Apolar chromatography on Sephadex LH-20 combined with high-speed counter-current chromatography

Journal of Chromatography A, 1117 (2006) 67–73

Apolar chromatography on Sephadex LH-20 combined with high-speed counter-current chromatography High yield strategy for structurally closely related analytes—Destruxin derivatives from Metarhizium anisopliae as a case study Christoph Seger a,∗ , Karin Eberhart a,b , Sonja Sturm a , Hermann Strasser b , Hermann Stuppner a a

Institute of Pharmacy, Center of Molecular Biosciences, Leopold Franzens University Innsbruck, Innrain 52, A-6020 Innsbruck, Austria b Institute of Microbiology, Leopold Franzens University Innsbruck, Technikerstraße 25, A-6020 Innsbruck, Austria Received 10 January 2006; received in revised form 19 March 2006; accepted 20 March 2006 Available online 4 April 2006

Abstract A novel high yield isolation procedure for lipophilic cyclic peptide derivatives is presented. Destruxin (dtx) A, B, D, E, and E-diol retrieval from Metarhizium anisopliae culture broth was achieved with a three-step purification protocol. After liquid–liquid extraction column chromatography over Sephadex LH-20 served as enrichment step. High-speed counter-current chromatography (HSCCC) was used for the final purification. Within the first chromatographic step dtx D and dtx E-diol were separated in purities exceeding 90%. The separation of dtx A, B, and E was achieved from an enriched Sephadex LH-20 fraction by a HSCCC protocol using light petroleum–ethyl acetate–methanol–water = 2:5:2:5 (v/v) as eluent system. These derivatives were obtained in purities above 98% and total yields exceeding 40%. © 2006 Elsevier B.V. All rights reserved. Keywords: Metarhizium anisopliae; Destruxins; Sephadex-LH20; HSCCC; High-speed counter-current chromatography

1. Introduction A general prerequisite for analytical, pharmacological, or toxicological research is the availability of highly purified analytes in sufficient amounts. Screening for bioactivities, as well as preclinical or clinical trials of secondary natural products is often hampered by the lack of the required gram quantities of these metabolites [1]. This need can be considered as one of the most prominent driving forces in preparative natural product chemistry. A multitude of strategies is known for the production, isolation and purification of the desired products [2,3]. Biotechnological processes like plant tissue cultures or submerged culture fermentation of anamorphic fungi are exploited to allow large scale production of biomass and metabolites of interest [4]. Liquid–liquid or liquid–solid extraction protocols are used

∗

Corresponding author. Tel.: +43 512 507 5344; fax: +43 512 507 2939. E-mail address: [email protected] (C. Seger).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.03.055

to obtain crude extracts. Subsequent application of preparative chromatographic techniques, usually in multiple steps, produces the desired analytes in sufficient purities. However, achieved yields are often unsatisfactory low. Thus, whenever confronted with the need to obtain large analyte amounts, strategies to overcome multiple step protocols and/or low yield separation techniques have to be envisioned. In the case of destruxin (dtx) derivatives, members of a lipophilic cyclic hexadepsipeptide compound class, are hardly commercially available. Currently, only dtx A can be purchased – at prices exceeding 300 D /mg. Most of the dtx derivatives have been described from Metarhizium anisopliae, an entomopathogenic anamorphic fungus used as biological pest control agent [5–7] (Fig. 1). Furthermore, a broad range of relevant biological activities have been reported for members of this compound class [5,6]. Approaches toward pure dtxs included isolation from fungal culture broth and synthetical work, with the later one hampered by low yield reactions [6]. Isolation involved either silica gel or preparative scale reversed phase chromatography steps, resulting in rather

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Fig. 1. Formula scheme of major dextruxin derivatives.

