Bioactive polyketides isolated from agar-supported fermentation of Phomopsis sp. CMU-LMA, taking advantage of the scale-up device, Platotex Emilie Adelin a , Marie-Thérèse Martin a , Sylvie Cortial a , Pascal Retailleau a , Saisamorn Lumyong 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 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand article info Article history: Received 17 August 2012 Received in revised form 8 February 2013 Available online xxxx Keywords: Fungal secondary metabolites Sch-642305 Phomopsis Polyketide Antimicrobial DnaG primase abstract Phomopsis sp. CMU-LMA was cultivated on agar-supported fermentation (Ag-SF) using the scale-up pro- totype Platotex. In total nine compounds were isolated from the ethyl acetate extract of the culture. Among them, compounds LMA-P1, Sch-642305, DHTO and LMA-P2 had already been reported in our pre- vious work on liquid state fermentation. The trihydroxybenzene lactone cytosporone D and dothiorelone A has been recently isolated from Phomopsis and Magnaporthe species. In addition, three compounds were isolated consisting in the reduced methoxy derivative of Sch-642305 (1), a hydroxylated derivative of LMA-P2 (2) and a linear ethyl ester polyketide (3) similar to the previously reported LMA-P3. Antimicro- bial activity and inhibition of Escherichia coli DnaG primase were investigated. Cytosporone D inhibited the E. coli DnaG primase, a Gram-negative antimicrobial target, with an IC 50 of 0.25 mM. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The production of secondary metabolites in filamentous fungi is regulated by environmental stimuli that trigger the production of specific transcriptional factors. Those factors concomitantly regu- late morphological evolution of the fungus including sexual devel- opment and sporulation (Fox and Howlett, 2008; Hoffmeister and Keller, 2007; Strauss and Reyes-Dominguez, 2011). Among them, the nature and composition of the growth support play a crucial role. Agar-supported cultivation is the most widely used technique for microbial isolation and preliminary antibiotic or enzyme bioas- says. Taking into consideration that in nature fungi grow almost exclusively on solid supports, the solid-state fermentation has been widely investigated and has shown some improvement in enzyme and bioactive metabolite production (Zeng and Chen, 2009; Kagliwal et al., 2009). However, although this technique has been exploited for enzyme and secondary metabolite production, the technology had not managed to overcome major scale up bottle- necks (Adelin et al., 2011a). Subsequently, solid cultivation is widely substituted by liquid state fermentation in scale-up proce- dures (Gerea et al., 2012). In order to solve this limitation, we re- cently focused on agar-supported fermentation (Ag-SF) and built- up a fully automated scale-up device called Platotex (Ouazzani et al., 2007; Adelin et al., 2011b). This prototype offers 2 m 2 of cultivation surface in highly controlled conditions. In an ongoing effort to identify new bioactive microbial second- ary metabolites, we cultivated Phomopsis sp. CMU-LMA on Ag-SF, taking advantage from Platotex capacity. Subsequent investigation of the produced biomass leads to the isolation and the character- ization of three new polyketide compounds, together with six known compounds we previously reported (Adelin et al., 2011b). Some of those compounds exhibited weak antimicrobial activity, while the cytosporone D inhibited the Gram-negative antimicro- bial target primase DnaG from Escherichia coli. 2. Results and discussions The fungal strain Phomopsis sp. CMU-LMA was isolated from healthy tissue of Alpinia malacensis as previously reported (Adelin et al., 2011b). Regarding the high productivity of this strain and the diversity of the isolated compounds, we extended our investi- gations to agar-supported fermentation (Ag-SF). Freshly recovered mycelium suspension of 3 days-old agar was used to inoculate the ten 0.2 m 2 plates of the Platotex. Each Platotex plate was filled with 0.75 L of agar medium before sterilization. Inoculation procedure and Platotex operations were performed according to the recently described procedure (Adelin et al., 2011a). After inoculation, Phomopsis sp. CMU-LMA was cultivated 6 days at 30 °C, under 0.2 bar pressure and 12 L/min airflow. Platotex in use and recov- 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.02.012 ⇑ 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 SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Please cite this article in press as: Adelin, E., et al. Bioactive polyketides isolated from agar-supported fermentation of Phomopsis sp. CMU-LMA, taking advantage of the scale-up device, Platotex. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.02.012
