Isolation and Characterization of Antibacterial Carotane Sesquiterpenes from Artemisia argyi Associated Endophytic Trichoderma virens QA-8

Carotane sesquiterpenes are commonly found in plants but are infrequently reported in the fungal kingdom. Chemical investigation of Trichoderma virens QA-8, an endophytic fungus associated with the inner root tissue of the grown medicinal herb Artemisia argyi H. Lév. and Vaniot, resulted in the isolation and characterization of five new carotane sesquiterpenes trichocarotins I–M (1–5), which have diverse substitution patterns, and seven known related analogues (6–12). The structures of these compounds were established on the basis of a detailed interpretation of their NMR and mass spectroscopic data, and the structures including the relative and absolute configurations of compounds 1–3, 5, 9, and 10 were confirmed by X-ray crystallographic analysis. In the antibacterial assays, all isolates exhibited potent activity against Escherichia coli EMBLC-1, with MIC values ranging from 0.5 to 32 µg/mL, while 7β-hydroxy CAF-603 (7) strongly inhibited Micrococcus luteus QDIO-3 (MIC = 0.5 µg/mL). Structure-activity relationships of these compounds were discussed. The results from this study demonstrate that the endophytic fungus T. virens QA-8 from the planted medicinal herb A. argyi is a rich source of antibacterial carotane sesquiterpenes, and some of them might be interesting for further study to be developed as novel antibacterial agents.


Structural Elucidation of the New Compounds
Compound 1 was purified as colorless crystals and its molecular formula was determined as C15H26O3 on the basis of HRESIMS data, implying three degrees of unsaturation. The 1 H and 13 C NMR data of 1 indicated the presence of four methyls, three methylenes, five methines (including one olefinic and two oxygenated), and three non-protonated carbons (including one olefinic and one oxygenated) (Tables 1 and 2 and Figures S1 and S2 in the Supplementary Material). Comprehensive analysis of its 1 H and 13 C NMR data suggested that compound 1 belonged to carotane sesquiterpenes with structural similarity to that of CAF-603 (6, Figure 1) and differed from 6 mainly at the five-membered ring [14,15]. Compared to CAF-603 (6), resonances for the methylene

