran-5(1H)-one (8) [20], (S)-1,2-dihydro-6,8-dihydroxy-1-methylcyclo-penta[c][2]-benzopyran-3,5-dione (9) [20], (S)-1-deoxyrubralactone (10) [30], (8R,9R,10aR)-altenuene (16) [31], (8S,9S,10aR)-altenuene (17) [31], alternariol 9-O-methyl ether (18) [32,33], alternariol (19) [33,34], 3′-hydroxyalternariol-5-O-methyl ether (20) [34], 3-O-demethylaltenuisol (21) [35], 4-hydroxyalternariol (22) [36,37], alterlactone (23) [34], alteryulactone (24) [38], purpactin A (30) [39], penicillide (31) [39,40], and dehydroisopenicillide (32) [41]. Among them, compounds 1–24 are altenusin or alternariol derivatives with a similar polyketide origin. Based on the previously reported references, the possible biosynthetic routes of these polyketides are proposed here (Figure 9) [20,21,29,42,43,44,45]. All of them are derived from the heptaketide intermediate through the iterative condensation of acetyl-CoA and malonyl-CoA via PKSs, followed by a multi-step reaction of aldol-type cyclization, lactonization, methylation, hydroxylation, reduction, transmethylation, etc.

2.2. Biological Activity

Partially isolated compounds were evaluated for their in vitro antimicrobial activities against some pathogenic bacteria and fungi. Eleven bacterial strains including four human pathogens (methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonia) and seven aquatic bacteria (Vibrio alginolyticus, Aeromonas hydrophilia, Micrococcus luteus, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio harveyi) together with six plant-pathogenic fungi (Ceratobasidium cornigerum, Penicillium digitatum, Physalospora piricola, Valsa mali, Colletotrichum gloeosporioides, and Fusarium oxysporum) were used as the tested strains. As shown in

Table 6. Antibacterial activity of isolated compounds (MIC, μg/mL).

Bacteria	Compounds
	Chloramphenicol	7	9	10	18	19	20	21	22	24	30	31
MRSA	8	64	-	-	-	64	64	-	-	-	64	-
PA	4	16	-	32	-	16	4	-	-	-	32	32
EC	0.25	32	32	0.5	0.5	8	4	32	32	8	4	64
KP	8	64	64	64	-	64	64	-	64	64	64	64
VAl	1	-	64	-	-	32	64	64	32	-	-	64
AH	0.5	32	32	0.5	4	8	4	-	0.5	0.5	8	-
ML	1	32	32	-	-	8	4	-	-	-	8	-
VAn	1	64	-	-	-	8	64	-	-	-	16	-
VP	1	32	32	1	2	8	4	64	0.5	64	32	32
VV	4	-	32	16	-	8	4	-	-	-	-	-
VH	2	32	16	0.5	32	16	8	-	32	64	8	-

* MRSA: methicillin-resistant S. aureus, PA: P. aeruginosa, EC: E. coli, KP: K. pneumonia, VAl: V. alginolyticus, AH: A. hydrophilia, ML: M. luteus, VAn: V. anguillarum, VP: V. parahaemolyticus, VV: V. vulnificus, VH: V. harveyi. -: no activity.

, Tables S1 and S2, eleven compounds, 7, 9–10, 18–22, 24, 30, and 31, exhibited comparable broad-spectrum antibiotic effects with MIC values ranging from 0.5 to 64 μg/mL.

Notably, compound 10 displayed significant inhibitory activities against four bacteria, namely E. coli, A. hydrophilia, V. parahaemolyticus, and V. harveyi, with MIC values in the range of 0.5–1 μg/mL, better than or equivalent to the positive control chloramphenicol (MIC = 0.25–2 μg/mL). Compound 18 showed considerable antibacterial activities against human pathogen E. coli and aquatic pathogenic bacteria V. parahaemolyticus that were comparable to those of the positive control, chloramphenicol. Moreover, compounds 20, 22, and 24 displayed inhibitory effects on the growth of P. aeruginosa with V. vulnificus, A. hydrophilia with V. parahaemolyticus, and A. hydrophilia, respectively, with MIC values like those of chloramphenicol from 0.5 to 4 μg/mL. Unfortunately, all measured compounds did not show any significant antifungal activity at a concentration of 64 µg/mL.

