Article pubs.acs.org/jnp
Natural Occurrence of Organofluorine and Other Constituents from Streptomyces sp. TC1 Nanjundan Jaivel,† Chokkalingam Uvarani,‡ Ramasamy Rajesh,† Devadasan Velmurugan,§ and Ponnusamy Marimuthu*,† †
Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, 641 003, Tamil Nadu, India Department of Chemistry, School of Chemical Sciences, Bharathiar University, Coimbatore, 641 046, Tamil Nadu, India § Department of Crystallography and Biophysics, University of Madras, Chennai, 600 025, Tamil Nadu, India ‡
S Supporting Information *
ABSTRACT: Antioxidant-directed fractionation of an ethyl acetate extract of Streptomyces sp. TC1 resulted in the isolation of a novel secondary metabolite with an aromatic organofluorine scaffold (1), an atypical tripod-type triallyl phenol (2), and a leucine residue comprised polyamine (3). Their structures were established by comprehensive spectroscopic analysis of 1D and 2D NMR data, and compound 1 was confirmed by 19F NMR and single-crystal X-ray diffraction studies. The absolute configuration of compound 3 was assigned by comparison of its ECD spectra and quantum chemical ECD calculations. Of these, compound 1 displayed antioxidant and DNA and protein binding properties. ficiency syndrome, stroke, arteriosclerosis, diabetes, and gastric ulcer.13−15 Anticancer agents exhibiting antioxidant and cytotoxic activities are expected to provide better medical efficacy due to their delaying onset of carcinogenesis by inhibiting the growth of cancer cells, without injuring neighboring cells.16,17 DNA is the primary pharmacological target of many antitumor compounds, and hence, binding studies of small molecules to DNA are useful in the development of DNA molecular probes and new therapeutic reagents. The nature and magnitude of binding parameters are helpful in studying protein−drug binding interactions, as they would significantly influence absorption, distribution, metabolism, and excretion properties of a given drug.18,19 This study was undertaken in order to determine the interactive behavior of compounds with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA), while evaluating their cytotoxic properties. In our continuing effort to discover new secondary metabolites from natural sources20 with the aforementioned biological properties, we have identified compounds 1−3 from Streptomyces sp. TC1. This paper describes the isolation and structure elucidation of compounds 1−3 along with their antioxidant, cytotoxic, and DNA and protein binding properties.
T
he emergence of drug resistance, new diseases, novel drug targets, and the concept of personalized medicine, along with a declining rate of new biologically active chemical entities, has created an urgent demand for new lead compounds.1 Terrestrial actinomycetes, especially those belonging to the Streptomyces genus, are prolific sources for natural product discovery with potential pharmaceutical activity.2 Aromatic organofluorine compounds have attracted a great deal of attention in pharmaceutical, agrochemical, and material sciences due to the unique physical and chemical features brought by the fluorine atom of the molecule.3 Despite the relatively high abundance of fluorine in the earth’s crust, organofluorine compounds are unusual among natural products.4 The first natural organofluorine compound identified, fluoroacetate, was from the South African plant Dichapetalum cymosum;5 other natural fluorine-containing compounds include fatty acid homologues6 and primary metabolites, i.e., 4-fluorothreonine (Streptomyces cattleya),7 nucleocidin (Streptomyces calvus),8 and derivatives of 5fluorouracil (Phakellia f usca).9 In addition the presence of a tert-butyl moiety is atypical in natural products. For instance, compounds containing a tert-butyl functionality have been identified from Lyngbya majuscule,10 L. bouillonii,11and Stylocheilus longicauda.12 However the occurrence of a tertbutyl moiety with an organofluorine compound (1) is unprecedented. Herein, we report a rare naturally occurring organofluorine metabolite (1) along with a triallyl phenol (2) and a polyamine (3). Oxidative stress induced by reactive oxygen and nitrogen species is reported to be responsible for degenerative diseases including cancer, heart diseases, malaria, acquired immunode© 2014 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Compound 1 was obtained as white crystals. The HRFABMS of 1 showed the molecular ion peak at m/z 280.1839. The mass Received: May 7, 2013 Published: January 3, 2014 2
