Note pubs.acs.org/jnp
Parvifloranines A and B, Two 11-Carbon Alkaloids from Geijera parvif lora Qingyao Shou,*,† Linda K. Banbury,† Dane E. Renshaw,† Joshua E. Smith,† Xiaoxiang He,† Ashley Dowell,† Hans J. Griesser,‡ Michael Heinrich,†,§ and Hans Wohlmuth† †
Southern Cross Plant Science, Southern Cross University, PO Box 157, Lismore NSW 2480, Australia Ian Wark Research Institute, University of South Australia, Mawson Lakes SA 5095, Australia § Centre for Pharmacognosy and Phytotherapy, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom ‡
S Supporting Information *
ABSTRACT: Two novel alkaloids (parvifloranines A and B), possessing an unusual 11-carbon skeleton linked with amino acids, were isolated from Geijera parvif lora, an endemic Australian Rutaceae. Their structures were elucidated by extensive spectroscopic measurements including 2D NMR analyses. Parvifloranine A was found to be a mixture of two enantiomers, (S)-1 and (R)-1, in a ratio of 1:4, based on their separation using a chiral column. Parvifloranine B is also believed to be a mixture of enantiomers. Proposed biosynthetic pathways are discussed. Parvifloranine A inhibited the synthesis of nitric oxide in LPSstimulated RAW 264.7 macrophages with an IC50 value of 23.4 μM. coumarin at δH 6.22 (d, J = 9.5 Hz, H-3) and δH 7.84 (d, J = 9.5 Hz, H-4); three protons appearing as an ABX spin system [δH 6.92 (d, J = 2.3 Hz, H-8), δH 6.95 (dd, J = 8.6, 2.3 Hz, H-6), and δH 7.52 (d, J = 8.6 Hz, H-5)] on the B ring; five methylenes containing an oxymethylene at δH 4.94 (d, J = 5.6 Hz); two methylenes attached to a nitrogen atom [δH 4.06, 4.16 (d, J = 13.9 Hz, H-8″), δH 3.17, 3.57 (m, H-5‴)]; two aliphatic methylenes [δH 1.89, 2.05 (m, H-4‴), δH 2.07, 2.40 (m, H3‴)]; one trisubstituted double bond at δH 6.61 (t, J = 5.6 Hz, H-2′); a methine attached to a nitrogen atom at δH 3.82 (m, H2‴); three methyls containing one olefinic methyl at δH 2.11 (s, H-4′); and two aliphatic methyls at δH 1.39 (s, H-6″) and δH 1.40 (s, H-7″). The coumarin moiety was fully established based on 1H and 13C chemical shifts and proton coupling patterns. An HMBC correlation between δH 4.94 (d, J = 5.6 Hz, H-1′) and δc 163.3 (C-7) (Figure 1) of the coumarin skeleton was observed, indicating the presence of a side chain attached to C-7. A close comparison of the carbon chemical shift (Table 1) of 1 with geiparvarin (a major coumarin in this species) suggested a geiparvarin derivative with a pyrrolidine ring substituent. In the 1H−1H COSY spectrum of 1, homonuclear coupling correlations of H1-2‴/H2-3‴/H2-4‴/H2-5‴ and HMBC
T
he genus Geijera Schott (Rutaceae) consists of six species, which are native to Australia, New Guinea, and New Caledonia.1 G. parvif lora Lindl. (Rutaceae), commonly called wilga or native willow, is endemic to eastern mainland Australia and widespread in Queensland and New South Wales, with a southern limit in northwestern Victoria.2 Previous chemical investigations have identified coumarins,3,4 alkaloids,4 and hydrocarbons5 from its leaves and fruits, and the composition of the essential oil has also been reported.6 As part of our efforts to explore new compounds with biological activity from Australian native plants, we isolated and characterized two novel alkaloids, parvifloranines A (1) and B (2), from G. parvif lora. Parvifloranine A was tested to assess its cytotoxicity and capacity to inhibit the synthesis of nitric oxide