Bicyclic and tetracyclic diterpenes from a Trichoderma symbiont of Taxus baccata Emilie Adelin a , Claudine Servy a , Marie-Thérèse Martin a , Guillaume Arcile a , Bogdan I. Iorga a , Pascal Retailleau a , Mercedes Bonfill b , Jamal Ouazzani a,⇑ a Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles ICSN, Centre National de la Recherche Scientifique, CNRS, Avenue de la Terrasse 91198, Gif-sur-Yvette cedex, France b Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain article info Article history: Received 15 July 2013 Received in revised form 9 October 2013 Available online xxxx Keywords: Fungal secondary metabolites, Harzianes Trichoderma Diterpenes abstract Trichoderma atroviridae UB-LMA is an endophytic fungus isolated from Taxus baccata trees. Liquid-state fermentation coupled to in situ solid phase extraction (SPE) was applied, and four compounds were dis- covered. Compounds 2–4 belong to the harziane tetracyclic diterpene family. Bicylic compound 1 may represent the biosynthetic precursor of this scarce family of compounds. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Taxol, also known as paclitaxel, is a powerful anticancer drug iso- lated from the bark of the pacific yew, Taxus Brevifolia (Brown, 2003; Wani et al., 1971). Taxol, as a major therapeutic discovery, was lar- gely investigated at the synthetic (Holton et al., 1994; Nicolaou et al., 1994), biosynthetic (Croteau et al., 2006; Heinig and Jennew- ein, 2009) and biological levels (Brown, 2003). The genes and en- zymes involved in the biosynthesis of taxol were characterized (Guerra-Bubb et al., 2012), starting from the cyclization of geranyl- geranyl diphosphate by the taxadiene synthase (TS) to form the taxadiene precursor (Chow et al., 2007). The subsequent functional- ization steps are highly regulated and compartmentalized into the cell. Since 1993, and the first report on Taxus endopyhtic fungus that could synthesize taxol and other taxanes derivatives (Stierle et al., 1993), many strains were reported and were claimed to offer a cred- ible alternative to Taxus extraction or taxol hemi-synthesis. Among them Alternaria, Aspergillus, Cladosporium, Fusarium, Monochaetia, Pestlotia, Pestalotiopsis, Pithomyces, Penicillium, Xylaria, Guignardia and Colletotrichum (Flores-Bustamante et al., 2010; Miao et al., 2009; Soca-Chafre et al., 2011; Strobel et al., 1996; Xiong et al., 2013; Zhang et al., 2009; Zhao et al., 2009). Unfortunately, no signif- icant improvement of taxol production by fungi was obtained 20 years after the first discovery, causing a great debate and rising uncertainty. The first question concerns the horizontal gene transfer from the yew to the endophytic fungi. Such a transfer is conceivable between related organisms for one to few genes but seems unlikely for distant organisms with more than twenty non-clusterized genes. Another intriguing questions concern the weak questionable analytical evidences for taxol production by fungi and the non- reproducibility of the published results (Zhou et al., 2010). Recent reports that tend to demystify taxol production by fungi are not contradicted by indisputable evidence (Heinig et al., 2013; Staniek et al., 2009). We have recently isolated a Trichoderma atroviridae strain from the bark of Taxus baccata trees. This strain did not produce taxane compounds, as confirmed by extensive HPLC–MS analysis and comparison with authentic taxane compounds. However, cultiva- tion combined with in situ solid-phase extraction (SPE) led to a set of harziane-related diterpenes. Besides the previously reported harziandione, we isolated four new harziane related compounds 1– 4. Among them, compound 1 is a potential derivative of geranyl- geranyl diphosphate and may represent the biosynthetic precursor of this scarce family of compounds. Compounds 1–4 were evalu- ated for their antimicrobial and cytotoxic activities. No significant antimicrobial activity was detected while weak cytotoxic activity was measured with a maximum IC 50 of 25 lM for compound 3, against KB and HCT-116 cancer cell lines. 2. Results and discussions The fungal strain Trichoderma atroviridae UB-LMA was isolated as a symbiotic endophyte from the bark of Taxus baccata. The strain 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.10.016 ⇑ Corresponding author. Tel.: +33 1 69 82 30 01; fax: +33 1 69 07 72 47. E-mail address: [email protected](J. Ouazzani). Phytochemistry xxx (2013) xxx–xxx Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Please cite this article in press as: Adelin, E., et al. Bicyclic and tetracyclic diterpenes from a Trichoderma symbiont of Taxus baccata. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.10.016