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Table 1. 1H and 13C NMR Assignments and HMBC Correlations for Compounds 1 and 2 1 position 1 2 3 4 5 6 1′ 1a′ 2′ 3′ 4′ 1″ 2″ 3″ 4″−6″ 7″ 8″ 9″ 1‴ 1a‴ a
δC (APT) 152.2, 147.1, 139.6, 130.7, 124.8, 136.1, 34.9, 29.3,
C C CH C CH C C CH3
30.7, CH2 35.9, CH2 177.2, C
34.3, C 30.3, CH3
δH (J = Hz)
2
6.55, s
1′, 2″
7.01, s
1″, 1‴, 1
1.24, s
2.89, t (9, 7.5) 2.66, t (9, 7.5)
1.43, s
δC
HMBC 151.8, 135.6, 116.1, 123.5, 143.2, 124.0, 30.1,
C C CH CH C CH C
δH (J = Hz)
HMBC
6.59, d (7.9) 7.06, dd (8.3, 2.4)
5, 2 6, 1
7.30, d (2.4)
4, 1, 1″
2.04, t (7.5, 8.3) 5.80, m 4.95, m 2.04, t (7.5, 8.3) 1.26−1.42, m 1.26−1.42, m 1.26−1.42, m 1.26−1.42, m 1.26−1.42, m 0.86, t (7.3)
3′, 4′, 1′ 2′ 2′ 2″, 5
1″, 2
2″, 5, 4, 3″ 1″, 4, 3″
33.8, CH2 139.4, CH 114.2, CH2 31.9, CH2 29.1,a CH2 29.3,a CH2 28.9−30.1,a CH2 31.9, CH2 22.7, CH2 14.1, CH3
9″ 8″, 7′
1‴, 5, 6
Assignments may be interchanged.
Figure 1. Selected 2D correlations of compounds 1, 2, and 3.
124.8) revealed the presence of propanoic acid at the C-4 position of compound 1. The deshielded quaternary carbon at δ 152.2 and the HMBC correlations of H-5 (δ 7.01) to C-1″ (δ 30.7), C-1‴ (δ 34.3), and C-1 (δ 152.2) revealed that C-1 might have been substituted with a hydroxy group. The absence of a hydroxy proton in 1H NMR spectrum and the inertness of compound 1 with methyl iodide in K2CO3/KOH suggested the substitution at C-1 with a fluorine atom. Consequently, the intense absorption band at 1215 cm−1 in its IR spectrum delineated the presence of a C−F bond in compound 1. Finally, single-crystal X-ray diffraction studies (Figure 2) and a strong resonance at δF −168.01 in the 19F NMR spectrum confirmed the existence of a fluorine atom in compound 1, and it was defined as 3-(3,5-di-tert-butyl-4-fluorophenyl)propanoic acid. Compound 2 was obtained as a yellow-colored oil with a molecular formula of C25H38O as determined by HRFABMS (m/z 353.1248 [M − H]+) and verified by its NMR data. The 13 C NMR (APT) spectrum displayed six aromatic carbons at δ
and NMR data provided its molecular formula as C17H25FO2. The IR spectrum displayed intense absorption bands at 3629, 1701, and 1215 cm−1, indicating the presence of −OH, CO, and C−F functionalities. The 13C NMR spectrum exhibited 17 carbon signals, consisting of six methyl, two methine, two methylene, and seven quaternary carbons as determined by an APT experiment. The 1H NMR spectrum (Table 1) exhibited two methine protons at δ 7.01 and 6.55, two methylene protons [δ 2.89 (t, J = 9, 7.5 Hz), δ 2.66 (t, J = 9, 7.5 Hz)], and a sharp singlet of two peaks (each 9H, s) at δ 1.43 and 1.24, suggesting that compound 1 was a tetrasubstituted phenyl system with tert-butyl moieties. The tert-butyl substitutions at C-2 and C-6 were inferred by the HMBC correlations of C-1′/CH3 (δ 1.24) to C-1′ (δ 34.9) and C-2 (δ 147.1) and C-1‴/CH3 (δ 1.43) to C-1‴ (δ 34.3), C-6 (δ 136.1), and C-5 (δ 124.8), respectively. The 1H−1H COSY interaction (Figure 1) of methylene protons H-1″ (δ 2.89) to H-2″ (δ 2.66) and their HMBC correlations with C-3″ (δ 177.2), C-4 (δ 130.7), and C-5 (δ 3
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and α-methine resonances (δC 170.8 and 166.2, δC/H 53.2/4.07 and 68.7/4.60, respectively) indicated that 3 was a peptide. The 13 C NMR (APT) spectrum showed 11 signals including two methyl, three methylene, four methine, and two amide carbonyl carbons, which were assigned on the basis of the HSQC spectrum. The characteristic resonances of leucine22 were observed at δ 1.00 (3H, d, J = 6.2), 0.96 (3H, d, J = 5.3), 1.54 (1H, m), 2.07 (1H, m), 1.76 (1H, m), and 4.07 (1H, dd, J = 9.8, 4.4) in the 1H NMR spectrum and at δ 21.4/23.4 (−CH3), 53.2/24.9 (−CH), 38.7 (−CH2), and 166.2 (−CONH2) in the 13 C NMR spectrum (Table 2). This was further confirmed by Table 2. 1H and 13C NMR Assignments and HMBC Correlations for Compound 3 position 1 2 3α 3β 4 5 6 1′ 2′ 3α′ 3β′ 4′ 5α′ 5β′
Figure 2. ORTEP representation of compound 1.