The structure–activity relationship (SAR) for the above polyketides is discussed here. The addition of a ketonic carbonyl or methoxy group in the structures of methylcyclopenta[c][2]-benzopyran could improve their antibacterial effects (10 vs. 7–9). Then, a comparison of the inhibitory activities of compounds 18–24 indicated that their activities were influenced by the number and position of methyl, hydroxyl, and methoxy groups, as well as the expansion of the lactone ring. Furthermore, a correlation was observed between the acetylation of the side chain and antibacterial efficacy for penicillide-type compounds (30 vs. 31).

3. Materials and Methods

3.1. General Experimental Procedures

The general experimental procedures in this study were similar to those previously reported [6,14,15,46].

3.2. Fungal Material

The fungus strain CS-258 was isolated from the marine mussel sample collected from a cold seep in the northeast of the South China Sea in May 2020, and its strain identification was conducted by a BLAST search in GenBank. This strain sequence data had been uploaded to GenBank to obtain an accession number of No. PP065775. The strain was deposited at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

3.3. Fermentation, Extraction, and Isolation

The fresh mycelium of Talaromyces sp. CS-258 was cultured on potato dextrose broth medium for 3 days and then incubated in autoclaved 1 L Erlenmeyer flasks with rice culture medium (70 g rice, 0.2 g corn steep liquor, 0.5 g yeast extract, 0.3 g peptone, 0.6 g gourmet powder, and 100 mL seawater collected from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS) for 30 days at room temperature. After incubation, the EtOAc crude extract was obtained by exhaustive concentration with MeOH and successive extraction with EtOAc/H₂O four times.

The crude extract was fractionated by silica gel column vacuum liquid chromatography (VLC) elution with increasing polarity gradient of petroleum ether (PE)/EtOAc and CH₂Cl₂/MeOH to yield nine fractions (Fr.A–Fr.I). Fr.E was further split by column chromatography (CC) using Sephadex LH-20 to produce three subfractions from E-1 to E-3. Fr.E-1 was rechromatographed over silica gel elution with a slow CH₂Cl₂/MeOH gradient from 200:1 to 20:1 to yield three mixtures of E.1.1–E.1.3. Then, mixture E.1.1 was followed by semipreparative HPLC separation (Elite ODS-BP column, 5 μm, 10 × 250 mm, MeOH-H₂O, 75:25, 3 mL/min) to afford compounds 12 (6.0 mg, t_R 8 min) and 10 (10.2 mg, t_R 10 min). Mixtures of E.1.2 and E.1.3 were also subjected to semipreparative HPLC purification to yield 7 (3.3 mg, t_R 20 min, 70% MeOH-H₂O), 30 (2.0 mg, t_R 18 min, 75% MeOH-H₂O), and 31 (5.1 mg, t_R 28 min, 65% MeOH-H₂O). Compound 4 (21.1 mg) was obtained via recrystallization from Fr.E-2. Another semipreparative HPLC collection with 75% MeOH-H₂O was applied for Fr.E-3 to give compounds 18 (4.2 mg, t_R 22 min), 19 (3.2 mg, t_R 16 min), and 20 (4.0 mg, t_R 18 min).