Please cite this article in press as: Adelin, E., et al. Bicyclic and tetracyclic diterpenes from a Trichoderma symbiont of Taxus baccata. Phytochemistryhttp://dx.doi.org/10.1016/j.phytochem.2013.10.016
Emilie Adelin a, Claudine Servy a, Marie-Thérèse Martin a, Guillaume Arcile a, Bogdan I. Iorga a,Pascal Retailleau a, Mercedes Bonfill b, Jamal Ouazzani a,⇑a Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles ICSN, Centre National de la Recherche Scientifique, CNRS, Avenue de la Terrasse 91198, Gif-sur-Yvettecedex, Franceb Laboratori de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
a r t i c l e i n f o
Article history:Received 15 July 2013Received in revised form 9 October 2013Available online xxxx
Trichoderma atroviridae UB-LMA is an endophytic fungus isolated from Taxus baccata trees. Liquid-statefermentation coupled to in situ solid phase extraction (SPE) was applied, and four compounds were dis-covered. Compounds 2–4 belong to the harziane tetracyclic diterpene family. Bicylic compound 1 mayrepresent the biosynthetic precursor of this scarce family of compounds.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Taxol, also known as paclitaxel, is a powerful anticancer drug iso-lated from the bark of the pacific yew, Taxus Brevifolia (Brown, 2003;Wani et al., 1971). Taxol, as a major therapeutic discovery, was lar-gely investigated at the synthetic (Holton et al., 1994; Nicolaouet al., 1994), biosynthetic (Croteau et al., 2006; Heinig and Jennew-ein, 2009) and biological levels (Brown, 2003). The genes and en-zymes involved in the biosynthesis of taxol were characterized(Guerra-Bubb et al., 2012), starting from the cyclization of geranyl-geranyl diphosphate by the taxadiene synthase (TS) to form thetaxadiene precursor (Chow et al., 2007). The subsequent functional-ization steps are highly regulated and compartmentalized into thecell. Since 1993, and the first report on Taxus endopyhtic fungus thatcould synthesize taxol and other taxanes derivatives (Stierle et al.,1993), many strains were reported and were claimed to offer a cred-ible alternative to Taxus extraction or taxol hemi-synthesis. Amongthem Alternaria, Aspergillus, Cladosporium, Fusarium, Monochaetia,Pestlotia, Pestalotiopsis, Pithomyces, Penicillium, Xylaria, Guignardiaand Colletotrichum (Flores-Bustamante et al., 2010; Miao et al.,2009; Soca-Chafre et al., 2011; Strobel et al., 1996; Xiong et al.,2013; Zhang et al., 2009; Zhao et al., 2009). Unfortunately, no signif-icant improvement of taxol production by fungi was obtained20 years after the first discovery, causing a great debate and risinguncertainty. The first question concerns the horizontal gene transfer
from the yew to the endophytic fungi. Such a transfer is conceivablebetween related organisms for one to few genes but seems unlikelyfor distant organisms with more than twenty non-clusterized genes.Another intriguing questions concern the weak questionableanalytical evidences for taxol production by fungi and the non-reproducibility of the published results (Zhou et al., 2010). Recentreports that tend to demystify taxol production by fungi are notcontradicted by indisputable evidence (Heinig et al., 2013; Stanieket al., 2009).
We have recently isolated a Trichoderma atroviridae strain fromthe bark of Taxus baccata trees. This strain did not produce taxanecompounds, as confirmed by extensive HPLC–MS analysis andcomparison with authentic taxane compounds. However, cultiva-tion combined with in situ solid-phase extraction (SPE) led to aset of harziane-related diterpenes. Besides the previously reportedharziandione, we isolated four new harziane related compounds 1–4. Among them, compound 1 is a potential derivative of geranyl-geranyl diphosphate and may represent the biosynthetic precursorof this scarce family of compounds. Compounds 1–4 were evalu-ated for their antimicrobial and cytotoxic activities. No significantantimicrobial activity was detected while weak cytotoxic activitywas measured with a maximum IC50 of 25 lM for compound 3,against KB and HCT-116 cancer cell lines.