151.8 (C-1), 135.6 (C-2), 116.1 (C-3), 123.5 (C-4), 143.2 (C5), and 124.0 (C-6), indicating the presence of a trisubstituted aromatic ring. The remaining carbon bearing eight methylene groups appeared at δ 22.7 to 31.9, and a methyl group at 14.1 consistent with a nonyl group, as well the methine and methylene peaks at δ 139.4, 114.2, and 33.8, was attributed to an allyl moiety. The 1H NMR spectrum (Table 1) showed ABX-type aromatic signals at δ 7.30 (1H, d, J = 2.4 Hz), 7.06 (1H, dd, J = 8.3, 2.4 Hz), and 6.59 (1H, d, J = 7.9 Hz), signals for an allyl group at δ 5.80 (3H, m), 4.95 (6H, m), and 2.04 (6H, t, J = 7.5, 8.3 Hz), and signals for nonyl methylene protons between δ 1.26 and 1.42 and a methyl group resonating at δ 0.86, also supporting compound 2 as a trisubstituted benzene. The protons integrating for three sets of allylic groups and the HMBC correlations of H-2′ (δ 2.04) to C-3′ (δ 139.4), C-4′ (δ 114.2), C-1′ (δ 30.1), and C-1 (δ 151.8) showed the substitution of a symmetric triallyl moiety at C-2. This was further confirmed by 1H−1H COSY interactions (Figure 1) of H-3′ with H-2′ and H-4′. The HMBC interactions of H-6 (δ 7.30) with C-1 (δ 151.8), C-1″ (δ 31.9), and C-4 (δ 123.5) suggested the substitution of OH and nonyl moieties at C-1 and C-5, respectively. The 13C NMR assignments of C-1‴, C-8″, and C-9‴ of nonyl linear alkyl side chain were also ascertained based on COSY, HSQC, and HMBC experiments, and it was compared with the known 2methoxy-3-nonyl resorcinol.21 However the signals at δ 28.9 to 30.1 assigned for six methylenes (C-2″ to C-7″) were not unequivocal; hence they may be reassigned. Accordingly, the existence of fragment ion peaks at m/z 218 [M − 135]+ and 241 [M − 112]+ confirmed the triallyl and nonyl groups in compound 2. On the basis of the aforementioned data, compound 2 was identified as a new phenolic analogue and defined as 2-(1,1-diallylbut-3-enyl)-5-nonylphenol. Compound 3 was obtained as colorless crystals. The HRFABMS of 3 showed the molecular ion peak at m/z 260.1492. This mass spectrum along with NMR data provided its molecular formula as C11H24O3N4. The absorptions at 3415 and 1670 cm−1 in the IR spectrum revealed the presence of hydroxy, amide, and carbonyl functional groups. Together with these IR absorptions, typical NMR chemical shifts for amide
δC(APT) 166.2, 53.2, 38.7, 38.7, 24.9, 21.4, 23.4, 170.8, 68.7, 37.5, 37.5, 57.5, 54.5, 54.5,
C CH CH2 CH2 CH CH3 CH3 C CH CH2 CH2 CH CH2 CH2
δH (J = Hz)
HMBC
4.07, 1.54, 2.07, 1.76, 0.96, 1.00,
dd (9.8, 4.4) m m m d (5.3) d (6.2)
3, 4, 1 1, 4, 6 1, 5, 4, 2
4.60, 2.16, 2.39, 4.50, 3.56, 3.70,
t (5.3) m dd (13.7, 6.9) dd (12.4, 6.4) brd (13.3) dd (13.3, 4.4)
5′, 5′, 1′, 3′,