low overall yields [8–16]. Being confronted with the need to obtain larger amounts of destruxin derivatives as reference standards for analytical purposes and for ongoing pharmacological investigations, the development of a novel dtx preparation protocol was pursued. After removing hydrophilic fungal metabolites by liquid–liquid extraction, column chromatography using a beaded cross-linked dextran gel (Sephadex LH-20) was envisioned as first purification step. This material has been tailored for the size exclusion separation of small organic molecules and is usually employed in methanol or methanol/water mixtures. It has been and is routinely used for a broad variability of natural products, among these series of fungal cyclic peptides like virotoxins [17]. However, if used in with more apolar mobile phases as acetone, ethyl acetate, dichloromethane, or chloroform, analyte separation is dominated by partition between stationary and mobile phase. Thus, these chromatographic systems tend to separate analytes according to their lipophilicity instead of their molecular weight [2,18–22]. High-speed counter-current chromatography (HSCCC) was chosen as final purification step. This support free liquid–liquid chromatography technique has been applied successfully to a vast range of natural products [23–28], including lipophilic carotenoids [29], fungal macrolide type antibiotics [30–32], and cyclosporins – cyclic oligopeptide derivatives of fungal origin with immunosuppressant activities [33]. HSCCC is known for the reduced risk of sample loss and ease of system upscaling [3,34–36]. Its theory is well understood and solvents mixtures are known for almost any analyte polarity [34,35].

2. Experimental 2.1. Reagents Acetonitrile (gradient grade), dichloromethane, ethyl acetate, heptane, hexane, methanol (all of analytical grade), light petroleum (b.p. 60–90 ◦ C), agar, Sabouraud 2% glucose (S2G) medium and Tween-80 were purchased from Merck (Darmstadt, Germany). Deuterated chloroform for NMR analysis (99.6% deuteration) was purchased from Eurisotop (Saarbr¨ucken, Germany). Nitrogen (99.995%) for mass spectrometry was produced by a nitrogen generator (Peak Scientific Instruments, Fountain Crescent, UK). Water for the HPLC was produced by reverse osmosis followed by distillation. 2.2. HPLC–DAD and HPLC–DAD–MS–MS conditions All chromatographic analyses were performed either with a method presented recently [5] or with an accelerated assay. The original method was utilized for quantitative analyses and HPLC–DAD–MS–MS coupling, whereas the novel assay was used for qualitative HPLC–DAD monitoring of chromatographic fractions. A HP 1100 liquid chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with diode array detection (DAD), an automatic injector, an auto sampler and a column oven was used for all assays. Separations were performed on a Zorbax SB-C18 column (150 mm × 2 mm),

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particle size 3.5 ␮m (Agilent Technologies) with a solvent gradient of water (A) and acetonitrile (B). A LiChroCART 4-4 (Merck) column (4 mm × 4 mm) filled with LiChrospher 100 RP-18 (particle size 5 ␮m) material was used as guard column. The accelerated assay with a total duration of 12 min (compared to 20 min of the original method) used a solvent gradient program of: t = 0 min 70% A; t = 4 min 2% A; t = 5.3 min 2% A; t = 5.4 min 70% A. Between runs the column was equilibrated with 70% A for 4 min. The system was operated at a flow rate of 0.3 ml/min at room temperature (thermo-stated, 23 ◦ C). The injection volume was 2 ␮l. Chromatograms were recorded at 210 nm. DAD spectra between 190 and 600 nm were stored for all peaks exceeding a threshold of 0.1 mAU. For HPLC–DAD–MS–MS experiments the liquid chromatograph described above was coupled to an Esquire 3000plus ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany). Experiments were performed in positive ESImode using experimental parameters as described previously [5]. 2.3. Cultivation of M. anisopliae and extract preparation Conidia, harvested from 14 to 20 days old cultures of single spore isolates of M. anisopliae var. anisopliae (Metsch.) Sorokin, BIPESCO 5, and re-suspended in sterile 0.05% (w/v) aqueous Tween 80, were used to inoculate 1000 ml Erlenmeyer flasks containing 500 ml S4G liquid medium. The final concentration per flask was of 5 × 104 conidia/ml. Cultures were incubated at 25◦ on a gyratory shaker (150 rpm; 80% relative humidity). After 5 days this culture was used to inoculate (2.5% (v/v)) a 14 l stirred tank reactor (Bio Engineering NLF22, Wald, Switzerland) containing 10 l S4G liquid medium (pH 6) and 0.05% (v/v) antifoam agent (Clerol FBA 5050, Henkel, D¨usseldorf, Germany). The thermo-stated (23.6 ◦ C) culture was aerated with 1 volume air/volume liquid/minute (vvm) and stirred with 300 rpm. The culture broth was harvested after 7 days of grows. The mycelium was separated by filtration through cotton cloth. A final purification of the culture broth was achieved by filtering through two layers of filtration gauze (pore size 20 ␮m, Schleicher & Sch¨ull, Dassel, Germany). The obtained culture filtrate was stored at −20 ◦ C until needed. Dtx enrichment from this filtrate was achieved by extraction of aliquots with dichloromethane (3 volumes/volume culture filtrate, five repetitions). Combined extracts were washed twice with 50 ml water, dried with sodium sulphate and evaporated to dryness. Recycling of dichloromethane allowed keeping the total solvent consumption below 5 l dichloromethane/10 l culture filtrate. For comparison purposes an alternative extraction protocol presented recently was utilized [37]. In this case the culture filtrate was mixed with an equal volume of acetonitrile and 5% (w/v) sodium chloride. After the salt dissolved completely, two phases were separated (four repetitions). Again, combined extracts were washed twice with 50 ml water, dried with sodium sulphate and evaporated to dryness. All preparative acetonitrile or dichloromethane operations were performed under a well-vented hood to minimize the impact on the laboratory staff.

