Unsaturated fatty acids and a prenylated tryptophan derivative from a rare actinomycete of the genus Couchioplanes

A genome mining survey combined with metabolome analysis of publicly available strains identified Couchioplanes sp. RD010705, a strain belonging to an underexplored genus of rare actinomycetes, as a producer of new metabolites. HPLC-DAD-guided fractionation of its fermentation extracts resulted in the isolation of five new methyl-branched unsaturated fatty acids, (2E,4E)-2,4-dimethyl-2,4-octadienoic acid (1), (2E,4E)-2,4,7-trimethyl-2,4-octadienoic acid (2), (R)-(−)-phialomustin B (3), (2E,4E)-7-hydroxy-2,4-dimethyl-2,4-octadienoic acid (4), (2E,4E)-7-hydroxy-2,4,7-trimethyl-2,4-octadienoic acid (5), and one prenylated tryptophan derivative, 6-(3,3-dimethylallyl)-N-acetyl-ʟ-tryptophan (6). The enantiomer ratio of 4 was determined to be approximately S/R = 56:44 by a recursive application of Trost’s chiral anisotropy analysis and chiral HPLC analysis of its methyl ester. Compounds 1–5 were weakly inhibitory against Kocuria rhizophila at MIC 100 μg/mL and none were cytotoxic against P388 at the same concentration.

At present, only 5 out of 29 valid genera in this family [18] -Actinoplanes, Dactylosporangium, Micromonospora, Salinispora, and Verrucosispora -have mainly been investigated [11] and the remaining 24 are still untouched or underexplored. However, in silico genome mining identified multiple secondary metabolite biosynthetic gene clusters in selected strains from minor actinomycetes genera, implying their comparable biosynthetic capacities to those of the already proven genera [19]. Encouraged by these reports, we examined the metabolites of Pseudosporangium sp. RD062863, a strain available at the culture collection of the Biological Resource Center, National Institute of Technology and Evaluation (NBRC) [20], and discovered a novel cyclopeptide pseudosporamide along with three new oligomycin-class polyketide [21]. In addition, the same approach to the different family (Pseudonocardiaceae) yielded mycetoindole, a new class of dehydrotryptophan derivative from Actinomycetospora [22].