Fr.F was subjected to reversed-phase CC using Lobar LiChroprep RP-18 from 10% to 90% MeOH-H₂O to produce three subfractions, Fr.F-1 to F-3. Fr.F-1 was sequentially recrystallized to obtain compound 2 (100.0 mg). Fr.F-2 was subjected to Sephadex LH-20 CC to afford compound 24 (65.9 mg) and two mixed components. Then, the former component was purified by HPLC separation with MeOH-H₂O (52:48) and identified as 8 (13.7 mg, t_R 25 min). The latter was subjected to preparative TLC (developing solvents: CH₂Cl₂−MeOH, 20:1) to obtain 27 (3.1 mg). Treated with successive CC on Sephadex LH-20 and HPLC with 58% MeOH-H₂O, Fr.F-3 was found to produce compounds 9 (16.5 mg, recrystallization), 21 (15.3 mg, t_R 16 min), 22 (9.5 mg, t_R 18 min), and 23 (4.9 mg, recrystallization) totally.

Fr.G was subjected to further CC over Lobar LiChroprep RP-18 in the MeOH−H₂O solvent system and thus gave 6 subfractions, Fr.G-1 to Fr.G-6. Subsequently, compounds 25 (4.0 mg, t_R 14 min) and 26 (2.5 mg, t_R 12 min) were provided from Fr.G-1 by Sephadex LH-20 (MeOH) and semipreparative HPLC elution with 42% MeOH−H₂O; meanwhile, compound 1 (11.0 mg, t_R 16 min) was obtained from another mixture of Fr.G-1 via HPLC in 30% MeOH−H₂O. Fr.G-2 was subjected to a series of CC on Sephadex LH-20 and the HPLC separation system of 42% MeOH/H₂O and 45% MeOH/H₂O to afford 11 (6.7 mg, t_R 18 min) and 5 (24.6 mg, t_R 15 min). Fr.G-3 was further purified by Sephadex LH-20 and then by preparative TLC (developing solvents: CH₂Cl₂−MeOH, 15:1) and by HPLC with 80% MeOH/H₂O to obtain compounds 13 (90.0 mg, t_R 27 min), 14 (14.6 mg, t_R 35 min), and 15 (4.8 mg, t_R 25 min). The remaining subfractions, Fr.G-4 to Fr.G-6, were partitioned using nearly the same method through CC over Sephadex LH-20, silica gel, and semipreparative HPLC to produce compounds 16 (5.5 mg, from Fr.G-4, t_R 10 min, 52% MeOH/H₂O), 17 (5.0 mg, from Fr.G-6, t_R 28 min, 51% MeOH/H₂O), and 6 (6.0 mg, from Fr.G-5).

After CC on reversed-phase RP-18 and Sephadex LH-20, Fr.I was fractionated and purified to give compound 29 (3.2 mg, by silica gel column and following Sephadex LH-20), 28 (2.0 mg, by preparative TLC), 3 (13.0 mg, by semipreparative HPLC collection of 46% MeOH-H₂O at t_R 35 min), and 32 (2.4 mg, by preparative TLC and Sephadex LH-20).

3.4. Spectroscopic Data

(3R,5′R)-5-hydroxytalaroflavone (1): brown oil; ${\left[\alpha \right]}_D^{25}$+42.9 (c 0.44, MeOH); UV (MeOH, 0.20 mg/mL) λ_max (log ε) 216 (3.39), 259 (2.94), 297 (2.58) nm; ECD (0.20 mg/mL, MeOH) λ_max (Δε) 208 (−11.78), 222 (+12.43), 240 (+7.56) nm; ¹H and ¹³C NMR data, see Table 1; (+)-HRESIMS m/z 263.055012 [M + H]⁺ (calcd for C₁₃H₁₁O₆, 263.055014).

Talaroisochromenol A (3): brown oil; ${\left[\alpha \right]}_D^{25}$+105.9 (c 0.06, MeOH); UV (MeOH, 0.20 mg/mL) λ_max (log ε) 209 (3.13), 261 (2.92), 310 (2.64) nm; ECD (0.20 mg/mL, MeOH) λ_max (Δε) 213 (−3.47), 261 (−1.62) nm; ¹H and ¹³C NMR data, see Table 1; (+)-HRESIMS m/z 261.0398 [M + H]⁺ (calcd for C₁₃H₉O₆, 261.0405).