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2.4. Apolar chromatography on Sephadex LH-20 For gel chromatography the stationary phase bed was prepared by equilibrating 150 g stationary phase (Sephadex LH-20, Sigma–Aldrich, Vienna, Austria) over night in dichloromethane:acetone = 85:15 (v/v). After transferring the slurry to the column (100 cm × 4 cm, Kronlab, Dinkslaken, Germany) the bed was allowed to settle and was washed with 1 l dichloromethane–acetone = 1:1 (v/v). Prior to the separation equilibration 1 l dichloromethane was performed. The sample (3.5 g crude extract) was dissolved in 15 ml dichloromethane, filtered through cotton batting and applied to the chromatographic system. Elution was carried out with 1000 ml dichloromethane followed by 500 ml dichloromethane–acetone = 85:15 (v/v) and 500 ml dichloromethane–acetone = 1:1 (v/v) and 500 ml acetone at a flow rate of 1.2 ml/min. The resulting eluate was collected (fraction size 6 ml) into test tubes using a fraction collector (SuperFrac, Pharmacia Biotech, Amersham Biosciences). Column chromatography and fraction collection were performed under a well-vented hood. All fractions were transferred to pre-weighed glasses and evaporated to dryness (SpeedVac Plus SC 210A, Thermo Savant, USA). After weighing samples for HPLC–DAD analysis were prepared by dissolving a fraction aliquot in gradient grade methanol (c = 1 mg/ml). Subsequently, all fractions were analyzed by HPLC–DAD and fractions with same content were pooled. 2.5. High-speed counter-current chromatography 2.5.1. Apparatus Preparative HSCCC was carried out using a Model CCC1000 multilayer coil counter-current chromatograph equipped with a 325 ml coil column and an electronic controller (PharmaTech-Research, Baltimore, MD, USA). The solvent was pumped into the column by a HPLC pump (LC-10AD-VP, Shimadzu). A manual sample injection valve with a 10 ml sample loop was used to inject the sample into the system. For HSCCC optimisation a high-speed counter-current chromatograph (P.C., Potomac, MD, USA) with an 80 ml coil column and a 1 ml sample loop was used. The mobile phase was pumped through a Gilson pump (Model 302, Villiers-la-Bel, France) with a manometric module (Model 803C, Gilson). 2.5.2. Selection of appropriate two-phase solvent systems Ten ml of an investigated solvent system (Table 1) were merged in a test tube to which 5 mg crude extract was added. After the tube was shaken with a Vortex mixer for 30 s to distribute the sample, two layers formed within 20 s. The two phases were separated manually, transferred to glass vials, dried (SpeedVac Plus SC 210A, Thermo Savant) and re-dissolved in 500 ␮l gradient grade methanol. Samples were analyzed by HPLC–DAD and the peak areas for dtx A, dtx B, and dtx E were recorded. Partition coefficients KA , KB , and KE were expressed as the peak area of the respective dtx derivative dtx A, dtx B and dtx E in the upper phase divided by that in the lower phase. An average partition coefficient KABE was derived from the dtx A, dtx B, and dtx E peak area sums in the respective phases.

