Eight New Peptaibols from Sponge-Associated Trichoderma atroviride

Eight new and four known peptaibols were isolated from a strain of the fungus, Trichoderma atroviride (NF16), which was cultured from an Axinellid sponge collected from the East Mediterranean coast of Israel. The structures of the pure compounds were determined using HRMS, MS/MS and one- and two-dimensional NMR measurements. The isolated compounds belong to the trichorzianines, a family of 19-residue linear hydrophobic peptides containing a high proportion of α-aminoisobutyric acid (Aib), an acetylated N-terminus and a C-terminal amino alcohol. These new peptaibols exhibited antimicrobial activity against environmental bacteria isolated from the Mediterranean coast of Israel.

of the peptaibols [14]. These two fragments were advantageous in the structural elucidation of 1 by MS/MS ( Figure 1). Table 1. Trichorzianines (TA) 1-12 isolated from Trichoderma atroviride (NF16). Positions marked in grey differ between compounds. TA1938, trichorzianine 1938. Aib, α-amino-isobutyric acid. The 1 H and 13 C NMR spectra (Table 2 and Supplementary Figures S1 and S2) revealed that 1 is composed of the amino alcohol, phenylalaninol (Pheol), acetate, the non-proteinogenic amino acids, α-amino-isobutyric acid (Aib) and isovaline, and the proteinogenic amino acids, leucine, isoleucine, serine, glutamine, glutamic acid, proline and alanine. According to the MS/MS data (Figure 1), Aib appeared seven times in the molecule. The presence of the amino alcohol and isovaline and a high proportion of Aib indicated that the compound belonged to the peptaibol family. The amino acid sequence of the peptide, which was partially determined by the MS/MS, was confirmed and completed by analysis of the 2D NMR data (Table 2 and Supplementary Figures S3-S6). Initially, the structure of the amino acids were determined through interpretation of the COSY, TOCSY, HSQC and HMBC correlations, followed by their connection into the complete planar peptide structure through the HMBC and ROESY correlations and MS/MS data. Leu-NH,4 12 Aib-NH, 11 Leu-NH,3a,3b,5, 13 Pro-5a 3a 39.2 t 1.56 m 11 Leu-2,5,6 11 Leu-2,3b,5 3b 1.70 m 11 Leu-NH,2,3a,5 4 23.9 d 1.73 m 11 6 11 Leu-NH,6 5 20.5 q 0.76 d 11 Leu-6,3b 11 Leu-2,3a,3b 6 23.0 q 0.82 d 11 Leu-5,3b 11 Leu-4 NH 7.59 d (8.0) 10 Ser-2, 11 Leu-2,3b,4 12 3a,4,5b,14 Leu-NH, 12 Aib-NH 13 Pro-4,5a, 12 Aib-NH,3  According to the MS/MS spectra (Figure 1), the difference of 85 mass units appears seven times, in the positions 1, 4, 7 (or 6), 8, 9, 12 (or 11) and 15. The difference of 213 mass units in positions 6-7, which was explained by a couple of amino acids, glutamine (128) and Aib (85), could also be explained by the amino acids, valine or isovaline (99) and asparagine (114). The difference of 198 mass units in positions 11-12, which was explained by the amino acids, leucine (123) and Aib (85), could also be explained by the presence of two Vxx (valine or isovaline). However, according to the NMR data, valine and asparagine were not present in the molecule, and the only isovaline was assigned to position 5 according to the MS/MS and NMR data analysis. In the NMR spectra, signals typical of α-amino-isobutyric acid (Aib) were observed, including eight singlet amide signals resonating at δ H 7.47 (assigned to the isovaline), 7.89, 8.56, 7.55, 7.64, 7.77, 7.86 and 7.87 ppm, multiple carbon signals resonating at δ C 55. 8-56.3, typical of carbons α to carboxyl; all of them, except one, (δ C 56.0, assigned to 6 glutamine) are quaternary according to the HSQC spectrum and multiple overlapping singlet methyl signals in the region of 1.30-1.49 ppm of the 1 H NMR spectrum. According to the HSQC map, some of the protons of these methyl signals were bound to carbons resonating between 22.6 and 23.4 or between 25.8 and 26.7 ppm, while one to a carbon at 24.2 ppm. In addition to the Aib methyls, a proton signal of a single methyl of isovaline, one of the methylene protons of the isoleucine and methyl signals of 2 alanine and 3 alanine, resonate in the region of 1.30-1.49 ppm. Four methyl carbons, resonating between 22.6 and 23.4 ppm, were assigned to 11 leucine, 14 leucine, acetate and 5 isovaline, in addition to the seven Aib methyls. In the region of 25.8-26.7 ppm, three methylene carbons of 13 proline, 6 glutamine and 17 glutamic acid resonate beside the Aib methyl groups. Due to signal overlap, some geminal methyl groups and carbon α to carboxyls of Aib could not be distinguished unambiguously and remain interchangeable. Carboxyamides and amide protons were assigned according to HMBC correlations with amide protons and carboxyamide carbons of the neighboring amino acids, when the amino acids were connected to the full peptide chain.
