- Email: [email protected]
Two new 2,5-diketopiperazines produced by Streptomyces sp. SC0581
Phytochemistry Letters 20 (2017) 89–92
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Two new 2,5-diketopiperazines produced by Streptomyces sp. SC0581 a,b
a
Li Yang , Ahmed Mahal , Youhua Liu ⁎ Xiaoyi Weia,
a,b
a
a
a
a
, Hanxiang Li , Ping Wu , Jinghua Xue , Liangxiong Xu ,
MARK
a Key Laboratory of Plant Resources Conservation and Sustainable Utilization/Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, People’s Republic of China b University of Chinese Academy of Sciences, Yuquanlu 19A, Beijing 100049, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords: 2,5-diketopiperazines DOPA Streptomyces sp. Antioxidant
Two new diketopiperazines, cyclo(L-Phe-L-NMe-DOPA) (2) and cyclo[L-Phe-L-(NMe-3-(NMe-3-O-α-L-rhamnopyranosyl)-DOPA] (3), along with a known diketopiperazine (1), were isolated from the cultures of Streptomyces sp. SC0581. Their structures were elucidated by extensive spectroscopic analysis, single-crystal X-ray crystallographic analysis, and chemical correlation. Compounds 1 − 3 exhibited more potent ABTS radical cation scavenging activity (IC50 values: 3.7 − 14.6 μM) than L-ascorbic acid (IC50: 17.7 μM). Compounds 2 and 3 also showed remarkable DPPH radical scavenging activity.
1. Introduction
reported (Liu et al., 2007). In the present study, colorless crystals (mp 160 − 162 °C) of this compound were obtained in MeOH and subjected to X-ray diffraction analysis for the first time, with which its complete structure (Fig. 2), including the absolute configuration, was confirmed (Fig. 1). Compound 2 was obtained as a white powder. Its (−)-HRESIMS presented a pronounced ion peak at m/z 339.1339 [M − H]−, corresponding to a molecular formula of C19H20N2O4, with one more oxygen atom than that of 1. Its 1H NMR and 13C NMR data were closely similar to those of 1, except that the proton and carbon resonances for the p-hydroxyphenyl group in the spectra of 1 were replaced by those indicating the presence of a 3,4-dihydroxyphenyl group [δH 6.32 (H, dd, J = 8.0, 2.1 Hz), δH 6.67 (1H, d, J = 8.0 Hz), δH 6.51 (H, d, J = 2.1 Hz); δC 116.8 (CH), 118.5 (CH), 121.8 (CH), 128.2 (C), 145.7 (C), and 146.6 (C)] in 2, suggesting a 2,5-diketopiperazine composed of a phenylalanine and an NMe-DOPA. The planar structure of 2 was supported by 2D NMR data (Fig. 3). With regard to the stereochemistry, compound 2 showed a negative specific rotation value (−110.0) close to that of 1 (−164.0), allowing assignment of the same absolute configuration (3S,6S) as 1. Consequently, 2 was elucidated as cyclo(LPhe-L-NMe-DOPA). Compound 3, obtained as a white powder, had a molecular formula of C25H30N2O8 on the basis of the deprotonated ion peak at m/z 485.1935 observed in the (−)-HRESIMS. The 1H NMR and 13C NMR spectra of 3 were comparable to those of 2 except for the presence of additional signals (δH 5.34, 4.12, 3.95, 3.50, 3.83, 1.31; δC 101.6, 71.9, 72.2, 73.9, 70.8, 18.0) attributable to a rhamnopyranosyl moiety (Fan
2,5-Diketopiperazines, cyclic dipeptides produced by condensation of two α-amino acids, have been found in a variety of natural resources including bacteria, fungi, marine sponge, plants, and mammals (Huang et al., 2010, 2014). They possess a series of intriguing chemical properties, such as resistance to proteolysis, rigid backbone, constrained conformation, stereochemistry controlled at up to four positions, and 2 hydrogen bond donor and 2 hydrogen bond acceptor sites important for binding to various biological targets (Borthwick, 2012). In addition, they have been reported to exhibit diverse biological activities including antibacterial, antifungal, antiviral activities, cytotoxicity, and anti-inflammatory (Martins and Carvalho, 2007). Owing to these chemical and biological properties, 2,5-diketopiperazines have attracted much attention of researchers in synthetic chemistry, medicinal chemistry, and pharmacology. In the course of our searching for novel bioactive metabolites from soil-derived actinomycetes (Feng et al., 2007), two new 2,5-diketopiperazines (2 and 3), together with the known diketopiperazine cyclo(L-Phe-L-NMe-Tyr) (1), were isolated from the cultures of Streptomyces sp. SC0581. Herein, we report the isolation, structure elucidation, and antioxidant activity of these compounds. 2. Results and discussion The known compound cyclo(L-Phe-L-NMe-Tyr) (1) was identified by comparison of its spectroscopic data and specific rotation with those
⁎
Corresponding author. E-mail address: [email protected] (X. Wei).
http://dx.doi.org/10.1016/j.phytol.2017.04.012 Received 5 January 2017; Received in revised form 21 March 2017; Accepted 13 April 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 20 (2017) 89–92
L. Yang et al.
Table 1 1 H (500 MHz) and position
2
13
C (125 MHz) NMR Data of Compounds 2 and 3.
a
3b
δC, type 2 3 5 6 7a 7b 8 9 10 11 12 13 14a 14b 15 16,20 17,19 18 1-NCH3 4-NH 1′ 2′ 3′ 4′ 5′ 6′
Fig. 1. Structures of compounds 1 − 3.
a
Fig. 2. X-ray structure of 1.
