Cytotoxic Peptidic Metabolites Isolated from the Soil-Derived Fungus Trichoderma atroviride

Abstract

Twelve undescribed peptidic compounds, bukhansantaibols A–K (1–10) and bukhansantaibals A–B (11–12), were isolated from the soil fungus Trichoderma atroviride through LC-MS and bioactivity-guided purification. Their structures were elucidated by the analysis of 1D and 2D NMR spectra, HRESIMS, and acid hydrolysis using modified Marfey’s method. All compounds were evaluated for their cytotoxic activity against HCT-8 (colon cancer) and SK-OV-3 (ovarian cancer) cells. Among them, compounds 1–5 exhibited significant inhibitory effects, with IC₅₀ values ranging from 2.1 to 19.6 μM.

1. Introduction

Peptaibols are fungal secondary metabolites produced by non-ribosomal peptide synthetases (NRPSs) and constitute a class of linear peptides characterized by N-terminal acetylation, the incorporation of non-proteinogenic amino acids, and a C-terminal amino alcohol [1,2]. Peptaibols are mainly produced by fungi belonging to the order Hypocreales, which includes culturable genera such as Trichoderma and Escovopsis [3]. Recently, the weeding behaviour of the leaf-cutting fungus-growing ant Trachymyrmex septentrionalis was reported to be triggered by the detection of peptaibols [4]. The production of peptaibol compounds during mycoparasitic interactions has inspired studies on their antimicrobial properties, which have been experimentally validated in various systems [5,6]. Their proposed mechanism of action—formation of transmembrane ion channels leading to cell lysis—has also prompted investigations into their cytotoxic potential against a range of cancer cells [7,8,9].

Through inter-university collaboration research, we aimed to discover novel peptaibols with cell membrane-permeable properties from soil fungi. As a result of this effort, BHM16-2 strain from the BHM collection was identified to produce peptaibols, as confirmed by our in-house HPLC analysis and dereplication process. Large-scale cultivation of this fungus was carried out, and 12 new peptidic molecules were purified using multiple rounds of column chromatography and preparative HPLC purification. The isolated compounds were subsequently evaluated for their cytotoxic activities against HCT-8 (colon cancer) and SK-OV-3 (ovarian cancer) cell lines. Herein, we report the isolation, structure elucidation, and bioactivity of these 12 novel peptaibols.

2. Results and Discussion

In an effort to discover novel cytotoxic molecules, the Natural Products Drug Discovery laboratory at Duksung Women’s University has established a fungal library comprising approximately 2000 fungal strains. Crude extracts of these strains have been subjected to in-house HPLC analysis for preliminary dereplication [10,11]. Among these, the BHM collection (Bukhansan Mountain collection) comprises approximately 200 soil fungi strains isolated from 33 different locations in the Bukhansan Mountain area, located in northern Seoul, South Korea. Within this collection, the strain BHM16-2, isolated near Insubong Peak, was identified as a peptaibol-producing fungus. Given the well-known membrane-permeable properties of peptaibols and their associated antimicrobial and cytotoxic activities, we performed further dereplication using HRESIMS data, which revealed that BHM16-2 produces a new class of peptaibols. Based on this dereplication, BHM16-2 was selected for large-scale cultivation, and reproducibility of the novel peptaibol production was confirmed in the large-scale extract by the HRESIMS analysis. A total of 12 metabolites (1–12) were isolated, and their structures were elucidated. Several of these compounds featured modifications at the C-terminal amino acid residues. Notably, all isolates were determined to be new natural molecules. Herein, we report the structure elucidation of the 12 novel peptidic compounds and the evaluation of their cytotoxic activities against cancer cells.

Compound 1 (Structure shown in

Table 1. Structure of compounds 1–6.

Compound	Mass	R1	R2	R3	R4
1	1948.1146	L-Trp	L-Ser	L-Ile	L-Gln
2	1948.1146	L-Trp	L-Ser	L-Leu	L-Gln
3	1909.1037	L-Phe	L-Ser	L-Ile	L-Gln
4	1932.1197	L-Trp	L-Ala	L-Ile	L-Gln
5	1949.0986	L-Trp	L-Ser	L-Ile	L-Glu
6	1910.0877	L-Phe	L-Ser	L-Ile	L-Glu

) was isolated as a white amorphous powder, and its molecular formula was determined as C₉₁H₁₄₉N₂₃O₂₄ by the HRESIMS data (m/z 975.0645 [M + 2H]²⁺; calcd 975.0646). The ¹H and ¹³C-NMR spectrum of 1 exhibited characteristic features of a peptaibol, including 24 exchangeable amide proton signals in the range of δ_H 7.28–9.93, α-proton signals in the range of δ_H 4.06–5.02, and an acetyl CH₃ at δ_H 2.07, and 23 methyl group proton signals within δ_H 0.85–1.83 (

Table 2. ¹H (600 MHz) data for compound 1 in pyridine-d₅ (δ in ppm, J in Hz).

	Position	δH		Position	δH
Ac	CH3	2.07, s	Leu11	N-H	8.19, m
Trp1	N-H	9.45, d (4.5)		α	4.93, m
	α	5.02, m		β	2.00, m; 2.23, m
	β	3.63, m; 3.70, m		γ	2.16, m
	1	11.94, d (2.4)		δ1	0.99, d (6.5)
	2	7.49, d (2.4)		δ2	0.98, d (6.5)
	4	7.84, d (8.0)	Aib12	N-H	8.53, s
	5	7.19, m		α-Me1	1.80, s
	6	7.28, m		α-Me2	1.74, s
	7	7.56, m	Pro13	α	4.57, m
Gly2	N-H	9.93, t (5.6)		β	1.74, m; 2.16, m
	α	4.06, m; 4.17, m		γ	1.75, m; 1.97, m
Ala3	N-H	8.48, d (5.4)		δ	3.99, m; 4.07, m
	α	4.49, m	Val14	N-H	8.04, m
	β	1.55, d (7.3)		α	4.07, m
Aib4	N-H	8.52, s		β	2.57, m
	α-Me1	1.80, s		γ1	1.20, d (6.5)
	α-Me2	1.71, s		γ2	1.14, d (6.5)
Aib5	N-H	8.27, s	Aib15	N-H	7.94, s
	α-Me1	1.72, s		α-Me1	1.80, s
	α-Me2	1.71, s		α-Me2	1.71, s
Gln6	N-H	8.31, d (4.7)	Ile16	N-H	7.73, d (4.9)
	α	4.36, m		α	4.28, m
	β	2.69, m; 2.79, m		β	2.23, m
	γ	2.77, m; 2.89, m		γ1	1.40, m
	CONH2	7.74, s; 8.14, s		γ2	1.07, d (6.7)
Aib7	N-H	8.43, s		δ	0.86, t (7.2)
	α-Me1	1.75, s	Gln17	N-H	8.38, d (5.5)
	α-Me2	1.70, s		α	4.69, m
Aib8	N-H	8.07, s		β	2.81, m; 2.88, m
	α-Me1	1.81, s		γ	2.75, m; 2.97, m
	α-Me2	1.74, s		CONH2	7.60, s; 8.02, s
Aib9	N-H	8.39, s	Gln18	N-H	8.18, m
	α-Me1	1.82, s		α	5.04, m
	α-Me2	1.72, s		β	2.73, m; 3.00, m
Ser10	N-H	8.46, d (4.4)		γ	2.81, m; 2.97, m
	α	4.70, m		CONH2	7.50, s; 8.10, s
	β	4.37, m; 4.52, m	Valol19	N-H	7.76, d (9.4)
				α	4.27, m
				β	2.38, m
				γ1	1.19, d (6.8)
				γ2	1.04, d (6.8)
				β’	4.04, m; 4.08, m

and

Table 3. ¹³C NMR (150 MHz) data for compound 1 in pyridine-d₅.

