Characterization of a silent sesquiterpenoid biosynthetic pathway in Streptomyces avermitilis controlling epi-isozizaene albaflavenone biosynthesis and isolation of a new oxidized epi-isozizaene metabolite.

The genome-sequenced, Gram-positive bacterium Streptomyces avermitilis harbours an orthologue (SAV_3032) of the previously identified epi-isozizaene synthase (SCO5222) in Streptomyces coelicolor A3(2). The sav3032 is translationally coupled with the downstream sav3031 gene encoding the cytochrome P450 CYP170A2 analogous to SCO5223 (CYP170A1) of S. coelicolor A3(2), which exhibits a similar translation coupling. Streptomyces avermitilis did not produce epi-isozizaene or any of its oxidized derivatives, albaflavenols and albaflavenone, under in any culture conditions examined. Nonetheless, recombinant SAV_3032 protein expressed in Escherichia coli catalysed the Mg²+-dependent cyclization of farnesyl diphosphate to epi-isozizaene. To effect the production of epi-isozizaene in S. avermitilis, the sav3032 gene was cloned and placed under control of a copy of the native S. avermitilis promoter rpsJp (sav4925). The derived expression construct was introduced by transformation into a large-deletion mutant of S. avermitilis SUKA16 and the resulting transformants accumulated epi-isozizaene. The previously characterized oxidized epi-isozizaene metabolites (4R)- and (4S)-albaflavenols and albaflavenone, as well as a previously undescribed doubly oxidized epi-isozizaene derivative were isolated from cultures of S. avermitilis SUKA16 transformants in which sav3032 was coexpressed with the P450-encoding sav3031. This new metabolite was identified as 4β,5β-epoxy-2-epi-zizaan-6β-ol which is most likely formed by oxidation of (4S)-albaflavenol.

Introduction

Terpenoid compounds are used as antibiotics, hormones, flavour and/or odour constituents, and pigments. Some of these compounds also possess other physiologically or commercially important properties (Glasby, 1982; Sacchettini and Poulter, 1997) and many have been shown to play an important role in intra‐species and inter‐species interactions. Terpenoid metabolites are produced by a wide variety of terrestrial and marine plants and by fungi, while only a relatively minor fraction have been isolated from bacterial species. Sesquiterpene synthases catalyse the cyclization of farnesyl diphosphate to any of 300 sesquiterpene hydrocarbons or alcohol. These enzymes are widely distributed among plants and fungi. Only relatively recently bacterial sesquiterpene synthases, primarily from Streptomyces, have been identified. The first to be characterized was pentalenene synthase from Streptomyces exfoliatus (Cane ), which generates the parent hydrocarbon of the antibiotic pentalenolactone, a metabolite that has been isolated from numerous species of Streptomyces. Analysis of the genome sequences of Streptomyces coelicolor A3(2) and Streptomyces avermitilis has revealed additional terpene synthases encoding the biosynthesis of germacradienol and geosmin, and of epi‐isozizaene and the derived antibiotic albaflavenone. Recent genomics‐based investigations have revealed that bacterial terpenoid metabolism may be more widespread than previously assumed. Following the discovery of the gene encoding formation of the bacterial monoterpene (2‐methylisoborneol synthase) in a variety of species of Streptomyces, a protein‐family search using a hidden Markov model revealed that bacterial terpene synthases, classified into three groups – monoterpene, sesquiterpene and diterpene synthases – are in fact widely distributed in many bacteria, especially Actinomytales microorganisms, all of which are therefore predicted to have the ability to produce wide variety of terpenoid metabolites (Komatsu ).