b
δH, mult. (J in Hz)
166.4, C 57.2, CH 167.1, C 64.0, CH 37.4, CH2 128.2, C 118.5, CH 146.6, C 145.7, C 116.8, CH 121.8, CH 42.0, CH2 138.3, C 130.8, CH 129.5, CH 127.7, CH 33.7, CH3
3.88, m 4.03, t (4.7) 2.74, d (14.1, 4.7) 2.46, d (14.1, 4.7) 6.51, d (2.1)
6.67, 6.32, 2.43, 1.69,
d (8.0) dd (8.0, 2.1) dd (13.6, 4.6) dd (13.6, 8.2)
7.00, 7.27, 7.20, 2.81, 7.82,
d (7.4) t (7.4) t (7.4) s brs
δC, type 167.9, C 58.0, CH 168.6, C 64.7, CH 37.8, CH2 128.5, C 120.9, CH 148.3, C 146.2, C 117.6, CH 125.6, CH 41.8, CH2
δH, mult. (J in Hz)
4.01, dd (8.9, 3.8) 4.14, dd (4.6, 5.0) 2.93, dd (14.3, 4.6) 2.57, dd (14.3, 5.0) 6.92, d (2.0)
6.86, 6.70, 2.73, 1.61,
d (8.0) dd (8.1, 2.0) dd (13.6, 3.8) dd (13.6, 8.9)
137.7, C 130.7, CH 129.8, CH 128.0, CH 33.9, CH3
7.05, 7.32, 7.22, 2.93,
d (7.4) t (7.4) t (7.4) s
101.6, CH 71.9, CH 72.2, CH 73.9, CH 70.8, CH 18.0, CH3
5.34, 4.12, 3.95, 3.50, 3.83, 1.31,
d (1.7) dd (3.5, 1.7) dd (9.5, 3.5) t (9.5) dd (9.5, 6.2) d (6.2)
DMSO-d6 used as solvent. CD3OD used as solvent.
Table 2 Antioxidant activity of compounds 1 − 3.
Fig. 3. Key 1H–1H COSY (bold lines) and HMBC (arrows) correlations of 2.
compound
ABTS (IC50, μM)
DPPH (IC50, μM)
FRAP (mmol/g)
1 2 3 L-ascorbic acid
3.7 ± 0.13 14.6 ± 0.70 7.1 ± 0.83 17.7 ± 0.40
> 250 37.2 ± 0.96 27.7 ± 0.09 36.2 ± 0.18
0.21 ± 0.002 7.15 ± 0.079 6.87 ± 0.014 13.31 ± 0.030
Values represent means ± standard deviation (SD) (n = 4).
et al., 2015). The glycosylation site was located at C-10 based on the long-range correlation from H-1′ to C-10 (δC 148.3) observed in the HMBC spectrum. The coupling constant (J = 1.7 Hz) of the anomeric proton (H-1′) indicated an α orientation of the glycosidic linkage. The L configuration of the rhamnose moiety was determined using the method described by Tanaka et al. (2007). The 3S,6S configurations of 3 were assigned by the same negative sign of specific rotation value of 1 and 2, and on the basis of the co-occurrence of 2 and 3. Thus, the structure of 3 was defined as cyclo[L-Phe-L-(NMe-3-O-α-L-rhamnopyranosyl)-DOPA]. Antioxidants play significant role in inhibiting or delaying the oxidation of susceptible cellular substrates and they are important for prevention of many oxidative stress-mediated diseases. Consequently, the antioxidant activity of compounds 1 − 3 was evaluated using ABTS radical cation and DPPH radical scavenging tests and FRAP assay. The results were listed in Table 2. The isolated compounds (1 − 3) displayed ABTS radical cation scavenging activity with IC50 values ranging from 3.7 to 14.6 μM, which were all superior to that of reference compound, L-ascorbic acid (IC50 value: 17.7 μM). In the DPPH radical scavenging assay, compounds 2 and 3 exhibited the activity comparable to that of L-ascorbic acid, but compound 1 was inactive. In FRAP assay, compounds 2 and 3 showed only a mild activity. Tyrosinase inhibitory activity was also evaluated for compounds 1 − 3 using the previously described method (Kim et al., 2016), but none of them was found to be active at the highest test concentration (250 μM).
3. Experimental 3.1. General experimental procedures Optical rotations were recorded on a Perkin-Elmer 343 spectropolarimeter. UV spectra were measured with a Perkin EImer Lambda 650 UV/VIS spectrometer. 1D and 2D NMR experiments were performed on a Bruker Avance III 500 Hz instrument. HR-ESIMS data were recorded on a Bruker maXis Q-TOF mass spectrometer. Preparative HPLC was run on a Shimadzu Shim–packed Pro-ODS column (20 mm × 25 cm). For column chromatography, silica gel 60 (100–200 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), YMC ODS (75 μm, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used. TLC was carried out on HSGF254 silica gel plates (0.2 mm, Yantai Jiangyou silica gel Development Co. Ltd., Yantai, China); spots were visualized by spraying with 10% H2SO4 in EtOH followed by heating. 3.2. Biological material and fermentation The actinomycetes strain Streptomyces sp. SC0581 was isolated from a soil sample collected in Dinghu Mountain Biosphere Reserve, Guangdong, China, and authenticated based on morphological characteristics and DNA sequence analysis of the ITS region (GenBank accession no: KX687558). A voucher strain is deposited in the culture 90
Phytochemistry Letters 20 (2017) 89–92
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3.5. Determination of absolute configuration of sugar moiety
collection of South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. The strain was grown on the potato dextrose agar at 28 °C for 10 days. Six pieces of culture plugs of the strain were inoculated into each of two 500 mL Erlenmeyer flasks containing 150 mL YMG medium consisting of 0.4% glucose, 1.0% malt extract, 0.4% yeast extract. The flasks were shaken on a rotatory (150 rpm) at 28 °C for 2 days. Then, the cultures were transferred into three 3 L flasks containing 1 L of YMG with the same incubation condition. Finally, 10 mL culture broth was transferred into each of two hundreds 500-mL flasks containing 60 mL of YMG medium and 60 g of wheat grains and incubated in the dark at 28 °C for 40 days.