	Position	δC		Position	δC		Position	δC
Ac	CH3	22.7	Aib7	C=O	177.0	Val14	C=O	174.0
	C=O	172.5		α	56.7		α	63.4
Trp1	C=O	175.0		α-Me1	26.9		β	29.4
	α	57.1		α-Me2	23.7		γ1	19.2
	β	27.9	Aib8	C=O	176.4		γ2	19.1
	2	124.3		α	56.9	Aib15	C=O	177.2
	3	110.3		α-Me1	26.9		α	56.7
	3a	128.1		α-Me2	23.7		α-Me1	27.0
	4	118.7	Aib9	C=O	177.7		α-Me2	22.7
	5	119.1		α	56.8	Ile16	C=O	174.3
	6	121.8		α-Me1	27.1		α	61.6
	7	112.0		α-Me2	23.0		β	35.9
	7a	137.4	Ser10	C=O	171.5		γ1	26.5
Gly2	C=O	171.9		α	60.4		γ2	15.7
	α	44.4		β	62.0		δ	11.1
Ala3	C=O	175.2	Leu11	C=O	173.6	Gln17	C=O	173.6
	α	52.4		α	52.2		α	56.3
	β	16.4		β	40.2		β	27.4
Aib4	C=O	175.8		γ	24.7		γ	32.8
	α	56.8		δ1	20.8		CONH2	174.6
	α-Me1	26.4		δ2	23.1	Gln18	C=O	172.8
	α-Me2	22.6	Aib12	C=O	175.0		α	55.5
Aib5	C=O	177.2		α	56.9		β	28.3
	α	56.5		α-Me1	23.3		γ	32.9
	α-Me1	26.9		α-Me2	27.1		CONH2	174.6
	α-Me2	22.7	Pro13	C=O	175.0	Valol19	α	57.3
Gln6	C=O	174.8		α	64.0		β	29.2
	α	57.4		β	29.0		γ1	19.2
	β	27.2		γ	26.3		γ2	19.9
	γ	32.5		δ	49.3		β’	63.0
	CONH2	174.5

, Figures S2 and S3) [12]. The primary amino acid sequence of 1 was elucidated based on the ROESY correlations between sequential amide protons. In particular, the connectivity among Aib¹², Pro¹³, and Val¹⁴ was supported by the ROESY correlations between the NH proton of Aib¹² (δ_H 8.53) and the δ-protons of Pro¹³ (δ_H 3.99 and 4.07), as well as between a proton of Pro¹³ (δ_H 4.57) and the NH proton of Val¹⁴ (δ_H 8.04). The spin systems of individual amino acid residues were further confirmed by TOCSY experiments (Figure 1). The presence of the non-proteinogenic amino acids Aib and Valol was validated through analysis of COSY and HMBC spectra. The COSY correlations among geminal methylene protons (δ_H 4.04 and 4.08), methine protons (δ_H 2.38 and 4.27), and methyl protons (δ_H 1.19 and 1.04) confirmed the presence of a Valol residue at the C-terminal end. In addition, the HMBC correlations from the α-methyl protons of Aib to the corresponding α-carbons, along with the correlations from neighbouring NH protons to the carbonyl carbon of Aib, further substantiated the assignment and position of each Aib residue (Figure 1). The presence of an N-terminal acetyl group was established based on the HMBC correlations among the acetyl methyl proton (δ_H 2.07), the NH proton (δ_H 9.45), and the α-proton of Trp¹ (δ_H 5.02) with the acetyl carbonyl carbon (δ_C 172.5). The absolute configurations of the amino acid residues were determined by modified Marfey’s analysis [13] which identified Trp, Ala, Gln, Ser, Leu, Pro, Val, Ile, and Valol as possessing the L-configuration (Figure S13). The negative Cotton effect around 205 nm was observed on the electronic circular dichroism (ECD) spectrum, suggesting that compound 1 adopts a right-handed helical conformation (Figure S11) [14]. Based on these results, the complete structure of compound 1 was determined to be N-acetyl-_L-Trp¹-Gly²-_L-Ala³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ser¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Ile¹⁶-_L-Gln¹⁷-_L-Gln¹⁸-_L-Valol¹⁹, and it was designated as bukhansantaibol A (1).

Compound 2 was obtained as a white amorphous powder and exhibited the same exact mass as 1 (m/z 975.0643 [M + 2H]²⁺; calcd 975.0646), consistent with the molecular formula C₉₁H₁₄₉N₂₃O₂₄. The ¹H and ¹³C-NMR spectra of 2 closely resembled those of 1 (Tables S1 and S2, and Figures S14 and S15). However, detailed analysis of the COSY and HMBC spectra revealed a substitution of the Ile¹⁶ residue in 1 with a Leu residue in 2 (Table 1). This substitution was supported by the TOCSY correlations corresponding to a leucine spin system, including the NH proton (δ_H 7.89), α-methine proton (δ_H 4.55), β-methylene protons (δ_H 1.91 and 2.01), γ-methine proton (δ_H 2.16), and two δ-methyl doublets (δ_H 0.96 and 0.86), indicating the presence of Leu at position 16. Furthermore, modified Marfey’s analysis yielded only the _L-FDLA derivative of Leu, confirming the exclusive incorporation of _L-Leu at this position (Figure S25). The ECD spectrum also indicated a right-handed helical conformation, consistent with compound 1 (Figure S23). Accordingly, the structure of compound 2 was determined to be N-acetyl-_L-Trp¹-Gly²-_L-Ala³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ser¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Leu¹⁶-_L-Gln¹⁷-_L-Gln¹⁸-_L-Valol¹⁹, and it was designated as bukhansantaibol B (2).

Compound 3 was obtained as a white amorphous powder, and its molecular formula was determined to be C₈₉H₁₄₈N₂₂O₂₄ based on the HRESIMS data (m/z 955.5593 [M + 2H]²⁺; calcd 955.5591). The ¹H-NMR spectrum of 3 closely resembled that of 1, except for distinct aromatic proton signals at δ_H 7.34 (2H, m), 7.27 (2H, m), and 7.23 (1H, m), consistent with the presence of a phenyl moiety (Table S3 and Figure S26). The TOCSY correlations from the amide proton (δ_H 9.58) to the β-methylene protons (δ_H 3.33 and 3.43) confirmed the spin system of a phenylalanine residue. The position of this phenylalanine residue at the N-terminus was supported by HMBC correlations from the N-acetyl methyl proton (δ_H 2.07) and the amide proton of phenylalanine (δ_H 9.58) to the acetyl carbonyl carbon (δ_C 172.2). Furthermore, modified Marfey’s analysis verified the substitution of the N-terminal Trp with Phe and confirmed the L-configuration of all amino acid residues (Figure S37). In addition, the ECD spectrum indicated a right-handed helical conformation (Figure S35). Based on these findings, the structure of compound 3 was determined to be N-acetyl-_L-Phe¹-Gly²-_L-Ala³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ser¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Ile¹⁶-_L-Gln¹⁷-_L-Gln¹⁸-_L-Valol¹⁹, and it was designated as bukhansantaibol C (3).