Results and Discussion
The producing strain RD010705 was shake-cultured in A16 liquid medium at 30 °C for 8 days, and the whole culture was extracted with 1-butanol. The extract (4.4 g from 3 L) was sequentially fractionated by column chromatographies on silica gel and ODS, and the resulting fractions were purified by reverse-phase HPLC to give 1 (5.2 mg), 2 (2.3 mg), 3 (1.0 mg), 4 (6.3 mg), and 5 (8.0 mg).
The molecular formula of 1 was determined to be C 10 H 16 O 2 on the basis of its NMR and HR-ESI-TOFMS data (m/z 191.1044 [M + Na] + , Δ + 0.1 mmu). Three degrees of unsaturation, calculated from the molecular formula, a UV absorption maximum at 264 nm, and IR absorption bands at 1679 and 2800-3200 cm −1 , suggested dienone and hydroxy functionalities. The 1 H, 13 C,  and HSQC data allowed to assign ten carbon signals to one carbonyl carbon (δ C 174.0), two each of non-protonated sp 2 carbons (δ C 132.5, 123.9), sp 2 methines (δ C 145.7, 138.5), and sp 3 methylenes, and three methyls ( Table 1 and Table 2). A sequence of COSY correlations was detected from a triplet methyl H-8 to an olefinic methine H-5 via two methylenes H-7 and H-6, which revealed a propyl-substituted olefin moiety ( Figure 2). In addition, HMBC correlations (Table 2) from the allylic methyl H-10 to C-5, C-4, and C-3 and from the other allylic methyl H-9 to C-3, C-2, and C-1 elucidated the carbon connectivity from C-5 to C-1, thus completing the planar struc-ture. The double bond geometries were determined to be both E based on NOESY correlations between H-3 and H-5 and between H-9 and H-10 ( Figure 2). Therefore, 1 was determined to be (2E,4E)-2,4-dimethyl-2,4-octadienoic acid.
The 1 H NMR spectrum of 2 was similar to those of 1 in overall ( Table 1 and Table 2). One major difference was a replacement of the triplet methyl peak in 1 by a six proton-equivalent doublet methyl peak, which suggested an isopropyl-terminated structure. This was proven by a COSY correlation between this methyl proton (H-8/H-9) and a methine proton (H-7) and   Table 1 and Table 2). The connectivity of the former two fragments were intervened by an sp 2 quaternary carbon (C-4) substituted by an allylic methyl group (H-11) based on HMBC correlations from H-11 to C-3, C-4, and C-5. Another intervention by a methylene unit (C-8) to connect the second and third fragments was supported by HMBC correlations from H-7 to C-9, H-8 to C-6, and H-10 to C-8, thus completing an alkyl chain part. The remaining atomic composition was CHO 2 , and despite the lack of evidentiary HMBC correlations, placing a carboxylic acid functionality at the open end (C-2) was reasonable in consideration of the chemical shift of the unused carbon (δ H 177.1, C-1) and the molecular formula. A NOESY correlation between H-11 and H-12 supported an E-configuration for the C-4/C-5 double bond. The established planar structure was identical to that of a fungal metabolite phialomustin B [26], for which specific rotation, enumerated 1 H and 13 C NMR data, and high-resolution ESIMS data were presented. While an S-configuration was assigned for phialomustin B based on its positive specific rotation ( [α] D 25 -76.0, c 0.05, MeOH), though the rotatory power in CHCl 3 not as large as expected presumably due to the sample scarcity, supported an R-configuration. Therefore, 3 was speculated to be (R)-(−)-phialomustin B.
The molecular formula of 4, determined to be C 10 H 16 O 3 on the basis of HR-ESITOF-MS data (m/z 207.0992 [M + Na] + , Δ 0.0 mmu), was one-oxygen larger than that of 1. The 1 H NMR spectrum was mostly similar to those of 1 (Table 1 and Table 2), except for the presence of an oxymethine resonance (δ H 3.96/δ C 67.8) in place of the shielded methylene at C-7, which suggested hydroxylation on the same carbon. This was supported by COSY correlations establishing the connectivity from H-5 to H-8, and completely the same HMBC and NOESY correlations for the remaining part to those observed for 1 and 2 ( Figure 2). To address the absolute configuration, 4 was esterified with TMS-diazomethane and the resulting methyl ester 4' was acylated with (R)-or (S)-α-methoxyphenylacetic acid (MPA). To our surprise, however, 1 H NMR spectra of both the acylation products were substantially the same but contained several duplicated signals with contrasting peak intensities, indicating 4 to be an enantiomeric mixture of unequal quantities ( Figure 3). Indeed, two 1 H resonance sets with slightly different chemical shifts were obtained, which were assigned to be a pair of MPA-acylated diastereomers (4'a and 4'b; 4'c and 4'd) by careful interpretation of a COSY spectrum of the (R)-MPA acylation product mixture (Supporting Information File 1, Figure S25). Furthermore, the slightly excessive 4'a and 4'c were identified to be MPA derivatives of (S)-4, and hence less excessive 4'b and 4'd to be those of (R)-4, by recursive application of Trost's chiral anisotropy rule to the 1 H chemical shift differences between the diastereomer pairs. The enantiomer ratio of 4 was estimated to be S/R = 56:44 or near by chiral phase HPLC analysis of 4' on a cellulose tribenzoate-coated silica gel column ( Figure 4). Thus, 4 was concluded to be an enantiomeric mixture of (2E,4E)-7-hydroxy-2,4-dimethyl-2,4octadienoic acid with an approximate enantiomer ratio of S/R = 56:44.
The 1 H and 13 C NMR spectra of 5 were quite similar to those of 4 (Table 1 and Table 2), except for the lack of the oxymethine proton resonance and replacement of the doublet methyl resonance by a singlet signal with a six-proton intensity. These differences, together with a one methylene-larger molecular formula (C 11 H 18 O 3 ), suggested a methyl substitution at the carbinol carbon (δ C 71.7, C-7). This was verified by HMBC correlations from the singlet methyl proton (H-8/H-9) to the carbinol carbon. Thus, 5 was identified to be (2E,4E)-7hydroxy-2,4,7-trimethyl-2,4-octadienoic acid.
The same strain also produced another new compound, 6-(3,3dimethylallyl)-N-acetyl-ʟ-tryptophan (6), when fermented in  (Table 3) revealed the composition of this molecule to be two shielded carbonyls, five other sp 2 nonprotonated carbons, five sp 2 methines, one sp 3 methine, two sp 3 methylenes, three singlet methyls, and two amino protons. The remaining OH group should be a part of a carboxylic acid functionality considering the lack of oxygenated carbons besides carbonyls, and two degrees of unsaturation, not accounted for by double bonds, were consistent with the indole ring.
As expected, assembling the above components by COSY and HMBC correlations established 6-prenylated N-acetyltryptophan ( Figure 5). The N-acetylation was evident from HMBC correlations from the amide (NH-11) and acetyl methyl protons (H-13) to the amide carbon (C-12), while prenylation at C-6 was supported by HMBC correlations from H-5 and H-7 to C-1' and from H-1' to C-6. The absolute configuration was determined by chiral anisotropy analysis after derivatization with each of the phenylglycine methyl ester (PGME) enantiomers [27], which gave positive Δδ H (S-R) values for NH-1, H-2, H-4, H-5 and H-8 and negative values for H-9, NH-11 and H-13 ( Figure 6). Thus, an S-configuration, corresponding to an ʟ-chirality for tryptophan, was assigned.

