Communication pubs.acs.org/jnp
Induced Production of Halogenated Epidithiodiketopiperazines by a Marine-Derived Trichoderma cf. brevicompactum with Sodium Halides Hiroyuki Yamazaki,*,† Henki Rotinsulu,†,‡ Reika Narita,† Ryosuke Takahashi,† and Michio Namikoshi† †
Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan Research Institute, University of Pembangunan Indonesia, Bahu, Manado 95115, Indonesia
‡
S Supporting Information *
ABSTRACT: Marine-derived Trichoderma sp. TPU199 (cf. T. brevicompactum) produced gliovirin (1), pretrichodermamide A (2), and trichodermamide A (3) in a freshwater medium. Compounds 1 and 2 are rare epidithiodiketopiperazines possessing an unusual disulfide linkage. In the seawater medium, the strain biosynthesized the 5-chloro-5-deoxy derivatives (4 and 5) of 3 and 2. The production of 5 was proportional to the concentration of seawater (NaCl). Therefore, 5-bromo-5-deoxy (6) and 5-deoxy-5-iodo (7) derivatives were biosynthesized in the freshwater media supplemented with NaBr and NaI, respectively. The structure of a new iodo derivative (7) was elucidated on the basis of its spectroscopic data.
H
Because the strain TPU199 was isolated from a marine environment, sterilized natural seawater was also used to culture this fungus. The chlorinated derivatives of 3 and 2, trichodermamide B (4)7 and DC1149B (5),8 respectively, were produced in the seawater medium. The production of 4 and 5 was proportional to the ratio of natural seawater in the culture medium. Therefore, the culture conditions of the strain TPU199 and induced production of other halogenated metabolites were examined. The production of 1 and 3 in the freshwater medium under agitation (condition A, Supporting Information) was higher than that in the static culture (condition B) (Figure S1). Compounds 1−3 were isolated by preparative HPLC from culture broth fermented for 7 days under agitation (condition A). The structures of 1−3 were identified by comparing their spectroscopic data with those of the reported values for gliovirin,5 pretrichodermamide A,6 and trichodermamide A,7 respectively. The production of 1 decreased as the concentration of seawater increased to 50% (condition C) and 100% (condition D), and two new peaks (4 and 5) were detected (Figure S2). The production of 5 increased as the content of 1 decreased in a manner that depended on the ratio of seawater. Compounds 4 and 5 were isolated from the culture broth of the natural seawater medium (condition D) by ODS column chromatography followed by preparative HPLC. The 1H and 13C NMR spectra of compounds 4 and 5 were very similar to those of trichodermamide A (3) and pretrichodermamide A (2). The molecular formulas of 4 and 5 were deduced as C20H19ClN2O8
alogenated natural products have been shown to exhibit a wide variety of biological activities including antimicrobial, antiviral, anti-inflammatory, cytotoxic, antifeedant, and insecticidal activities and enzyme-inhibitory properties.1 Some of these halogenated compounds have been used as medicines and agrochemicals. For example, vancomycin is used in the treatment of several infectious diseases including drug-resistant strains. Many kinds of halogenated natural products have thus far been obtained from marine organisms such as mollusks, sponges, ascidians, cyanobacteria, algae, and microorganisms, possibly because the concentration of halogen ions in oceans is higher than that in terrestrial environments.2,3 Among these marine organisms, fungi have been identified as a rich source of natural organohalides.4 Trichoderma sp. TPU199 (cf. T. brevicompactum) was isolated from an unidentified red alga collected in Palau in 2003 and identified by the ITS1 rDNA sequence in a BLAST search. The separation of broth extract cultured in a freshwater medium led to the isolation of the rare epidithiodiketopiperazines gliovirin (1)5 and pretrichodermamide A (2),6 which possess a disulfide bond between the α- and β-positions of two amino acid residues, together with the related compound trichodermamide A (3).7
Received: July 30, 2015 © XXXX American Chemical Society and American Society of Pharmacognosy
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DOI: 10.1021/acs.jnatprod.5b00669 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Communication
DC1149R (6) was prepared from gliovirin (1) by a reaction with HBr. Compound 1 was therefore transformed into 6 with HBr in this study to confirm the structure of 6. Because the NMR data for 6 were not assigned in the patent,8 the complete assignment of the NMR signals is listed in Table 1 (CD3OD) and Table S1 (DMSO-d6). This is the first study to isolate 6 as a fermentation product of a fungus.