Compound 4 was isolated as a white amorphous powder, and the HRESIMS analysis established its molecular formula as C₉₁H₁₄₉N₂₃O₂₃ (m/z 967.0672 [M + 2H]²⁺; calcd 967.0671), indicating a deficiency of one oxygen atom compared to 1 (Table 1). Comparison of the 1D and 2D NMR spectra with those of 1 revealed the substitution of Ser¹⁰ with Ala¹⁰ in compound 4 (Tables S5 and S6, and Figures S38–S44). Furthermore, modified Marfey’s analysis detected only alanine, not serine, supporting the presence of two alanine residues in 4 (Figure S49). The ECD spectrum also supported a right-handed helical conformation, consistent with the structural characteristics observed in related peptaibols (Figure S47). Thus, the structure of 4 was determined as N-acetyl-_L-Trp¹-Gly²-_L-Ala³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ala¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Ile¹⁶-_L-Gln¹⁷-_L-Gln¹⁸-_L-Valol¹⁹ and named as bukhansantaibol D (4).

Compound 5 showed the molecular formula of C₉₁H₁₄₈N₂₂O₂₅ by the HRESIMS (m/z 975.5563 [M + 2H]²⁺; calcd 975.5566). The ¹H and ¹³C-NMR spectrum of 5 closely resembled those of 1 (Tables S7 and S8), with a molecular weight difference of 0.984 Da. A detailed analysis of the molecular formula revealed that 5 contains one additional oxygen atom and one fewer nitrogen and hydrogen atom compared to 1, indicating a substitution of one glutamine (Gln) residue to glutamic acid (Glu). The position of Glu substitution was determined by the MS/MS fragmentation (Figure 2).

In the full MS¹ spectrum of 5, in-source fragmentation produced b₁₂ (m/z 1195.6460) and y₇ (m/z 755.4657) ion (Figure 2(A1)). Notably, the y₇ ion of 5 was 0.984 Da higher than the corresponding y₇ ion in 1 (m/z 754.4819), supporting the replacement of Gln with Glu. Further MS/MS analysis of the y₇ ion revealed a Glu-containing y₇/b₅ ion at m/z 524.3025 (Figure 2(A2)), further validating the site of substitution. In addition, comprehensive NMR analysis, including COSY, TOCSY, and ROESY spectrum confirmed the amino acid sequence. With modified Marfey’s analysis (Figure S61) and ECD spectral analysis (Figure S59), the structure of compound 5 and was determined to be N-acetyl-_L-Trp¹-Gly²-_L-A We appreciate your feedback. We have revised the figure labels accordingly.la³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ser¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Ile¹⁶-_L-Glu¹⁷-_L-Gln¹⁸-_L-Valol¹⁹ and named as bukhansantaibol E (5).

Compound 6 exhibited an m/z value of 956.0508 in the HRESIMS data, corresponding to the molecular formula of C₈₉ H₁₄₇N₂₁O₂₅ ([M + 2H]²⁺, calcd 956.0511). The ¹H-NMR spectrum of 6 was similar to that of 5, except for differences observed in the aromatic proton region. In place of the characteristic indole signals of tryptophan, aromatic signals corresponding to a phenyl group (δ_H 7.38, 7.28, and 7.22) were observed, suggesting substitution of tryptophan with phenylalanine. (Table S9 and Figure S62). Moreover, a mass difference of 0.984 Da between compounds 3 and 6 indicated Glu substitution, mirroring the relationship observed between compounds 1 and 5. The MS/MS fragmentation analysis further confirmed the substitution of Glu at position 17 in 6 (Figure 2B). Comprehensive analysis of the COSY, TOCSY, and ROESY correlations established its amino acid connectivity. Furthermore, the HMBC correlations between the acetyl methyl protons (δ_H 2.09), the amide proton of phenylalanine (δ_H 9.60), and the acetyl carbonyl carbon (δ_C 172.3) confirm the presence of an N-terminal acetyl group attached to the phenylalanine residue. The absolute configurations of all amino acid residues, as well as the right-handed helical conformation of 6, were determined by modified Marfey’s analysis and the ECD spectral data, respectively (Figures S71 and S73). Based on these results, the structure of 6 was elucidated as N-acetyl-_L-Phe¹-Gly²-_L-Ala³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ser¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Ile¹⁶-_L-Glu¹⁷-_L-Gln¹⁸-_L-Valol¹⁸ and named as bukhansantaibol F (6).

Compound 7 (Structure shown in

Table 4. Structure of compounds 7–10.

Compound	Mass	R1	R2
7	1718.9719	L-Trp	OH (S)
8	1718.9719	L-Trp	OH (R)
9	1732.9876	L-Trp	OCH3 (S)
10	1693.9767	L-Phe	OCH3 (S)

) was determined to have a molecular formula of C₈₁H₁₃₀N₂₀O₂₁ based on the HRESIMS data (m/z 851.4886 [M + 2H – H₂O]²⁺; calcd 851.4880). Comparison of the ¹H and ¹³C-NMR spectra of 1 and 7 revealed the absence of characteristic signals corresponding to Valol residue in 7 (

Table 5. ¹H (900 MHz) data for compound 7 in pyridine-d₅ (δ in ppm, J in Hz).

	Position	δH		Position	δH
Ac	CH3	2.02, s	Ser10	N-H	8.45, m
Trp1	N-H	9.44, d (4.6)		α	4.71, m
	α	5.02, m		β	4.37, m; 4.53, m
	β	3.62, m; 3.68, m	Leu11	N-H	8.16, m
	1	11.95, d (2.2)		α	5.01, m
	2	7.41, d (2.2)		β	2.05, m; 2.23, m
	4	7.82, d (8.0)		γ	2.14, m
	5	7.18, m		δ1	0.99, d (6.6)
	6	7.28, m		δ2	0.98, d (6.6)
	7	7.54, m	Aib12	N-H	8.41, s
Gly2	N-H	9.99, m		α-Me1	1.80, s
	α	4.04, m; 4.14, m		α-Me2	1.78, s
Ala3	N-H	8.52, m	Pro13	α	4.61, m
	α	4.51, m		β	1.75, m; 2.21, m
	β	1.53, d (7.3)		γ	1.68, m; 1.91, m
Aib4	N-H	8.52, s		δ	3.96, m; 4.02, m
	α-Me1	1.69, s	Val14	N-H	8.28, d (6.0)
	α-Me2	1.72, s		α	4.22, m
Aib5	N-H	8.23, s		β	2.61, m
	α-Me1	1.69, s		γ1	1.29, m
	α-Me2	1.70, s		γ2	1.19, m
Gln6	N-H	8.31, d (4.6)	Aib15	N-H	7.94, s
	α	4.36, m		α-Me1	1.83, s
	β	2.68, m; 2.77, m		α-Me2	1.82, s
	γ	2.77, m; 2.87, m	Ile16	N-H	7.53, d (5.3)
	CONH2	7.74, s; 8.14, s		α	4.98, m
Aib7	N-H	8.35, s		β	2.57, m
	α-Me1	1.82, s		γ1	1.61, m; 1.95 m
	α-Me2	1.78, s		γ2	1.13, d (6.8)
Aib8	N-H	8.06, s		δ	0.95, t (7.4)
	α-Me1	1.80, s	cycGln17	N-H	8.19, d (7.5)
	α-Me2	1.72, s		α	4.75, m
Aib9	N-H	8.39, s		β	2.25, m; 2.52, m
	α-Me1	1.82, s		γ	2.52, m; 3.04, m
	α-Me2	1.83, s		β’	5.67, t (3.6)
				CONH	8.80, d (3.6)

and

Table 6. ¹³C NMR (225 MHz) data for compound 7 in pyridine-d₅.