Talaroisochromenol B (5): yellow oil; ${\left[\alpha \right]}_D^{25}$+33.3 (c 0.24, MeOH); UV (MeOH, 0.20 mg/mL) 248 (3.08), 330 (2.18) nm; ECD (0.20 mg/mL, MeOH) λ_max (Δε) 210 (+1.54), 238 (+1.24), 291 (−0.38) nm; ¹H and ¹³C NMR data, see Table 1; (+)-HRESIMS m/z 265.0714 [M + H]⁺ (calcd for C₁₃H₁₃O₆, 265.0707).

Talaroisochromenol C (11): white solid; ${\left[\alpha \right]}_D^{25}$+4.3 (c 0.24, MeOH); UV (MeOH, 0.25 mg/mL) λ_max (log ε) 213 (3.20), 229 (2.96), 265 (2.68), 305 (2.56) nm; ¹H and ¹³C NMR data, see Table 2; (+)-HRESIMS m/z 251.0556 [M + H]⁺ (calcd for C₁₂H₁₁O₆, 251.0550).

(+)-(11): ${\left[\alpha \right]}_D^{25}$+60.0 (c 0.20, MeOH); ECD (0.30 mg/mL, MeOH) λ_max (Δε) 206 (+6.83), 226 (–1.93), 268 (−3.68) nm.

(−)-(11): ${\left[\alpha \right]}_D^{25}$−45.0 (c 0.20, MeOH); ECD (0.25 mg/mL, MeOH) λ_max (Δε) 208 (−4.41), 226 (+1.70), 268 (+2.69) nm.

(8R,9R,10aR)-5-hydroxyaltenuene (13): brown oil; ${\left[\alpha \right]}_D^{25}$−23.7 (c 0.30, MeOH); UV (MeOH, 0.25 mg/mL) λ_max (log ε) 242 (3.24), 282 (2.73), 323 (2.47) nm; ECD (0.25 mg/mL, MeOH) λ_max (Δε) 236 (+10.43), 282 (−5.59) nm; ¹H and ¹³C NMR data, see Table 3; (+)-HRESIMS m/z 279.0870 [M + H]⁺ (calcd for C₁₄H₁₅O₆, 279.0863).

(8R,9R,10aS)-5-hydroxyaltenuene (14): brown oil; ${\left[\alpha \right]}_D^{25}$+43.6 (c 0.18, MeOH); UV (MeOH, 0.20 mg/mL) λ_max (log ε) 244 (3.34), 282 (2.81), 325 (2.49) nm; ECD (0.20 mg/mL, MeOH) λ_max (Δε) 237 (−2.85), 284 (+1.31) nm; ¹H and ¹³C NMR data, see Table 3; (+)-HRESIMS m/z 279.0869 [M + H]⁺ (calcd for C₁₄H₁₅O₆, 279.0863).

(8R,9S,10aR)-5-hydroxyaltenuene (15): brown oil; ${\left[\alpha \right]}_D^{25}$−85.7 (c 0.21, MeOH); UV (MeOH, 0.30 mg/mL) λ_max (log ε) 244 (3.17), 282 (2.64), 325 (2.32) nm; ECD (0.30 mg/mL, MeOH) λ_max (Δε) 236 (+12.30), 282 (−7.29) nm; ¹H and ¹³C NMR data, see Table 3; (+)-HRESIMS m/z 279.0861 [M + H]⁺ (calcd for C₁₄H₁₅O₆, 279.0863).

Nemanecin D (25): yellow oil; ${\left[\alpha \right]}_D^{25}$−11.5 (c 0.26, MeOH); UV (MeOH, 0.40 mg/mL) λ_max (log ε) 209 (2.98), 320 (3.10) nm; ECD (0.40 mg/mL, MeOH) λ_max (Δε) 214 (+1.73), 345 (−1.67) nm; ¹H and ¹³C NMR data, see Table 4; (+)-HRESIMS m/z 255.1228 [M + H]⁺ (calcd for C₁₃H₁₉O₅, 255.1227).