6, 3 5, 4, 3 4′ 2′ 4′ 1′
3′, 4′, 2′
the HMBC correlations of H-2 (δ 4.07) with C-3 (δ 38.7), C-4 (δ 24.9), and C-1 (166.2) and H-3α/H-3β (δ 1.54/2.07) with C-4 (δ 24.9), C-5 (δ 21.4), C-1 (166.2), and C-2 (δ 53.2). The analysis of 1H NMR resonances for H-3α′/H-3β′ (δ 2.16/2.39) and H-4′ (δ 4.50) and for H-5α′/H-5β′ (δ 3.56/3.70) together with the HMBC correlations of H-2′ (δ 4.60) with C-4′ (δ 57.5) and C-5′ (δ 54.5), H-4′ (δ 4.50) with C-3′ (δ 37.5) and C-1′ (δ 170.8), and H-3α′ (δ 2.16) with C-4′ (δ 57.5) and C-1′ (δ 170.8) established the presence of a 4,5-diamino-2hydroxypentanoic acid, which was linked to the leucine moiety. This linkage was supported by the 1H−1H COSY correlations of oxymethine H-2′ (δ 4.60) and aminomethine H-4′ (δ 4.50) with H-3β′ (δ 2.39) and H-5β′ (δ 3.70), respectively. A detailed analysis of COSY, HSQC, and HMBC correlations was found to be in agreement with the proposed structure 3. The determination of relative configuration was based mainly on the results of NOESY experiments and on the observed 1H−1H coupling constants. The coupling constant observed for H-3β (δ 2.07) and H-2 (δ 4.07) is J = 9.8, indicating the αconfiguration of H-2; thus the leucine residue was determined to be L configured.22 The NOESY correlation (Figure 1) from H-2′ (δ 4.60) to H-3α′ (δ 2.16) and from H-4′ (δ 4.50) to H5β′ (δ 3.70) along with the coupling constant (J4′‑5β′ = 12.4 Hz) indicated the substituents at C-2′ and C-4′ were trans configured. For the determination of the absolute configuration, quantum chemical CD calculation23 was performed using timedependent density functional theory, and the results were compared with the experimental ECD spectrum. The corresponding minimum geometries were further fully optimized (Figure S3 in the Supporting Information) by using DFT at the B3LYP/6-31G* level as implemented in the Gaussian 03 program package. The calculated ECD spectrum 4
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of the stable conformer was subsequently compared with the measured value. The measured CD curve of 3 showed Cotton effects at λmax (Δε) 210 (−225), 237 (+72), and 262 (−75) nm, which matched with the calculated ECD curve of (2S,2′S,4′S)-3 and was opposite of that of (2R,2′R,4′R)-3 (Figure 3). Thus, the absolute configuration of 3 was unambiguously established to be (2S,2′S,4′S)-4,5-diamino-2hydroxypentanoic acid (1-carbamoyl-3-methylbutyl)amide.
Table 4. Binding Constant (Kb) and Photometric Properties of Compounds 1−3 in Contact with CT-DNA
a
compound
λmax (nm)
hypochromicity Δε (%)
1 2 3 EtBra
275 273 271 302
20.1 19.6 19.8 76.2
Kb × 104 (M−1) 3.13 2.23 2.36 7.26
± ± ± ±
0.04 0.01 0.02 0.12
EtBr, ethidium bromide was used as positive control.