in LPSstimulated RAW 264.7 macrophages and for activity against bacteria associated with infections of wounds, biomedical devices, and implants. Parvifloranine A (1) was obtained as a yellow oil. It had a molecular formula of C25H27NO7 as determined by HRESIMS at m/z 476.1686 [M + Na]+ (calcd 476.1685), indicating 13 degrees of unsaturation. The IR spectrum showed absorption bands of a lactone group (1731 cm−1) and a carboxylic group (3392 and 1696 cm−1). The 13C NMR spectrum (Table 1) revealed 25 resonances ascribed to three methyl groups, five methylenes, seven methines, and 10 quaternary carbons. The 1 H NMR spectrum (Table 1) displayed H-3 and H-4 of a © 2013 American Chemical Society and American Society of Pharmacognosy
Received: May 9, 2013 Published: July 12, 2013 1384
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Table 1. NMR Spectroscopic Data for Parvifloranine A, Parvifloranine B, and Geiparvarin (δ in ppm, J in Hz)a parvifloranine A δH
no. 2 3 4 5 6 7 8 4a 8a 1′ 2′ 3′ 4′ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 2‴ 3‴
6.22, d (9.5) 7.84, d (9.5) 7.52, d (8.6) 6.95, dd (2.3, 8.6) 6.92, d (2.3)
4.94, d (5.6) 6.61, t (5.6) 2.11, s
1.39, s 1.40, s a, 4.16, d (13.9) b, 4.06, d (13.9) 3.82, m a, 2.40, m
parvifloranine B δC
163.3 113.9 145.8 130.8 114.3 163.3 102.9 114.6 157.1 66.4 136.5 131.1 14.7 187.6 105.1 208.3 89.4 23.1 23.2 48.1 70.7 30.4
b, 2.07, m 4‴ 5‴ 6‴ a1
a, 2.05, m b, 1.89, m a, 3.57, m b, 3.17, m
6.27, 7.90, 7.58, 7.01,
geiparvarin
δH
δC
δC
d (9.5) d (9.5) d (8.4) overlap
163.3 113.9 145.9 130.8 114.4
161.5 113.6 143.4 130.6 113.1
163.4 102.9 114.6 157.3 66.4 136.4 130.9 14.6 185.9 105.4 208.5 89.2 23.1 23.1 40.9
161.1 100.4 113.1 156.0 65.4 129.1 128.9 13.9 183.0 101.7 207.3 88.8 23.2 23.2
6.99, brs
4.98, d (5.6) 6.60, t (5.6) 2.14, s
1.44, s 1.44, s a, 4.19, d (13.9) b, 4.07, d (13.9) 3.93, dd (3.4, 9.7) a, 3.01, dd (3.4, 17.0) b, 2.74, dd (9.7, 17.0)
HRESIMS at m/z 493.1590 [M + Na]+ (calcd 493.1587), indicating 13 degrees of unsaturation. Detailed analyses of 1D and 2D NMR data of 2 (Table 1) revealed a high degree of similarity between the structures of 1 and 2 in the geiparvarin moiety. In comparison with 1, the chemical shift of C-8″ (from δC 48.1 to 40.9) and C-2‴ (from δC 70.1 to 60.0) suggested a secondary amine at position 1‴ instead of a tertiary amine. Three protons at δH 3.93 (1H, dd, J = 3.4, 9.7 Hz), δH 3.01 (1H, dd, J = 3.4, 17.0 Hz), and δH 2.74 (1H, dd, J = 9.7, 17.0 Hz) appeared as an ABX system, assignable to H-2‴ and H2-3‴ of an asparagine substituent. The two carbonyl groups at δC 175.8 (C-4‴) and 175.0 (C-5‴) and the HMBC correlations of H-3‴ with C-2‴, C-4‴, and C-5‴ (Figure 1) together with two nitrogen atoms in 2 indicated the presence of an asparagine substituent. The mass fragmentation pattern was also consistent with the postulated structure of compound 2 (Figure 4, Supporting Information). Thus the structure of compound 2 was confirmed, and it was named parvifloranine B. The absolute configurations of 1 and 2 were deduced by comparison of the experimental and calculated ECD spectra generated for enantiomers (S)-1 and (S)-2 using Gaussian09.8 The experimental CD spectra of 1 and 2 were similar to mirror images of the ECD curves of (S)-1 and (S)-2, respectively (Figure 2). Therefore, the absolute configuration of both 1 and
60.0 34.6
24.7 56.2
175.8
173.2
175.0
H (500 MHz) and 13C (125 MHz) data in methanol-d4.