and C 20 H 21 ClN 2 O 8 S 2 from HREIMS and HRFABMS, respectively. The structures of 4 and 5 were confirmed by comparisons of NMR data and specific rotations for 4 and 5 with those of the reported values for trichodermamide B6 and DC1149B.7 The Cl atoms in the structures of 4 and 5 were expected to be incorporated from NaCl in seawater. Therefore, the effects of NaCl concentrations (1.5% and 3.0%) in the freshwater medium on the production of 4 and 5 were examined. The relative ratio of the peak 5 to 2 was increased and the production of 1 decreased as the concentration of NaCl increased from 1.5% (condition E) to 3.0% (condition F), which was similar to the production of these compounds in the natural seawater medium (3.5% NaCl) (Figures S2 and S3). As the strain TPU199 utilized the Cl− ion of NaCl in the freshwater medium, other sodium halides (NaBr, NaI, and NaF) were supplemented in the culture medium (conditions G−I, Supporting Information) in order to investigate the production of halogenated metabolites (Figure 1). The F− ion is generally poisonous to fungi; therefore, the strain TPU199 did not grow well in the 3.0% NaF medium (condition I).
Table 1. 1H and 13C NMR Data for Compounds 6 and 7 in CD3OD 6 C# 1 2 3
169.4, C 71.5, C 35.9, CH2
4 5
71.5, C 59.8, CH
6
130.8, CH
7
129.7, CH
8
66.1, CH
9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 7′OCH3 8′OCH3
Figure 1. Effects of sodium halides on metabolites of Trichoderma sp. TPU199. Strain was cultured under agitation for 1 week in the freshwater medium with or without 3.0% NaCl, NaBr, NaI, or NaF (conditions A or F−I, respectively).
δC, type
88.0, 167.6, 61.5, 46.6, 117.4, 149.2, 137.5, 154.8, 104.5, 124.2, 56.3,
CH C CH CH C C C C CH CH CH3
61.1, CH3
7 δH mult. (J in Hz)
2.43, dd (16.0, 1.5) 2.28, d (16.0) 4.96, m
δC, type
δH mult. (J in Hz)
169.7, C 69.3, C 39.7, CH2
2.46, d (15.5) 2.27, d (15.5)
69.3, C 36.1, CH
5.64, dt (10.5, 2.5) 5.83, dt (10.5, 2.2) 4.60, m
129.8, CH
4.16, m
85.6, 168.2, 61.5, 46.9, 117.4, 149.2, 137.4, 154.8, 104.5, 124.2, 56.3,
4.52, d (1.9) 4.69, d (1.9)
6.53, d (8.7) 7.47, d (8.7) 3.84, s 3.78, s
133.0, CH 65.9, CH CH C CH CH C C C C CH CH CH3
61.1, CH3
5.09, dd (5.0, 5.0) 5.98, dd (9.8, 5.0) 5.49, d (9.8) 3.22, dd (10.0, 5.0) 4.33, d (10.0) 4.47, d (1.7) 4.69, d (1.7)
6.53, d (9.2) 7.47, d (9.2) 3.84, s 3.78, s
The molecular formula of 7 was established as C20H21IN2O8S2 from HRFABMS.10 The 1H and 13C NMR data for 7 were similar to those for 6 (Table 1), except for the signals around the C-5 position. The 13C chemical shift at C-5 (δC 36.1) of 7 was more shielded than that of 6 (Table 1). The configuration of 6 and 7 at C-5 was tentatively assigned the same as that of 2, since these metabolites would be produced through the same pathway. Thus, the structure of 7 was assigned as an iodide variant of 2. A literature search revealed that 7 was a new compound and named iododithiobrevamide. Although Br derivatives have been reported to be produced by fermentation with inorganic bromides,11 it is rare and interesting to obtain an I derivative by fermentation with NaI. The absolute configuration of pretrichodermamide A (2) was previously determined by an X-ray crystallographic analysis,6 and, most recently, the absolute configuration of a new Nmethyl derivative of 5 (adametizine A) was reported by an Xray diffraction experiment utilizing the heavy atom method.12 The electronic circular dichroism (ECD) spectra of compounds 5−7 showed very similar curves to those of 2 (Figure 2) and the reported ECD spectrum of adametizine A.12 Consequently,