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Table 1 Partition coefficients K for dtx E (KE ), dtx A (KA ), dtx B (KB ) and total partition coefficient KABE of the solvent systems 1–20 tested during HSCCC-optimisation-phase.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Solvent system

Volume ratio

KE

KA

KB

KABE

Hexane–dicholoromethane–acetonitrile Hexane–ethanol–water Light petroleum–ethyl acetate–methanol–water Hexane–ethyl acetate–methanol–water Light petroleum–ethyl acetate–methanol–water Light petroleum–ethyl acetate–methanol–water Hexane–ethyl acetate–methanol–water Hexane–ethyl acetate–methanol–water Hexane–ethyl acetate–methanol–water Heptane–ethyl acetate–methanol–water Heptane–ethyl acetate–methanol–water Hexane–dichloromethane–acetonitrile Hexane–dichloromethane–acetonitrile Light petroleum–dichloromethane–acetonitrile Heptane–dichloromethane–acetonitrile Hexane–ethyl acetate–acetonitrile Dichloromethane–methanol–isopropanol–water Hexane–ethanol–water Hexane–methyl tertiary butyl ether–acetonitrile Hexane–acetonitrile–methanol

20:7:13 10:7:3 12:18:15:10 6:24:15:10 6:10:10:3 2:5:2:5 1:2:1:2 12:18:15:10 2:3:3:2 2:3:3:2 3:12:12:3 5:2:3 4:2:3 4:1:3 5:2:3 4:1:3 4:3:3:3 6:5:1 10:1:10 8:5:2

0.1 0.02 0.1 0.3 0.1 0.5 0.3 0.1 0.05 0.02 0.03 0.1 0.1 0.04 0.1 0.1 0.1 0.1 0.05 0.1

0.1 0.03 0.3 0.5 0.1 1.8 0.7 0.2 0.1 0.1 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.1 0.1 0.1

0.1 0.02 0.4 0.6 0.1 6.1 1.3 0.2 0.2 0.1 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.1

0.1 0.02 0.3 0.5 0.1 1.5 0.7 0.2 0.1 0.1 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.1

2.5.3. HSCCC separations Components of the solvent system chosen by the above described procedure were mixed and equilibrated by repeatedly shaking in a separation funnel at room temperature. The separated phases were collected and degassed for 10 min in an ultrasonic bath before use. Sample solutions were prepared by dissolving the sample in ethyl acetate (800 mg sample/9 ml ethyl acetate). After filling the column with the chosen stationary phase, the mobile phase was pumped into the “head” end of the inlet column at a flow rate of 1.0 ml/min at a rotation of 1000 rpm. If the upper phase was used as stationary phase and the lower phase as mobile phase, the elution mode for the HSCCC separation was head to tail and vice versa. Once the mobile phase eluted at the tail outlet and hydrodynamic equilibrium was reached, the sample solution was injected from the sample loop into the column through the sample port. The effluent was collected in 10 ml fractions portioned by a fraction collector (SuperFrac, Pharmacia Biotech). An aliquot of 500 ␮l of each fraction was transferred to HPLC vials without further purification. All fractions were analyzed by HPLC–DAD and fractions with same content were pooled. Finally, all fractions were dried (SpeedVac Plus SC 210A, Thermo Savant) for subsequent weight determination.