The structure determination of acetyl-1 Aib started with the methyl protons (δ H 1.93, s, δ C 23.1) that exhibited an HMBC correlation with the carboxyamide (δ C 171.1), which, in turn, was connected to a singlet amide proton resonating at δ H 8.56. The amide proton and two singlet methyl groups (δ H 1.32 and 1.35) exhibited HMBC correlations with a carboxyamide (one of the three carbons resonating at δ C 175. 7-175.9) and to a quaternary carbon α to a carboxyamide, resonating at δ C 55.8), assigning the amino acid as 1 Aib. The amide proton exhibited ROESY correlations to the methyl signals (δ H 1.35, δ C 26.5 and δ H 1.32, δ C 24.1). The carbon of the second methyl group exhibited an HMBC correlation with the protons of the first methyl (δ H 1.35). These correlations allowed the assignment of the acetyl-1 Aib sub-structure.
The assignment of the structure of 2 alanine and 3 alanine started with COSY correlations of the protons of two doublet methyl groups (δ H 1.31, δ H 1.33) with the α-protons resonating at δ H 4.02 and 3.99, respectively, which, in turn, were connected to amide protons resonating at 8.25 and 7.68 ppm, respectively. This structure was reinforced by HMBC correlations (see Table 2). HMBC correlations connected the methyl groups and α-protons (δ H 1.31, 1.33, 4.02, 3.99) to carboxyamide carbons resonating at δ C 174.5, culminating in the structure of the two alanine residues.
A singlet amide proton resonating at δ H 7.87 exhibited an HMBC correlation with the quaternary carbon resonating at δ C 56.0 ppm and ROESY and HMBC correlations with two singlet methyl residues (δ H 1.35, δ C 23.2 and δ H 1.43, δ C 26.1), assigning the signals, except for the carboxyamide carbon of 4 Aib. The other Aib residues in positions 7, 8, 9, 12 and 15 were assigned in a similar way.
The methyl protons (δ H 0.74 t, δ C 7.4) exhibited COSY correlation to methylene protons (δ H 2.20 and 1.62) and HMBC correlation to the methylene carbon (δ C 25.6). HMBC correlations of a quaternary carbon at δ C 58.6, typical of a carbon α to carboxyamide, with the methyl and the methylene protons, established it as the isovaline C-2 carbon, which, in turn, was coupled by HMBC correlations to an amide proton (δ H 7.47) and with a single methyl group (δ H 1.35). Several singlet methyl groups, most belonging to Aib residues, resonated at 1.35 ppm, interfering with the assignment of this methyl carbon by HSQC. However, this carbon was assigned by HMBC correlation of the amide proton (δ H 7.47) with a carbon resonating at 22.7 ppm. HMBC correlations of the amide (δ H 7.47), one of the methylene protons (δ H 2.20), and amide proton of the neighboring amino acid, 6 glutamine with a carboxyamide carbon resonating at δ C 174.6 ppm, established it as the 5 isovaline carboxyamide.