	Position	δC		Position	δC		Position	δC
Ac	CH3	22.7	Gln6	C=O	176.2	Pro13	C=O	174.7
	C=O	172.4		α	57.4		α	63.9
Trp1	C=O	174.2		β	27.0		β	29.0
	α	57.0		γ	32.6		γ	26.3
	β	27.9		CONH2	174.5		δ	49.4
	2	124.0	Aib7	C=O	176.9	Val14	C=O	173.4
	3	110.3		α	56.9		α	62.7
	3a	128.1		α-Me1	26.9		β	29.4
	4	118.7		α-Me2	23.2		γ1	19.8
	5	119.1	Aib8	C=O	176.2		γ2	19.3
	6	121.8		α	56.7	Aib15	C=O	175.3
	7	111.9		α-Me1	26.8		α	57.2
	7a	137.4		α-Me2	23.3		α-Me1	26.9
Gly2	C=O	174.8	Aib9	C=O	177.6		α-Me2	23.1
	α	44.3		α	57.2	Ile16	C=O	172.2
Ala3	C=O	175.1		α-Me1	27.9		α	58.9
	α	52.2		α-Me2	23.5		β	36.7
	β	16.4	Ser10	C=O	171.3		γ1	25.3
Aib4	C=O	176.7		α	60.3		γ2	16.3
	α	56.5		β	62.0		δ	11.9
	α-Me1	26.8	Leu11	C=O	173.9	cycGln17	α	50.4
	α-Me2	23.2		α	59.0		β	22.7
Aib5	C=O	177.1		β	40.3		γ	28.9
	α	56.6		γ	24.7		δ	171.0
	α-Me1	23.2		δ1	20.8		β’	78.9
	α-Me2	26.3		δ2	23.3
			Aib12	C=O	174.5
				α	56.9
				α-Me1	23.0
				α-Me2	26.9

, and Figures S74 and S75). Furthermore, COSY correlation among δ_H 5.67 (CH)/4.75 (CH)/2.25 and 2.52 (CH₂)/2.52 and 3.04 (CH₂), along with HMBC correlation from δ_H 5.67 to amide carbonyl carbon (δ_C 171.0) suggested that the C-terminal Gln residue underwent intramolecular cyclization (Figure 3 and Figures S74–S80).

To the best of our knowledge, this is the first report of C-terminal cyclo-Glnol moiety (2-hydroxy-3-amino-6-piperidone) among peptaibol derivatives. Similar cyclization of C-terminal Asn or Gln residues has been observed during protein splicing in Pyrococcus abyssi, resulting in the formation of C-terminal aminosuccinimide or aminoglutarimide, respectively [15,16]. It could be proposed that this occurs via a nucleophilic attack by the γ-amide nitrogen on the adjacent carbonyl carbon of the peptide bond. A subsequent reduction step may then lead to the formation of the unique cyclic structure observed at the C-terminus of compound 7. The absolute configurations of the amino acid residues and the right-handed helical conformation were confirmed by modified Marfey’s analysis and ECD spectrum, respectively (Figures S83 and S85). Based on the L-configuration of Gln¹⁷, the configuration of hydroxy group was determined through analysis of the coupling constant. In the MM2-minimized models, the two possible configurations, (S,S) and (S,R) exhibited the dihedral angles of 52.7° and −69.2°, respectively, between the α-proton and β’-proton (Figure 4). Given that the measured J value of the triplet methine proton at δ_H 5.67 was 3.6 Hz, the smaller dihedral angle associated with (S,S) configuration is more consistent with the observed data, according to the Karplus equation [17]. Based on these results, the structure of 7 was established as N-acetyl-_L-Trp¹-Gly²-_L-Ala³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ser¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Ile¹⁶-_L-cycGlnol¹⁷ and named as bukhansantaibol G (7).

Compound 8 showed the identical molecular formular with 7, as determined by the HRESIMS (m/z 851.4886 [M + 2H – H₂O]²⁺; calcd 851.4880). The ¹H and ¹³C-NMR spectra were highly similar to those of 7, and analysis of the 2D NMR spectra confirmed that 7 and 8 shared the same planar structure. However, subtle differences in the chemical shifts of the C-terminal cyclo-Glnol residue suggested that 8 is a diastereomer of 7; δ_H 9.14 in 8 vs. 8.80 in 7; δ_C 75.8/δ_H 5.45 in 8 vs. δ_C 78.9/δ_H 5.67 in 7; δ_C 21.7/δ_H 1.96, 2.73 in 8 vs. δ_C 22.7/δ_H 2.25, 2.52 in 7; δ_C 30.9/δ_H 2.53, 2.59 in 8 vs. δ_C 28.9/δ_H 2.52, 3.04 in 7. Notably, the coupling constant 2.5 Hz between the α-proton and β’-proton in 8 was smaller than that observed in 7 (3.6 Hz). Based on the dihedral angle derived from the previously mentioned MM2-minimized model (Figure 4) and interpretation of the Karplus equation, this difference supported the assignment of an (S,R)-configuration for 8. The absolute configurations of amino acid residues were confirmed by modified Marfey’s analysis (Figure S97). In addition, ECD spectrum revealed the right-handed helical conformation (Figure S95). Accordingly, the compound was named as bukhansantaibol H (8).

Compounds 9 and 10 were assigned the molecular formulas C₈₂H₁₃₂N₂₀O₂₁ and C₈₀H₁₃₁N₁₉O₂₁, respectively, based on the HRESIMS (Table 4). The ¹H-NMR, COSY, and TOCSY spectra of 9 and 10 revealed similar cyclic-Glnol moiety at the C-terminal with 7 (Tables S13–S16). Additionally, a methoxy singlet (δ_H 3.29, 3H, s) was observed in 9 and 10 (Figures S89 and S100). Compound 9 exhibited indole proton signals characteristic of tryptophan (δ_H 11.96, 7.41, 7.83, 7.18, 7.28, 7.55; δ_C 124.2, 110.2, 128.1, 118.7, 119.1, 121.8, 111.9, 137.4), while compound 10 showed aromatic proton signals consistent with a phenyl ring (δ_H 7.30, 7.26, 7.27; δ_C 137.5, 129.3, 128.6, 126.9), indicating substitution of tryptophan with phenylalanine. Combined with the result of modified Marfey’s analysis (Figures S109 and S121), the structure of compounds 9 and 10 elucidated as N-acetyl-_L-Trp¹-Gly²-_L-Ala³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ser¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Ile¹⁶-_L-cycGlnol¹⁷-Me (9) and N-acetyl-_L-Phe¹-Gly²-_L-Ala³-Aib⁴-Aib⁵-_L-Gln⁶-Aib⁷-Aib⁸-Aib⁹-_L-Ser¹⁰-_L-Leu¹¹-Aib¹²-_L-Pro¹³-_L-Val¹⁴-Aib¹⁵-_L-Ile¹⁶-_L-cycGlnol¹⁷-Me (10). In addition, compounds 9 and 10 found to have a right-handed helical conformation based on ECD data (Figures S107 and S119). These compounds are assigned to trivial name as bukhansantaibol I (9) and bukhansantaibol J (10).