The genome sequence of the industrially important Gram‐positive bacterium S. avermitilis, which is used for the production of the potent anthelminthic macrolide avermectin (Omura ; Ikeda ; Nett ), revealed the presence of at least four genes encoding sesquiterpene synthases. One of these genes, sav2998, was found to encode pentalenene synthase (Tetzlaff ) while sav2163 was shown to be a germacradienol/geosmin synthase (Cane ). Recently sav76 has been explained to encode a new sesquiterpene (avermitilol) synthase (Chou ). The remaining gene, sav3032, shows a high level of similarity at the predicted amino acid level to epi‐isozizaene (1) synthase, a sesquiterpene synthase recently identified in S. coelicolor A3(2) (Lin ). Epi‐isozizaene synthase has itself been shown to catalyse the committed step in the biosynthesis of the antibiotic albaflavenone (3), an α, β‐unsaturated sesquiterpene ketone with an earthy, camphor‐like odour that was originally isolated from the highly odorous Streptomyces species S. albidoflavus (Gürtler ). In S. coelicolor A3(2) the albaflavenone biosynthetic gene cluster consists of two adjacent ORFs, sco5222 and sco5223. The 1086 bp sco5222 gene encodes a protein of 361 amino acids that catalyses the cyclization of farnesyl diphosphate (2) to epi‐isozizaene (1) (Lin ; Lin and Cane, 2009; Aaron ). The sco5223 gene, which shares a four‐nucleotide ATGA transcriptional overlap with sco5222 at its 5′‐end, has been demonstrated to catalyse the two‐step allylic oxidation of epi‐isozizaene (1) to albaflavenone (3) through the intermediacy of an epimeric mixture of albaflavenols (4a and 4b) (Zhao ).

Although S. avermitilis also harbours a two‐gene cluster (sav3032–sav3031, nt 3 788 761–3 791 219) homologous to the sco5222–sco5223 pair in S. coelicolor A3(2), neither albaflavenone nor related compounds have been isolated from S. avermitilis. We now report the experimental demonstration of the biochemical function of the S. avermitilis sav3032–sav3031 cluster, which are shown to encode the predicted epi‐isozozaene synthase and the cytochrome P450, epi‐isozizaene mono‐oxygenase, resulting in the biosynthesis of albaflavenone and a previously uncharacterized sequiterpene metabolite, 4β,5β‐epoxy‐2‐epi‐zizaan‐6β‐ol (5).

Results

Orthologue of epi‐isozizaene synthase in S. avermitilis

The gene sav3032, encoding an orthologue of the recently characterized S. coelicolor A3(2) epi‐isozizaene synthase gene, is located at 3 790 128–3 791 219 nt of the S. avermitilis genome. The predicted 363 aa gene product of sav3032 shows 82% identity and 87% positive matches to S. coelicolor A3(2) epi‐isozizaene synthase (361 aa; SCO5222). Notably, sav3032 is also translationally coupled by a four‐nucleotide ATGA overlap to the downstream sav3031 gene that encodes an apparent cytochrome P450 of 456 aa with 80% identity and 89% positive matches to CYP170A1 of S. coelicolor A3(2) (461 aa; SCO5223) (Lamb ). It is therefore very likely that S. avermitilis also is competent to produce albaflavenone, and that the biochemical pathway is identical to that which has been established in S. coelicolor A3(2) (Lin ; Zhao ; Lin and Cane, 2009; Aaron ).

Both terpene synthases, SAV_3032 and SCO5222, harbour the two universally conserved Mg2+‐binding domains, the aspartate‐rich motif ‐DDRHD‐, beginning at Asp101 in SAV_3032, and the characteristic triad NDLCSLPKE, at Asn242 of SAV_3032 (Komatsu ). The universally conserved cysteine residue found in the C‐terminal region of all cytochrome P450s and which binds the proximal face of the protohaem co‐factor is found in SAV_3031 (‐RKCPS‐) at Cys405 and in SCO5223 (‐RKCPS‐) at Cys410 respectively. Both of these cytochrome P450s also harbour an unusual aspartate‐rich motif (‐AKDDNGDPI‐), which has not been found in bacterial cytochrome P450s, and which has recently been implicated in the conversion of farnesyl diphosphate to a mixture of acyclic farnesene isomers by recombinant SCO5223 (Zhao ).

In vitro Mg2+‐dependent cyclization of farnesyl diphosphate to epi‐isozizaene catalysed by recombinant SAV_3032 protein

To confirm the predicted biochemical function of the SAV_3032 protein, the recombinant protein was expressed using a standard Escherichia coli T7‐RNA polymerase‐based expression host–vector system. Although the C‐terminal His6‐tagged SAV_3032 was initially obtained as inclusion bodies, the insoluble protein was readily resolubilized and refolded, then purified to homogeneity by ion‐exchange chromatography to yield 3 mg of SAV_3032 protein from 1 l of culture. Incubation of the purified soluble recombinant SAV_3032 protein with farnesyl diphosphate (2) in the presence of Mg2+ ions and analysis of the resulting products by GC‐MS revealed the formation of a major sesquiterpene hydrocarbon product, C15H24, m/z 204 (Fig. 1, retention time 10.13 min), identical in both retention time and mass spectral fragmentation pattern by direct comparison with authentic epi‐isozizaene, generated by SCO5222 epi‐isozizaene synthase (Lin ). This result unambiguously demonstrates that SAV_3032 catalyses the cyclization of farnesyl diphosphate (2) to epi‐isozizaene (1).