Transcript
Phytochemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioactive polyketides isolated from agar-supported fermentation of Phomopsis sp.CMU-LMA, taking advantage of the scale-up device, Platotex
Emilie Adelin a, Marie-Thérèse Martin a, Sylvie Cortial a, Pascal Retailleau a, Saisamorn Lumyong 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 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
a r t i c l e i n f o
Article history:Received 17 August 2012Received in revised form 8 February 2013Available online xxxx
Please cite this article in press as: Adelin, E., etadvantage of the scale-up device, Platotex. Phy
a b s t r a c t
Phomopsis sp. CMU-LMA was cultivated on agar-supported fermentation (Ag-SF) using the scale-up pro-totype Platotex. In total nine compounds were isolated from the ethyl acetate extract of the culture.Among them, compounds LMA-P1, Sch-642305, DHTO and LMA-P2 had already been reported in our pre-vious work on liquid state fermentation. The trihydroxybenzene lactone cytosporone D and dothioreloneA has been recently isolated from Phomopsis and Magnaporthe species. In addition, three compounds wereisolated consisting in the reduced methoxy derivative of Sch-642305 (1), a hydroxylated derivative ofLMA-P2 (2) and a linear ethyl ester polyketide (3) similar to the previously reported LMA-P3. Antimicro-bial activity and inhibition of Escherichia coli DnaG primase were investigated. Cytosporone D inhibitedthe E. coli DnaG primase, a Gram-negative antimicrobial target, with an IC50 of 0.25 mM.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The production of secondary metabolites in filamentous fungi isregulated by environmental stimuli that trigger the production ofspecific transcriptional factors. Those factors concomitantly regu-late morphological evolution of the fungus including sexual devel-opment and sporulation (Fox and Howlett, 2008; Hoffmeister andKeller, 2007; Strauss and Reyes-Dominguez, 2011). Among them,the nature and composition of the growth support play a crucialrole. Agar-supported cultivation is the most widely used techniquefor microbial isolation and preliminary antibiotic or enzyme bioas-says. Taking into consideration that in nature fungi grow almostexclusively on solid supports, the solid-state fermentation has beenwidely investigated and has shown some improvement in enzymeand bioactive metabolite production (Zeng and Chen, 2009;Kagliwal et al., 2009). However, although this technique has beenexploited for enzyme and secondary metabolite production, thetechnology had not managed to overcome major scale up bottle-necks (Adelin et al., 2011a). Subsequently, solid cultivation iswidely substituted by liquid state fermentation in scale-up proce-dures (Gerea et al., 2012). In order to solve this limitation, we re-cently focused on agar-supported fermentation (Ag-SF) and built-up a fully automated scale-up device called Platotex (Ouazzani
et al., 2007; Adelin et al., 2011b). This prototype offers 2 m2 ofcultivation surface in highly controlled conditions.
In an ongoing effort to identify new bioactive microbial second-ary metabolites, we cultivated Phomopsis sp. CMU-LMA on Ag-SF,taking advantage from Platotex capacity. Subsequent investigationof the produced biomass leads to the isolation and the character-ization of three new polyketide compounds, together with sixknown compounds we previously reported (Adelin et al., 2011b).Some of those compounds exhibited weak antimicrobial activity,while the cytosporone D inhibited the Gram-negative antimicro-bial target primase DnaG from Escherichia coli.