The structure determination of 6 glutamine and 18 glutamine was initiated through COSY correlations of the two pairs of singlet amide protons resonating at 7.17 and 6.76 ppm and at 7.10 and 6.65 ppm, respectively. These amide protons exhibited HMBC correlations to two carbonyl signals resonating at δ C 173.6, thus establishing two primary amides. These amides were connected through HMBC correlations of the amide protons resonating at 6.65 ppm and 6.76 ppm to two methylene carbons resonating at 31.7-31.6 ppm ( 6 C-4 and 18 C-4, δ H 2.15, 2.23 and 2.05, 1.97, respectively), which, in turn, exhibited HMBC correlations with two pairs of methylene protons, ( 6 Gln-3, δ H 1.97, 2H, δ C 26.2) and ( 18 Gln-3, δ H 1.70, 1.83 ppm, δ C 27.7). One of the C-3 carbons (26.2 ppm) exhibited HMBC correlations to the pair of methylene-4 protons resonating at 2.15 and 2.23 ppm, while the second C-3 carbon (27.7 ppm) exhibited HMBC correlation with the methylene protons at 1.97 ppm and 2.05 ppm (H-4a, H-4b), allowing the differentiation between the two spin systems. HMBC correlations of C-3 carbons at 26.2 and 27.7 ppm with the methine protons resonating at δ H 3.78 (δ C 56.0) and δ H 4.06 (δ C 53.1), respectively, established the methines at position 2 of these amino acids. This assignment was further supported by the COSY correlations of H 2 -3 with H-2 of both spin systems. The α-amide protons were assigned through the COSY correlation of the α-proton, resonating at 3.78 ppm with the amide proton resonating at 7.74 ppm, and that at 4.06 ppm with that at 7.47 ppm, and reinforced by the HMBC correlations of C-2 carbons with these amide protons. HMBC correlations of the carbon resonating at 174.0 ppm with the proton resonating at 3.78 ppm, and of the carbon resonating at 170.8 ppm with protons resonating at 4.06 ppm, 1.70 ppm and 1.83 ppm, established those carbons as the carboxyamides of the glutamine residues.
The structure elucidation of 10 serine was based on COSY correlations of the oxymethylene protons (δ H 3.73, 3.78; δ C 61.2) with a proton resonating at 4.02 ppm (δ C 58.9), which, in turn, was connected to an amide proton resonating at δ H 7.74. HMBC correlations of the protons resonating at δ H 4.02 and 3.73 with the carbon resonating at δ C 170.6 established the latter as a serine carboxyamide. The hydroxyl proton was not observed in the NMR spectrum of TA1938, but was observed at chemical shifts of 4.70-4.85 ppm in some of the other peptaibols (TA1895, TA1909, TA1909A, TA1896 and TA.Vb).
The structure elucidation of the 13 proline was initiated with COSY correlation of the aminomethylene protons resonating at δ H 3.44 and 3.69 (H-5a and H-5b) with methylene protons resonating at δ H 1.86 (H-4a and -4b). The later protons were coupled through COSY correlations to protons of additional methylene δ H 1.59 and 2.22 (H-3a and H-3b), which, in turn, exhibited COSY correlations with a methine proton (δ H 4.21, t). The chemical shifts of this proton and the carbon to which it was attached (δ C 63.1, established through HSQC experiment) indicated their location α to a carboxyamide and amine. HMBC correlations between the methine carbon (δ C 63.1 d, C-2) and H-3b (δ H 2.22), C-3 (δ C 28.7, CH 2 ) and H-2, C-5 (δ C 48.7) and H-3b reinforced this structure. H-2 (δ H 4.21) was connected through HMBC correlation to a carbon that resonated at δ C 173.4, which was assigned as C-1 of this residue. No HMBC or ROESY correlations were present, which could prove the ring closure. However, the chemical shifts of the H-5a, H-5b and C-5 (δ H 3.44 and 3.69, δ C 48.7) indicated their vicinity to a nitrogen atom. Besides, none of the carbons or protons of this residue exhibited correlations with any of the amide protons, indicating a tertiary amide. In addition, the chemical shifts of the carbons and protons were similar to those of proline in TA1909 (2), in which a 3 J HMBC correlation between C-2 (δ C 63.0) and H-5b (δ H 3.71) was observed, indicating the closure of a pyrrolidine ring. Based on the above evidences, the structure of this amino acid was established as proline.
The structure elucidation of 14 leucine started with COSY correlations of two methyl groups resonating at δ H 0.92 and 0.82 (H 3 -5 and H 3 -6, respectively) with the same methine proton that resonated at δ H 1.68 (H-4). In the HMBC spectrum, H 3 -5 and -6 exhibited correlations with a methine carbon (δ C 24.8, C-4) and a methylene carbon, (δ C 38.7, C-3, δ H 1.51 and 1.78, H-3a and H-3b, respectively). H-3a and H-3b exhibited COSY correlations with a proton resonating at δ H 3.91 (H-2), which, in turn, was correlated with the amide proton resonating at δ H 7.72. HMBC correlation connected H-2 and one of three carbons resonating at δ C 173. 7-173.8. This HMBC correlation was weak, but the connectivity was reinforced by the HMBC correlation of this carbon with the amide proton δ H 7.64 of the adjacent amino acid, 15 Aib.