2. Results and discussions
The fungal strain Trichoderma atroviridae UB-LMA was isolatedas a symbiotic endophyte from the bark of Taxus baccata. The strain
2 E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx
was identified on the basis of ITS DNA sequence (genbank refer-ence BankIt1582103, 10260002.seq, KC171717). Cultivation instandard PDB medium was combined with in situ solid-phaseextraction (SPE) with Amberlite XAD-16 (Le Goff et al., 2013; Ade-lin et al., 2012). In addition to harziandione, which has been previ-ously isolated (Mannina et al., 1997), the resin extract offered fournew compounds 1–4, as shown in Fig. 1. Compounds 2–4 possess aharziane tetracyclic diterpene skeleton. Only two representativesof this structural class have been reported so far, including harzi-andione (Ghisalberti et al., 1992) and harzianone (Miao et al.,2012), which were isolated from Trichoderma species.
Compound 1 was isolated as a white powder. HRESIMS analysisgave the molecular formula C20H32O2 on the basis of m/z 303.2326[M�H]� (calculated for C20H31O2 303.2324), which requires 5 de-grees of unsaturation. The 1H NMR spectrum (Table 1) and HSQCdata displayed four methyl singlets. The 13C (Table 2) and DEPT-145 NMR spectra showed resonances for 20 carbons, includingsix sp2-hybridized C-atoms corresponding to four quaternary dou-ble bond carbons (C-9, C-6, C-13 and C-5 at dC 137.8, 136.0, 132.5,and 127.1, respectively), two methines (C-14 and C-10 at dC 134.0and 130.6, respectively), as confirmed by the IR absorption bandsat 1663 and 1438 cm�1, and one quaternary carbon (C-1 at dC
37.3). 13C NMR and HSQC spectra also showed the presence ofone oxy-methine carbon (C-11 at dC 68.0), one oxygen-boundmethylene (C-19 at dC 60.7), one methine carbon (C-2 at dC 42.7),six methylene groups (C-12, C-8, C-15, C-4, C-3 and C-7 at dC
44.3, 38.7, 33.8, 31.3, 27.4 and 26.0, respectively) and four methylgroups (C-17, C-16, C-18 and C-20 at dC 33.0, 24.6, 21.9 and 17.1).Detailed interpretation of the 1H–1H COSY and 1H–13C HMBC spec-tra (Fig. 2) indicated that 1 has a bicyclic core composed of a cyclo-hexene ring and a cyclododecadiene ring (Fig. 1).
Compound 2 was obtained as a yellow oil. The molecularformula was assigned as C20H34O by HRESIMS (m/z 273.2592[M+H–H2O]+, calculated for C20H33 273.2582), requiring 4 degreesof unsaturation. The 1H NMR (Table 1) spectrum and HSQC datadisplayed four methyl singlets and one methyl doublet. The 13C(Table 2) and DEPT-145 NMR spectra confirmed the presence of ahydroxylated C-9 (dC 74.4), and three aliphatic quaternary carbons(C-6, C-1 and C-13 at dC 49.2, 44.9, and 40.5). Furthermore, thesespectra displayed four methine carbons (C-10, C-14, C-2 and C-5at dC 56.2, 51.1, 42.8 and 29.1), seven methylene groups (C-12, C-8, C-15, C-7, C-3, C-4 and C-11 at dC 38.4, 36.7, 26.0, 25.7, 25.6,24.1 and 17.5) and five methyl groups (C-20, C-16, C-19, C-17and C-18 at dC 25.6, 25.3, 23.2, 21.9 and 20.0). Careful examination
Fig. 1. Compounds isolated from Tr
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of the NMR data, particularly 1H–1H COSY and 1H–13C HMBC corre-lations (Fig. 2), confirmed the presence of a harzianone-type tetra-cyclic scaffold consisting of a 6/5/7/4-fused tetra-cyclic ringsystem. Compared to harziandione, the two carbonyl groups werereplaced by two methylenes and the double bond was hydrated.The structure of compound 2 is presented in Fig. 1. The absoluteconfiguration was unambiguously assigned by molecular modelingcalculations as reported later in the manuscript.