3. Results 3.1. Submerged batch cultivation, destruxin production and crude extract preparation In submerged batch cultivation dtx A, B, and E could be detected by HPLC–DAD from the first day on. M. anisopliae BIPESCO5 reached a maximum of production of dtx A and B at day seven while the production of dtx E decreased after day five. The highest concentrations were 197 mg dtx A, 84 mg dtx B, and 67 mg dtx E per litre culture broth, respectively. Because of the observed dtx E degradation in culture broth [5,41,42] fermentation was stopped on day 7. Fig. 2A represents

2.6. Dtx identification Dtxs A, B, and E were identified using reference material. Further congeners were identified by their relative retention times and their MS–MS fragmentation pattern [5,38,39]. The identity of the isolated major dtx derivatives dtx A, B, E, Ediol, and D was further confirmed by standard one- and twodimensional NMR performed on a Bruker-DRX300 NMR spectrometer using experimental parameters as previously described [40].

Fig. 2. HPLC–DAD chromatograms (210 nm) of (A) the M. anisopliae culture broth and the extracts obtained by liquid–liquid partition (B) with acetonitrile (ACN) or (C) dichloromethane (DCM). Separations were performed on a Zorbax SB-C18 column (150 mm × 2.0 mm, particle size 3.5 ␮m) using a water–acetonitrile gradient (t = 0 min 5% ACN; t = 6 min 50% ACN; t = 8 min 98% ACN; t = 12 min 98% ACN, flow rate 0.3 ml/min). The separation system was kept at room temperature and the injection volume was 2 ␮l.

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Fig. 3. Fractions of apolar Sephadex LH-20 chromatography; weights against fraction numbers. Pooled fractions CF 1–CF 4 are shown in grey.

the HPLC chromatogram of M. anisopliae culture broth before harvest. With increasing incubation time, pH decreased (day 1: pH 5.25; day 7: pH 3.74). Fungal biomass increased to 30 g dry weight/l culture broth until day 2 and stayed constant afterwards. Liquid–liquid extraction of the culture broth was carried out to obtain a crude extract enriched with dtxs. Thus, after filtration over two layers of filtration gauze the culture filtrate (7 l) was extracted using either dichloromethane or alternatively a novel procedure based on acetonitrile [37]. The HPLC–DAD chromatogram comparison showed that both extraction procedures provided a high dtx A, B and E concentration (Fig. 2B and C). However, the acetonitrile based protocol left more nondestruxin impurities (peaks at tR = 1.5–5.0 min) in the obtained extract compared to the dichloromethane approach. Therefore, dichloromethane extraction can be still considered the superior method to isolate dtxs from liquid culture broth. After five extraction steps no considerable amount of dtxs was left in the culture broth. Thus, the whole fermenter content was extracted five times, yielding 3.5 g crude extract enriched with dtxs from 10 l submerged batch fermentation. 3.2. Apolar chromatography on Sephadex LH-20 The dichloromethane extract was subjected to SephadexLH20 chromatography utilized as pre-purification step. Elution started with dichloromethane followed by dichloromethane blended with increasing amounts of acetone. Every obtained fraction was weighed and analyzed by HPLC–DAD. Combining fraction weight distribution and fraction chromatograms allowed distinguishing co-eluting analyte groups (Fig. 3). Fractions of similar content were pooled to combined fractions (CF 1–4). Residual non-destruxin type culture broth constituents eluted before the first destruxin fraction. CF 1 (120 ml, 2.1 g) and CF 2 (228 ml, 0.4 g) were eluted with 100% dichloromethane whereas CF 3 (30 ml, 11 mg) and CF 4 (48 ml, 38 mg) were collected during the gradient step dichloromethane–acetone = 85:15 (v/v). HPLC–DAD chromatograms of the pooled fractions CF 1 and CF 2 are presented in Fig. 4. HPLC–DAD–MS–MS experiments were performed for peak identification and analyte assignments are based on literature comparisons [5,38,39]. Major peaks of

Fig. 4. HPLC–DAD chromatograms (210 nm) of the major combined fractions (CF 1 and CF 2) obtained from apolar Sephadex LH-20 chromatography. Separations were performed on a Zorbax SB-C18 column (150 mm × 2.0 mm, particle size 3.5 ␮m) using a water–acetonitrile gradient (t = 0 min 30% ACN; t = 4 min 98% ACN; t = 5.3 min 98% ACN; t = 5.4 min 30% ACN, flow rate 0.3 ml/min) The separation system was kept at room temperature and the injection volume was 2 ␮l.