Structural Elucidation of the New Compounds
Compound 1 was purified as colorless crystals and its molecular formula was determined as C 15 H 26 O 3 on the basis of HRESIMS data, implying three degrees of unsaturation. The 1 H and 13 C NMR data of 1 indicated the presence of four methyls, three methylenes, five methines (including one olefinic and two oxygenated), and three non-protonated carbons (including one olefinic and one oxygenated) (Tables 1 and 2 and Figures S1 and S2 in the Supplementary Material). Comprehensive analysis of its 1 H and 13 C NMR data suggested that compound 1 belonged to carotane sesquiterpenes with structural similarity to that of CAF-603 (6, Figure 1) and differed from 6 mainly at the five-membered ring [14,15]. Compared to CAF-603 (6), resonances for the methylene group at δ H /δ C 1.39/50.3 (CH 2 -2) of 6 disappeared in that of 1. Instead, signals corresponding to an oxymethine group at δ H /δ C 3.09/78.4 (CH-2) were observed in the NMR spectra of 1. Additionally, a signal for an additional hydroxy group was observed at δ H 4.16 (s, OH-2) in the 1 H NMR spectrum of 1. These data suggested that the methylene group CH 2 -2 in 6 was replaced by an oxyme-thine group in 1. COSY and HMBC data ( Figure 2) supported the above deduction. The planar structure of 1 was thus determined. group at δH/δC 1.39/50.3 (CH2-2) of 6 disappeared in that of 1. Instead, signals corresponding to an oxymethine group at δH/δC 3.09/78.4 (CH-2) were observed in the NMR spectra of 1. Additionally, a signal for an additional hydroxy group was observed at δH 4.16 (s, OH-2) in the 1 H NMR spectrum of 1. These data suggested that the methylene group CH2-2 in 6 was replaced by an oxymethine group in 1. COSY and HMBC data ( Figure 2) supported the above deduction. The planar structure of 1 was thus determined. The relative configuration of 1 was determined by the observed NOEs from H3-15 to the protons of OH-2 and OH-4, from H-10α to the proton of OH-2, and from H-5 to H-2, H-3, and H-10β. The Cu Kα radiation single-crystal X-ray diffraction experiment resulted in Flack parameter 0.1(4) of 1, which allowed the establishment of its absolute configuration as 1S, 2R, 3R, 4S, and 5S ( Figure 4). Thus, the structure of 1 was identified and named as trichocarotin I.    The relative configuration of 1 was determined by the observed NOEs from H 3 -15 to the protons of OH-2 and OH-4, from H-10α to the proton of OH-2, and from H-5 to H-2, H-3, and H-10β. The Cu Kα radiation single-crystal X-ray diffraction experiment resulted in Flack parameter 0.1(4) of 1, which allowed the establishment of its absolute configuration as 1S, 2R, 3R, 4S, and 5S ( Figure 4). Thus, the structure of 1 was identified and named as trichocarotin I.
Trichocarotin J (2) was originally isolated as an amorphous powder. Its molecular formula was also determined as C 15 H 26 O 3 based on the HRESIMS data. The 1 H NMR spectrum of 2 indicated the presence of an olefinic methyl moiety, an aliphatic singlet methyl, and an isopropyl unit, which are typical structural features of the known carotane sesquiternene members. The 1 H, 13 C, and DEPT NMR data of 2 (Tables 1 and 2 and Figures  S8 and S9 in the Supplementary Material) showed almost identical spectral patterns to those of 7β-hydroxy CAF-603 (7) [15], with some minor variations for the chemical shifts of C-5 through C-9, and C-14 as well. Detailed inspection of the NMR data suggested that 2 is a diastereomer of 7, epimeric at C-7. This was supported by the observed NOEs from H-5 to H-3, H-6β, H-7, and H-10β, and from H 3 -15 to the protons of OH-3 and OH-4 ( Figure 3). Upon slow evaporation of the solvent (MeOH:H 2 O = 8:1) by storage in a refrigerator, quality single crystals of compound 2 were obtained. The structure and absolute configuration of 2 were further confirmed by a single-crystal X-ray diffraction experiment using Cu Kα radiation ( Figure 4). The Flack parameter 0.06(4) allowed for the establishment of the absolute configuration of 2 as 1R, 3R, 4S, 5S, and 7R. Trichocarotin J (2) was originally isolated as an amorphous powder. Its molecular formula was also determined as C15H26O3 based on the HRESIMS data. The 1 H NMR spectrum of 2 indicated the presence of an olefinic methyl moiety, an aliphatic singlet methyl, and an isopropyl unit, which are typical structural features of the known carotane sesquiternene members. The 1 H, 13 C, and DEPT NMR data of 2 ( Tables 1 and 2 and Figures S8 and S9 in the Supplementary Material) showed almost identical spectral patterns to those of 7β-hydroxy CAF-603 (7) [15], with some minor variations for the chemical shifts of C-5 through C-9, and C-14 as well. Detailed inspection of the NMR data suggested that 2 is a diastereomer of 7, epimeric at C-7. This was supported by the observed NOEs from H-5 to H-3, H-6β, H-7, and H-10β, and from H3-15 to the protons of OH-3 and OH-4 ( Figure 3). Upon slow evaporation of the solvent (MeOH : H2O = 8 : 1) by storage in a refrigerator, quality single crystals of compound 2 were obtained. The structure and absolute configuration of 2 were further confirmed by a single-crystal X-ray diffraction experiment using Cu Kα radiation ( Figure 4). The Flack parameter 0.06(4) allowed for the establishment of the absolute configuration of 2 as 1R, 3R, 4S, 5S, and 7R.    Trichocarotin J (2) was originally isolated as an amorphous powder. Its molecular formula was also determined as C15H26O3 based on the HRESIMS data. The 1 H NMR spectrum of 2 indicated the presence of an olefinic methyl moiety, an aliphatic singlet methyl, and an isopropyl unit, which are typical structural features of the known carotane sesquiternene members. The 1 H, 13 C, and DEPT NMR data of 2 (Tables 1 and 2) showed almost identical spectral patterns to those of 7β-hydroxy CAF-603 (7) [15], with some minor variations for the chemical shifts of C-5 through C-9, and C-14 as well. Detailed inspection of the NMR data suggested that 2 is a diastereomer of 7, epimeric at C-7. The molecular formula of trichocarotin K (3) was determined to be C 15 H 26 O 3 by HRESIMS. Its 1 H and 13 C NMR data revealed the presence of four methyls, four methylenes, four methines (one oxygenated), and three non-protonated (one oxygenated and one ketone) carbons (Tables 1 and 2 and Figures S15 and S16 in the Supplementary Antibiotics 2021, 10, 213 5 of 10 Material). A detailed comparison of NMR data revealed that compound 3 differed from CAF-603 (6) [14,15] mainly at the seven-membered ring, and that resonances corresponding to the double bond (C-8 and CH-9) in 6 disappeared in that of 3, while signals for an aliphatic methine (CH-8) and for a keto group (C-9) were observed in the NMR spectra of 3 (Tables 1 and 2). HMBC correlations from H-14 to C-7, C-8, and C-9 confirmed this deduction. Other COSY and HMBC correlations ( Figure 2) further confirmed the planar structure of 3.
The key NOE correlations from H 3 -15 to H-8, H-10α, and to the protons of OH-3 and OH-4, and from H-3 to H-5 determined the relative configuration of 1 (Figure 3). Upon slow evaporation of the solvent (MeOH) by storing the sample in a refrigerator, quality single crystals of 3 were obtained, and the absolute configuration of 3 was thus determined as 1S, 3R, 4S, 5S, and 8S by X-ray diffraction analysis ( Figure 5).
Antibiotics 2021, 10, x FOR PEER REVIEW 5 of 11 The molecular formula of trichocarotin K (3) was determined to be C15H26O3 by HRESIMS. Its 1 H and 13 C NMR data revealed the presence of four methyls, four methylenes, four methines (one oxygenated), and three non-protonated (one oxygenated and one ketone) carbons (Tables 1 and 2 and Figures S15 and S16 in the Supplementary Material). A detailed comparison of NMR data revealed that compound 3 differed from CAF-603 (6) [14,15] mainly at the seven-membered ring, and that resonances corresponding to the double bond (C-8 and CH-9) in 6 disappeared in that of 3, while signals for an aliphatic methine (CH-8) and for a keto group (C-9) were observed in the NMR spectra of 3 (Tables 1 and 2). HMBC correlations from H-14 to C-7, C-8, and C-9 confirmed this deduction. Other COSY and HMBC correlations (Figure 2) further confirmed the planar structure of 3.
The key NOE correlations from H3-15 to H-8, H-10α, and to the protons of OH-3 and OH-4, and from H-3 to H-5 determined the relative configuration of 1 (Figure 3). Upon slow evaporation of the solvent (MeOH) by storing the sample in a refrigerator, quality single crystals of 3 were obtained, and the absolute configuration of 3 was thus determined as 1S, 3R, 4S, 5S, and 8S by X-ray diffraction analysis ( Figure 5). Compound 4 was isolated as a colorless oil and its molecular formula was determined as C15H24O2 by HRESIMS. Its 1 H and 13 C NMR data revealed the presence of four methyls, two methylenes, six methines (one oxygenated and three olefinic), and three non-protonated (one oxygenated and one olefinic) carbons (Tables 1 and 2 and Figures  S22 and S23 in the Supplementary Material). These data suggested that 4 had the same carbon skeleton as that of CAF-603 (6) [14,15]. Actually, compound 4 was a C-6 and C-7 deprotonated product of CAF-603. This was verified by chemical shifts and COSY correlations of H-6 with H-5 and H-7 ( Figure 2). In the NOESY experiments, the NOE from H-3 to H-5 indicated the co-facial orientation of these groups ( Figure 3). However, no other diagnostic NOEs were observed, and thus the relative configuration could not be characterized by NOESY experiments. According to the literature reports [14][15][16], the relative configuration of all the congeners of CAF-603 was deduced to be same at the five-membered ring. The absolute configuration of 4 was tentatively deduced as 1R, 3R, 4S, 5S on the basis of biogenic considerations. Thus, the structure of compound 4 was characterized and was named trichocarotin L.
Trichocarotin M (5), initially obtained as a colorless waxy solid, was determined to possess the molecular formula C15H26O3, based on HRESIMS data. The 1 H and 13 C NMR data of 5 (Tables 1 and 2 and Figures S29 and S30 in the Supplementary Material) closely resembled those of 14-hydroxy CAF-603 (trichocarane B), whose relative configuration was determined by NOESY spectrum [17], but its absolute configuration was not clarified. The relative configuration of 5 was deduced to be the same as that of 14-hydroxy CAF-603 based on the NOESY data ( Figure 3). However, the optical rotation of com-  Compound 4 was isolated as a colorless oil and its molecular formula was determined as C 15 H 24 O 2 by HRESIMS. Its 1 H and 13 C NMR data revealed the presence of four methyls, two methylenes, six methines (one oxygenated and three olefinic), and three non-protonated (one oxygenated and one olefinic) carbons (Tables 1 and 2 and Figures S22 and S23 in the Supplementary Material). These data suggested that 4 had the same carbon skeleton as that of CAF-603 (6) [14,15]. Actually, compound 4 was a C-6 and C-7 deprotonated product of CAF-603. This was verified by chemical shifts and COSY correlations of H-6 with H-5 and H-7 ( Figure 2). In the NOESY experiments, the NOE from H-3 to H-5 indicated the co-facial orientation of these groups ( Figure 3). However, no other diagnostic NOEs were observed, and thus the relative configuration could not be characterized by NOESY experiments. According to the literature reports [14][15][16], the relative configuration of all the congeners of CAF-603 was deduced to be same at the five-membered ring. The absolute configuration of 4 was tentatively deduced as 1R, 3R, 4S, 5S on the basis of biogenic considerations. Thus, the structure of compound 4 was characterized and was named trichocarotin L.
Trichocarotin M (5), initially obtained as a colorless waxy solid, was determined to possess the molecular formula C 15 H 26 O 3 , based on HRESIMS data. The 1 H and 13 C NMR data of 5 (Tables 1 and 2 and Figures S29 and S30 in the Supplementary Material) closely resembled those of 14-hydroxy CAF-603 (trichocarane B), whose relative configuration was determined by NOESY spectrum [17], but its absolute configuration was not clarified. The relative configuration of 5 was deduced to be the same as that of 14-hydroxy CAF-603 based on the NOESY data ( Figure 3). However, the optical rotation of compound 5 ([α]25 D +16 (c 0.25, CHCl 3 )) has an opposite sign to that of 14-hydroxy CAF-603 ([α]25 D -28.0 (c 0.20, CHCl 3 )) [17]. Thus, trichocarotin J (5) might be the enantiomer of 14-hydroxy CAF-603. To further confirm this deduction we turned our efforts to a crystallographic study of this compound. Upon slow evaporation of the solvent (MeOH:H 2 O = 15:1) by storage in a refrigerator, quality single crystals of compound 5 were obtained, and the relative and absolute configuration of 5 was therefore established by a single-crystal X-ray diffraction experiment using Cu Kα radiation ( Figure 5). The Flack parameter 0.3(2) of 5 allowed the unambiguous confirmation of the absolute configuration of 5 as 1R, 3R, 4S, and 5S, and named trichocarotin M, which is an enantiomer of 14-hydroxy CAF-603. In addition to new compounds 1-5, seven known analogues including CAF-603 (6) [14,15], 7β-hydroxy CAF-603 (7) [15], trichocarotins E-H (8-11) [16], and trichocarane A (12) [17] were also isolated and identified. Their structures were elucidated by comparing their NMR data with those reported in the literature. It deserve to mention that the absolute configurations of compounds 9 and 10 were previously assigned solely from a biogenetic perspective [16], but in the present study the single-crystal X-ray diffraction ( Figure 6) was used to confirm their absolute configurations.
14-hydroxy CAF-603. To further confirm this deduction we turned our efforts to a crystallographic study of this compound. Upon slow evaporation of the solvent (MeOH : H2O = 15 : 1) by storage in a refrigerator, quality single crystals of compound 5 were obtained, and the relative and absolute configuration of 5 was therefore established by a single-crystal X-ray diffraction experiment using Cu Kα radiation ( Figure 5). The Flack parameter 0.3(2) of 5 allowed the unambiguous confirmation of the absolute configuration of 5 as 1R, 3R, 4S, and 5S, and named trichocarotin M, which is an enantiomer of 14-hydroxy CAF-603.