Compound 3 (2 mg) was hydrolyzed with 5 mL of 2 M aqueous HCl at 100 °C for 4 h. After removal of the solvent under reduced pressure, the resulting hydrolysate was dissolved in 5 mL of distilled water, and then partitioned with 5 mL of EtOAc for three times. The aqueous layer, after concentrated to dryness, was dissolved in 2 mL of pyridine containing 1 mg/mL L-cystein methyl ester hydrochloride and heated at 60 °C for 1 h. Then, O-tolylisothiocyanate (4 μL) was added and kept at 60 °C for additional 1 h. The reaction mixture was concentrated to dryness under vacuum. The residue was dissolved in MeOH and analyzed by HPLC at 40 °C on an SPOLAR C18 column (4.6 × 250 mm, 5 μm) using CH3CN/H2O/HCOOH (25:75:0.1, v/v/v) at a flow rate of 0.8 mL/min for 60 min. Peaks were detected at 250 nm. The absolute configuration of rhamnose was clarified by comparison of retention time of its derivative with that of standard L-rhamnose derivative (tR = 35.1 min) prepared in the same manner (Tanaka et al., 2007; Xiao et al., 2016; Fan et al., 2015).
3.3. Extraction and isolation The solid cultures were extracted with 95% EtOH three times. The resultant crude extract was sequentially partitioned with petroleum ether, EtOAc, and n-BuOH. The EtOAc- and n-BuOH-soluble fractions were combined and subjected to silica gel column chromatography (CC) with CHCl3/MeOH mixtures of increasing polarity (100:0 to 70:30) to afford twenty fractions (Frs. 1 − 20). Fr. 2 was further separated by ODC CC using aqueous MeOH (20% − 70%) to give ten subfractions (Frs. 2-1 − 2-10). Fr. 2-2, obtained on elution with 40% aqueous MeOH, yielded crystals in MeOH, which were collected by filtration to obtain 1 (30 mg). Fr. 6 was also subjected to ODC CC eluted with aqueous MeOH (20 − 70%) to give six subfractions (Frs. 6-1 − 6-6). Fr. 6-3 was purified by preparative HPLC (flow rate 5 mL/min) using 30% aqueous MeOH to give 2 (10 mg, tR = 72.8 min). Fr. 10 was applied to ODC CC using aqueous MeOH (10 − 60%) to give 5 subfractions (Frs. 10-1 − 10-5). Fr. 10-2 was purified by preparative HPLC (flow rate 5 mL/min) using 28% aqueous MeOH to give 3 (13 mg, tR = 54.2 min).
3.6. Antioxidant assays 3.6.1. ABTS radical cation scavenging assay ABTS radical cation scavenging activity was tested by previously reported method (Re et al., 1999; Ma et al., 2014). 7 mM ABTS in water was mixed with 2.45 mM potassium persulfate and kept in the dark at room temperature for 12–16 h. Then, the ABTS radical cation solution was diluted with buffered saline (PBS), pH 7.4, to obtain an absorbance of 0.7 ± 0.02 units at 734 nm. Test compounds were dissolved in DMSO and two-fold diluted to serial concentrations. Compound solution (5 μL) and ABTS radical cation solution (195 μL) were mixed and added to 96-well plates. L-ascorbic acid was used as reference compound. The control wells contained DMSO instead of compound solution, and the blank wells were added with PBS in place of ABTS radical cation solution. After a mixing time of 15 s and an incubation period of 6 min at room temperature in the dark, OD value of each well was read at 415 nm. The percentage inhibition was calculated using the formula as below:
3.3.1. Cyclo(L-Phe-L-NMe-DOPA) (2) White powder; [α] D20 −110.0 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 202 (4.60), 282 (3.57); 1H and 13C NMR data, see Table 1; (−)-HRESIMS m/z 339.1339 [M − H] − (calcd for C19H19N2O4, m/z 339.1345).
ABTS%+ scavenging effect (%) = [1–(ODcompound − ODblank)/ (ODcontrol − ODblank)] × 100
3.3.2. Cyclo[L-Phe- L-(NMe-3-O-α-L-rhamnopyranosyl)-DOPA] (3) White amorphous powder; [α] D20 −112.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.17), 283 (3.12); 1H and 13C NMR data, see Table 1; (−)-HRESIMS m/z 485.1935 [M − H] − (calcd for C25H29N2O8, m/z 485.1924).