Figure 1. Key HMBC correlations for compounds 1 and 2.
correlations of H-2‴/C-3‴, C-4‴, C-6‴ and H-3‴/C-2‴, C-4‴, C-5‴, C-6‴ (Figure 1) together with the shifts of these protons and carbon resonances established a 2-pyrrolidinecarboxylic acid moiety. The HMBC correlations of H-8″ with C-2″, C-3″, C-4″, C-2‴, and C-5‴ supported the assignment that the 2pyrrolidinecarboxylic acid substituent was attached to C-3″ through a methylene. The mass spectrum of 1 revealed fragment ion peaks at m/z 408 and 339, which confirmed the presence of a 2-pyrrolidinecarboxylic acid (Figure 4, Supporting Information). The E configuration of the 2′−3′ double bond was assigned based on the carbon chemical shift of C-4′ at δC 14.7 compared with δC 22.3 of the E configuration.7 Thus the structure of 1 was established, and it was named parvifloranine A. Parvifloranine B (2) was obtained as a light yellow oil. Its molecular formula was determined to be C24H26N2O8 by
Figure 2. Experimental CD spectra of 1 and 2 and calculated ECD spectra of (S)-1 and (S)-2.
2 was tentatively deduced to be 2‴R. However, the calculation showed that the strength of the predicted CD was much larger than the experimental CD, indicating that the compounds might be mixtures of two enantiomers. This led us to conduct a chiral analysis, and compound 1 was confirmed to be a mixture of two enantiomers, (S)-1 and (R)-1, in a ratio of 1:4, based on its separation using a chiral column (Figure 3). Unfortunately, 1385
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Packard 8453 polarimeter at room temperature. IR spectra were recorded on a Bruker Tensor 27 FT IR spectrometer. CD spectra were measured on a Jasco J-815 CD spectropolarimeter at room temperature. High-resolution electrospray ionization (HRESIMS) accurate mass measurements were carried out on a Bruker microTOF-Q instrument with a Bruker ESI source. NMR spectra were acquired on a Bruker AVANCE 500 MHz spectrometer with TMS as the internal standard. Column chromatography (CC) separations were carried out using silica gel (Silica-Amorphous, precipitated, 200−425 mesh, Sigma-Aldrich), Sepra C18-E (50 μm, 65A; Phenomenex Torrance, CA, USA), Sephadex LH-20 (Sigma), and MCI gel CHP20P (Supelco, Bellafonte, PA, USA). Preparative HPLC was performed on a Gilson 322 system with a UV/vis-155 detector and an FC204 fraction collector using a Phenomenex Luna 5 μm (150 × 21.2 mm i.d.) C-18 column. Plant Material. The leaves of G. parvif lora were collected near Lightning Ridge, New South Wales, Australia (S 29°11′; E 147°52′), in December 2011 and identified by one of the authors (H.W.). A voucher specimen (PHARM110063) has been deposited in the Medicinal Plant Herbarium at Southern Cross University. Extraction and Isolation. The dried leaves of G. parvif lora (2 kg) were powdered and extracted with 95% ethanol at room temperature. The EtOH extract was suspended in H2O and partitioned