Compound 3 was obtained as a white powder. The 1H NMR (Ta-ble 1) along with HSQC spectrum displayed four methyl singlets(dH 1.8, 1.3, 1.0 and 0.8) and one methyl doublet (dH 1.0) (Table 1).The 13C (Table 2) NMR and DEPT-145 spectra showed the presenceof five quaternary carbons (C-10, C-9, C-6, C-1 and C-13 at dC 145.2,130.6, 50.8, 45.8 and 41.7), three methines (C-14, C-2, C-5 at 54.5,43.0 and 29.2), a hydroxymethine C-11 (dC 69.3), six methylenes(C-12, C-7, C-8, C-15, C-3 and C-4 at dC 48.2, 31.2, 28.7, 26.2, 25.9and 25.3) and five methyls (C-16, C-17, C-19, C-20 and C-18 at dC
26.0, 22.5, 22.1, 21.1 and 20.6). The NMR data are closely relatedto those of harziandione except the replacement of the ketoneC-3 by a methylene and the ketone C-11 by a hydroxyl group at69.3 ppm. The structure of compound 3 in Fig. 1 was supportedby 2D NMR 1H–1H COSY and 1H–13C HMBC correlations shown inFig. 2. Fortunately, suitable crystals for X-ray diffraction wereobtained, and the structure and absolute stereochemistry wereestablished (Fig. 3). Herein is presented the first X-ray structurefor this unique family of compounds that unambiguously fixesthe absolute stereochemistry at carbons 2, 5, 6, 13 and 14, whichare common to harziandione and compounds 2, 3 and 4 (See alsoFigs. SI-21 and SI-22).
Compound 4 was obtained as colorless oil. The molecular for-mula was determined to be C20H30O2 on the basis of HRESIMS peakat m/z 303.2325 [M+H]+ (calculated for C20H31O2 303.2324), indi-cating that the molecule possessed 6 degrees of unsaturation. The13C (Table 2) and DEPT-145 NMR spectra displayed two units ofunsaturation corresponding to the substituted double bond (C-9at dC 153.3 and C-10 at dC 149.1), and the carbonyl group C-11 atdC 200.3, corroborated by an IR absorption band at 1729 cm�1.Additionally, the 13C and DEPT-145 NMR spectra confirmed thepresence of three quaternary carbons (C-1, C-6 and C-13 at dC
50.5, 46.1 and 40.2), three methines (C-14, C-2 and C-5 at dC
51.8, 42.8 and 29.3), one oxy-methylene (C-20 at dC 67.0), sixmethylenes (C-12, C-7, C-15, C-3, C-4 and C-8 at dC 58.8, 30.6,27.4, 25.6, 25.3 and 24.4) and four methyl groups (C-16, C-17, C-19, C-18 at dC 25.8, 22.5, 21.8 and 20.6). The 1H and HSQC NMR
ichoderma atroviridae UB-LMA.
enes from a Trichoderma symbiont of Taxus baccata. Phytochemistry (2013),
Table 11H NMR (600 MHz, CDCl3) data for compounds 1–4.
No. Harziandione 1 2 3 4
1 – – – – –2 2.30 d (7.9) 1.66 m 1.54 m 1.59 m 1.68 m3 – 2.20 m 1.88 m 1.90 m 1.96 m
1.58 m 1.23 m 1.25 m 1.31 m4 2.91 m 2.30 m 2.08 m 2.00 m 2.07 m
2.10 m 1.90 dd (7.0; 17.5) 1.13 dd (7.0; 14.6) 1.17 dd (7.0; 14.6) 1.29 m5 2.94 m - 1.89 m 2.30 m 2.42 m6 – – – – –7 1.93 dd (6.4; 14.1) 2.26 m 1.64 m 1.75 m 1.86 m
1.43 dt (1.4; 13.2) 2.20 m 1.39 m 1.28 dt (2.1; 13.2) 1.22 dt (2.1; 13.2)8 2.46 t (6.4; 15.0) 2.56 dt (4.5; 13.0) 1.66 m 2.33 m 2.38 m
2.06 m 2.00 d (13.0) 1.59 m 1.68 ddd (1.9; 6.4; 15.7) 1.95 m9 – – – – –10 - 4.89 d (9.9) 1.99 dt (2.9; 8.8) – –11 - 4.43 td (3.3; 10.5) 2.18 m 4.87 m –
1.83 m12 2.59 d (17.0) 2.64 dt (2.4; 12.0) 1.62 m 2.13 dd (7.7; 10.5) 2.58 d (17.0)
2.46 d (17.0) 1.98 t (12.0) 1.61 m 1.36 dd (7.0; 10.5) 2.47 d (17.0)13 – – – – –14 2.49 m 5.50 dd (2.5; 12.2) 2.32 dd (8.8; 11.5) 2.09 dd (8.8; 11.5) 2.15 dd (8.8; 11.5)15 2.05 m 2.80 ddd (6.4; 12.3; 15.7) 1.72 m 1.77 m 1.87 m
1.54 dd (8.8; 13.5) 1.95 d (15.7) 1.27 dd (8.8; 13.7) 1.22 dd (8.8; 13.5) 1.39 dd (8.8; 13.5)16 1.02 s 0.85 s 0.86 s 0.82 s 0.83 s17 1.00 s 0.98 s 0.98 s 1.00 s 1.04 s18 1.14 d (6.9) 1.72 s 0.99 d (7.6) 0.99 d (7.5) 1.04 d (7.5)19 1.54 s 4.21 d (11.8) 1.43 s 1.32 s 1.54 s
3.98 d (11.8)20 2.15 s 1.64 s 1.43 s 1.78 s 4.40 d (18.0)
4.37 d (18.0)
Table 213C NMR (150 MHz, CDCl3) data for compounds 1–4.