3.6.2. DPPH radical scavenging assay DPPH radical scavenging assay was performed according to a previously described method (Brand-Williams et al., 1995; Ma et al., 2014). Test compounds were dissolved in DMSO and two-fold diluted to serial concentrations. The mixture of test compound solution including L-ascorbic acid as positive reference (5 μL) and 195 μL of 0.1 mM DPPH was added to 96-well plates. The control wells contained DMSO instead of compound solution, and the blank wells were added with MeOH in place of DPPH radical solution. The plates were incubated at room temperature for 30 min in the dark, then absorbance of each well was measured at 517 nm. The DPPH radical scavenging activity in percentage of compounds was calculated as follows:
3.4. X-ray diffraction analysis of 1 The single-crystal X-ray diffraction data for 1 were collected on Agilent Xcalibur Nova single-crystal diffractometer using Cu Kα radiation. Crystal data of 1: C19H20N2O3, M = 324.37, 0.42 × 0.40 × 0.36 mm3, monoclinic, space group: P 1 21 1, a = 8.82950(13) Å, b = 11.04848(14) Å, c = 8.89756(13) Å, α = γ = 90°, β = 107.8744(15)°, V = 826.08(2) Å3, Z = 2, ρ = 1.304 Mg/m3, F000 = 344. Cu Kα radiation, λ = 1.54184 Å, T = 150(2) K, theta range for data collection: 5.22 to 66.98°; 11303 reflections collected, 2909 unique (Rint = 0.0200), completeness to theta = 66.98° (99.5%), max. and min. transmission: 0.7813 and 0.7515. The structure was refined by full-matrix least-squares on F2. Final GooF = 1.172, final R indices [I > 2sigma(I)]: R1 = 0.0315, wR2 = 0.0804, R indices (all data): R1 = 0.0319, wR2 = 0.0809; absolute structure parameter: Hooft = 0.09(16); largest diff. peak and hole: 0.220 and −0.339 e.Å−3. Crystallographic data for the structure of 1 have been deposited at the Cambridge Crystallographic Data Centre: reference number CCDC 1522635. This data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html or from CCDC, 12 Union Road, Cambridge, CB21EZ, UK; (fax: +44-(0) 1223 336033 or e-mail: [email protected]).
DPPH scavenging effect (%) = [1–(ODcompound − ODblank)/ (ODcontrol − ODblank)] × 100
3.6.3. FRAP assay FRAP assay was carried out followed by previously described procedure (Benzie and Strain, 1999; Ma et al., 2014). FRAP solution was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl, and 20 mM aqueous ferric chloride (FeCl3) solution in the ratio of 10:1:1 (v/v/v). Test compounds were dissolved in DMSO. Compound solution (20 μL) was mixed with 180 μL of FRAP and added to 96-well plates. L-ascorbic acid was used as a reference 91
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L. Yang et al.
of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–27. Borthwick, A.D., 2012. 2,5-Diketopiperazines: synthesis, reactions, medicinal chemistry, and bioactive natural products. Chem. Rev. 112, 3641–3716. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci. Technol. 28, 25–30. Fan, L., Liao, C.H., Li, S.G., Huang, X.J., Hu, X.P., Song, X., Fan, C.L., Wang, Y., Ye, W.C., Kang, Q.R., Zheng, K., Liu, L.P., Jiang, Y.C., Fan, X.M., Zhang, J., Li, Y., Zeng, Y., He, Z.D., 2015. Phenylethanoid and secoiridoid glycosides from the leaves of Ligustrum purpurascens. Phytochem. Lett. 13, 177–181. Feng, N., Ye, W.H., Wu, P., Huang, Y.C., Xie, H.H., Wei, X.Y., 2007. Two new antifungal alkaloids produced by Streptoverticillium morookaense. J. Antibiot. 60, 179–183. Huang, R.M., Zhou, X.F., Xu, T.H., Yang, X.W., Liu, Y.H., 2010. Diketopiperazines from marine organisms. Chem. Biodivers. 7, 2809–2829. Huang, R.M., Yi, X.X., Zhou, Y., Su, X., Peng, Y., Gao, C.H., 2014. An update on 2,5diketopiperazines from marine organisms. Mar. Drugs 12, 6213–6235. Kim, D.W., Woo, H.S., Kim, J.Y., Ryuk, J.A., Park, K.H., Ko, B.S., 2016. Phenols displaying tyrosinase inhibition from Humulus lupulus. J. Enzym. Inhib. Med. Chem. 31, 742–747. Liu, Y.N., Xue, J.H., Feng, N., Wu, P., Liu, X.Z., Wei, X.Y., 2007. A new cyclodipeptide from the cultures of Geotrichum candidum. Chin. Chem. Lett. 18, 1081–1083. Ma, Q., Xie, H.H., Li, S., Zhang, R.F., Zhang, M., Wei, X.Y., 2014. Flavonoids from the pericarps of Litchi chinensis. J. Agric. Food. Chem. 62, 1073–1078. Martins, M.B., Carvalho, I., 2007. Diketopiperazines: biological activity and synthesis. Tetrahedron 63, 9923–9932. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237. Tanaka, T., Nakashima, T., Ueda, T., Tomii, K., Kouno, I., 2007. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 55, 899–901. Xiao, Y.Y., Xie, H.H., Zhao, L., Gou, P., 2016. Acyl flavone and lignan glucosides from Leontopodium leontopodioides. Phytochem. Lett. 17, 247–250.