Figure 1

GC‐MS analysis of n‐pentane extracts from incubation of farnesyl diphosphate and C‐terminal His6‐tagged SAV_3032 protein in the presence of Mg2+.

The pH optimum for the SAV_3032‐catalysed cyclization was found to be pH 7.0. The steady‐state kinetic parameters were determined by carrying out a series of 60 min incubations at 30°C in pH 7.0 PIPES using a range of concentrations of farnesyl diphosphate, giving a kcat of (2.58 ± 0.06) × 10−5 s−1 and a Km (farnesyl diphosphate) of 125 ± 11 nM. Substitution of 10 mM Mn2+ for Mg2+ gave relative rates of formation of 1 of 32%. Other divalent cations were significantly less effective: Zn2+ (5%), Fe2+ (1%), Fe3+ (5%), Co2+ (1%), Cu2+ (2%) and Ni2+ (1%).

Production of epi‐isozizaene and related compounds from S. avermitilis

Although several attempts were made to produce epi‐isozizaene (1) and its oxidized derivatives from S. avermitilis, none of the metabolites could be detected in either the S. avermitilis wild‐type or its derivates under any culture conditions examined. Since both DNA microarray and real‐time PCR analyses indicated that sav3032 was not expressed under any of these conditions, we placed sav3032 under the control of an alternative promoter, rpsJp, which is a constitutive promoter of the gene encoding ribosomal protein S10 of S. avermitilis and we had previously shown can efficiently control the expression of genes encoding monoterpene synthase, 2‐methylisoborneol synthase, in S. avermitilis SUKA16 (Komatsu ). Transformants of S. avermitilis SUKA16 (pKU460::rpsJp‐sav3032) indeed produced epi‐isozizaene (1) as a major component, as established by GC‐MS comparison with authentic epi‐isozizaene (Fig. 2A). To detect further oxidized derivatives of epi‐isozizaene, sav3032 was coexpressed with sav3031 which encodes the cytochrome P450 CYP170A2. While the measured titres of epi‐isozizaene in the resulting transformants were significantly reduced, several additional GC‐MS peaks sesquiterpenoid components were observed. As shown in Fig. 2B, the peaks at 11.38 min and 11.44 min corresponded to the allylic oxidation products (4S)‐albaflavenol (4a) and (4R)‐albaflavenol (4b), respectively, by comparison with authentic albaflavenols prepared by allylic oxidation of epi‐isozizaene with SeO2 (Lin ). A compound eluting at 11.90 min was also identical to the further oxidation product, albaflavenone (3), by direct comparison with an authentic sample. In addition to these three previously identified derivatives of epi‐isozizaene, a fourth metabolite (5) that eluted at 11.83 min with parent M+m/z 236 did not match the EI‐MS fragmentation pattern of any compounds in public MS databases.

Figure 2

GC‐MS analysis of n‐hexane extracts from 120 h cultures of S. avermitilis SUKA16 carrying pKU460::rpsJp–sav3032 (A) or pKU460::rpsJp–sav3032–sav3031 (B), with EI‐MS of component at 11.83 min corresponding to compound 5. In traces A‐1 and B‐1, the region from 11.30 to 11.95 min is expanded for chromatograms A and B. The peaks eluted from 11.54 to 11.77 min (A‐1 and B‐1) and from 12.0 to 13.0 (A and B) were identical to short‐ and medium‐chain fatty acids.

Structure elucidation of the new compound (5)

Fermentation products were extracted with methanol from cultured mycelia and the organic extract was purified by silica gel column chromatography. Ultimately 2.6 mg of the new compound (5) was obtained from 5 l of culture as a colourless oil with a mild odour. The molecular formula C15H24O2 of 5 was obtained from HRMS (FAB), indicating that the structure results from the addition two oxygen atoms to epi‐isozizaene (1). Comparative analysis of EI‐MS fragments between albaflavenols (4a, 4b) and 5 indicated that the new compound (5) was not a simple hydroxylation product of albaflavenols (4a, 4b), based on the absence of a characteristic dehydration fragmentation peak at m/z 218. In other respects, the new compound (5) showed an EI‐MS pattern similar to but distinct from all other known epi‐isozizaene derivatives.