Table 5. Quenching Constant (Kq) and Apparent Binding Constant (Kapp) of Ethidium Bromide Bound to CT-DNA compound
hypochromicity Δε (%)
Kq × 104 (M−1)
Kapp × 105 (M−1)
1 2 3
30.1 15.5 6.3
1.71 ± 0.13 0.68 ± 0.02 0.26 ± 0.07
1.60 ± 0.12 0.34 ± 0.06 0.13 ± 0.26
with a blue shift. To study the quenching process further, fluorescence data were analyzed with the Stern−Volmer and Scatchard equations.27 From the Stern−Volmer and Scatchard plots (Figure 4) the calculated quenching constant (Kq), binding constant (Kbin), and number of binding sites (n) are given in Table 6. The values of Kq and Kbin suggested that compound 3 was found to have stronger interactions with BSA than the other isolates.28 The synchronous fluorescence spectra of BSA with various concentrations of test compounds resulted in a small decrease in fluorescence intensity at Δλ = 15 nm and considerable decrease in fluorescence intensity at Δλ = 60 nm (Figures S7 and S8) for compounds 1 and 2, suggesting that the interaction of these compounds with BSA affects the conformation of the tryptophan significantly more than tyrosine microregions. However compound 3 exhibited considerable decreases in fluorescence intensity at both the microregions. Thus, the strong interaction between the compounds and BSA suggested that these compounds can be readily stored in protein and will be released at appropriate targets. The isolated compounds were tested for cytotoxicity against selected human cancer cell lines, HeLa, AGS, and HCT116, and no significant inhibitory activity was observed.
Figure 3. Measured and calculated ECD spectra of compound 3.
The antioxidant (FRAP and metal chelating) and radical scavenging (ABTS+•, DPPH•, OH•, and O2−•) ability of compounds 1−3 were determined by in vitro methods, and results were tabulated (Table 3). Compound 1 exhibits potent primary antioxidant capacity by having substantial reducing power (6031.1 ± 37.5 mmol Fe(II)/g), total antioxidant capacity (6736.5 ± 132.1 μmol trolox equivalent/g), and radical scavenging abilities (DPPH•, OH•, and O2−• with IC50 values of 8.4 ± 0.2, 25.8 ± 1.0, and 8.4 ± 0.3 μM, respectively), but its secondary antioxidant power (metal chelating 4.5 ± 0.2 mg EDTA equivalent/g) was found to be weak. The DNA binding properties of 1−3 were evaluated by absorption, fluorescence, and competitive ethidium bromide (EtBr) displacement spectrometric assays (Figures S4, S5, and S6). The calculated intrinsic binding constants from the absorption titration (Table 4) along with the quenching constant24 (Kq) and the apparent binding constant (Kapp) values (Table 5) from the ethidium bromide displacement assays25 suggested the moderate interaction of compounds with CT-DNA.26 Qualitative analysis of the binding of chemical compounds to BSA is usually detected by fluorescence spectra. The addition of compounds 1−3 to a solution of BSA resulted in a significant decrease in the fluorescence intensity (Figure 4) accompanied
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol IV polarimeter at a wavelength of 50 nm. ECD spectra were recorded at 25 °C on a Jasco-715 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 1600 FT IR spectrophotometer as KBr pellets, and the absorption frequencies are expressed in reciprocal centimeters (cm−1). Electronic absorption spectra were recorded using a Jasco V-630 spectrophotometer. Emission spectra were measured on a Jasco FP 6600
Table 3. Antioxidant Data of Compounds 1−3a IC50 (μM) sample 1 2 3 rutin BHT
FRAP 6031.1 4373.3 3787.8 7794.4 8345.6
± ± ± ± ±
b
37.5 20.4 27.8 22.1 87.8
metal chelating 4.5 8.4 11.9 18.5 15.4
± ± ± ± ±
0.2 0.6 0.5 0.6 0.6
c
ABTS 6736.5 4016.9 4093.9 10 106.7 6052.7
+•d
± ± ± ± ±
132.1 86.5 91.9 2.3 3.2
DPPH 8.4 14.9 34.7 4.7 4.5
± ± ± ± ±
•
0.2 0.3 4.5 0.3 0.2
OH• 25.8 31.1 35.5 19.4 11.5
± ± ± ± ±
1.0 0.9 0.6 5.3 3.1
O2−• 8.4 32.8 34.6 4.7 8.6
± ± ± ± ±
0.3 0.2 4.5 0.7 4.1
a “±” represents standard error of these bioassays. bmmol Fe(II)/g. cmg EDTA equivalent/g. dμmol trolox equivalent/g; rutin and BHT (butylated hydroxytoluene) were used as positive controls.