(S)-MTPA Ester (25a): ¹H NMR (Figure S65, selected signals, pyridine-d₅, 500 MHz) δ_H: 5.60 (1H, m, H-10), 2.63 (1H, dd, H-9_α), 2.60 (1H, dd, H-9_β), 1.31 (3H, d, H-11); ¹H-¹H COSY and HRMS spectra, see Figures S66 and S67.

(R)-MTPA Ester (25b): ¹H NMR (Figure S68, selected signals, pyridine-d₅, 500 MHz) δ_H: 5.51 (1H, m, H-10), 2.54 (2H, m, H-9), 1.36 (3H, d, H-11); ¹H-¹H COSY and HRMS spectra, see Figures S69 and S70.

Nemanecin E (26): yellow oil; ${\left[\alpha \right]}_D^{25}$−48.0 (c 0.40, MeOH); UV (MeOH, 0.25 mg/mL) λ_max (log ε) 209 (2.89), 319 (2.76) nm; ECD (0.40 mg/mL, MeOH) λ_max (Δε) 214 (+0.70), 345 (−1.18) nm; ¹H and ¹³C NMR data, see Table 4; (+)-HRESIMS m/z 255.1226 [M + H]⁺ (calcd for C₁₃H₁₉O₅, 255.1227).

(S)-MTPA Ester (26a): ¹H NMR (Figure S79, selected signals, pyridine-d₅, 500 MHz) δ_H: 5.58 (1H, m, H-10), 2.62 (1H, dd, H-9_α), 2.50 (1H, dd, H-9_β), 1.38 (3H, d, H-11); ¹H-¹H COSY and HRMS spectra, see Figures S80 and S81.

(R)-MTPA Ester (26b): ¹H NMR (Figure S82, selected signals, pyridine-d₅, 500 MHz) δ_H: 5.53 (1H, m, H-10), 2.68 (1H, dd, H-13_α), 2.54 (1H, dd, H-9_β), 1.28 (3H, d, H-15); ¹H-¹H COSY and HRMS spectra, see Figures S83 and S84.

2,5-dimethy-8-iodochromone (27): yellow oil; UV (MeOH, 0.05 mg/mL) λ_max (log ε) 210 (3.56), 247 (3.52), 296 (3.19) nm; ¹H and ¹³C NMR data, see Table 5; (+)-HRESIMS m/z 316.9668 [M + H]⁺ (calcd for C₁₁H₁₀O₃I, 316.9669).

6-hydroxy-4-methoxycoumarin (28): yellow oil; UV (MeOH, 0.05 mg/mL) λ_max (log ε) 222 (3.33), 270 (2.96), 331 (2.67) nm; ¹H and ¹³C NMR data, see Table 5; (+)-HRESIMS m/z 193.0493 [M + H]⁺ (calcd for C₁₀H₉O₄, 193.0495).

Talarofurolactone A (29): yellow oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$−110.3 (c 0.15, MeOH); UV (MeOH, 0.25 mg/mL) λ_max (log ε) 220 (3.01) nm; ECD (0.25 mg/mL, MeOH) λ_max (Δε) 211 (−4.27) nm; ¹H and ¹³C NMR data, see Table 5; (+)-HRESIMS m/z 213.1122 [M + H]⁺ (calcd for C₁₁H₁₇O₄, 213.1121), m/z 230.1389 [M + NH₄]⁺ (calcd for C₁₁H₂₀O₄N, 230.1387), m/z 235.0942 [M + Na]⁺ (calcd for C₁₁H₁₆O₄Na, 235.0941).