The 1H‐ and 13C NMR spectra of 5 (described in Materials and Methods in Supporting information) showed signals for a secondary methyl signal (δH 0.99, δC 21.53) and for three methyl signals (δH 0.887, δC 20.44; δH 1.04, δC 21.77; δH 0.893, δC 27.69), suggesting that the structure of 5 corresponds to typical tricyclic sesquiterpene (Gürtler ). In the 1H‐1H COSY spectrum of 5, the connection between the methyl signal at δ 0.99 (d, J = 7.3 Hz, H‐12) and the methine at δ 3.49 (1H, broad singlet, H‐4) through the H‐2 methine (δ 1.74) and H‐3 methylene (δ 1.98, δ 1.68), and the methylene signals at H‐10 (δ 1.93, δ 1.50) and H‐11 (δ 1.77, δ 1.20) through H‐9 (δ 2.27, δ 1.61) and H‐8 (δ 1.74) could be traced. These results indicated that the partial structure of 5 is similar to that of albaflavenols (4a, 4b) (Zhao ). In the 13C NMR and HMBC spectra of 5, typical geminal methyl signals at C15 (δ 21.77) and C14 (δ 27.69) connected to C7 (δ 40.52) were similar to those of albaflavenols (4a, 4b), except for quaternary carbon signals at C5 (δ 76.50) and C6 (δ 74.98) compared with those of the quaternary olefinic carbon at C5 (δ 147.5) and C6 (δ 135.5) of 4a. In consideration of the four degrees of unsaturation and the 13C NMR chemical shifts of 5, the carbon chemical shifts at δ 65.61 (C4) and δ 76.50 (C5) were assigned to epoxy carbons, instead of the characteristic secondary hydroxyl carbon signal at δ 70.8 (C4) and quaternary carbon at δ 147.5 (C5) of 4a. Among the four methyl carbons, a remaining tertiary methyl (δH 0.887, δC at 20.44 C13) and hydroxyl group were clearly connected to C6 (δ 74.98) in the HMBC spectrum of 5. To combine MS and NMR spectral data, structure 5 was determined as 4,5‐epoxy‐2‐epi‐zizaan‐6‐ol shown in Fig. 3A. The complete stereochemistry was assigned from the NOESY spectrum of 5, which displayed correlation peaks between the 12α‐methyl (δ 0.99) and H‐3α (δ 1.68), H‐4α (δ 3.49) and 13α‐methyl (δ 0.887), and 13α‐methyl (δ 0.887) and 15α‐methyl (δ 1.04), thereby establishing the relative configuration of the 12α‐methy (2‐epi), 4β, 5β‐epoxy and 6β‐hydroxy substituents, respectively, in 5 (Fig. 3B). The absolute configuration could then be assigned with confidence based on the derivation from epi‐isozizaene, giving the assignment of 5 as 4β,5β‐epoxy‐2‐epi‐zizaan‐6β‐ol.

Figure 3

COSY, HMBC (A) and NOESY (B) analysis of 5. Bold lines in (A) represent 1H‐1H correlations. Arrows in (A) shows key HMBC correlations (1H <‐> 13C), while dashed arrows in (B) represent NOESY correlations.