5
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Figure 4. Emission spectra of BSA (2 μM; λexi = 280 nm; λemi = 345 nm) in the presence of increasing amounts of compounds 1 (A), 2 (B), and 3 (C) (0, 0.5, 1, 1.5, 2, and 2.5 μM). The arrow shows the fluorescence quenching upon the increasing concentration of the compounds. (D) Scatchard plots of the fluorescence titration of compounds 1−3 with BSA. into 1000 mL of production medium contained in 3 L Erlenmeyer flasks (25 in number). The production medium having the composition of soluble starch 1.0%, casein 0.03%, KNO3 0.2%, NaCl 0.2%, K2HPO4 0.2%, CaCO3 0.002%, MgSO4·7H2O 0.005%, and FeSO4·7H2O 0.001% with pH 8.0 and the production flasks were incubated for 7 days at 28 °C. Extraction and Isolation. The fermentation broth (25 L) of strain TC1 was centrifuged, and the supernatant was extracted with EtOAc. The organic layer was evaporated under reduced pressure to give a residue (2.3 g), and it was subjected to column chromatography over silica gel (60−120 mesh) with the gradient elution of n-hexane− CHCl3 (0−100%), which afforded several fractions, which were pooled based on their analytical TLC results. The fractions obtained with the mixture of n-hexane−CHCl3 (88:12 v/v) were washed using n-hexane, affording compound 2 (12 mg, 0.52%). Further, a gradient elution using a mixture of n-hexane−CHCl3 (46:54 v/v) yielded compound 1 (25 mg, 1.09%) as white crystalline in nature, which was subsequently crystallized using a mixture of CHCl3 and MeOH. The final fraction obtained from n-hexane−CHCl3 (22:78 v/v) was subjected to preparative TLC using CHCl3−MeOH (92:8 v/v) as the mobile phase in order to purify compound 3 (7 mg, Rf = 0.42, 0.3%). Compound 1: white crystal; mp 158−160 °C; UV (DMSO) λmax (log ε) 275 (5.74), 310 (5.79) nm; IR (KBr) νmax 3629, 1701, 1215 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100
Table 6. Quenching Constant (Kq), Binding Constant (Kbin), Number of Binding Sites, and Photometric Properties of Compounds 1−3 in Contact with BSA Protein compound
hypochromicity Δε (%)
Kq × 105 (M−1)
Kbin × 105 (M−1)
n
1 2 3
52.9 45.4 88.5
4.06 ± 0.39 0.68 ± 0.30 6.46 ± 0.13
1.21 ± 0.12 0.26 ± 0.14 2.36 ± 0.09
1.3 0.97 1.6
spectrofluorometer. Mass spectrometric studies were carried out on a JEOL JMS600H HRFABMS. 1D and 2D NMR experiments were performed on a Bruker Advance-3 spectrometer at 400 MHz. Microorganism. The Streptomyces sp. TC1 was isolated from a soil sample obtained from Western Ghats of Tamil Nadu, Coimbatore, India. The strain was identified as Streptomyces sp. based on 16S rRNA sequence analysis, and it has been deposited in GenBank with accession number KC954629. Comparative BLAST search of the 16S rRNA gene sequence of strain TC1 was found to be 96% similar to that of Streptomyces sp. DL-28 (Gene Bank entry: JQ855896). The phylogenetic tree obtained by applying the neighbor-joining method is presented in the Supporting Information (S11). Fermentation. A seed culture of Streptomyces sp. TC1 was prepared by inoculating a loop of biomass into a 200 mL Erlenmeyer flask containing 100 mL of Kenknight’s broth and then incubating at 28 °C for 3 days. A 100 mL amount of this inoculum was transferred 6
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absorbance was taken at 30 °C after 30 min of initial mixing. The unit of total antioxidant activity was defined as the concentration of Trolox having equivalent antioxidant activity expressed as μmol/g. DPPH• Radical Scavenging Assay. The DPPH • radical scavenging activity was measured according to the method of Siddhuraju et al.34 The concentration of compound necessary to decrease initial concentration of DPPH• by 50% (IC50) under the specified experimental condition was calculated. Hydroxy Radical Scavenging Activity. The hydroxy radical scavenging activity of compounds was