3.5. X-ray Crystallographic Analysis of Compounds 2, 11, 13, and 14

Compounds 2, 11, 13, and 14 were dissolved in MeOH and crystallized after slow evaporation in a refrigerator. All X-ray single-crystal diffraction data were recorded at 293(2) K on an Agilent Xcalibur Eos Gemini CCD plate diffractometer equipped with graphite monochromatic CuKα radiation. The data were revised for absorption with the program SADABS [47]. Direct methods of the SHELXTL software and subsequent refinement via full-matrix least-squares difference Fourier techniques were applied to solve the structures accurately [48,49]. Additional refinements for non-hydrogen, H, and O atoms were carried out as previously reported [9,14]. All crystallographic data have been deposited in the Cambridge Crystallographic Data Centre (CCDC) and can be obtained on application to the director free of charge.

Crystal data for 2: m.p. 224.0–248.0 °C, C₁₄H₁₂O₆, fw = 276.24, monoclinic, space group C2/c, unit cell dimensions a = 23.8978(11) Å, b = 6.9420(3) Å, c = 14.9300(7) Å, V = 2467.83(19) ų, α = 90^◦, β = 94.895(2)^◦, γ = 90^◦, Z = 8, d_calcd = 1.487 mg/m³, crystal size 0.16 × 0.15 × 0.14 mm³, μ = (CuKα) 1.002 mm⁻¹, F(000) = 1152.0. Independent reflections: 2260 [R_int = 0.0427, R_sigma= 0.0248]. The final indices gave R₁ = 0.0405 and wR₂ = 0.1032 [I > 2σ(I)]. CCDC number was deposited as 2327879.

Crystal data for 11: m.p. 181.3–184.0 °C, C₁₂H₁₀O₆, fw = 250.20, triclinic, space group P-1, unit cell dimensions a = 7.300(2) Å, b = 7.4441(2) Å, c = 9.9268(2) Å, V = 516.92(2) ų, α = 70.6080(10)^◦, β = 86.7490(10)^◦, γ = 87.6150(10)^◦, Z = 2, d_calcd = 1.607 mg/m³, crystal size 0.16 × 0.15 × 0.14 mm³, μ = (CuKα) 1.126 mm⁻¹, F(000) = 260.0. Independent reflections: 1869 [R_int = 0.0291, R_sigma= 0.0280]. The final indices gave R₁ = 0.0347 and wR₂ = 0.0996 [I > 2σ(I)]. CCDC number was deposited as 2327875.

Crystal data for 13: m.p. 244.1–247.2 °C, C₁₄H₁₄O₆, fw = 278.25, monoclinic, space group P2₁/n, unit cell dimensions a = 9.0780(2) Å, b = 6.90080(10) Å, c = 19.2001(4) Å, V = 1202.71(4) ų, α = 90^◦, β = 90.6920(10)^◦, γ = 90^◦, Z = 4, d_calcd = 1.537 mg/m³, crystal size 0.18 × 0.16 × 0.15 mm³, μ = (CuKα) 1.028 mm⁻¹, F(000) = 584.0. Independent reflections: 2185 [R_int = 0.0319, R_sigma= 0.0248]. The final indices gave R₁ = 0.0328 and wR₂ = 0.0873 [I > 2σ(I)]. CCDC number was deposited as 2327876.

Crystal data for 14: m.p. 273.0–278.0 °C, C₁₄H₁₄O₆ +(CH₃)₂SO, fw = 356.38, monoclinic, space group P2₁/c, unit cell dimensions a = 8.8828(3) Å, b = 20.8250(7) Å, c = 8.9113(3) Å, V = 1595.86(9) ų, α = 90^◦, β = 104.511(2)^◦, γ = 90^◦, Z = 2, d_calcd = 1.483 mg/m³, crystal size 0.16 × 0.15 × 0.12 mm³, μ = (CuKα) 2.143 mm⁻¹, F(000) = 752.0. Independent reflections: 2917 [R_int = 0.0489, R_sigma = 0.0318]. The final indices gave R₁ = 0.0315 and wR₂ = 0.0877 [I > 2σ(I)]. CCDC number was deposited as 2327877.