Compounds 11 and 12 (Structure shown in

Table 7. Structure of compounds 11–12.

Compound	Mass	R	Compound	Mass	R
11	1014.6114	L-Leu	12	1014.6114	L-Ile

) were both assigned the molecular formular of C₅₀H₈₂N₁₀O₁₂ based on HRESIMS (m/z 1015.6174 [M + H]⁺; calcd 1015.6186). The ¹H-NMR spectrum of 11 displayed characteristic signals including one aldehyde proton (δ_H 9.90), ten amide protons (δ_H 8.04–9.56), two aromatic protons (δ_H 7.48 and 7.17), four α-protons (δ_H 4.21–4.57), and eighteen methyl protons (δ_H 0.78–2.06) (

Table 8. ¹H (600 MHz) and ¹³C (150 MHz) data for compound 11 in pyridine-d₅.

	Position	δC	δH		Position	δC	δH
Ac	CH3	23.0	2.06, s	Leu6	N-H	-	8.13, d (6.4)
	C=O	171.7	-		C=O	174.8	-
Aib1	N-H	-	9.56, s		α	55.8	4.49, m
	C=O	177.4	-		β	39.8	1.96, m; 2.11, m
	α	56.7	-		γ	24.8	2.11, m
	α-Me1	26.5	1.63, s		δ1	21.2	0.93, d (6.2)
	α-Me2	24.2	1.73, s		δ2	22.7	0.99, d (6.1)
Ile2	N-H	-	8.93, d (4.0)	Aib7	N-H	-	8.24, s
	C=O	172.7	-		C=O	176.2	-
	α	61.4	4.21, m		α	56.7	-
	β	35.4	1.98, m		α-Me1	26.5	1.84, s
	γ1	26.8	1.67, m		α-Me2	23.4	1.85, s
	γ2	15.6	1.02, d (6.8)	Ala8	N-H	-	8.18, m
	δ	11.1	0.78, t (7.4)		C=O	173.4	-
Aib3	N-H	-	8.29, s		α	52.4	4.52, m
	C=O	177.1	-		α-Me1	16.9	1.74, d (2.1)
	α	56.7	-	Aib9	N-H	-	8.12, s
	α-Me1	26.8	1.72, s		C=O	176.1	-
	α-Me2	23.0	1.75, s		α	56.7	-
Tyr4	N-H	-	8.04, d (6.0)		α-Me1	26.8	1.96, s
	C=O	173.6	-		α-Me2	24.4	1.97, s
	α	59.4	4.60, m	Aibal10	N-H	-	8.06, s
	β	36.7	3.54, m; 3.53, m		HC=O	201.8	9.90, s
	γ	127.9	-		α	59.2
	δ	130.5	7.48, d (6.0)		α-Me1	21.6	1.66, s
	ε	116.1	7.17, m		α-Me2	21.7	1.59, s
	ζ	157.4	-
Aib5	N-H		8.35, s
	C=O	176.9	-
	α	56.6	-
	α-Me1	26.6	1.84, s
	α-Me2	23.2	1.78, s

). The TOCSY correlation enabled the identification of spin systems from amide proton to side chain protons for most amino acid residues, except Aib. Sequential connectivity of the amino acids was further elucidated through the ROESY correlations between amide protons (Figure 5). The HMBC correlation between an amide proton (δ_H 9.56), two singlet methyl groups (δ_H 1.63, 1.73) and the corresponding α-carbon (δ_C 56.7), together with the correlations between the same amide proton (δ_H 9.56), the acetyl methyl proton (δ_H 2.06), and the acetyl carbonyl carbons (δ_C 171.7) confirmed the presence of N-terminal acetylated Aib residue. Additional Aib residues were similarly assigned by the HMBC data. Furthermore, HMBC correlations from the aldehyde proton (δ_H 9.90) and two singlet CH₃ (δ_H 1.59 and 1.66) to α-carbon (δ_C 59.2) supported the presence of aldehyde moiety of C-terminal Aib residue (Aibal, 2-amino-2-methylpropanal). Based on the etymology of peptaibol, we propose the term ‘peptaibal’ as a new class name for this sequence, which terminates with an aldehyde group instead of an alcohol at the C-terminus. Compound 12 exhibited similar 1D and 2D-NMR spectra with 11, with the primary difference being the substitution of the Ile⁶ in 11 with Leu⁶ in 12. ECD spectral analysis revealed that both compounds have a right-handed helical conformation (Figures S131 and S143). Along with the result of modified Marfey’s analysis (Figures S133 and S145), the structures of 11 and 12 were determined as N-acetyl-Aib¹-_L-Ile²-Aib³-_L-Tyr⁴-Aib⁵-_L-Leu⁶-Aib⁷-_L-Ala⁸-Aib⁹-Aibal¹⁰ and N-acetyl-Aib¹-_L-Ile²-Aib³-_L-Tyr⁴-Aib⁵-_L-Ile⁶-Aib⁷-_L-Ala⁸-Aib⁹-Aibal¹⁰, respectively, and named as bukhansantaibal A (11) and bukhansantaibal B (12).

These peptaibals 11 and 12 are presumed to represent intermediates formed during the biosynthetic conversion of a carboxylic acid to an alcohol in peptaibol biosynthesis. The assembly of peptaibol is mediated by NRPS multi-modular enzyme, which iteratively condenses amino acid onto a peptide carrier protein module (PCP)-tethered amino acyl intermediate. Upon completion of chain elongation, the thioesterase domain (TE) typically catalyzes the release of the mature peptide [2]. A reductase domain (thioreductase, TR), located adjacent to the PCP or TE domains, enables the release of the peptide as either an aldehyde or an alcohol [18,19]. Alcohol formation involves a two-step reduction process catalyzed by the TR domain with NADPH as a cofactor, necessitating the reintroduction of an aldehyde intermediate for the second reduction. In certain NRPS systems, structural variations allow only the initial two-electron reduction step to occur, resulting in the predominant formation of aldehyde products [20,21]. While most peptaibol are reduced to alcohol, the isolation of peptaibols bearing C-terminal aldehydes is particularly noteworthy. Further biochemical characterization of the associated enzymatic machinery may elucidate the mechanism underlying aldehyde formation in these systems.

All compounds were evaluated for their inhibitory effects against HCT-8 (colon cancer) and SK-OV-3 (ovarian cancer) cell lines (

Table 9. IC₅₀ values (μM) of compounds 1–12 against HCT-8 and SK-OV-3 cancer cells ^a.