2. Results and discussions
The fungal strain Phomopsis sp. CMU-LMA was isolated fromhealthy tissue of Alpinia malacensis as previously reported (Adelinet al., 2011b). Regarding the high productivity of this strain andthe diversity of the isolated compounds, we extended our investi-gations to agar-supported fermentation (Ag-SF). Freshly recoveredmycelium suspension of 3 days-old agar was used to inoculate theten 0.2 m2 plates of the Platotex. Each Platotex plate was filled with0.75 L of agar medium before sterilization. Inoculation procedureand Platotex operations were performed according to the recentlydescribed procedure (Adelin et al., 2011a). After inoculation,Phomopsis sp. CMU-LMA was cultivated 6 days at 30 �C, under0.2 bar pressure and 12 L/min airflow. Platotex in use and recov-
ted from agar-supported fermentation of Phomopsis sp. CMU-LMA, takingoi.org/10.1016/j.phytochem.2013.02.012
Fig. 1. Platotex during the cultivation period (left). Removal of the frame supporting the cultivation plates (center and right).
2 E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx
ered plates at the end of the cultivation step are shown in Figs. 1and 2.
Agar layers recovery was easily performed due to the mirrorpolishing of the plate surface. The agar (6.7 kg) was extracted withethyl acetate on the specific high-pressure static extractor Zipper-tex, developed in our laboratory (Ouazzani et al., 2008). The 10 LZippertex-cell allowed the extraction of 3 kg of agar by three con-secutive extractions with 3 L of ethyl acetate, thereby limiting thecontamination of the target compounds with the medium constit-uents. 1.66 g of crude extract was obtained corresponding to830 mg/m2 of cultivation surface or 222 mg/L. This yield is higherthan the 120 mg/L obtained from LSF. In addition of being moreeconomic in terms of medium, solvent and power, Ag-SF lead togreater diversity of metabolites compared to LSF (Fig. 3).
The compounds isolated in this study belong to the polyketidefamily (Fig. 4). They could be classified in four groups; the substi-tuted 10 membered lactones Sch-642305 (Chu et al., 2003), LMA-P1 (Adelin et al., 2011b) and compound (1); the six membered lac-tones LMA-P2 (Adelin et al., 2011b), DHTO, cytosporone D (Bradyet al., 2000), and compound (2); the benzo-ethyl ester dothioreloneA (Xu et al., 2004) and the linear ethyl ester compound (3). 1H and13C NMR data of the new compounds are presented in Table 1.
Compound 1 has molecular formula C15H24O5 as determined byHRESIMS m/z 285.1705 [M+H]+ (calcd for C15H25O5 285.1702). TheIR spectrum of (1) showed characteristic bands at 3311, 1716 and1700 cm�1, corresponding to hydroxyl, carbonyl and ester func-tional groups. 13C NMR data (Table 1) revealed the existence of acarbonyl group (dc 209.2) and a lactone carbonyl (dc 173.6). The13C NMR spectrum also showed a methoxy group (dc 80.8) con-
Fig. 2. Platotex plates at the end of the cultivation period. The mirror polishing ofthe plates makes easy the agar recovery (left).
Please cite this article in press as: Adelin, E., et al. Bioactive polyketides isolaadvantage of the scale-up device, Platotex. Phytochemistry (2013), http://dx.d
firmed by the 1H NMR integration of the singlet at 3.35 ppm, twooxymethines (dc 74.3 and 72.4), two methines (dc 46.4 and 36.9),six methylene groups (dc 40.5, 38.6, 33.5, 25.8, 22.4, 21.8) andone methyl group at dc 19.8 ppm. 1H–1H COSY and 1H–13C HMBCcorrelations showed that compound (1) had the same skeleton asSch-642305 except the double bond between C-2 and C-3; this lat-ter bears the methoxy group. H-2A and H-2B showed a small cou-pling constant with H-3 (J = 3.6 Hz and J = 2.4 Hz respectively)confirming that H-3 is in an equatorial position. NOESY spectrumshowed correlation between H-2B/H-3, H-2B/H-6, H-3/H-4 andH-4/H-5 (see Supplementary data S3 and S4 for details). All theseinformation confirmed that the methoxy group was in an axial po-sition, above the average plan of the molecule and in a trans posi-tion relative to the hydroxy group in C-4. The structure ofcompound (1) is presented in Fig. 4.
