Chinese Chemical Letters  2016, Vol. 27 Issue (7): 1044-1047   PDF    
Neopeapyran, an unusual furo[2, 3b]pyran analogue and turnagainolide C from a soil Streptomyces sp. S2236
Zhou Haoa, Yang Ya-Bina, Duan Rong-Tinga, Yang Xue-Qionga, Zhang Ju-Chenga, Xie Xiao-Guanga, Zhao Li-Xingb, Ding Zhong-Taoa     
a Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China ;
b Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, China
Abstract: Neopeapyran (1), an unusual furo[2, 3b]pyran analogue, together with a new cyclopeptide, turnagainolide C (2), were isolated from Streptomyces sp. S2236 associated with the rhizosphere soil of Panax notoginseng. The planar structure and relative configuration of neopeapyran (1)) were elucidated on the basis of spectroscopic techniques, while the absolute configuration was determined by TDDFT calculation. The absolute configuration of turnagainolide C (2) was determined by partial hydrolysis, together with the advanced Marfey's method and spectroscopic analysis. The antimicrobial activities of these two compounds were also investigated.
Key words: Streptomyces sp.     Furo[2, 3b]pyran analogue     Cyclopeptide     Structure elucidation     Antimicrobial activity    
1. Introduction

The actinomycetes of genus Streptomyces have attracted the attention of the scientific community due to their enormous biosynthetic capabilities of producing secondary metabolites showing novel scaffolds and biologically actives [1-3]. In particular, terrestrial soil-derived Streptomyces species have been extensively studied because they can potentially provide bioactive natural products [1, 4]. They have been ascertained to afford clinically useful antibiotics, such as streptomycin and neomycin [5, 6]. Although actinomycetes are a major source of microbial compounds, the chemical redundancy of compounds isolated from these actinomycetes has become one of the current challenges in the discovery of novel secondary metabolites with biologically actives [7]. Investigation of unique niches harbouring chemically new actinomycetes, rather than typical environments, is one approach to overcome this problem [8, 9]. Many recent investigations have exposed compelling evidence that the Streptomyces species derived from unique habitats might lead to the discovery of novel natural products with significant biological activity, such as the rhizosphere soil of hosts [8, 10], saltern [3], sulphur mine [11], and so on.

As part of our programme to discover structurally unique and biologically active secondary metabolites from Streptomyces associated with the rhizosphere soil of medicine plants, Streptomyces sp. S2236 had drawn the most interest of us which was isolated from the rhizosphere soil of Panax notoginseng in Wenshan, Yunnan Province, China. The strain was most likely a Streptomyces sp. based on the 99.22% 16S rRNA sequence similarity with the Streptomyces neopeptinius KNF 2047T (EU258679) . Our chemical investigation of this strain yielded two new natural products, neopeapyran (1), an unusual furo[2, 3b]pyran analogue, and a new cyclopeptide, turnagainolide C (2) (Fig. 1). The planar structure and relative configuration of compound 1 were elucidated on the basis of spectroscopic techniques, while the absolute configuration was determined by TDDFT calculation. The planar structure of compound 2 was determined by spectroscopic analysis, and the absolute configuration of constituent amino acid residues was determined by the advanced Marfey's method. Differentiation of L-Val and D-Val in the sequence was established by the advanced Marfey's analysis of fragment peptides obtained from the partial hydrolysate. In the antimicrobial assays, compound 2 displayed moderate antimicrobial activities against Candida albicans, Escherichia coli and Staphylococcus aureus. Herein, we report the discovery and characterization of these two compounds and their antimicrobial activities.

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Figure 1. Structures of compounds 1, 2.

2. Experimental 2.1. Biological material and cultivation

The bacterial strain S2236 was isolated using modified ISP4- medium from the rhizosphere soil of P. notoginseng in Wenshan, Yunnan Province, China. The strain was most likely a Streptomyces sp. based on the 99.22% 16S rRNA sequence similarity with the S. neopeptinius KNF 2047T (EU258679) , and it was identified as Streptomyces sp. S2236. The strain has been preserved at Yunnan Institute of Microbiology, Yunnan University, China. This bacterium was cultivated on 40 L scale using 1 L Erlenmeyer flasks containing 250 mL of the seed medium (yeast extract 0.4%, glucose 0.4%, malt extract 1.0%, decavitamin 0.01%, pH 7.2) and the fermentation medium (soluble starch 1%, glucose 1%, peptone 0.5%, yeast extract 0.5%, soybean flour 0.3%, NaCl 0.4%, K2HPO4 0.05%, MgSO4.7H2O 0.05%, CaCO3 0.2%, pH 7.0) at 28 ℃ for 7 on rotary shaker (250 rpm).