No.	HCT-8 (Colon Cancer)	SK-OV-3 (Ovarian Cancer)
1	2.1 ± 0.2	3.4 ± 0.2
2	11.7 ± 0.8	13.8 ± 1.0
3	18.5 ± 0.6	19.6 ± 0.1
4	7.7 ± 0.3	5.7 ± 0.04
5	2.6 ± 1.0	3.5 ± 0.2
6	>50	>50
7	>50	>50
8	>50	>50
9	>50	>50
10	>50	>50
11	>50	>50
12	>50	>50
Etoposide b	33.7 ± 1.5	37.3 ± 1.8

^a Results are presented as mean ± standard deviation from triplicate experiments. ^b Etoposide was used as positive control.

). Among them, compounds 1–5 exhibited notable cytotoxicity, with IC₅₀ values ranging from 2.1 to 19.6 μM. In particular, compound 1, a 19-residue peptaibol, exhibited potent activity against both HCT-8 and SK-OV-3 cells, with IC₅₀ values of 2.1 and 3.4 μM, respectively. Compound 5, which differ from compound 1 by substitution of one Gln at position 17, showed comparable cytotoxicity, with IC₅₀ values of 2.6 and 3.5 μM against HCT-8 and SK-OV-3 cells, respectively. In contrast, substitution of the C-terminal Valol with a cyclic Glnol moiety, as seen in compounds 7–10, resulted in a marked loss of cytotoxicity. The importance of the C-terminal Valol group was further supported by the inactivity of compounds 11 and 12, which bear a C-terminal aldehyde instead of amino alcohol.

In conclusion, twelve previously undescribed peptidic compounds were isolated from the culture extract of Trichoderma atroviride, obtained from soil samples through LC-MS and bioactivity-guided purification. Notably, compounds 7–10 and 11–12 were characterized by the presence of a unique C-terminal cyclic Glnol moiety and aldehyde group, respectively. Given their structural diversity and potent cytotoxicity against cancer cell lines, peptaibols from Trichoderma species, including the strain examined in this study, represent a promising source for the discovery of novel anticancer drug leads.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotation was detected using a JASCO DIP-1000 polarimeter. The optical densities (O.D) based on the UV spectra were measured using a JASCO UV-550 spectrophotometer (JASCO, Tokyo, Japan). Electronic circular dichroism (ECD) spectra were recorded on a ChirascanVX spectrometer (Applied Photophysics Ltd., Leatherhead, UK). Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCE 600, 800, and 900 MHz spectrometers (Bruker, Billerica, MA, USA). LC-MS/MS analysis was performed using a UHPLC-HR-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) system equipped with a diode array detector and an YMC-Triart C₁₈ column (2.0 μm, 100 × 2.1 mm, i.d., flow rate 0.3 mL/min, YMC Co., Kyoto, Japan). Column chromatography was performed using silica gel (230–400 mesh, Merck, Darmstadt, Germany) and HP20ss gel (Mitsubishi Chemical, Tokyo, Japan). The preparative HPLC was performed using Waters HPLC system equipped with pumps, a 996 photodiode-array detector (Waters Corporation, Milford, MA, USA), and a Luna 5 μm C₁₈ column (100 Å, 250 × 21.2 mm I.D., Phenomenex, Torrance, CA, USA) with a flow rate of 10 mL/min. The semi-preparative HPLC were conducted on the same Waters HPLC system using a Luna 5 μm C₁₈ (100 Å, 250 × 10 mm I.D., Phenomenex, Torrance, CA, USA) and a Kinetex 5 μm F5 (100 Å, 250 × 10 mm I.D., Phenomenex, Torrance, CA, USA) with a flow rate of 4 mL/min. All solvents used were of ACS grade or better.

3.2. Fungal Material

The Trichoderma atroviride isolate (soil collection from the territory of Mountain Bukhansan; Deposit No.: BHM16-2) was obtained from a soil sample collected near Insubong Peak in the area of Bukhansan Mountain, South Korea (37.6597281, 126.9845630) in February 2023. The fungi were identified based on the ribosomal internal transcribed spacer (ITS) region (Macrogen, Korea). The resulting sequence data were compared to fungal sequences contained in GenBank, which revealed 100% identity matches to isolates described as Trichoderma atroviride (GenBank accession no. KJ786757.1). The sequence data were deposited in GenBank (Trichoderma atroviride: GenBank accession no. PV809826).

3.3. Fermentation

To carry out large-scale cultivation for the isolation and purification of the bioactive constituents, the fungi were recovered from cryogenic storage (stored in a vial at −80 °C as mycelium with 20% aqueous glycerol). After recovery on Rose Bengal medium plates (15 g of agar, 10 g of malt extract, 1 g of yeast, 0.05 g of chloramphenicol, 0.025 g Rose Bengal, 1 L of DI H₂O), fungal mycelia were aseptically cut into small pieces (~1 cm²) to prepare the inoculum for large-scale fermentation. The fermentation was performed in 1 L flask containing bilayered Cheerios breakfast cereal as the cultivation medium, supplemented with a 0.3% sucrose solution and 0.005% chloramphenicol. Three mycelial fragments were aseptically inoculated onto the surface of Cheerios cereal solid medium in each of ten 1L flasks. The cultures were incubated at room temperature for 3 weeks.

3.4. Extraction and Isolation of Compounds 1–12

Cultured fungi were extracted with EtOAc (0.5 L × 3) for 12 h at room temperature. The extract was filtered and evaporated under reduced pressure to obtain the EtOAc soluble residue, fraction A (3 g). Fraction A was subjected to silica gel vacuum liquid column chromatography (VLC), eluted sequentially with dichloromethane (100%, fraction B), dichloromethane-MeOH (9:1, fraction C), and MeOH (100%, fraction D). Fraction C (1.5 g) was separated using HP20ss gel VLC, eluted sequentially with aqueous MeOH (30% MeOH for fraction E, 50% MeOH for fraction F, 70% MeOH for fraction G, 90% MeOH for fraction H, and 100% MeOH for fraction I), and dichloromethane (CH₂Cl₂)-MeOH (1:1, fraction J). Fraction D (1.0 g) was also subjected to the same procedure using HP20ss gel VLC, yielding fractions K (30% MeOH), L (50% MeOH), M (70% MeOH), N (90% MeOH), O (100% MeOH), and P (CH₂Cl₂/MeOH 1:1).

Fraction H (244 mg) was fractionated by semi-preparative HPLC (C₁₈, isocratic 60% MeOH in H₂O, flow rate of 4 mL/min) to obtain 13 subfractions (H-1~H-13). Compound 11 (2.3 mg, t_R = 12.4 min) was purified from subfraction H-9 (4.4 mg) by semi-preparative HPLC using C₁₈ (isocratic 65% ACN in H₂O, flow rate of 4 mL/min). Compound 12 (3.1 mg, t_R = 14.0 min) was obtained from subfraction H-13 (8.7 mg) via semi-preparative HPLC (C₁₈, isocratic 60% ACN in H₂O, flow rate of 4 mL/min).