Antibacterial Activities of the Isolated Compounds
Compounds were assayed for their antibacterial activities against human pathogens Escherichia coli EMBLC-1 and Micrococcus luteus QDIO-3. Statistical analysis by ANOVA for various concentrations when assessed for E. coli and M. luteus showed potent inhibitory growth both at concentrations ≥ MICs when compared to that at concentrations < MICs, and the MICs were obtained. Significance of all the statistical tests was predetermined at p < 0.05. As a result, each of these compounds showed strong inhibitory activity against E. coli, with MIC values ranging from 0.5 to 16 µg/mL, and the activity of compounds 3-5, 8, and 11 are as active as that of the positive control (chloramphenicol, MIC = 0.5 µg/mL) ( Table 3). In addition, compounds 6-8 showed potent activity against M. luteus with MIC values of 4, 0.5, and 2 µg/mL, respectively, and the activity of compound 7 was stronger than that of chloramphenicol (MIC = 1 µg/mL).
Structure-activity relationship analysis revealed that the OH substitution at C-11 increased the activities against both E. coli and M. luteus (8 vs 6), while the OH group at C-14 increased the activity against E. coli and decreased the activity against M. luteus (5 vs 6). Similarly, the 8,9-epoxy group at the seven-membered ring also increased the activity against E. coli and decreased the activity against M. luteus (12 vs 6).