2.2. Extraction and isolation

After 7 days of growth, the mycelia were removed from the cultures (40 L) by filtration. The filtrate was extracted with ethyl acetate (EtOAc, 3 × 40 L), and the solvent was removed under vacuum. The EtOAc extract (31.0 g) was separated into four fractions (Fr 1-Fr 4) by a chromatographic column (3 cm × 50 cm) on silica gel (200-300 mesh), eluting with stepwise CHCl3/MeOH gradient (CHCl3, CHCl3/MeOH = 30:1 v/v, CHCl3/MeOH = 10:1 v/v, MeOH, 1.5 L each). The Fr 2 (18.5 g, eluted with CHCl3/ MeOH = 30:1 v/v) was placed in a silica gel column (2 cm× 30 cm) and elutedwith petroleum ether/ethyl acetatemixture (10:1) to ethyl acetate, then MeOH, which gave three fractions (Fr 2-1 to Fr 2-3) . Fr 2-1 (3.4 g) was separated by a chromatographic column (1 cm× 150 cm) on Sephadex LH-20 (MeOH) and then subjected to further elution on a silica gel column (1 cm × 15 cm) with CHCl3/MeOH (50:1-9:1) to afford 2 (10.2mg). Fr 2-2 (2.1 g) was subjected to further elution on a silica gel column (1 cm× 20 cm) with CHCl3/MeOH (40:1-9:1) and then separated by a chromatographic column (1 cm× 150 cm) on Sephadex LH-20 (MeOH) to give 1 (2.1 mg).

2.3. Determination of absolute configuration by the advanced Marfey's method

Details may be found in Supporting information and the references cited therein [12, 13].

2.4. Partial hydrolysis and the advanced Marfey's analyses of the fragments

Each of 2 (0.5 mg) was hydrolyzed in 6 mol/L HCl at 100 ℃ for 3 h or 4 h. The dried hydrolysates were redissolved in 50 mL of MeOH. 20 mL portions were separated by RP-HPLC (Agilent Eclipse XDB-C18 column 4.6 mm × 150 mm 5 μm) using a gradient elution from 10 to 100% MeCN containing 0.2% AcOH. After the assignments of peptide sequences by extensive MS/MS analyses, the fractions were identified as Ala-Val-Hppa, Ile-Val-Hppa, Val- Ile, together with other fragments (Fig. 2). Then 2.0 mg of 2 was hydrolyzed in 6 mol/L HCl at 100 ℃ for 3 h. The hydrolysate was redissolved in 100 mL of MeOH, and each of 20 mL portions was separated by RP-HPLC (Agilent Eclipse XDB-C18 column 4.6 mm × 150 mm, 5 μm) using the same gradient elution, and the eluate was collected according to the retained time which was analyzed by the LC-MS/MS data. The fractions of Ala-Val-Hppa, Ile- Val-Hppa and Val-Ile were hydrolyzed in 6 mol/L HCl at 110 ℃ overnight. The solution was dried, converted to the FDLA derivative, and analyzed as described previously.

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Figure 2. Total ion current (TIC) chromatogram of the partial hydrolysate of 2. (A) The hydrolysis time was 3 h, (B) the hydrolysis time was 4 h.