Since fractions N (179 mg) and O (106 mg) shared most of metabolites, as observed in their HPLC chromatograms, they were combined and subsequently subjected to further separation (fraction NO). Fraction NO (285 mg) was subjected to semi-preparative HPLC (C₁₈, isocratic 55% MeOH in H₂O with 0.1% formic acid, flow rate of 4 mL/min) to obtain 13 subfractions (NO-1~NO-13). Subfraction NO-1 (10.2 mg) was further purified by semi-preparative HPLC (F5, isocratic 40% ACN in H₂O, flow rate of 4 mL/min) to give compounds 10 (1.6 mg, t_R = 12.9 min) and 9 (2.0 mg, t_R = 14.8 min). Subfraction NO-2 (4.9 mg) was separated by semi-preparative HPLC (F5, isocratic 35% ACN in H₂O, flow rate of 4 mL/min), yielding six subfractions (NO-2-1~NO-2-6). Compound 7 (1.2 mg, t_R = 23.2 min) was purified from subfraction NO-2-4 (1.6 mg) by semi-preparative HPLC (C₁₈, isocratic 45% ACN in H₂O, flow rate of 4 mL/min), and compound 8 (1.3 mg, t_R = 18.9 min) was purified from subfraction NO-2-5 (1.5 mg) by semi-preparative HPLC (C₁₈, isocratic 45% ACN in H₂O, flow rate of 4 mL/min). Subfraction NO-4 (27.2 mg) was subjected to semi-preparative HPLC (C₁₈, isocratic 50% ACN in H₂O, flow rate of 4 mL/min), giving compound 2 (3.4 mg, t_R = 18.2 min). Compound 1 (6.0 mg, t_R = 19.3 min) was purified from subfraction NO-5 (30.3 mg) by semi-preparative HPLC (F5, isocratic 37% ACN in H₂O, flow rate of 4 mL/min). Subfraction NO-6 (17.4 mg) was separated using semi-preparative HPLC (F5, isocratic 35% ACN in H₂O, flow rate of 4 mL/min) to yield compounds 3 (5.0 mg, t_R = 61.4 min) and 4 (1.4 mg, t_R = 95.2 min). Compound 5 (28.9 mg, t_R = 23.7 min) was purified from subfraction NO-7 (37.0 mg) by semi-preparative HPLC (C₁₈, isocratic 52% ACN in H₂O with 0.1% formic acid, flow rate of 4 mL/min), and compound 6 (16.0 mg, t_R = 23.1 min) was purified from subfraction NO-8 (22.1 mg) by semi-preparative HPLC (C₁₈, isocratic 52% ACN in H₂O with 0.1% formic acid, flow rate of 4 mL/min).

Bukhansantaibol A (1): white amorphous powder; [α]²⁵_D −6.8 (c 0.05, MeOH); UV (c 0.05, MeOH) λ_max (log ε) 202 (4.67), 219 (4.52), 280 (3.65) nm; ECD (c 0.5, CH₃CN) λmax (Δε) 208 (−67.9), 221 (−48.1); ¹H-NMR (600 MHz, pyridine-d₅) and ¹³C-NMR (150 MHz, pyridine-d₅), see Table 2 and Table 3; HRESIMS m/z 975.0645 [M + 2H]²⁺ (calcd for C₉₁H₁₅₁N₂₃O₂₄, 975.0646).

Bukhansantaibol B (2): white amorphous powder; [α]²⁵_D −6.7 (c 0.05, MeOH); UV (c 0.05, MeOH) λ_max (log ε) 201 (4.54), 220 (4.37), 280 (3.48) nm; ECD (c 0.3, CH₃CN) λmax (Δε) 207 (−32.6), 221 (−22.1); ¹H-NMR (600 MHz, pyridine-d₅) and ¹³C-NMR (150 MHz, pyridine-d₅), see Tables S1 and S2; HRESIMS m/z 975.0643 [M + 2H]²⁺ (calcd for C₉₁H₁₅₁N₂₃O₂₄, 975.0646).

Bukhansantaibol C (3): white amorphous powder; [α]²⁵_D −7.4 (c 0.10, MeOH); UV (c 0.10, MeOH) λ_max (log ε) 201 (4.46) nm; ECD (c 0.5, CH₃CN) λmax (Δε) 207 (−39.5), 223 (−27.8); ¹H-NMR (600 MHz, pyridine-d₅) and ¹³C-NMR (150 MHz, pyridine-d₅), see Tables S3 and S4; HRESIMS m/z 955.5593 [M + 2H]²⁺ (calcd for C₈₉H₁₅₀N₂₂O₂₄, 955.5591).

Bukhansantaibol D (4): white amorphous powder; [α]²⁵_D −7.5 (c 0.05, MeOH); UV (c 0.05, MeOH) λ_max (log ε) 200 (4.46), 219 (4.19), 280 (3.33) nm; ECD (c 0.5, CH₃CN) λmax (Δε) 208 (−25.5), 224 (−16.8); ¹H-NMR (800 MHz, pyridine-d₅) and ¹³C-NMR (200 MHz, pyridine-d₅), see Tables S5 and S6; HRESIMS m/z 967.0672 [M + 2H]²⁺ (calcd for C₉₁H₁₅₁N₂₃O₂₃, 967.0671).

Bukhansantaibol E (5): white amorphous powder; [α]²⁵_D −7.5 (c 0.10, MeOH); UV (c 0.10, MeOH) λ_max (log ε) 204 (4.45), 219 (4.33), 281 (3.57) nm; ECD (c 0.6, CH₃CN) λmax (Δε) 208 (−24.6), 220 (−17.0); ¹H-NMR (600 MHz, pyridine-d₅) and ¹³C-NMR (150 MHz, pyridine-d₅), see Tables S7 and S8; HRESIMS m/z 975.5563 [M + 2H]²⁺ (calcd for C₉₁H₁₅₀N₂₂O₂₅, 975.5566).

Bukhansantaibol F (6): white amorphous powder; [α]²⁵_D −10.3 (c 0.10, MeOH); UV (c 0.10, MeOH) λ_max (log ε) 201 (4.57), 256 (3.54) nm; ECD (c 0.6, CH₃CN) λmax (Δε) 208 (−42.7), 222 (−30.9); ¹H-NMR (600 MHz, pyridine-d₅) and ¹³C-NMR (150 MHz, pyridine-d₅), see Tables S9 and S10; HRESIMS m/z 956.0508 [M + 2H]²⁺ (calcd for C₈₉H₁₄₉N₂₁O₂₅, 956.0511).

Bukhansantaibol G (7): white amorphous powder; [α]²⁵_D −6.7 (c 0.12, MeOH); UV (c 0.12, MeOH) λ_max (log ε) 200 (4.06), 219 (3.81), 280 (2.84) nm; ECD (c 0.3, CH₃CN) λmax (Δε) (Δε) 207 (−28.2), 224 (−19.0); ¹H-NMR (900 MHz, pyridine-d₅) and ¹³C-NMR (225 MHz, pyridine-d₅), see Table 5 and Table 6; HRESIMS m/z 851.4886 [M + 2H − H₂O]²⁺ (calcd for C₈₁H₁₃₀N₂₀O₂₀, 851.4880).

Bukhansantaibol H (8): white amorphous powder; [α]²⁵_D −3.8 (c 0.12, MeOH); UV (c 0.12, MeOH) λ_max (log ε) 201 (4.27), 219 (4.01), 280 (3.18) nm; ECD (c 0.6, CH₃CN) λmax (Δε) 209 (−22.8), 223 (−17.0); ¹H-NMR (800 MHz, pyridine-d₅) and ¹³C-NMR (200 MHz, pyridine-d₅), see Tables S11 and S12; HRESIMS m/z 851.4886 [M + 2H − H₂O]²⁺ (calcd for C₈₁H₁₃₀N₂₀O₂₀, 851.4880).