The structure elucidation of 16 isoleucine started with COSY correlation of protons of a doublet methyl, (H 3 -6, resonating at δ H 0.86, δ C 15.7) with a methine proton, H-3 (δ H 1.88). Methine-3 carbon (δ C 35.6) exhibited HMBC correlations with the protons of CH 3 -6, the protons of an additional triplet methyl group (δ H 0.82) and protons of a methylene (δ H 1.20 and 1.47, δ C 25.0 by HSQC). COSY correlations of the methylene protons with the methyl protons at δ H 0.82 determined the ethyl segment. COSY correlations coupled the ethyl moiety through the methine proton at 1.88 ppm to a downfield shifted methine (H-2, δ H 3.93, C-2, δ C 58.9) and the later to an amide proton resonating at δ H 6.96. H-2 presented an HMBC correlation to a carbon resonating at δ C 170.6 that was assigned as the carboxyamide carbon of the isoleucine residue. In the 13 C NMR spectrum, three carbons resonate at 173.7-173.8 ppm. Two of them were attributed to 11 leucine and 14 leucine. The third one exhibited an HMBC correlation to a proton that resonated at 2.37 ppm (H-4b, δ C 30.4, δ H-4a 2.25). The protons of the later methylene were coupled through COSY correlations to protons that resonated at δ H 1.97 and 1.87 (H-3a and H-3b, δ C 26.5), which, in turn, were connected to an α-proton resonating at 4.06 ppm and to an amide proton (δ H 7.68). HMBC correlations of the carbon resonating at δ C 171.3 with the protons at δ H 4.06, 1.97 and 1.87 established it as the glutamic acid carboxamide, establishing the structure of 17 glutamic acid.
The four aromatic carbons presented in the 13 C spectrum were assigned to 19 phenylalaninol. The carbon at δ C 139.2 was identified as a quaternary carbon and the other three carried protons (δ C 126.0, δ H 7.12), (δ C 128. 2, δ H 7.18) and (δ C 129.4, δ H 7.21). The signal intensity of the carbons and integration of the protons indicated that two pairs of symmetric aromatic protons resonate at 7.18 and 7.21 ppm and one proton resonates at δ H 7.12 ppm, in accordance with a mono-substituted phenyl ring. COSY correlations coupled the later proton with the protons resonating at δ H 7.18 (H-6,6′, δ C 128.2) and to those resonating at δ H 7.21 (H-5,5′, δ C 129.4). This was reinforced by HMBC correlations (see Table 2). The assignment of the aliphatic part of the amino alcohol was based on HMBC correlations of the aromatic carbons, C-4 and C-5, with the two methylene protons, δ H 2.84 and 2.61 ppm (H-3a, H-3b), which were coupled by COSY correlations to a methine proton resonating at δ H 3.86 (H-2). This methine proton was connected to an amide proton (δ H 7.27 ppm) and to the protons of an oxymethylene resonating at δ H 3.29 and 3.32 (H-1a and H-1b, δ C 62.7). The hydroxyl proton of the phenylalaninol did not appear in the NMR spectra of 1, but was present in other trichorzianine: TA1895, TA1909, TA1909A, TA1896 and TA.Vb.
The assembling of the amino acids to the planar peptide structure was based on HMBC correlations from the carboxyamide carbon of an amino acid to the amide protons of the adjacent amino acid, by NOE correlations (from ROESY experiment) of the αor amide proton of an amino acid with the αor amide proton of the adjacent amino acid and by interpretation of MS/MS data. The structure of the peptide with the most significant HMBC and NOE correlations that led to the structure elucidation are summarized in Figure 2. The sequence Ac-1 Aib-2 Ala-3 Ala-4 Aib-5 Iva/Val was inferred from MS/MS data. 1 Aib carboxyamide carbon resonated (δ C 175.7-175.9) closely with those of 7 Aib and 8 Aib, excluding an unequivocal proof of the HMBC correlation of 2 Ala-NH with 1 Aib-C-1. The carboxyamides carbons of 2 Ala and 3 Ala resonated at the same chemical shift (δ C 174.5), not allowing the confirmation of their connection by HMBC correlation, but NOE correlation of 2 Ala-NH with 3 Ala-NH confirmed their vicinity. NOE correlation of 3 Ala-H-2 with 4 Aib-NH and HMBC correlation of 4 Aib-C-1 with 5 Iva-NH established the 3 Ala-4 Aib-5 Iva partial structure. The connection of 5 Iva with 6 Gln could not be proven either by HMBC or NOE correlations, since the carboxyamides of 5 Iva and 9 Aib (δ C 174.6) and the doublet amide protons of 6 Gln and 10 Ser (δ H 7.74) resonated at the same chemical shifts. The correlation of these chemical shifts (δ C 174.6 with δ H 7.74) in the HMBC map remained unequivocal, while no correlations between 5 Iva and 6 Gln were observed in the ROESY map. The MS/MS data suggested that 5 Iva was connected either to Gln or Aib. HMBC and especially NOE correlations (Table 2) confirmed the 6 Gln-7 Aib-8 Aib-9 Aib substructure, and in conjugation, with the MS/MS data, the 5 Iva-6 Gln-7 Aib-8 Aib-9 Aib-10 Ser sequence could be secured. 