No. Harziandione 1 2 3 4
1 49.7, C 37.3, C 44.9, C 45.8, C 50.5, C2 59.4, CH 42.7, CH 42.8, CH 43.0, CH 42.8, CH3 217.0, C 27.4, CH2 25.6, CH2 25.9, CH2 25.6, CH2
Fig. 3. ORTEP diagram showing the crystal state conformation of compound 3.
E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx 3
spectra displayed three methyl singlets (H-19, H-16 and H-17 at dH
1.5, 1.0 at 0.8 ppm) and one methyl doublet H-18 (dH 1.0). Com-pared to harziandione, the C-3 ketone signal of the latter com-pound is replaced by a methylene, and the C-20 methyl group is
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replaced by an oxy-methylene in the structure of 4. 2D NMR1H–1H COSY and 1H–13C HMBC correlations are presented inFig. 2, and are in agreement with the structural proposal (seeTable 2).
nes from a Trichoderma symbiont of Taxus baccata. Phytochemistry (2013),
Fig. 4. The four possible diastereoisomers of compound 2.
Fig. 6. Carbocation rearrangements leading to taxadiene or harziene scaffolds.
4 E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx
Compounds 1–4 were evaluated for Gram-positive and Gram-negative antibacterial activity and cytotoxic effects against threecancer cell lines (KB, HTC116 and MCF7). None of the compoundsdisplayed antibacterial activity, while compound 3 exhibited weakcytotoxicity against KB and HCT-116 cancer cell lines with an IC50
of 25 lM (S2).Molecular modeling calculations of harziandione and com-
pounds 1–4 provided deeper insight into their three-dimensionalstructure and conformational stability (S3). Along with the NMR,the unambiguous assignment of stereochemistry was achieved inseveral key positions. Conformational analysis and DFT calcula-tions for all the possible diastereomers of 1 (Fig. SI-1) led to onlyone or two conformers in each case. All the lowest energy conform-ers were correctly identified; however, minor differences were ob-served for higher energy conformers (Table SI-1), whichhighlighted the limitations of currently available software in theconformational analysis of macrocycles. A similar procedure wasapplied to four diasteromers of 2 with variable configurations atpositions 9 and 10 (Fig. 4 and Fig. 5), which could not be assignedfrom the NMR and crystallographic data. Diastereomers 2B and 2Dwere discarded because they should present NOE interactions be-tween H-10 and H-14, which were not observed experimentally.In conformer 1 of diastereomer 2A, H-7 should show NOE interac-
Fig. 5. The conformers of compou
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tions with both methyls 16 and 19, whereas only the interactionwith methyl 16 was observed experimentally. For H-7 in con-former 2, NOE interactions with both methyls 16 and 17 are ex-pected, but only the interaction with methyl 16 was evidenced inthe NMR spectrum. Consequently, diastereomer 2A was also elim-inated. The remaining diastereomer 2C was compatible with all theavailable NMR information and also has the lowest energy (Fig. 5);therefore, the stereochemistry at positions 9 and 10 in compound 2was unambiguously assigned as R and S, respectively.
nd 2 and associated energies.
enes from a Trichoderma symbiont of Taxus baccata. Phytochemistry (2013),
E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx 5
Diterpenoids are all derived from geranylgeranyl diphosphate(GGDP). The key cyclization of GGDP to various diterpenoid scaf-folds is catalyzed by diterpene synthases also called diterpene cyc-lases (Smanski et al., 2012). Fungal diterpene cyclases catalyze theprotonation of the double bond of GGDP; the resulting carbocationundergoes a cascade of cyclization by a combination of steric andelectrostatic forces within the catalytic site (Toyomasu et al.,2007). Based on sequence homologies, Trichoderma atroviridae gen-ome has an expansion of one geranylgeranyl diphosphate synthase(protein ID. G9PBF7) and three terpene cyclases (Protein ID.G9NEN0, G9P1I4, G9P454).