compound. The plates were incubated at room temperature for 30 min in the dark, then absorbance of each well was measured at 593 nm. Twenty μL of ferrous sulfate (FeSO4) at six different concentrations (1000, 500, 250, 125, 62.5, 31.25 μM) plus 20 μL of 10 mM TPTZ and 100 μL of 300 mM acetate buffer (pH 3.6) were used as a calibration curve. The final results were expressed as millimoles of FeSO4/g of compound (mmol/g). Acknowledgments We are grateful to Mr. Yunfei Yuan, South China Botanical Garden, Chinese Academy of Sciences, for NMR spectroscopic measurements and Ms. Aijun Sun, South China Sea Institute of Oceanology, Chinese Academy of Sciences, for HRESIMS measurements. We acknowledge support from NSFC grant (No. 81172942) and a research project from the Bureau of Science and Technology of Guangzhou Municipality (grant no. 201510010015). A.M. acknowledges the Chinese Academy of Sciences for support through the CAS President’s International Fellowship Initiative (2016PM032). 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.phytol.2017.04.012. 1D and 2D NMR spectra and HRESIMS of 2 and 3. References Benzie, I.F.F., Strain, J.J., 1999. Ferric reducing antioxidant power assay: direct measure
92
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Two new 2,5-diketopiperazines produced by Streptomyces sp. SC0581 a,b
a
Li Yang , Ahmed Mahal , Youhua Liu ⁎ Xiaoyi Weia,
a,b
a
a
a
a
, Hanxiang Li , Ping Wu , Jinghua Xue , Liangxiong Xu ,
MARK
a Key Laboratory of Plant Resources Conservation and Sustainable Utilization/Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, People’s Republic of China b University of Chinese Academy of Sciences, Yuquanlu 19A, Beijing 100049, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords: 2,5-diketopiperazines DOPA Streptomyces sp. Antioxidant
Two new diketopiperazines, cyclo(L-Phe-L-NMe-DOPA) (2) and cyclo[L-Phe-L-(NMe-3-(NMe-3-O-α-L-rhamnopyranosyl)-DOPA] (3), along with a known diketopiperazine (1), were isolated from the cultures of Streptomyces sp. SC0581. Their structures were elucidated by extensive spectroscopic analysis, single-crystal X-ray crystallographic analysis, and chemical correlation. Compounds 1 − 3 exhibited more potent ABTS radical cation scavenging activity (IC50 values: 3.7 − 14.6 μM) than L-ascorbic acid (IC50: 17.7 μM). Compounds 2 and 3 also showed remarkable DPPH radical scavenging activity.
1. Introduction
reported (Liu et al., 2007). In the present study, colorless crystals (mp 160 − 162 °C) of this compound were obtained in MeOH and subjected to X-ray diffraction analysis for the first time, with which its complete structure (Fig. 2), including the absolute configuration, was confirmed (Fig. 1). Compound 2 was obtained as a white powder. Its (−)-HRESIMS presented a pronounced ion peak at m/z 339.1339 [M − H]−, corresponding to a molecular formula of C19H20N2O4, with one more oxygen atom than that of 1. Its 1H NMR and 13C NMR data were closely similar to those of 1, except that the proton and carbon resonances for the p-hydroxyphenyl group in the spectra of 1 were replaced by those indicating the presence of a 3,4-dihydroxyphenyl group [δH 6.32 (H, dd, J = 8.0, 2.1 Hz), δH 6.67 (1H, d, J = 8.0 Hz), δH 6.51 (H, d, J = 2.1 Hz); δC 116.8 (CH), 118.5 (CH), 121.8 (CH), 128.2 (C), 145.7 (C), and 146.6 (C)] in 2, suggesting a 2,5-diketopiperazine composed of a phenylalanine and an NMe-DOPA. The planar structure of 2 was supported by 2D NMR data (Fig. 3). With regard to the stereochemistry, compound 2 showed a negative specific rotation value (−110.0) close to that of 1 (−164.0), allowing assignment of the same absolute configuration (3S,6S) as 1. Consequently, 2 was elucidated as cyclo(LPhe-L-NMe-DOPA). Compound 3, obtained as a white powder, had a molecular formula of C25H30N2O8 on the basis of the deprotonated ion peak at m/z 485.1935 observed in the (−)-HRESIMS. The 1H NMR and 13C NMR spectra of 3 were comparable to those of 2 except for the presence of additional signals (δH 5.34, 4.12, 3.95, 3.50, 3.83, 1.31; δC 101.6, 71.9, 72.2, 73.9, 70.8, 18.0) attributable to a rhamnopyranosyl moiety (Fan
2,5-Diketopiperazines, cyclic dipeptides produced by condensation of two α-amino acids, have been found in a variety of natural resources including bacteria, fungi, marine sponge, plants, and mammals (Huang et al., 2010, 2014). They possess a series of intriguing chemical properties, such as resistance to proteolysis, rigid backbone, constrained conformation, stereochemistry controlled at up to four positions, and 2 hydrogen bond donor and 2 hydrogen bond acceptor sites important for binding to various biological targets (Borthwick, 2012). In addition, they have been reported to exhibit diverse biological activities including antibacterial, antifungal, antiviral activities, cytotoxicity, and anti-inflammatory (Martins and Carvalho, 2007). Owing to these chemical and biological properties, 2,5-diketopiperazines have attracted much attention of researchers in synthetic chemistry, medicinal chemistry, and pharmacology. In the course of our searching for novel bioactive metabolites from soil-derived actinomycetes (Feng et al., 2007), two new 2,5-diketopiperazines (2 and 3), together with the known diketopiperazine cyclo(L-Phe-L-NMe-Tyr) (1), were isolated from the cultures of Streptomyces sp. SC0581. Herein, we report the isolation, structure elucidation, and antioxidant activity of these compounds. 2. Results and discussion The known compound cyclo(L-Phe-L-NMe-Tyr) (1) was identified by comparison of its spectroscopic data and specific rotation with those
⁎
Corresponding author. E-mail address: [email protected] (X. Wei).
http://dx.doi.org/10.1016/j.phytol.2017.04.012 Received 5 January 2017; Received in revised form 21 March 2017; Accepted 13 April 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 20 (2017) 89–92
L. Yang et al.
Table 1 1 H (500 MHz) and position
2
13
C (125 MHz) NMR Data of Compounds 2 and 3.
a
3b
δC, type 2 3 5 6 7a 7b 8 9 10 11 12 13 14a 14b 15 16,20 17,19 18 1-NCH3 4-NH 1′ 2′ 3′ 4′ 5′ 6′
Fig. 1. Structures of compounds 1 − 3.
a
Fig. 2. X-ray structure of 1.