Antibacterial Activities of the Isolated Compounds
Compounds were assayed for their antibacterial activities against human pathogens Escherichia coli EMBLC-1 and Micrococcus luteus QDIO-3. Statistical analysis by ANOVA for various concentrations when assessed for E. coli and M. luteus showed potent inhibitory growth both at concentrations ≥ MICs when compared to that at concentrations < MICs, and the MICs were obtained. Significance of all the statistical tests was predetermined at p < 0.05. As a result, each of these compounds showed strong inhibitory activity against E. coli, with MIC values ranging from 0.5 to 16 µg/mL, and the activity of compounds 3-5, 8, and 11 are as active as that of the positive control (chloramphenicol, MIC = 0.5 µg/mL) ( Table 3). In addition, compounds 6-8 showed potent activity against M. luteus with MIC values of 4, 0.5, and 2 µg/mL, respectively, and the activity of compound 7 was stronger than that of chloramphenicol (MIC = 1 µg/mL). Table 3. Antibacterial activity of compounds 1-12 (MIC, µg/mL). Structure-activity relationship analysis revealed that the OH substitution at C-11 increased the activities against both E. coli and M. luteus (8 vs . 6), while the OH group at C-14 increased the activity against E. coli and decreased the activity against M. luteus (5 vs. 6). Similarly, the 8,9-epoxy group at the seven-membered ring also increased the activity against E. coli and decreased the activity against M. luteus (12 vs. 6).