3. Results and discussion

Neopeapyran (1) was obtained as colourless oil, and the results of TCL chromogenic reactions certified the inexistence of N in compound 1. Its molecular formula of C13H20O5 was confirmed on the basis of a prominent ion peak at m/z 257.1388 [M+H]+ (calcd. for C13H21O5, 257.1389) observed in the HRESIMS spectrum (Fig. S7 in Supporting information) and 13C NMR spectra, and that indicated four degrees of unsaturation. IR spectrum exhibited absorption at 3433, 2923, 1632, 1450 and 1047 cm-1 which indicated that the structure of compound 1 contained at least one hydroxyl group and one olefin group. The 1H NMR and 13C NMR spectra of 1 displayed resonances that were assigned to one methyl (δC 18.2) , one methylene (δC 26.6) , three oxygen-bearing methylenes (δC 55.8, 78.0, 77.9) , six methines [among them four vinyl methines (δC 132.1, 130.8, 130.6, 130.3) ], and two quaternary carbons (δC 104.8, 88.4) (Table 1). The 1H-1H COSY correlations between H-10 and H-11, H-11 and H-12, H-12 and H-13, H-13 and H-14, revealed the carbon skeletons for C-10 connected to C-11, C-11 connected to C-12, C-12 connected to C-13, and C-13 connected to C-14. The HMBC correlations from H-14 to C-12 and C-13, together with J10, 11 = 15.4 Hz and J12, 13 = 15.1 Hz for H-10, 11 trans and H-12, 13 trans, respectively, indicated the presence of a (2E, 4E)-pentylene residue. A tetrahydrofuran moiety was deduced by the HMBC correlations from H-5 to C-6 and C-9, H-7 to C-5, C-6 and C-9, together with the chemical shifts of C-7 (δC 78.0) and C-9 (δC 104.8) . The HMBC correlations from H-15 to C-5, C-6 and C-7, together with the chemical shifts of C-6 (δC 88.4) and C-15 (δC 77.9) , indicated that C-6 was substituted by a hydroxymethyl and a hydroxyl. At the same time, the HMBC correlations from H-10 and H-11 to C-9, H-10 to C-5, indicated that the (2E, 4E)-pentylene residue was linked to C-9. The 1H-1H COSY correlations between H-2 and H-3, H-3 and H-4, H-4 and H-5, revealed the carbon skeletons for C-2 connected to C-3, C-3 connected to C-4, and C-4 connected to C-5, and the chemical shift of C-4 (δC 74.7) indicated that C-4 was substituted by a hydroxyl. However, the HMBC correlations from H-2 and H-4 to C-9, along with one additional degree of unsaturation and the molecular formula of C13H20O5 deduced by the HRESIMS spectrum, indicated the existed of a tetrahydropyrane moiety (Fig. 3), and the upfield shifts of H-2 (dH 3.63, 4.12) and C-2 (δC 55.8) cased by the shielding effect of the double bond between C-10 and C-11. Finally, the structure of compound 1 was deduced as shown in Fig. 1. The skeleton of furo[2, 3b]pyran with a (2E, 4E)-pentylene side chain in compound 1 was reported for the first time.

Table 1
NMR data for compound 1 (δ in ppm, J in Hz).a

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Figure 3. Key COSY, HMBC and ROESY correlations of 1 and 2.

The relative stereochemistry of 1 was deduced from the ROESY experiments (Fig. 3). ROESY correlations between H-4, H-5 and H-15, and no significant correlations between H-10 and H-4/H-5/ H-15, suggested that there were two possible isomers [(4S, 5R, 6R, 9R)-1 and (4R, 5S, 6S, 9S)-1] of compound 1. The absolute configuration of 1 was determined by comparison of quantum chemical TDDFT calculated and experimental specific rotations. Each isomer was optimized using DFT at the B3LYP/6-311 + G(d, p) level in the Gaussian 09 program. Then, the optimized isomer was calculated using TDDFT/GIAOs at the B3LYP/6-311 + G(d, p) in the Gaussian 09 program to generate its specific rotation. The calculated specific rotation of (4S, 5R, 6R, 9R)-1 was negative, and the calculated specific rotation of (4R, 5S, 6S, 9S)-1 was positive. Therefore, the absolute configuration of 1 was determined as (4S, 5R, 6R, 9R)-1 (Fig. 1) by compared with the experimental specific rotation of 1 which was [a]D 25 -5.8 (c 0.1, MeOH).

Turnagainolide C (2) obtained as a white powder, and its molecular formula of C30H44N4O6 was assigned by positive HRESIMS (m/z 579.3156 for [M+Na]+, calcd. for C30H44N4O6Na: 579.3159, Fig. S14 in Supporting information) and 13C NMR data (Table 2). The molecular formula of 2 indicated 11 degrees of unsaturation. IR spectrum exhibited intense N-H and CO absorption at 3392 and 1665 cm-1, respectively. The appearance of four amino carbonyl signals (δC 173.1, 172.4, 170.4, and 168.8) in 13C NMR spectrum and four NH signals (dH 7.59, 7.73, 8.09, and 8.56) in 1H NMR spectrum, suggesting the molecule contained a peptide component. The amino acid residues were identified as two valine groups (Vals), one alanine group (Ala), and one isoleucine group (Ile) by 1H-1H COSY, HSQC and HMBC spectra. The sequence of amino acid residues in 2 was determined from HMBC correlations between the carbonyl group and amide of the adjacent residues (Fig. 3). HMBC correlations from aH-Val(I) and NH-Val(I) to CO-Ile, from aH-Ile and NH-Ile to CO-Ala, from NH-Ala to CO-Val(Ⅱ), demonstrated the sequence of the four amino acid residues was - Val(I)-Ile-Ala-Val(Ⅱ)-. Compared with the molecular formula of 2 (C30H44N4O6), the molecule still had an elemental composition of C11H10O2 by subtracted the atoms attributed to the four amino acids residues (C19H34N4O4). This fragment was identified as a 3-hydroxy-5-phenylpent-4-enoic acid residue (Hppa) by 1H-1H COSY, HSQC and HMBC spectra (Fig. 3). The 16.0 Hz scalar coupling observed between H-4(Hppa) and H-5(Hppa) deduced the moiety was (E)-3-hydroxy-5-phenylpent-4-enoic acid residue. Finally, the HMBC correlations from aH-Val(Ⅱ) and NH-Val(Ⅱ) to CO-Hppa, from H-3(Hppa) to CO-Val(I) established that 2 was cyclo(Val(I)- Ile-Ala-Val(Ⅱ)-Hppa).