The HRESIMS of compound (2) gave an m/z at 265.1418[M+Na]+ corresponding to the molecular formula C13H22O4Na(calcd 265.1416). The IR absorption bands at 3292 and1698 cm�1 were attributed to a hydroxyl and a carbonyl groupsrespectively. 1H and 13C NMR data (Table 1) showed the existenceof a lactone carbonyl (dC 176.7), two olefinic methines (dC 141.9and 124.8), a hydroxylated quaternary carbon at 73.5 ppm, twooxymethines (dC 83.4 and 71.1), two sp3 carbons (dC 42.5, and38.8), a methylene (dC 34.2) and four methyl groups (dC 26.1,13.0, 11.7 and 7.0). The coupling constant between H-8 and H-9(J = 15.4 Hz) attested that the double bond between C-8 and C-9is trans. 1H–1H COSY and 1H–13C HMBC NMR spectra confirmedthat the structure of compound (2) is very close to those of LMA-P2 and may derive from it through an allylic hydroxylation. Thestereochemistry of C-10 in compound (2) was deduced from theORTEP diagram showing the crystal state conformation (Fig. 5).The stereochemistry is in agreement with the enzymatic hydroxyl-ation rules recently published (De Montellano, 2010), which sug-gested that the enzymatic hydroxylation usually proceeded withretention of configuration at the hydroxylated carbon. This meansthat the methyl C-13 position of LMA-P2 remains unchanged whilethe hydroxyl group replaces the hydrogen at C-10 position. Subse-quently, the C-10 (S) configuration in LMA-P2 became (R) in com-pound (2).
HRESIMS of compound (3) gave a m/z 325.1786 [M+Cl]� corre-sponding to the molecular formula C15H30O5Cl (calcd 325.1782). IRabsorption bands at 3421 and 2916 cm�1 were attributed to a hy-droxyl, and bands at 1746 and 1725 cm�1 to ester groups. 1H and13C NMR data (Table 1) disclosed the existence of a carbonyl (dC
173.6), three oxymethines (dC 73.9, 69.8 and 69.3), a methoxy
ted from agar-supported fermentation of Phomopsis sp. CMU-LMA, takingoi.org/10.1016/j.phytochem.2013.02.012
Fig. 3. Comparison of HPLC profiles obtained after extraction of Ag-SF culture (continuous line) and LSF (dashed lines). Medium, inoculum and culture duration were similarin both cases. Focus on the major compounds shows that in these conditions, only LMA-P1, Sch-642305 and DHTO were formed in LSF.
Fig. 4. Structures of the compounds isolated from Ag-SF culture of Phomopsis sp. CMU-LMA.
E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx 3
group (dC 52.8), a methyl group (dC 14.1) and nine methylenes (dC
39.9, 37.6, 31.9, 29.5, 29.5, 29.5, 29.5, 29.2, 25.8,). 1H–13C HMBCcorrelations did not support the presence of a cyclic ring as one de-gree of unsaturation is observed. The structure of compound 3 isshown in Fig. 4. Its stereochemistry was deduced after its conver-sion to DHTO by treatment with 3 equiv of K2CO3 in methanol for3 h, according to literature (Khan et al., 2010). In these conditions,the lactonization derivative of compound 3 and DHTO exhibitedthe same spectral and physical properties, suggesting that com-pound 3 could be the biological precursor of DHTO.