The isolation of compound 1 provided crucial evidence for aplausible new cyclization route of GGDP in fungi. We suggest acommon early biosynthetic step for taxadiene and harziene scaf-folds starting from a common precursor corresponding to thenon-functionalized analog of compound 1 (in bold on Fig. 6). Thecarbocation-mediated rearrangements of GGDP to taxadiene, cor-responding to the left part of Fig. 6, was recently reported on thebasis of theoretical calculations (Tantillo, 2011). The author con-cluded that the geometries of monocyclic and bicyclic intermedi-ates were compatible with intramolecular proton transfers,which have low energy barriers and do not require any catalyticactivation by the enzyme (Tantillo, 2011). Similar carbocation rear-rangements could lead to the junctions between C-6/C-14 and C-10/C-13 forming the characteristic 6/5/7/4-fused tetra-cyclic ringof harziene.
The crystal structure of Taxus brevifolia taxadiene synthase wasrecently solved (Köksal et al., 2011), and studies have shown thatGGDP binds to the three-Mg2+ cluster present in the C-terminalcatalytic site of the enzyme. Subsequent removal of PPi and intra-molecular proton transfer requires transannular ring closure with-out the assistance of an enzyme-bound base. This hypothesis is inagreement with the theoretical results (Tantillo, 2011), which ru-led out any active participation of the enzyme in the proton-ation–deprotonation steps.
3. Conclusion
Three novel harziane tetracyclic diterpenes (2–4) and their po-tential biosynthetic precursor 1 were isolated from Trichodermaatroviridae UB-LMA, a Taxus baccata symbiont. The proposed bio-synthetic pathway of 1 from GGDP shares the early steps of taxadi-ene biosynthesis. Compounds 2–4 may be derived from theharziene scaffold through a series of oxido-reduction reactions(S26). Although we possess many fungal strains containing the tax-adiene synthase gene (Mirjalili et al., 2012), we have not yet de-tected the concomitant production of taxadiene or its derivatives.According to the biosynthetic route proposed in Fig. 6 and the pres-ence, in Trichoderma atroviridae, of one geranylgeranyl diphosphatesynthase gene and three terpene cyclases genes, we are currentlyfocusing on the identification of the harziane cyclases.
4. Experimental section
4.1. General experimental procedure
1H and 13C spectra were recorded using a Bruker Avance-500 or600 instrument operating at 500 and 600 MHz respectively. TheBruker avance 600 MHz was equipped with a microprobe (1.7TXI). LC-ESI–MS analyses were performed on a simple-stage qua-drapoleWaters-Micromass� ZQ 2000 mass spectrometer equippedwith an ESI (electrospray ionization) interface coupled to an Alli-ance Waters 2695 HPLC instrument with PDA and ELS detection.The HRESIMS spectra were recorded on a Waters-Micromass�
mass spectrometer equipped with ESI-TOF (electrospray-time of
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flight). Optical rotations were measured at 25 �C on a JASCOP1010 polarimeter. IR spectra were obtained on a Perkin-ElmerSpectrum100 model instrument. The Antibase database was usedfor rapid dereplication and characterization of known compounds(Lang et al., 2008). HPLC chromatograph consisted of a Waters sys-tem including an autosampler 717, a pump 600, a photodiode array2998 and an evaporative light-scattering detector, ELSD 2420. TheHPLC analytical column used was a 3.5 lm, C-18 column (Sunfire150 � 4.6 mm) operating at 0.7 mL/min. The semipreparative col-umn was a 5 lm, C-18 (Sunfire 250 mm � 10 mm) operating at4 mL/min. On both columns, the gradient consisted of a linear gra-dient for 50 min from H2O to acetonitrile, both containing 0.1% for-mic acid. Silica gel 60 (6–35 and 35–70 lm) and analytical TLCplates (Si gel 60 F 254) were purchased from SDS (France). Pre-packed silica gel 80 g Redisep columns were used for flash chroma-tography using a Combiflash-Companion apparatus (Serlabo). Allother chemicals and solvents were purchased from SDS (France).