with CHCl3. The CHCl3 portion was evaporated under reduced pressure to afford a crude extract (167.5 g). The crude CHCl3 extract was subjected to MCI gel (CHP20P) column chromatography, eluted with a gradient of MeOH/H2O (80:20−100:0), to give 10 fractions (A−J). Fraction A was separated on a C18 column (5 × 40 cm) with MeOH/H2O (5:5, 7:3) to give fractions AI−AIV. AII (280 mg) was further purified by CC over Sephadex LH-20 to give parvifloranine A (40 mg). AI (130 mg) was fractionated by preparative HPLC [Gilson 322 system with a UV/vis-155 detector and a FC204 fraction collector, fitted with a Phenomenex Luna 5 μm (150 × 21.2 mm i.d.) C-18 column; mobile phase CH3CN and H2O containing 0.05% TFA (0−5 min: 20% CH3CN, 5−17 min: 20−60% CH3CN, 17−20 min: 60−20% CH3CN); flow rate 20 mL/min; UV detection at 210 and 280 nm] to give parvifloranine B (5 mg). Parvifloranine A (1): yellow oil; [α]25D −34 (c 1.04, MeOH); UV λmax (MeOH) (log ε) 205.0 nm (5.85), 318.0 nm (4.49); CD λmax (MeOH) Δε283 +4.39, Δε326 −5.10; IR (neat) νmax 3392, 2979, 1731, 1696, 1614, 1404, 1127, 1016, 838, 734 cm−1; 1H and 13C NMR (Table 1); APCIMS m/z 454.2 [M + H]+, 408.1, 339.1: HRESIMS m/ z 476.1686 [M + Na]+ (calcd 476.1685 for C25H27NNaO7). Parvifloranine B (2): light yellow oil; [α]25D −18 (c 0.05, MeCN); UV λmax (MeOH) (log ε): 203.0 (5.62) nm, 318.0 (4.47) nm; CD λmax (MeOH) Δε282 +3.00, Δε330 −3.72; IR (neat) νmax 3453, 1706, 1691, 1678, 1642, 1613, 1200, 1136, 838, 722 cm−1; 1H and 13C NMR (Table 1); APCIMS m/z 471.2 [M + H]+, 454.1, 407.9, 339.0; HRESIMS m/z 493.1590 [M + Na] + (calcd 493.1587 for C24H26N2NaO8). Computational Details. The conformational searches were performed using the MMFF94S force field, and the low-energy conformations (0−6 kcal/mol) were further optimized at the B3LYP/ 6-311+G(d) level in the gas phase. The methods have been widely used and also well summarized.11 Only the conformations with relative energy from 0 to 2.5 kcal/mol were used for CD computations at the B3LYP/6-311+G(d,p) level. Gibbs free energy was used in the CD prediction; UV correction was also performed. Chiral HPLC Analysis. Parvifloranine A (1, 0.5 mg) was dissolved in MeOH (2 mL) and filtered. Chiral HPLC analysis was carried out on an Agilent 1200 system (binary pump, column oven, autosampler, DAD), using a Lux 5u Cellulose-2 column (100 × 4.6 mm, 5 μm) and a mixture of MeCN/H2O/TFA (25:75:0.1) as eluent, applying a total flow rate of 1 mL/min and ambient temperature. Cytotoxicity Assay. Cytotoxicity in RAW 264.7 murine leukemic monocyte-macrophages (ATCC, Manassas, VA, USA) was assayed in 96-well plates using the ATPlite assay kit (PerkinElmer, Glen Waverley, Australia) with chlorambucil (Sigma C0253) as a positive control. Cells were grown in clear 96-well plates. The growth medium consisted of color-free Dulbecco’s modified Eagle’s medium containing
Figure 3. Chiral HPLC analysis of compound 1.