Compounds isolated from Ag-SF culture of Phomopsis sp. CMU-LMA were evaluated for their antimicrobial and E. coli DanG pri-mase inhibition as described in Section 4 and Supplementary data.E. coli DnaG primase is a key enzyme in DNA replication, and aGram-negative antimicrobial target (Agarwal et al., 2007; Maciaget al., 2010; Frick and Richardson, 2001). Among the evaluated
Please cite this article in press as: Adelin, E., et al. Bioactive polyketides isolaadvantage of the scale-up device, Platotex. Phytochemistry (2013), http://dx.d
compounds, Sch-642305 and cytosporone D exhibited antimicro-bial activity against Gram-positive bacteria but not againstGram-negative E. coli. Results from literature indicated that cyto-sporone D has broad antimicrobial activity against Staphylococcusaureus, Enterococcus faecalis, and Candida albicans with a MIC of2; 16.2 and 128 lg/ml respectively (Brady et al., 2000). As shownin Fig. 6, cytosporone D inhibits the E. coli DnaG primase, in a dosedependent manner, with an IC50 of 0.25 mM.
3. Conclusion
Phomopsis sp. CMU-LMA exhibited a high productivity anddiversity of secondary metabolites in Ag-SF, highlighting theadvantages of solid-state cultivation, and supporting the efficiencyof Platotex as a unique available Ag-SF scale-up fermentor. Besides
ted from agar-supported fermentation of Phomopsis sp. CMU-LMA, takingoi.org/10.1016/j.phytochem.2013.02.012
Table 11H and 13C NMR spectroscopic data for the new compounds isolated from Phomopsis sp. CMU-LMA. dC and dH were in ppm.
No. Compound 1 Compound 2 Compound 3
dC dH (J, Hz) dC dH (J, Hz) dC dH (J, Hz)
1 209.2 176.7 173.62 40.5 H-2A 2.57 m, H-2B 2.82 dd (3.6; 14.6) 42.5 2.56 qd (2.9; 7.1) 73.9 4.15 d (2.0)3 80.8 3.76 q (3.4) 71.1 3.68 t (2.2) 69.8 4.27 dt (2.8; 9.2)4 72.4 4.03 t (2.8) 38.8 1.83 m 39.9 1.65 ddd (3.3; 9.0; 14.5),
1.93 ddd (3.3; 9.0; 14.5)5 36.9 2.64 m 83.4 4.59 dd (8.4; 10.8) 69.3 3.97 m6 46.4 2.54 m 11.7 1.15 d (6.8) 37.6 1.54 m7 21.8 1.57; 1.74 m 13.0 0.92 d (6.8) 25.8 1.30 m8 25.8 1.35; 1.57 m 124.8 5.55 dd (8.4; 15.4) 29.5 1.30 m9 22.4 1.19; 1.74 m 141.9 5.73 d (15.4) 29.5 1.30 m10 33.5 1.35; 1.96 m 73.5 29.5 1.30 m11 74.3 5.10 m 34.2 1.46 q (7.5) 29.5 1.30 m12 173.6 7.0 0.79 t (7.5) 29.2 1.30 m13 38.6 2.55 m, 2.78 dd (5.5; 14.6) 26.1 1.16 s 31.9 1.30 m14 19.8 1.25 d (6.5) 14.1 0.91 t (6.6)15 56.9 3.35 s 58.8 3.87 s
Fig. 5. ORTEP diagram showing the solid-state structure of compound 2.
Fig. 6. Cytosporone D inhibition of E. coli DnaG primase.
4 E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx
Please cite this article in press as: Adelin, E., et al. Bioactive polyketides isolaadvantage of the scale-up device, Platotex. Phytochemistry (2013), http://dx.d
offering high yield and diversity of target compounds, Ag-SF is acheap, environmental friendly process, with low energy and waterconsumption. Ag-SF coupled to solid/liquid extraction confirmedthe potential of Zippertex, a specially designed high pressure staticextractor. This device limited the quantity of organic solvent anddid not produce any aqueous wastes. Both Platotex and Zippertexwere built according to the highest security and quality standards,while enabling integrated automation and software-based remotecontrol. Platotex and Zippertex were 3D-digitized using CATIA-V5software from Dassault-System, which facilitate the conceptionof higher capacity devices.