Conclusion
In this study, five unsaturated fatty acids (1-5) and one prenylated tryptophan derivative (6) were isolated as new natural products from Couchioplanes sp. RD010705. The α,γ-dimethylα,γ-dienoyl C 8 motif in 1, 2, 4, and 5 is only precedented by 64p-B (2,4-dimethyl-2,4-octadienamide) produced by manumycin-producing Streptomyces, though the physicochemical properties of which is yet to be disclosed [28]. The γ,εdimethyl-α,γ-dienoyl motif in 3 is seen in many antibiotics but that with a C 10 chain length is only precedented by manumycin C [29], TMC-1 C [30], and phialomustins [26] (Figure 7). In the family Micromonosporaceae, a strain belonging to the genus Plantactinospora is known to produce U-62162, a manumycintype metabolite with a methyl-branched C 9 unsaturated acyl chain [31]. Moreover, salinipyrones, produced by a Salinispora strain, were shown to be biosynthetic byproducts of the rosamicin polyketide synthase [32]. Though not a result from Micromonosporaceae, another example of truncated polyketides is citreodiol, a similarly methyl-branched unsaturated fatty acid ester, which is produced by type I polyketide synthase in a Streptomyces strain by a heterologous expression experiment [33]. These facts suggest that 1-5 could be byproducts from the biosynthesis of larger polyketides, but further investigation is necessary for their biosynthesis. Prenylated indoles are widely distributed among bacteria, fungi and plants, and all seven positions are subject of prenylation except for the bridgehead carbons [34]. Compound 6 is the acetylated derivative of 6-(3,3dimethylallyl)-ʟ-tryptophan from Streptomyces sp. SN-593 [35]. Further chemical exploration on the genus Couchioplanes will disclose its actual biosynthetic capacity in secondary metabolism.

Experimental General experimental procedures
Optical rotations were measured using a JASCO P-1030 polarimeter. UV spectra were recorded on a Shimadzu Isolation of compounds 1-5 Compounds 1-5 were obtained from a culture fermented in A16 production medium with a composition of glucose 2%, Pharmamedia ® (Traders Protein, Memphis, TN, USA) 1%, and CaCO 3 0.5%. The pH of the medium was adjusted to 7.0 before autoclaving. At the end of fermentation, 100 mL of 1-butanol was added to each flask, and the flasks were allowed to shake for 1 h. The mixture was centrifuged at 6,000 rpm for 10 min and the organic layer was separated from the aqueous layer containing the mycelium. The organic layer was concentrated in vacuo to give 4.4 g of an extract from a 3 L culture. The extract was subjected to silica gel column chromatography eluted with a step gradient of CHCl 3 Table 1 and

Isolation of 6
Compound 6 was obtained from a culture fermented in modified V22 production medium with a composition of soluble starch 1%, glucose 0.5%, NZ Amine, Type A 0.3%, yeast extract 0.2%, Tryptone 0.5% K 2 HPO 4 0.1%, MgSO 4 ·7H 2 O 0.05%, and CaCO 3 0.3%. The pH of the medium was adjusted to 7.0 before autoclaving. At the end of fermentation, 100 mL of 1-butanol was added to each flask, and the flasks were allowed to shake for 1 h. The mixture was centrifuged at 6,000 rpm for 10 min and the organic layer was separated from the aqueous layer containing the mycelium. The organic layer was concentrated in vacuo to give 5.2 g of an extract from a 6 L culture. The crude extract was chromatographed on a silica gel column similarly as above and the sixth fraction was fractionated by ODS column chromatography with a gradient of MeCN/0.1% HCO 2 H solution (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2 v/v). The fraction 4 (5:5) was evaporated, and purified by preparative HPLC operated at the same conditions as above to give 6-(3,3-dimethylallyl)-N-acetyl-ʟ-tryptophan (6, 45 mg, t R = 10.4 min). (6)   In the same manner as described for the preparation of 4'a and (S)-MPA ester of 7R enantiomer 4'd: 1 H NMR data was identical to those of 4'a.

Cytotoxicity assay
The cytotoxicity assay was carried out against P388 murine leukemia cells in the same manner as reported previously [36]. The IC 50 of a reference drug doxorubicin hydrochloride was 0.13 μM.

Supporting Information
Supporting Information File 1 Copies of NMR spectra.