Distribution of epi‐isozizaene synthase orthologues in other bacterial genomes

Since two verified epi‐isozizaene synthases exhibiting a high level of mutual amino acid sequence identity and similarity had thus been demonstrated from S. coelicolor A3(2) and S. avermitilis, we created a hidden Markov model for epi‐isozizaene synthase using the two amino acid sequences as well as those of predicted epi‐isozizaene synthases that were identified by screening public databases of bacterial proteins using this model. At least nine additional predicted epi‐isozizaene synthases were found as follows: Streptomyces albus J1074, S. lividans TK24, S. ghanaensis ATCC 14672, S. griseoflavus Tu4000, S. sviceus ATCC 29083, S. viridochromogenes DSM 40736, Streptomyces sp. SPB74 and SPB78, and Burkholderia pseudomallei 668 (Table S1). The albaflavenone biosynthetic gene clusters in both S. avermitilis and S. coelicolor A3(2) consist of two adjacent ORFs, epi‐isozizaene synthase and a downstream, translationally coupled gene encoding a cytochrome P450 belonging to the CYP170A family of mixed function monooxygenases. Indeed CYP170‐family cytochrome P450 genes were located immediately downstream of the predicted epi‐isozizaene synthase genes in all cases that were examined except that of B. pseudomallei (Table S1). These predicted epi‐isozizaene synthases (Fig. S1) all contained the canonical aspartate‐rich motif ‐WFF[AV]WDD[RQ]HD[RE]D‐ and the characteristic triad motif ‐A[WL]YNDLCSLPKE‐, respectively, separated by some 140 ± 5 amino acids. The three aromatic amino acid residues, F95, F96 and F198 in SCO5222, that appeared to be oriented to stabilize carbocation intermediates in the cyclization cascade through cation‐π interactions (Aaron ) were universally conserved in all predicted terpene synthases. Furthermore, the characteristic aromatic amino acid‐rich motif ‐VYWFHHES[GD]RY‐ was found in the C‐terminus regions of each of these predicted terpene synthases (Fig. S1).

To confirm the production of epi‐isozizaene or its oxidized derivatives in five Streptomyces strains, S. albus, S. ghanaensis, S. lividans, S. sviceus and S. viridochromogenes, each microorganism was cultivated and their n‐hexane extracts of mycelia were subjected to GC‐MS analysis using authentic samples (Table S2). The production of epi‐isozizaene was detected from mycelia in S. ghanaensis and S. lividans. Streptomyces albus produced not only epi‐isozizaene but also albaflavenols and albaflavenone in the mycelium.

Discussion

The S. avermitilis epi‐isozizaene synthase (SAV_3032) displays an unusually low kcat of 2.58 ± 0.06 × 10−5 s−1 compared with that of the SCO5222 protein (0.049 ± 0.001 s−1), and indeed all other previously characterized terpene synthases. This discrepancy probably results from the necessity of refolding the SAV_3032 protein from insoluble inclusion bodies, resulting in only a small fraction of the refolded SAV_3032 protein that is catalytically competent. The fact that the Km for farnesyl diphosphate (125 ± 11 nM) is very close to that of the S. coelicolor A3(2) synthase (147 ± 14 nM) suggests that the active protein is properly folded, as distinguished from the formation of a large proportion of partially active, misfolded recombinant protein. In any case, it is absolutely clear that the SAV_3032 protein catalyses the cyclization of farnesyl diphosphate (2) to epi‐isozizaene (1).

We have recently shown that mutants of S. avermitilis such as SUKA16, harbouring deletions of more than 1.4 Mb, including the gene clusters for the majority of the natural products produced by wild‐type S. avermitilis, are exceptionally useful for the production of a variety of both homologous and heterologous natural products, including terpenoids, based on the re‐introduction of the relevant biosynthetic gene clusters under control of strong, tunable promoters (Komatsu ). Thus although the sav3032 gene encoding epi‐isozizaene synthase was found to be silent in S. avermitilis under the culture conditions examined, placing sav3032 under the control of the alternative promoter, rpsJp, resulted in efficient expression of sav3032 and consequent generation of epi‐isozizaene. Coexpression of sav3032 with the sav3031 gene encoding the CYP170A‐family oxygenase (SAV_3031; CYP170A2) led to the production of the expected allylic oxidation products, the epimeric albaflavanols (4a, 4b) and albaflavenone (3), as well as a previously unknown compound, 4β,5β‐epoxy‐2‐epi‐zizaan‐6β‐ol (5). The results confirming that the two‐gene, translationally coupled S. avermitilis operon, sav3032–sav3031, is responsible for albaflavenone biosynthesis. The production of derived metabolites such as albaflanols, albaflavenone, and the newly discovered 4β,5β‐epoxy‐2‐epi‐zizaan‐6β‐ol (5) requires the coexpression of sav3031 encoding CYP170A2 since the SUKA16 transformant carrying pKU460::rpsJp–sav3032 produced only epi‐isozizaene. Formation of compound 5 from epi‐isozizaene (1) requires two successive oxidation reactions. In principle, compound 5 might be generated from (4S)‐albaflavenol by successive epoxidation and rearrangement, with ring opening of the initially formed epoxide involving net SN1‐like retention of configuration (Fig. 4), but this has yet to be demonstrated directly. Whatever the detailed mechanism, it is also likely that the second reaction is also catalysed by CYP170A2, although we cannot yet rule out the action of one of the 20 kinds other cytochrome P450s (Lamb ) or the putative epoxidase (sav1745) that are evident in the genome of S. avermitilis.