10 Ser was connected to 11 Leu based on the HMBC correlation of 10 Ser-carboxyamide with 11 Leu-NH. 11 Leu-and 14 Leu-carboxyamides resonated close one to the other (δ C 173.7-173.8), not allowing unambiguous assignment of their neighboring amino acid residues. However, NOE correlations of 12 Aib-NH with 11 Leu-NH and 13 Pro-5-Ha and 5-Hb established the 11 Leu-12 Aib-13 Pro sequence. HMBC correlation of 13 Pro-C-1 with 14 Leu-NH established the connectivity of the latter two amino acids. NOE correlations of 15 Aib-NH with 14 Leu-H-2 and 16 Ile-NH determined the 14 Leu-15 Aib- 16 Ile fragment. The rest of the sequence, 16 Ile-17 Glu-18 Gln- 19 Pheol, could be secured with correlations from the HMBC map, resulting in the full planar structure of 1.

Structure Elucidation of 2-12
The structures of compounds 2-12 were determined in a similar manner to that of TA1938 (1). Compounds 2-12 vary from 1 in the amino acid residues of positions 5, 9, 14 and 17. 1 H-and 13 C-NMR data of the new trichorzianines are summarized in Tables 3 and 4 Figures S7-S15), complete NMR data (Supplementary  Tables S2-S12 and Figures S16-S29) and the assemblage of the amino acids to the planar peptide structures (Supplementary Figures S30-S40) of compounds 2-12 are displayed in the supplementary material. The presence of Glu-OMe in compounds 4-7 was supported by the direct connectivity of the OMe-proton signal with the side-chain carboxyl carbon of Glu.

Determination of the Absolute Stereochemistry
Marfey's analysis [15] of TA1938 (1) using L-FDAA as derivatizing reagent established the L-configuration of Ile, Leu × 2, Glu × 3 (one from glutamic acid, two from glutamine), Pro and Ser. Advanced Marfey analysis [16] of TA1938 using L-FDAA and D-FDAA as derivatizing agents and analysis by LC/MS established the L-configuration of Ala × 2. Iva configuration was not established, and Aib is not chiral. Configuration of phenylalaninol was established as L in TA1909 (2) by Marfey's analysis preceded by Jones oxidation [17] and comparison to the Phe standard and assumed as L in other compounds, due to the similarity of their structures. Marfey's analysis of TA1910 (6) using L-FDAA as derivatizing reagent established the L-configuration of Ile, Leu, Glu × 3 (one from glutamic acid, two from glutamine), Pro, Ser and Val. Marfey's analysis of TA1895 (5) using L-FDAA as derivatizing reagent established the L-configuration of Val. The configurations of other amino acids were established by comparison of Marfey's chromatograms to those of TA1938 (1). Retention times were found similar, meaning that absolute configurations of amino acids are as in TA1938 (1). Marfey's analysis of TA1909 (2), TA1896 (4), TA1924 (5), TA1924A (7) and TA1909A (8)

Antibacterial Bioassay
The antibacterial activity of the isolated trichorzianines (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) was tested against five environmental bacteria and three laboratory bacterial strains (detailed in the Experimental Section). MIC (minimal inhibitory concentration) was designated as the lowest concentration in which bacterial growth was inhibited to 0%-10% and MEC (minimal effective concentration) as the lowest concentration in which bacterial growth was inhibited to 70%. The results that were obtained after 48 h of incubation are summarized in Figure 3. Examination of the trichorzianines activity ( Figure 3) shows a general pattern. The tested trichorzianines exhibited stronger activity against environmental bacteria than against the laboratory strains. When compared within each group (environmental/laboratory), Gram-positive bacteria were more sensitive than Gram-negative bacteria (E. coli was resistant to all the tested compounds at all concentrations). Upon comparison of pairs of compounds differing in a single amino acid, no correlation was found between change in the amino acid sequence and activity.

Lab strains
Environmental bacteria