4. Experimental section
4.1. General experimental procedure
1H and 13C spectra were recorded using a Bruker Avance-300and 600 instrument operating at 300 and 600 MHz respectively.LC–ESI-MS analysis were performed on a simple-stage quadrupoleWaters-Micromass� ZQ 2000 mass spectrometer equipped with anESI (electrospray ionization) interface coupled to an AllianceWaters 2695 HPLC instrument with PDA and ELS detection. TheHRESIMS spectra were recorded on a Waters-Micromass� massspectrometer equipped with ESI-TOF (electrospray-time of flight).Optical rotations were measured at 25 �C on a JASCO P1010 polar-imeter. IR spectra were obtained on a Perkin-Elmer Spectrum100model instrument. The Antibase database was used for rapid dere-plication and characterization of known compounds (Lang et al.,2008). HPLC chromatograph consisted of a Waters system includ-ing an autosampler 717, a pump 600, a photodiode array 2998and an evaporative light-scattering detector, ELSD 2420. The HPLCanalytical column used was a 3.5 lm, C-18 column (Sunfire150 mm � 4.6 mm) operating at 0.7 mL/min. The preparative 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 40 g Redisep were used for flash chromatographyusing a Combiflash-Companion apparatus (Serlabo). All otherchemicals and solvents were purchased from SDS (France).
4.2. Microorganisms
The fungal strain Phomopsis sp. CMU-LMA was isolated fromhealthy wild galanga tissue (A. malacensis). Fungal strain Phomopsis
ted from agar-supported fermentation of Phomopsis sp. CMU-LMA, takingoi.org/10.1016/j.phytochem.2013.02.012
E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx 5
sp. CMU-LMA was identified according to molecular techniquesand DNA sequencing of SSU and ITS rDNA, as described in previousstudies (Bussaban et al., 2001).
4.3. Cultivation on Platotex and extraction
The strain was cultivated on Platotex during 6 days at 30 �C and0.2 bar, according to prior optimization conducted on 25 � 25 cmagar PDB plates. Each Platotex plate contained 750 mL of sterilePDB medium complemented with 20% agar and inoculated with200 mL of inoculum (mycelium from 3 days old agar cultures).After cultivation period, the agar/biomass layers were recoveredfrom each Platotex plate. The agar (6.7 kg) was extracted by ethylacetate on the specific high-pressure static extractor Zippertex(Ouazzani et al., 2008). The 10 L Zippertex-cell allowed the extrac-tion of 3 kg of agar by three consecutive extractions with 3 L ofethyl acetate. The organic layers were pooled and dried over anhy-drous magnesium sulfate affording 1.6 g of crude extract.
4.4. Purification of secondary metabolites
3.0 g of the extract was submitted to flash chromatographyusing an n-heptane – ethyl acetate gradient (90:10–50:50) at30 mL/min during 80 min to afford nine fractions, according toTLC profiles. Fraction 1 (20 mg, Hpt–AcOEt 80:20) afforded LMA-P2. Fraction 4 (15 mg, Hpt–AcOEt 70:30) was further purified onpreparative C-18 column (Sunfire, 250 mm � 10 mm) using lineargradient for 50 min from H2O to acetonitrile, both containing0.1% formic acid (tr: 18.8 min) to give 10 mg of Sch-642305. Frac-tion 5 (121 mg, Hpt–AcOEt 70:30) was further purified on prepara-tive C-18 column (Sunfire, 250 mm � 10 mm) using linear gradientfor 50 min from H2O to acetonitrile, both containing 0.1% formicacid. This afforded 50 mg of Sch-642305, 15 mg of LMA P1 and40 mg of compound 1 (tr: 18.0, 18.8 and 19.9 min respectively).Fraction 6 (167 mg, Hpt–AcOEt 65:35) was further purified on C-18 column (Sunfire, 250 mm � 10 mm) using linear gradient for50 min from H2O to acetonitrile, both containing 0.1% formic acidaffording 22 mg of compound 3 and 95 mg of DHTO (tr: 23.8 and25.4 min respectively). Fraction 8 (552 mg, Hpt–AcOEt 50:50)was further purified by flash chromatography using isocratic n-heptane – ethyl acetate (70:30) at 20 mL/min during 60 min to af-ford 60 mg of compound 3, 45 mg of DHTO, 10 mg of dothioreloneA, and 3 mg of compound 2.