Bukhansantaibol I (9): white amorphous powder; [α]²⁵_D +2.0 (c 0.06, MeOH); UV (c 0.06, MeOH) λ_max (log ε) 200 (4.40), 219 (4.11), 280 (3.35) nm; ECD (c 0.6, CH₃CN) λmax (Δε) 207 (−34.0), 223 (−22.1); ¹H-NMR (800 MHz, pyridine-d₅) and ¹³C-NMR (200 MHz, pyridine-d₅), see Tables S13 and S14; HRESIMS m/z 1733.9958 [M + H]⁺ (calcd for C₈₂H₁₃₃N₂₀O₂₁, 1733.9949).

Bukhansantaibol J (10): white amorphous powder; [α]²⁵_D +3.0 (c 0.06, MeOH); UV (c 0.10, MeOH) λ_max (log ε) 200 (4.48) nm; ECD (c 0.6, CH₃CN) λmax (Δε) 207 (−22.6), 226 (−14.3); ¹H-NMR (900 MHz, pyridine-d₅) and ¹³C-NMR (225 MHz, pyridine-d₅), see Tables S15 and S16; HRESIMS m/z 1694.9852 [M + H]⁺ (calcd for C₈₀H₁₃₂N₁₉O₂₁, 1694.9840).

Bukhansantaibal A (11): white amorphous powder; [α]²⁵_D +4.6 (c 0.10, MeOH); UV (c 0.10, MeOH) λ_max (log ε) 200 (4.21), 224 (3.71), 277 (2.71) nm; ECD (c 0.5, CH₃CN) λmax (Δε) 210 (−20.4), 221 (−18.1); ¹H-NMR (600 MHz, pyridine-d₅) and ¹³C-NMR (150 MHz, pyridine-d₅), see Table 8; HRESIMS m/z 1015.6174 [M + H]⁺ (calcd for C₅₀H₈₃N₁₀O₁₂, 1015.6186).

Bukhansantaibal B (12): white amorphous powder; [α]²⁵_D +4.4 (c 0.10, MeOH); UV (c 0.10, MeOH) λ_max (log ε) 203 (4.40), 224 (4.01), 278 (3.09) nm; ECD (c 1.0, CH₃CN) λmax (Δε) 211 (−20.9), 221 (−17.7); ¹H-NMR (600 MHz, pyridine-d₅) and ¹³C-NMR (150 MHz, pyridine-d₅), see Table S17; HRESIMS m/z 1015.6174 [M + H]⁺ (calcd for C₅₀H₈₃N₁₀O₁₂, 1015.6186).

3.5. Absolute Configuration of Amino Acids in 1–12 Using Marfey’s Method

Compounds 1–12 (each 0.2 mg) were hydrolyzed at 110 °C for 24 h with 400 μL of 6 N HCl containing 1% 2-mercaptoethanol (2-ME) [22]. After cooling, the hydrolysates were evaporated to dryness under reduced pressure at room temperature. The dried residues were then dissolved in H₂O (100 μL) and 1 M NaHCO₃ (30 μL). 100 μL of 1% solution of N-α-(2,4-dinitro-5-fluorophenyl)-_D/L-leucinamide (_D/L-FDLA, Marfey’s reagent, Sigma, St. Louis, MO, USA) in acetone was added to each vial and incubated at 45 °C for 1 h. The mixture was neutralized with 1 N HCl (30 μL) and diluted with ACN (740 μL) before filtering with a PTFE filter (0.22 μm). LC/MS analysis (YMC-Triart C₁₈ column, 2.0 μm, 100 × 2.1 mm, i.d.) of the DLA derivatives was performed using a gradient system consisting of solvent A (H₂O with 0.1% formic acid) and solvent B (ACN with 0.1% formic acid). The gradient was programmed from 90: 10 to 40: 60 (A: B) over 34 min at a flow rate of 0.3 mL/min. The injection volume was 2 μL, and the column oven temperature was maintained at 40 °C. The retention times (min) of the _L and _D-DLA derivatives obtained from hydrolysis were as follows: 18.43 (_L-Ser-_L-DLA), 19.69 (_L-Glu-_L-DLA), 21.26 (_L-Ala-_L-DLA), 21.31 (_L-Pro-_L-DLA), 23.94 (_L-Val-_L-DLA), 24.28 (_L-Valol-_L-DLA), 25.83 (_L-Ile-_L-DLA), 26.03 (_L-Leu-_L-DLA), 26.05 (_L-Trp-_L-DLA), 26.35 (_L-Phe-_L-DLA), 19.02 (_L-Ser-_D-DLA), 20.67 (_L-Glu-_D-DLA), 23.18 (_L-Pro-_D-DLA), 23.89 (_L-Ala-_D-DLA), 28.26 (_L-Val-_D-DLA), 28.38 (_L-Trp-_D-DLA), 28.51 (_L-Valol-_D-DLA), 29.72 (_L-Phe-_D-DLA), 30.52 (_L-Ile-_D-DLA), and 30.72 (_L-Leu-_D-DLA). The _L-DLA or _D-DLA derivatives of authentic amino acid standards of _L-Ile and _L-allo-Ile were subjected to LC-MS analysis under the same condition. The retention times of the derivatives were 25.84 (_L-Ile-_L-DLA) and 25.71 (_L-allo-Ile-_L-DLA), respectively (Figure S146).

3.6. Cell Culture

HCT-8 (colorectal carcinoma), and SK-OV-3 (ovarian cancer) cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in RPMI-1640 medium (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and streptomycin, and incubated in a humidified atmosphere with 5% CO₂ at 37 °C [23].

3.7. Cell Viability Assay

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma Chemical Co., St. Louis, MO). Cells were seeded in 96-well plates and incubated at 37 °C for 24 h. Subsequently, cells were treated with isolated compounds at concentrations of 2, 20, and 100 μM for an additional 24 h. After treatment, 25 μL of MTT solution (5 mg/mL; Thermo Fisher Scientific, Waltham, MA, USA) was added to each well and incubated for 3 h. The resulting formazan crystals were dissolved in 100 μL of dimethyl sulfoxide, and the absorbance was measured at 550 nm using an ELx808 microplate reader (BioTek Instruments Inc., Winooski, VT, USA) [23].

4. Conclusions

Twelve new compounds, bukhansantaibols A–J (1–10) and bukhansantaibals A–B (11–12), were isolated from the soil fungus Trichoderma atroviride collected from the area of Bukhansan Mountain. Among them, six were identified as typical peptaibols (1–6), while the remaining six were elucidated highly unusual peptaibol derivatives that have not been previously reported: four compounds (7–10) featured cyclized alcohol-containing amino acids at the C-terminal, and two metabolites (11–12) possessed an aldehyde group at the C-terminal, which is considered an intermediate in the biosynthetic pathway of peptaibols. All isolated compounds were evaluated from their cytotoxicity against two human cancer cell, HCT-8 and SK-OV-3. As a result, the typical peptaibols exhibited greater cytotoxicity compared to the new class of molecules. Notably, compounds 1, 4, 5 showed IC₅₀ values of below 10 μM against both cell lines. These results were thought to be associated with the membrane-permeabilizing properties of peptaibols, and suggest that, in addition to their cytotoxicity against cancer cells, these compounds may serve as promising leads for the development of antimicrobial and antiprotozoal agents.