b
δH, mult. (J in Hz)
166.4, C 57.2, CH 167.1, C 64.0, CH 37.4, CH2 128.2, C 118.5, CH 146.6, C 145.7, C 116.8, CH 121.8, CH 42.0, CH2 138.3, C 130.8, CH 129.5, CH 127.7, CH 33.7, CH3
3.88, m 4.03, t (4.7) 2.74, d (14.1, 4.7) 2.46, d (14.1, 4.7) 6.51, d (2.1)
6.67, 6.32, 2.43, 1.69,
d (8.0) dd (8.0, 2.1) dd (13.6, 4.6) dd (13.6, 8.2)
7.00, 7.27, 7.20, 2.81, 7.82,
d (7.4) t (7.4) t (7.4) s brs
δC, type 167.9, C 58.0, CH 168.6, C 64.7, CH 37.8, CH2 128.5, C 120.9, CH 148.3, C 146.2, C 117.6, CH 125.6, CH 41.8, CH2
δH, mult. (J in Hz)
4.01, dd (8.9, 3.8) 4.14, dd (4.6, 5.0) 2.93, dd (14.3, 4.6) 2.57, dd (14.3, 5.0) 6.92, d (2.0)
6.86, 6.70, 2.73, 1.61,
d (8.0) dd (8.1, 2.0) dd (13.6, 3.8) dd (13.6, 8.9)
137.7, C 130.7, CH 129.8, CH 128.0, CH 33.9, CH3
7.05, 7.32, 7.22, 2.93,
d (7.4) t (7.4) t (7.4) s
101.6, CH 71.9, CH 72.2, CH 73.9, CH 70.8, CH 18.0, CH3
5.34, 4.12, 3.95, 3.50, 3.83, 1.31,
d (1.7) dd (3.5, 1.7) dd (9.5, 3.5) t (9.5) dd (9.5, 6.2) d (6.2)
DMSO-d6 used as solvent. CD3OD used as solvent.
Table 2 Antioxidant activity of compounds 1 − 3.
Fig. 3. Key 1H–1H COSY (bold lines) and HMBC (arrows) correlations of 2.
compound
ABTS (IC50, μM)
DPPH (IC50, μM)
FRAP (mmol/g)
1 2 3 L-ascorbic acid
3.7 ± 0.13 14.6 ± 0.70 7.1 ± 0.83 17.7 ± 0.40
> 250 37.2 ± 0.96 27.7 ± 0.09 36.2 ± 0.18
0.21 ± 0.002 7.15 ± 0.079 6.87 ± 0.014 13.31 ± 0.030
Values represent means ± standard deviation (SD) (n = 4).
et al., 2015). The glycosylation site was located at C-10 based on the long-range correlation from H-1′ to C-10 (δC 148.3) observed in the HMBC spectrum. The coupling constant (J = 1.7 Hz) of the anomeric proton (H-1′) indicated an α orientation of the glycosidic linkage. The L configuration of the rhamnose moiety was determined using the method described by Tanaka et al. (2007). The 3S,6S configurations of 3 were assigned by the same negative sign of specific rotation value of 1 and 2, and on the basis of the co-occurrence of 2 and 3. Thus, the structure of 3 was defined as cyclo[L-Phe-L-(NMe-3-O-α-L-rhamnopyranosyl)-DOPA]. Antioxidants play significant role in inhibiting or delaying the oxidation of susceptible cellular substrates and they are important for prevention of many oxidative stress-mediated diseases. Consequently, the antioxidant activity of compounds 1 − 3 was evaluated using ABTS radical cation and DPPH radical scavenging tests and FRAP assay. The results were listed in Table 2. The isolated compounds (1 − 3) displayed ABTS radical cation scavenging activity with IC50 values ranging from 3.7 to 14.6 μM, which were all superior to that of reference compound, L-ascorbic acid (IC50 value: 17.7 μM). In the DPPH radical scavenging assay, compounds 2 and 3 exhibited the activity comparable to that of L-ascorbic acid, but compound 1 was inactive. In FRAP assay, compounds 2 and 3 showed only a mild activity. Tyrosinase inhibitory activity was also evaluated for compounds 1 − 3 using the previously described method (Kim et al., 2016), but none of them was found to be active at the highest test concentration (250 μM).
3. Experimental 3.1. General experimental procedures Optical rotations were recorded on a Perkin-Elmer 343 spectropolarimeter. UV spectra were measured with a Perkin EImer Lambda 650 UV/VIS spectrometer. 1D and 2D NMR experiments were performed on a Bruker Avance III 500 Hz instrument. HR-ESIMS data were recorded on a Bruker maXis Q-TOF mass spectrometer. Preparative HPLC was run on a Shimadzu Shim–packed Pro-ODS column (20 mm × 25 cm). For column chromatography, silica gel 60 (100–200 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), YMC ODS (75 μm, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used. TLC was carried out on HSGF254 silica gel plates (0.2 mm, Yantai Jiangyou silica gel Development Co. Ltd., Yantai, China); spots were visualized by spraying with 10% H2SO4 in EtOH followed by heating. 3.2. Biological material and fermentation The actinomycetes strain Streptomyces sp. SC0581 was isolated from a soil sample collected in Dinghu Mountain Biosphere Reserve, Guangdong, China, and authenticated based on morphological characteristics and DNA sequence analysis of the ITS region (GenBank accession no: KX687558). A voucher strain is deposited in the culture 90
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3.5. Determination of absolute configuration of sugar moiety
collection of South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. The strain was grown on the potato dextrose agar at 28 °C for 10 days. Six pieces of culture plugs of the strain were inoculated into each of two 500 mL Erlenmeyer flasks containing 150 mL YMG medium consisting of 0.4% glucose, 1.0% malt extract, 0.4% yeast extract. The flasks were shaken on a rotatory (150 rpm) at 28 °C for 2 days. Then, the cultures were transferred into three 3 L flasks containing 1 L of YMG with the same incubation condition. Finally, 10 mL culture broth was transferred into each of two hundreds 500-mL flasks containing 60 mL of YMG medium and 60 g of wheat grains and incubated in the dark at 28 °C for 40 days.