On the other hand, the HPLC analysis of the metabolites cultured with 3.0% NaBr (condition G) showed compound 6 (peak 6 in Figure 1), and compound 7 (peak 7 in Figure 1) was detected in the chromatogram when the fungus was cultured in the 3.0% NaI medium (condition H). The UV spectra of these peaks (6 and 7) were very similar to those of 1, 2, and 5. Compounds 6 and 7 were isolated by an ODS column followed by preparative HPLC from the culture broths supplemented with 3.0% NaBr and NaI, respectively. Compound 6 showed two protonated molecule ion peaks at m/z 561 and 563 with a 1:1 ratio in the FABMS spectrum, and the molecular formula, C20H21BrN2O8S2, was determined from HRFABMS data.9 The 1H and 13C NMR spectra of 6 were very similar to those of 5, except for the signals around the C-5 position (Table S1), and the structure of 6 was assigned as the Br variant of 5. The structure of 6 has been reported as DC1149R together with DC1149B (5) in the patent,8 in which B
DOI: 10.1021/acs.jnatprod.5b00669 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (25870660) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan to H.Y., Takeda Science Foundation to H.Y., and the Foundation for Japanese Chemical Research to H.Y. We express our thanks to Mr. T. Matsuki and S. Sato of Tohoku Pharmaceutical University for measuring mass spectra and Mr. H. Fujiwara, K. Murakami, T. Kaneko, S. Konno, and R. Saito of Tohoku Pharmaceutical University for their technical assistance.
the absolute configurations of compounds 5−7 were assigned as (2R, 4R, 5R, 8R, 9S, 2′S, 3′S).
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In conclusion, the marine-derived fungus Trichoderma sp. TPU199 isolated from a red alga in Palau possessed the ability to utilize halide ions from sodium halides in the medium and produced halogenated epidithiodiketopiperazine derivatives. This modulation of fermentation media led to the isolation of 6 as a fungal metabolite for the first time and a new iodinated derivative (7). The absolute structures of the halogenated compounds (5−7) were assigned by comparisons of their ECD spectra with those of the related compounds. A number of natural products belong to the epidithiodiketopiperazine family, the members of which possess a disulfide bond between the αpositions of two amino acid residues.13 However, compounds 1, 2, and 5−7 are very rare examples among this family because the disulfide bond in these compounds was connected between the α- and β-positions of two amino acid residues. Therefore, the strain TPU199 is a remarkable fungus because it produces rare types of epidithiodiketopiperazines and utilizes halide ions in the medium.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00669. Experimental procedures, 1H and 13C NMR spectra of 6 and 7, and experimental data for the known compounds (PDF)
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REFERENCES
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Figure 2. ECD (upper traces) and UV (lower traces) spectra of compounds 2 and 5−7.
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AUTHOR INFORMATION
Corresponding Author
*Tel/fax: +81 22 727 0218. E-mail: yamazaki@tohoku-pharm. ac.jp. Notes
The authors declare no competing financial interest. C
DOI: 10.1021/acs.jnatprod.5b00669 J. Nat. Prod. XXXX, XXX, XXX−XXX