Plant and Fungal Materials
The fungus T. virens QA-8 was isolated from the fresh inner root tissue of the Compositae medical plants A. argyi collected at Qichun, Hubei Province, in central China in July 2014 and was identified by analysis of its ITS region of the rDNA. The primers used for PCR are ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ), and the total length of sequenced ITS is 616bp. The BLAST search showed that the amplified ITS sequence (GenBank accession no. MK224593) has 100% homology with other members of the genus T. virens (compared with MT256290.1). The strain QA-8 is cryopreserved at −80 • C in 20% aqueous glycerol at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the Chinese Academy of Sciences (IOCAS).

X-Ray Crystallographic Analysis of Compounds
Crystal data for compound 1:

Antibacterial Assays
Antibacterial evaluation against human pathogens E. coli EMBLC-1 and M. luteus QDIO-3 was carried out by the microplate assay with three repetitions [26]. E. coli and M. luteus (95 µL × 5 × 10 5 CFU/mL per well) were cultured at 37 • C in LB medium containing 1% peptone, 0.5% yeast extract, 1% NaCl and distilled water with 5 µL various concentrations of compounds 1-12 in each well of 96-well plates for 24 h. The pathogens used in the assays were obtained from the Institute of Oceanology, Chinese Academy of Sciences. Chloramphenicol was used as positive control.

Conclusions
In summary, five new carotane sesquiterpenes trichocarotins I-M (1-5) and seven known analogues (6)(7)(8)(9)(10)(11)(12) were identified from the culture extract of endophytic fungus Trichoderma virens QA-8. Their structures were elucidated by a detailed interpretation of the spectroscopic data and the structures and absolute configurations of compounds 1-3, 5, 9, and 10 were confirmed by X-ray crystallographic analysis. The crystal structures of the known compounds 9 and 10 are reported for the first time. The absolute configurations of this kind of sesquiterpene were barely presumed by biosynthesis in previously published reports [16], and some of them were not even determined [14,17], but in the present study single crystal X-ray diffraction was used to confirm their absolute configurations. Compounds 3-5, 8, and 11 showed inhibitory activity against E. coli (MIC = 0.5 µg/mL) and compound 7 showed the strongest activity against M. luteus (MIC = 0.5 µg/mL), which are similar to or stronger than that of the positive control. Our results suggested that some of these compounds could be interesting and could lead compounds into a further development of novel antibacterial agents.

Data Availability Statement:
The data presented in this study is included in the supplementary material and is available online at https://www.mdpi.com/2079-6382/10/2/213/s1.