CF 1 were identified as dtx A ([MH]+ 578 m/z), dtx B ([MH]+ 594 m/z) and dtx E ([MH]+ 594 m/z). Components of CF 2 were tentatively assigned as dtx C ([MH]+ 610 m/z), dtx Cl ([MH]+ 631 m/z), and a dtx derivative of the [MH]+ 580 m/z series (either desMe-dtx B, dihydro-dtx A or dtx B2 ). The main peak of CF 3 was identified as dtx E-diol ([MH]+ 612 m/z) and the major peak of the most polar fraction CF 4 is the carboxylic acid moiety bearing derivative dtx D ([MH]+ 624 m/z). Both analytes were further characterized by NMR spectroscopy allowing complete 1 H and 13 C NMR signal assignments. Comparison with literature data did further confirm the analyte identity [8,43]. 3.3. High-speed counter-current chromatography of CF 1 Prior to preparative separation of CF 1, a two-phase solvent system with suitable partition coefficients K for dtx A, B, and E had to be selected. Twenty different solvent systems were tested. Analyte specific K values KA , KB , KE as well as the total partition coefficient KABE were calculated by dividing the integrated peak areas of the respective dtx derivatives in the upper phase divided by that in the lower phase. Results are summarized in Table 1. Based on the test chromatograms solvent systems 4, 6, and 7 with sum partition coefficients KABE = 0.5, 1.5, and 0.7, respectively, were chosen as the best systems. For all three systems, analyte specific K values increased with the observed HPLC retention times of the analytes (KE < KA < KB ). When employing these solvent systems by using the lower phase as stationary phase and the upper phase as mobile phase, none of the three selected solvent systems attained a satisfactory separation of the analytes. However, using the lower phase as mobile phase and the upper phase as stationary phase (“head to tail” mode), solvent system 6 (light petroleum–ethyl acetate–methanol–water = 2:5:2:5) gave satisfactory separations, although optimal K values for

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Fig. 5. Fractions of HSCCC separation of CF1; weights against faction numbers. Pooled analyte fractions are shown in grey.

HSCCC performed in this mode should range between 0.5 and 1.0 [34]. HPLC–DAD based analysis of the weighed HSCCC fractions allowed to distinguish three major peaks representing dtx A, B, and E (Fig. 5). The retention time order dtx E < dtx A < dtx B is reflecting both the HPLC retention times and the analyte specific partition coefficients (KE < KA < KB ). Minute amounts of dtx E-diol were eluting shortly before dtx E. The dtx Ediol peak increased with prolonged sample storage and is most likely stemming from the abiotic degradation process of dtx E [5,44]. Two more minor dtx derivatives gave rise to additional peaks between the major dtx derivatives. Fractions showing identical compositions were pooled. The combined fractions were subjected to HPLC–DAD/MS measurements for analyte identification and purity control. Dtx A, B, and E were identified by comparison with reference material. Characterization by NMR spectroscopy allowed complete 1 H and 13 C NMR signal assignment. Literature data comparison confirmed the analyte identities [8,45,46]. Based on comparison with previous analyte identifications [5,38] the two minor derivatives were tentatively assigned as dtx A2, eluting between dtx E and A, and dihydro dtx A, eluting between dtx A and dtx B. NMR based structure confirmation of these analyte identification is subject of further investigations. The HSCCC separation of CF 1 was repeated four times using the same apparatus and identical conditions. The assay proved to be stable, retention volumes of the analytes were found to be within a relative standard deviation of <3.5%. A single HSCCC run starting from 0.8 g CF1 yielded about 150–220 mg of each major dtx derivative with HPLC–DAD/MS and NMR checked purities exceeding 98%. 4. Discussion The presented novel three steps isolation strategy for dtx derivatives combines liquid–liquid partition for crude extract production, apolar chromatography on Sephadex LH-20 for analyte enrichment, and a HSCCC protocol for final analyte purification. Chromatography over Sephadex LH-20 used