Table 2
NMR data for compound 2 (δ in ppm, J in Hz).a

1H NMR and 13C NMR spectra of 2 displayed the similar resonances as EGM-556 [14] and turnagainolides A-B [15], but the absolute stereochemistry of the amino acid residues in 2 were identified as L-Val, D-Val, L-Ile and D-Ala by the advanced Marfey's method. There was no correlations between aH-Ile and aH-Val(I), aH-Ala and aH-Val(Ⅱ) in ROESY spectrum, but observed the correlations between βH-Ile and aH-Val(I), gH-Ile(CH2) and aH-Val(I), bH-Ala and aH-Val(Ⅱ) in ROESY spectrum, together with contrasting theNMR spectra of 2 with turnagainolide A, established the configurations ofVal(I), Val(Ⅱ) and Ile as (R)-Val(I), (S)-Val(Ⅱ) and (2S, 3S)-Ile (Fig. 3). In the same way, the configuration of Hppa was determined as (3R)-Hppa by comparing the chemical shifts of Hppa residue with turnagainolide A, and there was no ROESY correlation between H-3(Hppa) and aH-Val(I). Therefore, the structure of 2 was determined as cyclo((R)-Val(I)-(2S, 3S)-Ile-(R)-Ala-(S)-Val(Ⅱ)-(3R)- Hppa), which had a different stereochemical structure from turnagainolides A-B, named as turnagainolide C.

In order to confirm the configuration elucidation of 2, we used LC-MS/MS analysis to set up proper hydrolysis conditions and get suitable peptide fragments by partial hydrolysis [16]. We hoped to isolate peptides to determine the chirality of each Val in the peptide sequence. To do this we chose harsher conditions of 6 mol/L HCl at 100 ℃ and set the hydrolysis time for 3 h on the basis of the LC-MS data. The LC-MS/MS data allowed us to identify fragments that contain Val residues of specific position, i.e., Ala-Val-Hppa, Ile-Val- Hppa and Val-Ile, together with other fragments (Fig. 2). Marfey's analysis of fragment Ala-Val-Hppa indicated that the Val(Ⅱ) was a L-Val. Due to the little amount of 2, we did not get enough fragments of Ile-Val-Hppa or Val-Ile to determined the absolute stereochemistry of Val(I). However, the Marfey's analysis result of partial hydrolysis of 2 was consistent with the determined structure. The extract of Streptomyces sp. S2236 and the isolates (1 and 2) were tested for their antimicrobial activites against C. albicans, E. coli, and S. aureus. The results were showed in Table 3. Both 1 and 2 showed activites against E. coli with MICs of 64 and 32 g/mL, respectively. At the same time, 2 also showed moderate antimicrobial activities against C. albicans and S. aureus.

Table 3
MICs of the extract and compounds from Streptomyces sp. S2236 (g/mL).

4. Conclusion

Two new compounds, neopeapyran (1) and turnagainolide C (2) were isolated from the fermentation broth of Streptomyces sp. S2236. The skeleton of furo[2, 3b]pyran with a (2E, 4E)-pentylene side chain has never been reported from natural resources or synthesis. The skeleton of 1 was reported for the first time. The relative configuration of 1 was elucidated by spectroscopic techniques, while the absolute configuration was determined by TDDFT calculated specific rotation. Compound 2 showed moderate antimicrobial activities against C. albicans, E. coli and S. aureus all with the MIC of 32 g/mL.

Acknowledgments

The work was funded by grants from the National Natural Science Foundation of China (Nos. 81360480, 81460536 and 81560571) .

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.03.018.

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