we were not able to perform a chiral separation of compound 2, because it proved to be unstable, as discussed below, but we speculate that it also contained two enantiomers. The origin of parvifloranines A and B can be rationalized biosynthetically and traced back to geiparvarin (Scheme 1, Supporting Information). Position 3″ of geiparvarin is nucleophilic due to the presence of 1″-oxygen and can participate in a Mannich-type reaction. Proline and asparagine can react with an aldehyde group to give Schiff bases, which upon attachment to the C (3″) carbon atom of geiparvarin could then lead to compounds 1 and 2 through a Mannich-type reaction.9 To the best of our knowledge, parvifloranines A (1) and B (2) represent a new 11-carbon skeleton linked with amino acids from a natural source, which is of interest in the context of chemotaxonomy, plant biochemistry, and synthetic chemical research. During our investigations it became clear that 2 was unstable when stored in an NMR tube with methanol-d4 for two months. Two byproducts sharing the same molecular weight as 2 but with different UV absorption spectra were observed. This might be due to the presence of an active secondary amine, which could be involved in the formation of a tetrahydropyridine ring through a Michael addition reaction (Figure 5, Supporting Information). The lack of stability of parvifloranine B needs to be further investigated. The nitric oxide (NO) inhibitory activity of 1 was evaluated in lipopolysaccharide-stimulated RAW 264.7 mouse macrophages using the Griess reaction.10 The IC50 (95% CI) value for NO inhibition was 23.4 (18.2−30.0) μM, indicating a moderate degree of inhibition. The EC50 value for in vitro cytotoxicity of compound 1 evaluated in the same cell line was 74.7 (55.9− 99.9) μM. The selectivity index for 1, i.e., the ratio between the EC50 value for cytotoxic activity and the IC50 value for the assay of interest, was 3.2. Compound 1 did not inhibit TNF-α production in LPS-stimulated RAW 264.7 cells or prostaglandin E2 production in calcium ionophore A23187-stimulated 3T3 Swiss albino mouse fibroblast cells. It also showed no measurable activity, within the limits of solubility (245 μg/ mL), against several bacteria relevant to human health and infection of wounds, biomedical devices, and implants, viz., Staphylococcus aureus (MRSA and MSSA strains), S. epidermidis, Pseudomonas aeruginosa, and Escherichia coli.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a Polarriz-D DPL polarimeter (Nippon Optical Works Co., Ltd., Tokyo, Japan). UV spectra were measured on a Hewlett1386
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10% (v/v) fetal bovine serum (FBS; Interpath, Heidelberg, Australia), L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (200 U/ mL), and streptomycin (200 μg/mL) (all from Invitrogen, Mulgrave, Australia). Cells were plated out at a concentration of 30 000 cells/well (90 μL cell suspension/well), test, control compounds dissolved in DMSO at six concentrations and further diluted 20-fold in media were added to the cell suspension at 10 μL/well, and the plates were incubated at 37 °C with 5% CO2 for 24 h. Following incubation, cell lysates were assayed for ATP with the ATPlite assay kit as per the manufacturer’s instructions. Briefly, all kit components were equilibrated to room temperature. Mammalian cell lysis solution (50 μL) was added to each well of the cell culture microplate, the plate was shaken on an orbital microplate shaker (500 rpm, 5 min), then substrate solution (50 μL/well) was added, and the plate was further shaken (500 rpm, 5 min). The plate was dark adapted for 10 min and the luminescence measured on a Wallac 1450 Microbeta luminescence counter (Wallac, Turku, Finland). Half-maximal effective concentration (EC50) values were calculated using GraphPad Prism version 4 (La Jolla, CA, USA). All samples and standards were assayed in triplicate. Nitrite (Griess) Assay. RAW 264.7 cells were cultivated as described above. Cell