4.2. Microorganisms
Fungal strain Trichoderma artroviridae UB-LMA was isolatedfrom the bark of a yew tree (Taxus baccata) and identified accord-ing to ITS DNA sequence (GeneBank reference BankIt 1582103,10260002.seq, KC171717).
4.3. Cultivation
The cultivation broth consisted of Potato Dextrose Broth (PDB),Difco (Potato Starch 4 g/L, Dextrose 20 g/L), completed with 30 g/Lof XAD-16 resin. The Potato Dextrose Agar (PDA) consisted of PDBsupplemented with agar at a final concentration of 20%. Prior to thecultivation step, Petri dishes were inoculated with a frozen myce-lium sample for 5 days. Four milliliters of medium were thenspread on the agar layer and scratched. The spores-mycelium mix-ture was used to inoculate 10 � 2 L Erlenmeyers containing 1 L ofPDB medium. The strain was then allowed to grow 5 days in a ro-tary shaker at 27 �C and 150 rpm. At the end of the incubation per-iod, resin was recovered by filtration and separated from residualmycelium by decantation.
4.4. Purification of secondary metabolites
Resin was extracted with 3 � 500 mL of dichloromethane to af-ford 702 mg of dry residue. The extract was submitted to silica gelflash chromatography using a gradient from n-heptane to a mix-ture of n-heptane–ethyl acetate 20:80 (Prepacked silica gel, 80 gRedisep column, 40 mL/min, 110 min) to afford 8 fractions. Frac-tion 2 (35.0 mg), fraction 3 (24.0 mg), fraction 5 (11.3 mg) and frac-tion 7 (15.0 mg) were further purified by preparative HPLC (C-18column Sunfire, 250 � 10 mm, from water to acetonitrile, bothcontaining 0.1% formic acid). We obtained 2 mg of isoharzandione(Rt. 6.5 min), 6 mg of compound 1 (Rt. 8.0 min), 4 mg of compound2 (Rt. 5.5 min), 22 mg of compound 3 (Rt. 5.8 min) and 7 mg ofcompound 4 (Rt. 6.0 min).
4.4.1. Compound 1White powder; ½a�25
D + 27.0 (c 0.1, MeOH); IR mmax 3333, 2970,2913; 1663; 1438; 1032; 1003 cm�1; HRESIMS m/z 303.2326[M�H]� (calculated from C20H31O2 303.2324).
4.4.2. Compound 2Yellow oil, ½a�25
D � 25,5 (c 0.6, MeOH); IR mmax 3369, 2887, 1453,1380, 1106, 1084, 931 cm�1; HRESIMS m/z 273.2592 [M+H–H2O]+
(calculated from C20H33 273.2582).
nes from a Trichoderma symbiont of Taxus baccata. Phytochemistry (2013),
6 E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx
4.4.3. Compound 3White powder; ½a�25
D � 3.0 (c 0.4, MeOH); IR mmax 3290, 2964,2929, 1447, 1377; 1118; 1089 cm�1; HRESIMS m/z 271.2431[M+H–H2O]+ (calculated from C20H31 271.2426).
4.4.4. Compound 4Colorless oil, ½a�25
D + 0.6 (c 0.5, MeOH); IR mmax 3412, 2931,1729, 1449, 1380, 1186, 1024 cm�1; HRESIMS m/z 303.2325[M+H]+ (calculated from C20H31O2 303.2324).
4.5. Molecular modeling
Three-dimensional coordinates of compounds 1–4 and herzadi-one were generated from SMILES using CORINA. (CORINA, version3.44, Molecular Networks GmbH, Erlangen, Germany (http://www.molecular-networks.com/products/corina).
The conformational analysis of these compounds was carriedout using MacroModel v9.5, as implemented in the SchrödingerSuite (http://www.schrodinger.com). Default values were used ex-cept the allowed energy window (42 kJ/mol) and the number ofevaluations per rotatable bond (500). The resulting conformerswere clustered using a 2.0 Å cut-off.