Compound 3 (2 mg) was hydrolyzed with 5 mL of 2 M aqueous HCl at 100 °C for 4 h. After removal of the solvent under reduced pressure, the resulting hydrolysate was dissolved in 5 mL of distilled water, and then partitioned with 5 mL of EtOAc for three times. The aqueous layer, after concentrated to dryness, was dissolved in 2 mL of pyridine containing 1 mg/mL L-cystein methyl ester hydrochloride and heated at 60 °C for 1 h. Then, O-tolylisothiocyanate (4 μL) was added and kept at 60 °C for additional 1 h. The reaction mixture was concentrated to dryness under vacuum. The residue was dissolved in MeOH and analyzed by HPLC at 40 °C on an SPOLAR C18 column (4.6 × 250 mm, 5 μm) using CH3CN/H2O/HCOOH (25:75:0.1, v/v/v) at a flow rate of 0.8 mL/min for 60 min. Peaks were detected at 250 nm. The absolute configuration of rhamnose was clarified by comparison of retention time of its derivative with that of standard L-rhamnose derivative (tR = 35.1 min) prepared in the same manner (Tanaka et al., 2007; Xiao et al., 2016; Fan et al., 2015).
3.3. Extraction and isolation The solid cultures were extracted with 95% EtOH three times. The resultant crude extract was sequentially partitioned with petroleum ether, EtOAc, and n-BuOH. The EtOAc- and n-BuOH-soluble fractions were combined and subjected to silica gel column chromatography (CC) with CHCl3/MeOH mixtures of increasing polarity (100:0 to 70:30) to afford twenty fractions (Frs. 1 − 20). Fr. 2 was further separated by ODC CC using aqueous MeOH (20% − 70%) to give ten subfractions (Frs. 2-1 − 2-10). Fr. 2-2, obtained on elution with 40% aqueous MeOH, yielded crystals in MeOH, which were collected by filtration to obtain 1 (30 mg). Fr. 6 was also subjected to ODC CC eluted with aqueous MeOH (20 − 70%) to give six subfractions (Frs. 6-1 − 6-6). Fr. 6-3 was purified by preparative HPLC (flow rate 5 mL/min) using 30% aqueous MeOH to give 2 (10 mg, tR = 72.8 min). Fr. 10 was applied to ODC CC using aqueous MeOH (10 − 60%) to give 5 subfractions (Frs. 10-1 − 10-5). Fr. 10-2 was purified by preparative HPLC (flow rate 5 mL/min) using 28% aqueous MeOH to give 3 (13 mg, tR = 54.2 min).
3.6. Antioxidant assays 3.6.1. ABTS radical cation scavenging assay ABTS radical cation scavenging activity was tested by previously reported method (Re et al., 1999; Ma et al., 2014). 7 mM ABTS in water was mixed with 2.45 mM potassium persulfate and kept in the dark at room temperature for 12–16 h. Then, the ABTS radical cation solution was diluted with buffered saline (PBS), pH 7.4, to obtain an absorbance of 0.7 ± 0.02 units at 734 nm. Test compounds were dissolved in DMSO and two-fold diluted to serial concentrations. Compound solution (5 μL) and ABTS radical cation solution (195 μL) were mixed and added to 96-well plates. L-ascorbic acid was used as reference compound. The control wells contained DMSO instead of compound solution, and the blank wells were added with PBS in place of ABTS radical cation solution. After a mixing time of 15 s and an incubation period of 6 min at room temperature in the dark, OD value of each well was read at 415 nm. The percentage inhibition was calculated using the formula as below:
3.3.1. Cyclo(L-Phe-L-NMe-DOPA) (2) White powder; [α] D20 −110.0 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 202 (4.60), 282 (3.57); 1H and 13C NMR data, see Table 1; (−)-HRESIMS m/z 339.1339 [M − H] − (calcd for C19H19N2O4, m/z 339.1345).
ABTS%+ scavenging effect (%) = [1–(ODcompound − ODblank)/ (ODcontrol − ODblank)] × 100
3.3.2. Cyclo[L-Phe- L-(NMe-3-O-α-L-rhamnopyranosyl)-DOPA] (3) White amorphous powder; [α] D20 −112.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.17), 283 (3.12); 1H and 13C NMR data, see Table 1; (−)-HRESIMS m/z 485.1935 [M − H] − (calcd for C25H29N2O8, m/z 485.1924).