suspensions (120 μL/well, 106 cells/mL) were added to the wells of a 96-well microplate and incubated for 20 h (37 °C, 5% CO2), after which test compounds (dissolved in DMSO and further diluted 20-fold in media) were added to the cell suspension at 10 μL/well. Following incubation for 1 h, LPS solution (10 μL/well, 10 μg/mL) was added and the plate incubated for a further 20 h. Following this incubation, the plate was centrifuged (1500g, 3 min), and 90 μL of the supernatant transferred to a clear flat-bottom assay plate (PerkinElmer, Glen Waverley, VIC, Australia) and assayed immediately for nitrite. Nitrite standards (0−100 μM) were prepared in media. A 90 μL amount of each standard and cell supernatant were transferred to a flat-bottom microplate (Greiner Bio-One, Frickenhausen, Germany) with 90 μL of Griess reagent (0.1% N-1naphthylethylenediamine dihydrochloride, 1% sulfanilic acid in 5% phosphoric acid) added to each well, followed by incubation (23 °C, 20 min) on an orbital plate shaker. Following incubation the absorbance was read at 550 nm in a Wallac Victor 2 plate reader (Wallac, Turku, Finland). Samples and controls were assayed in triplicate. Standard curves were calculated for nitrite standards, and R2 values determined to verify linearity. Mean and standard deviation were calculated for replicates. The nitric oxide (as measured by nitrite) production in sample wells was calculated as a percentage of the production in solvent control wells. Assays for antibacterial activity and inhibition of TNF-α and PGE2 were carried out as previously reported.12
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G. Macfarlane of the School of Chemistry and Molecular Biosciences at the University of Queensland, for determining the accurate mass of the compounds, and to Ms. H. Mon of the Ian Wark Research Institute, University of South Australia, for performing the bacterial assays.
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REFERENCES
(1) Mabberley, D. J. Mabberley’s Plant-book: A Portable Dictionary of Plants, Their Classification and Uses, 3rd ed.; Cambridge Press University: Cambridge, USA, 2008; p 353. (2) Duretto, M., F. Rutaceae. In Flora of Victoria, 4th ed.; Walsh, N. G., Entwisle, T. J., Eds.; Inkata Press: Melbourne, Australia, 1999; p 153. (3) Lahey, F.; Macleod, J. Aust. J. Chem. 1967, 20, 1943. (4) Dreyer, D. L.; Lee, A. Phytochemistry 1972, 11, 763. (5) Jones, R. V. H.; Sutherland, M. D. Aust. J. Chem. 1968, 21, 2255. (6) Brophy, J. J.; Goldsack, R. J.; Forster, P. I. J. Essent. Oil Res. 2005, 17, 169. (7) Chimichi, S.; Boccalini, M.; Salvador, A.; Dall’Acqua, F.; Basso, G.; Viola, G. ChemMedChem. 2009, 4, 769. (8) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratemann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuk, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 09 User’s Reference; Gaussian Inc.: Carnegie, PA, USA, 2009. (9) Dewick, P. M. Medicinal Natural Products. A Biosynthetic Approach, 2nd ed.; John Wiley & Sons, Ltd: London, UK, 2002; p 349. (10) Tsikas, D. J. Chromatogr. B 2007, 851, 51. (11) Zhu, H. J. Modern Organic Stereochemistry; Science Presses of China: Beijing, 2009; Chapters 1 and 6. (12) Shou, Q.; Banbury, L. K.; Renshaw, D. E.; Lambley, E. H.; Mon, H.; Macfarlane, G. A.; Griesser, H. J.; Heinrich, M.; Wohlmuth, H. J. Nat. Prod. 2012, 75, 1612.
ASSOCIATED CONTENT
S Supporting Information *
UV, IR, HRESIMS, and 1D and 2D NMR spectra of 1 and 2 are available free of charge via the Internet at http://pubs.acs. org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +61 2 66203572. Fax: +61 2 66223459. E-mail: qingyao.
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Wound Management Innovation CRC (established and supported under the Australian Government’s Cooperative Research Centres Program). The authors thank H. Zhu, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunan, China, for performing the CD calculations. Thanks also go to 1387
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