dichloromethane with an increasing amount of acetone as solvent system. Dtx A, B, and E were enriched in a fraction (CF 1) eluting with 100% dichloromethane. Directly thereafter another bulk of dtxs eluted and was pooled. Moreover two more polar fractions containing dtx E-diol and dtx D (purities of >90%) were obtained. CF 1 was worked up by a tailored HSCCC system allowing to obtain 78 mg dtx A, 59 mg dtx B, and 63 mg dtx E per liter culture filtrate with purities exceeding >98%. Recoveries from culture broth were found to be higher than 40%. A total of several 100 mg of these metabolites were obtained from a single laboratory scale fermenter within few weeks working time. Due to the lipophilic character of the analytes the use of dichloromethane for crude extract production and Sephadex LH20 chromatography was indispensable. However, work place pollution and environmental impact of using this chlorinated organic solvent was minimized by (i) the use of well vented hoods, (ii) the re-use of the extraction solvent, and (iii) the high yield isolation strategy. Acknowledgements This work was partially supported by the European Commission, Quality of Life and Management of Living Resources Programme (QoL), Key Action 1 on Food, Nutrition and Health, QLK1-2001-01391. The authors express their gratitude to E.P. Ellmerer (Institute of Organic Chemistry, University Innsbruck, Austria) for measuring the NMR spectra and to A. Vey (INRACNRS, St. Christol-les-Al`es, France) for the generous gift of reference materials. References [1] G.A. Cordell, Y.G. Shin, Pure Appl. Chem. 6 (1999) 1089. [2] K. Hostettmann, A. Marston, M. Hostettmann, Preparative Chromatography Techniques: Applications in Natural Product Isolation, SpringerVerlag, Berlin, 1986. [3] K. Hostettmann, A. Marston, Phytochem. Rev. 1 (2002) 275. [4] R. Verpoorte, A. Contin, J. Memelink, Phytochem. Rev. 1 (2002) 13. [5] C. Seger, S. Sturm, H. Stuppner, T.M. Butt, H. Strasser, J. Chromatogr. A 1061 (2004) 35. [6] M.S.C. Pedras, I.L. Zaharia, D.E. Ward, Phytochemistry 59 (2002) 579. [7] A. Vey, R. Hoagland, T.M. Butt, in: T.M. Butt, C.W. Jackson, N. Magan (Eds.), Fungi as Biocontrol Agents: Progress, Problems and Potential, CAB International, Wallingford, 2001, p. 311. [8] M. Pa¨ıs, B.C. Das, P. Ferron, Phytochemistry 20 (1981) 715. [9] R.I. Samuels, A.K. Charnley, S.E. Reynolds, Mycopathologica 104 (1988) 51. [10] S. Gupta, D.W. Roberts, J.A.A. Renwick, J. Liq. Chromatogr. 12 (1989) 383. [11] L. Buchwaldt, J.S. Jensen, Phytochemistry 30 (1991) 2311. [12] M. Wahlmann, B.S. Davidson, J. Nat. Prod. 56 (1991) 643. [13] A. Jegorov, V. Matha, P. Sedmera, D.W. Roberts, Phytochemistry 31 (1992) 2669. [14] S.B. Krasnoff, D.M. Gibson, G.N. Belofsky, K.B. Gloer, J.B. Gloer, J. Nat. Prod. 59 (1996) 485. [15] A. Jegorov, P. Sedmera, V. Havlicek, V. Matha, Phytochemistry 49 (1998) 1815. [16] J.W. Chen, B.L. Liu, Y.M. Tzeng, J. Chromatogr. A 830 (1999) 115. [17] H. Faulstich, A. Buku, H. Bodenm¨uller, T. Wieland, Biochemistry 19 (1980) 3334. [18] H. Henke, LaborPraxis 11 (1987) 644.

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