Please cite this article in press as: Adelin, E., et al. Bioactive polyketides isolaadvantage of the scale-up device, Platotex. Phytochemistry (2013), http://dx.d
C13H22O4, Mr = 242.31, colorless tab, 0.60 � 0.30 � 0.25 mm,triclinic, space group P1, a = 7.1900(2) Å, b = 7.2161(2) Å,c = 7.5447(5) Å, a = 62.419(4)�, b = 81.523(6)�, c = 84.311(6)�,V = 342.96(3) Å3, Z = 1, qcalcd = 1.173 g cm�3, F(000) = 132, k(CuKa) = 1.54187 Å, 2hmax = 136.5�, �8 6 h 6 7, �8 6 k 6 8,�9 6 l 6 9, 4879 measured reflections, 2122 independent,R(int) = 0.0351, l = 0.700 mm�1, Tmin = 0.67 and Tmax = 0.84, 165parameters were refined against all reflections, R1 = 0.038,wR2 = 0.093 (using all 2116 data) based on observed F values,R1 = 0.032, wR2 = 0.086 (1863 reflections with I > 2r(I)), extinctioncoefficient = 0.027(3), Dqmin and Dqmax = �0.119 and 0.114 e Å�3,GOF = 1.036 based on F2 for three restraints. Eight hundred andninety-two Friedel opposites (71% of coverage) were kept un-merged and the weak anomalous scattering contribution enhancedby the copper radiation was exploited to claim the compoundenantiopure as (3R,4R,5S,6R)-4-hydroxy-6-((R,E)-3-hydroxy-3-methylpent-1-en-1-yl)-3,5-dimethyltetrahydro-2H-pyran-2-onecompound. This was assumed unambiguously from convergent cri-teria as the value of the Flack parameter, x = 0.12 (19) derived fromSHELXL and those implemented within PLATON (e.g. Hooft param-eter y = 0.15 (11); 12 out of 13 DF P 0.5r show sign agreement be-tween calculated and observed differences; P2(true) andP3(true) = 1.00 and 0.974 (Bayesian statistics)).
This structure (including structure factors) has been depositedwith the Cambridge Crystallographic Data Centre as supplemen-tary publication number CCDC 923188. Copies can be obtained,free of charge, on application to CCDC, 12 Union Road, CambridgeCB2 1EZ, UK [fax: +44(0) 1223 336033 or e-mail:[email protected]].
4.6. Antibacterial and primase bioassays
Antibacterial activity was measured by the disk inhibition zonemethod against Bacillus subtilis ATCC 6633, Micrococcus luteus ATCC10240 and Escherichia coli ATCC 25922. Inhibition was compared to10 lg gentamicin and 30 lg chloramphenicol. E. coli DnaG primasecloning, purification and bioassay conditions were fully describedin Supporting Information.
ted from agar-supported fermentation of Phomopsis sp. CMU-LMA, takingoi.org/10.1016/j.phytochem.2013.02.012
6 E. Adelin et al. / Phytochemistry xxx (2013) xxx–xxx
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in theonline version, at http://dx.doi.org/10.1016/j.phytochem.2013.02.012.
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