The geometries of all conformers were optimized in gas-phaseusing the Gaussian 09 package (Gaussian 09, Revision A.2, Gauss-ian Inc., Wallingford CT, USA, http://www.gaussian.com). withthe Becke’s three-parameter hybrid exchange functional (B3LYP)Lee et al., 1988; Becke, 1993), and the 6-311+G(d,p) basis set (allthe energies presented in this paper were calculated at this level).Subsequent vibrational frequency calculations confirmed thatthese conformations were local minima. Structure images were ob-tained with Chimera (Pettersen et al., 2004) using the Pov-Ray ren-dering feature. CACTVS Chemoinformatics Toolkit (http://www.xemistry.com) was used for generating the PDF3D represen-tations of structures included in this study.
4.6. Crystal data for compound (3)
The crystal structure presented herein was solved from singlecolourless crystals suitable to X-ray diffraction, obtained by slowevaporation of chloroform solvent. Data were collected using a Rig-aku MM007 HF copper rotating-anode generator with Osmic con-focal optics and a rapid II Curved Image Plate at 193(2) K.
Crystal data of compound 3: C20H32O, Mr = 288.46, paleyellow stick, 0.35 � 0.19 � 0.15 mm, tetragonal, space group I 41,a = 29.7121(10) Å, b = 29.7121(10) Å, c = 7.9513(5) Å, V = 7019.5(6)Å3, Z = 16, Z0 = 2, rcalcd = 1.092 g cm�3, F(000) = 2560, l (CuKa) = 1.54187 Å, qmax = 68.21, �35 6 h 6 26, �35 6 k 6 21, �7 6l 6 9, 15744 measured reflections, 6028 independent,R(int) = 0.028, completeness to qmax = 99.2%, l = 0.484 mm�1,Tmin = 0.81 and Tmax = 0.93, 392 parameters were refined againstall reflections, R1 = 0.046, wR2 = 0.110 (using all (6026) data) basedon observed F values, R1 = 0.041, wR2 = 0.103 (3288 reflections withI > 2s(I)), Flack parameter = 0.01(3), extinction coeffi-cient = 0.0001(3), Drmin and Drmax = �0.126 and 0.167 e Å�3,GOF = 1.086 based on F2. After extensive refinement and softwarecalculations using likelihood methods (Hooft et al., 2008) the com-pound is assumed to be 100% enantiopure as (2S,5aR,6R,9-S,10aS,10bS)-3,6,10b,11,11-pentamethyl 2,4,5,6,7,8,9,10,10a,10b-decahydro-1H-5a,9-methanocyclobuta[a]heptalen-2-ol.
Crystallographic data for the structure (compound 3) reportedin this paper have been deposited with the Cambridge Crystallo-graphic Data Centre under the respective following deposit num-ber CCDC-913428. Copies of these data can be obtained, free ofcharge on application to the Director, CCDC 12 Union Road,Cambridge CB2 1EZ, UK (fax: +44 1223 336033; or e-mail:[email protected]).
Please cite this article in press as: Adelin, E., et al. Bicyclic and tetracyclic diterphttp://dx.doi.org/10.1016/j.phytochem.2013.10.016
4.7. Antibacterial and cytotoxic assays
Antibacterial activity was measured by the disk inhibition zonemethod against Bacillus subtilis ATCC.6633, Micrococcus luteusATCC.10240 and Echerichia coli ATCC.25922. Inhibition was com-pared to 10 lg gentamycin and 30 lg chloramphenicol.
tetrazolium-bromide; MTT]-based colorimetric assay was used tomeasure inhibition of the proliferation of the colonic epithelialcancer cell line HCT-116, the carcinoma cells KB and mammaryadenocarcinoma cells MCF7 as previously reported (Tempeteet al., 1995; Perri et al., 2010). All test compounds were formulatedin DMSO and applied to cells such that the final DMSO concentra-tion ranged from 1% to 3%.
Acknowledgment
This work was achieved in the frame of an MTA between Uni-versity of Barcelona, Spain and Institut de Chimie des SubstancesNaturelles, CNRS, Gif-sur-Yvette, France. Grants from the SpanishMEC (BIO2011-29856-CO2-01) and the Catalan Government(2009SGR1217).
Appendix A. Supplementary data
Full details of the cultivation, isolation, purification, analysis,bioassays, characterization data, aD, IR, 1D and 2D 1H and 13CNMR, mass spectrometry, crystallography, molecular modelingand PDF3D representations of theoretical compounds. Supplemen-tary data associated with this article can be found, in the onlineversion, at http://dx.doi.org/10.1016/j.phytochem.2013.10.016.
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