3.6. Antimicrobial Activity Assay

The antimicrobial evaluations against four human pathogens (methicillin-resistant S. aureus, P. aeruginosa, E. coli, and K. pneumonia) and seven aquatic bacteria (V. alginolyticus, A. hydrophilia, M. luteus, V. anguillarum, V. parahaemolyticus, V. vulnificus, and V. harveyi) as well as six plant-pathogenic fungi (C. cornigerum, P. digitatum, P. piricola, V. mali, C. gloeosporioides and F. oxysporum) were carried out in a 96-well microplate as described in our previous reports [6,46,50,51]. These microbial strains were provided by the Institute of Oceanology, Chinese Academy of Sciences, by purchase or isolation. Chloramphenicol and amphotericin B served as positive controls against bacteria and fungi, respectively, while DMSO was treated as the negative control. All measurements at various concentrations were performed in triplicate.

3.7. Details of Computational Methods

The computational methods for ECD calculation and DP4+ probability analysis were similar to those in the previous papers from our group [6,10,51], with some modifications detailed below. Molecular mechanics using the MM⁺ method was carried out for conformational searches in HyperChem software (Version 8.0, Hypercube, Inc., Gainesville, FL, USA). The energy-minimized conformers were generated and further optimized using DFT calculations at the B3LYP/6-31G(d) level in Gaussian 09 software (Version D.01; Gaussian, Inc.: Wallingford, CT, USA). Frequency calculations were carried out at the same level of theory to confirm the absence of imaginary frequencies and to obtain thermal corrections to the Gibbs free energies. These obtained conformers were subjected to ECD calculations using the TDDFT method at the CAM-B3LYP/TZVP, BH&HLYP/TZVP, or PBE0/TZVP level. The solvent effects of MeOH were evaluated at the same DFT level using the self-consistent reaction field (SCRF) method with the polarizable continuum model (PCM). The ECD spectrum was generated by the SpecDis program and finally drawn using Origin Pro 8.5 software [52,53,54,55,56,57]. The NMR shielding tensors were calculated by the DFT method at the mPW1PW91\6-31+G(d) PCM level in DMSO and then weighted according to Boltzmann’s distribution. GIAO (gauge-independent atomic orbital) NMR chemical calculations were performed using an equation described previously. The theoretical shielding tensors (¹H and ¹³C) and experimental chemical shifts were finally analyzed and compared using DP4+ probability [58,59,60].

3.8. The Modified Mosher’s Method

The modified Mosher’s method was conducted in NMR tubes following the details described in the previous papers [25,61].

4. Conclusions

In this study, the chemical investigation of cold-seep-derived fungus Talaromyces sp. CS-258 has resulted in the isolation and identification of 32 fungal polyketides including 11 new compounds and 1 new naturally isolated metabolite. Notably, a pair of enantiomers ((±)-11) and two groups of diastereoisomers (13–15 and 25/26) were obtained, the isolation of which presented challenges due to their difficulty in separation from each other. Furthermore, compound 27 was identified as a rare occurring natural iodo-chromone analog. The plausible biogenetic pathways of compounds 1–24, which belong to altenusin or alternariol families with polyketide origin bearing variable tricyclic ring skeletons, were proposed. The results of antimicrobial activities displayed that 11 polyketides exhibited broad-spectrum antibiotic effects against human and aquatic pathogenic bacteria. Among them, compounds 10 and 18 exhibited potent inhibition against E. coli, A. hydrophilia, and V. parahaemolyticus, while 22 and 24 effectively suppressed the growth of A. hydrophilia. These findings highlight the cold-seep-derived fungus Talaromyces sp. CS-258 as a promising source for bioactive metabolites, especially for fungal polyketides, with potential applications as antibiotic agents in medicinal development and agriculture.