Figure 4

Biosynthetic pathway in S. avermitilis SUKA16 carrying pKU460::rpsJp–sav3032–sav3031 from farnesyl diphosphate to albaflavenone (3) and proposed mechanism of formation of 4β,5β‐epoxy‐2‐epi‐zizaan‐6β‐ol (5). Abbreviations are as follows: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; MEP, 2‐C‐methylerythritol‐4‐phosphate.

A bioinformatics‐based search for epi‐isozizaene synthases in the bacterial protein databases has revealed that nine additional bacterial strains harbour orthologous gene encoding epi‐isozizaene synthase, all but one of which is translationally coupled to a downstream member of the CYP170A family of oxygenases. This deduction is supported by the demonstrated production of epi‐isozizaene by three of these Streptomyces strains, S. albus, S. ghanaensis and S. lividans. The formation of albalflavenols and albaflavenone by S. albus also indicates that the coupled P450 is active in this strain under general culture conditions.

Experimental procedures

Bacterial strains, vector plasmid and bioinformatics

The large‐deletion mutant S. avermitilis, SUKA16 (Komatsu ), was used for production of sesquiterpenoid compounds. Streptomyces ghanaensis ATCC 14672, S. sviceus ATCC 29083, S. viridochromogenes DSM 40736 were obtained from the culture collection of the RIKEN Bioresource Center, Japan. Streptomyces albus J1074 and S. lividans TK24 were from the John Innes Centre, UK. The integrating vector containing attP and int from actinophage ϕC31 used for the expression of the gene encoding the terpene synthase was described previously (Komatsu ). The S. avermitilis cosmid clones used are listed at http://avermitilis.ls.kitasato‐u.ac.jp. Culture condition and recombinant DNA procedures were described in Materials and Methods in Supporting information. The procedures for bioinformatics were described previously (Komatsu ).

Detection of sesquiterpenoid compounds from transformants of S. avermitilis SUKA16

The spores of S. avermitilis transformants were transferred into vegetative medium (Cane ) and the culture was grown with shaking at 30°C. A 0.1 ml portion of the culture was inoculated into 10 ml of production medium (Cane ) in a 125 ml flask. After growth at 28°C on a rotary shaker at 200 r.p.m., the mycelia were harvested by centrifugation and the mycelial products were extracted with 4 ml of methanol for 15 min at room temperature. Mycelia were removed by the centrifugation and the supernatant was extracted with 1 ml of n‐hexane. After separation of phases, the upper n‐hexane layer was collected and a portion of the n‐hexane extract was directly subjected to GC‐MS as described previously (Komatsu ).

Isolation and purification of 4β,5β‐epoxy‐2‐epi‐zizaan‐6β‐ol (5)

Spores of S. avermitilis SUKA16 carrying pKU460::rpsJp–sav3032–sav3031–sav3129–sav5765 (rpsJp indicates relatively strong and constitutively controlled promoter in S. avermitilis. sav3129 and sav5765 are genes encoding ferredoxin and ferredoxin reductase respectively. The construction of the plasmid was described in Materials and Methods in Supporting information) were cultured under the same conditions as described above. After cultivation for 5 days, the 5 l culture was filtered to harvest the mycelia which were washed with deionized water. The mycelial products were extracted with 500 ml of methanol and then the mycelia were removed by filtration. The methanol extract was extracted three times with 300 ml of n‐hexane. After separation of the phases, the n‐hexane layers were combined and concentrated under the reduced pressure to obtain 296 mg of a brownish oil. The crude concentrated extract was dissolved in a small volume of n‐hexane and subjected to silica gel (70–230 mesh) column chromatography (10 mm in diameter × 85 mm). The previously unknown compound (5) was eluted with n‐hexane‐EtOAc (10:1) and confirmed by GC‐MS analysis. Pure compound (5) (2.6 mg) was obtained using a second silica gel column (230–400 mesh) chromatography (10 mm in diameter × 60 mm) eluted with n‐hexane‐EtOAc (100:1).