3.6.2. DPPH radical scavenging assay DPPH radical scavenging assay was performed according to a previously described method (Brand-Williams et al., 1995; Ma et al., 2014). Test compounds were dissolved in DMSO and two-fold diluted to serial concentrations. The mixture of test compound solution including L-ascorbic acid as positive reference (5 μL) and 195 μL of 0.1 mM DPPH was added to 96-well plates. The control wells contained DMSO instead of compound solution, and the blank wells were added with MeOH in place of DPPH radical solution. The plates were incubated at room temperature for 30 min in the dark, then absorbance of each well was measured at 517 nm. The DPPH radical scavenging activity in percentage of compounds was calculated as follows:
3.4. X-ray diffraction analysis of 1 The single-crystal X-ray diffraction data for 1 were collected on Agilent Xcalibur Nova single-crystal diffractometer using Cu Kα radiation. Crystal data of 1: C19H20N2O3, M = 324.37, 0.42 × 0.40 × 0.36 mm3, monoclinic, space group: P 1 21 1, a = 8.82950(13) Å, b = 11.04848(14) Å, c = 8.89756(13) Å, α = γ = 90°, β = 107.8744(15)°, V = 826.08(2) Å3, Z = 2, ρ = 1.304 Mg/m3, F000 = 344. Cu Kα radiation, λ = 1.54184 Å, T = 150(2) K, theta range for data collection: 5.22 to 66.98°; 11303 reflections collected, 2909 unique (Rint = 0.0200), completeness to theta = 66.98° (99.5%), max. and min. transmission: 0.7813 and 0.7515. The structure was refined by full-matrix least-squares on F2. Final GooF = 1.172, final R indices [I > 2sigma(I)]: R1 = 0.0315, wR2 = 0.0804, R indices (all data): R1 = 0.0319, wR2 = 0.0809; absolute structure parameter: Hooft = 0.09(16); largest diff. peak and hole: 0.220 and −0.339 e.Å−3. Crystallographic data for the structure of 1 have been deposited at the Cambridge Crystallographic Data Centre: reference number CCDC 1522635. This data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html or from CCDC, 12 Union Road, Cambridge, CB21EZ, UK; (fax: +44-(0) 1223 336033 or e-mail: [email protected]).
DPPH scavenging effect (%) = [1–(ODcompound − ODblank)/ (ODcontrol − ODblank)] × 100
3.6.3. FRAP assay FRAP assay was carried out followed by previously described procedure (Benzie and Strain, 1999; Ma et al., 2014). FRAP solution was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl, and 20 mM aqueous ferric chloride (FeCl3) solution in the ratio of 10:1:1 (v/v/v). Test compounds were dissolved in DMSO. Compound solution (20 μL) was mixed with 180 μL of FRAP and added to 96-well plates. L-ascorbic acid was used as a reference 91
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of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–27. Borthwick, A.D., 2012. 2,5-Diketopiperazines: synthesis, reactions, medicinal chemistry, and bioactive natural products. Chem. Rev. 112, 3641–3716. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci. Technol. 28, 25–30. Fan, L., Liao, C.H., Li, S.G., Huang, X.J., Hu, X.P., Song, X., Fan, C.L., Wang, Y., Ye, W.C., Kang, Q.R., Zheng, K., Liu, L.P., Jiang, Y.C., Fan, X.M., Zhang, J., Li, Y., Zeng, Y., He, Z.D., 2015. Phenylethanoid and secoiridoid glycosides from the leaves of Ligustrum purpurascens. Phytochem. Lett. 13, 177–181. Feng, N., Ye, W.H., Wu, P., Huang, Y.C., Xie, H.H., Wei, X.Y., 2007. Two new antifungal alkaloids produced by Streptoverticillium morookaense. J. Antibiot. 60, 179–183. Huang, R.M., Zhou, X.F., Xu, T.H., Yang, X.W., Liu, Y.H., 2010. Diketopiperazines from marine organisms. Chem. Biodivers. 7, 2809–2829. Huang, R.M., Yi, X.X., Zhou, Y., Su, X., Peng, Y., Gao, C.H., 2014. An update on 2,5diketopiperazines from marine organisms. Mar. Drugs 12, 6213–6235. Kim, D.W., Woo, H.S., Kim, J.Y., Ryuk, J.A., Park, K.H., Ko, B.S., 2016. Phenols displaying tyrosinase inhibition from Humulus lupulus. J. Enzym. Inhib. Med. Chem. 31, 742–747. Liu, Y.N., Xue, J.H., Feng, N., Wu, P., Liu, X.Z., Wei, X.Y., 2007. A new cyclodipeptide from the cultures of Geotrichum candidum. Chin. Chem. Lett. 18, 1081–1083. Ma, Q., Xie, H.H., Li, S., Zhang, R.F., Zhang, M., Wei, X.Y., 2014. Flavonoids from the pericarps of Litchi chinensis. J. Agric. Food. Chem. 62, 1073–1078. Martins, M.B., Carvalho, I., 2007. Diketopiperazines: biological activity and synthesis. Tetrahedron 63, 9923–9932. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237. Tanaka, T., Nakashima, T., Ueda, T., Tomii, K., Kouno, I., 2007. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 55, 899–901. Xiao, Y.Y., Xie, H.H., Zhao, L., Gou, P., 2016. Acyl flavone and lignan glucosides from Leontopodium leontopodioides. Phytochem. Lett. 17, 247–250.
compound. The plates were incubated at room temperature for 30 min in the dark, then absorbance of each well was measured at 593 nm. Twenty μL of ferrous sulfate (FeSO4) at six different concentrations (1000, 500, 250, 125, 62.5, 31.25 μM) plus 20 μL of 10 mM TPTZ and 100 μL of 300 mM acetate buffer (pH 3.6) were used as a calibration curve. The final results were expressed as millimoles of FeSO4/g of compound (mmol/g). Acknowledgments We are grateful to Mr. Yunfei Yuan, South China Botanical Garden, Chinese Academy of Sciences, for NMR spectroscopic measurements and Ms. Aijun Sun, South China Sea Institute of Oceanology, Chinese Academy of Sciences, for HRESIMS measurements. We acknowledge support from NSFC grant (No. 81172942) and a research project from the Bureau of Science and Technology of Guangzhou Municipality (grant no. 201510010015). A.M. acknowledges the Chinese Academy of Sciences for support through the CAS President’s International Fellowship Initiative (2016PM032). 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.phytol.2017.04.012. 1D and 2D NMR spectra and HRESIMS of 2 and 3. References Benzie, I.F.F., Strain, J.J., 1999. Ferric reducing antioxidant power assay: direct measure
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