measured according to the method of Klein et al.35 Various concentrations (20, 40, 60, 80, and 100 μg) of compounds were added with 1.0 mL of iron−EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 mL of EDTA solution (0.018%), and 1.0 mL of DMSO (0.85% DMSO v/ v) in 0.1 M phosphate buffer (pH 7.4) sequentially. The reaction was initiated by adding 0.5 mL of ascorbic acid (0.22%) and incubated at 80−90 °C for 15 min in a water bath. After incubation the reaction was terminated by the addition of 1.0 mL of ice-cold trichloroacetic acid (TCA) (17.5% w/v). Subsequently, 3.0 mL of Nash reagent (mixture of ammonium acetate, glacial acetic acid, and acetyl acetone) was added and left at room temperature for 15 min. The reaction mixture without sample was used as a control. The intensity of the color formed was measured spectrophotometrically at 412 nm against reagent blank. O2−• Radical Scavenging Activity. The superoxide anion radical scavenging assay36 was based on the capacity of the compounds to inhibit formazan formation by scavenging the superoxide radicals generated in a riboflavin−light−nitroblue tetrazolium system. Each 3 mL reaction mixture contained 50 mM sodium phosphate buffer (pH 7.6), 20 μg of riboflavin, 12 mM EDTA, 0.1 mg of nitroblue tetrazolium, and 1 mL of test solution (20−100 μg/mL). The reaction was started by illuminating the reaction mixture with different concentrations of the compounds for 90 s. Instantly after illumination, the absorbance was measured at 590 nm. The entire reaction assembly was enclosed in a box lined with aluminum foil. Identical tubes with the reaction mixture kept in the dark served as blanks. Protein Binding Studies. The excitation wavelength of BSA at 280 nm and the emission at 345 nm were monitored for the protein binding studies.37 The excitation and emission slit widths and scan rates were maintained constant for all of the experiments. A stock solution of BSA was prepared in 50 mM phosphate buffer (pH = 7.2) and stored in the dark at 4 °C for further use. The compounds were dissolved in a mixed solvent of 5% DMSO and phosphate buffer for all of the experiments. Absorption titration experiments were performed with a fixed concentration of the compounds (25 μM) while gradually increasing the concentration of BSA (5−25 μM). For synchronous fluorescence spectra also, the same concentrations of BSA and the compounds were used, and the spectra were measured at two different Δλ values (difference between the excitation and emission wavelengths of BSA), such as 15 and 60 nm.
MHz), see Table 1; 19F NMR (CDCl3, 282 MHz) δ −168.01 (s); HRFABMS m/z 280.1834 [M]+ (calcd for C17H25FO2, 280.1839). X-ray Crystallographic Analysis of Compound 1. Crystal data: white crystals; C17H25FO2; Mr = 280.37; monoclinic, space group P21/ c; a = 6.116(3) Å; b = 18.564(5) Å; c = 14.758(4) Å; α = 90°; β = 93.350(5)°; γ = 90°; V = 1672.7(10) Å3; Z = 4; ρ = 1.113 g cm−3; μ = 0.08 mm−1; F(000) = 608; hmin,max = −8, 8; kmin,max = −24, 24; lmin,max = −19, 19; no. of unique reflns = 4229; no. of parameters = 181; R_all, R_obs = 0.093, 0.066; wR2_all, wR2_obs = 0.216, 0.195; Δρmin,max (e Å−3) = −0.38, 0.30; GoF = 1.07. Compound 2: yellow oil; UV (DMSO) λmax (log ε) 350 (5.84) nm; IR (KBr) νmax 3533, 1642, and 1460 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Table 1; HRFABMS m/ z 353.1248 [M − H]+ (calcd for C25H38O, 353.2923). Compound 3: colorless crystal; [α]20D +252.0 (c 0.45, CHCl3); UV (DMSO) λmax (log ε) 270 (5.72) nm; ECD (MeOH) λ (Δε) 210 (−225), 237 (+72), 262 (−75); IR (KBr) νmax 3415, 1670 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Table 2; HRFABMS m/z 260.1492 [M]+ (calcd for C11H24O3N4, 260.1848). Computational Section. The quantum chemical calculations were performed by using the density functional theory (DFT) as carried out in Gaussian 03.29 The molecular geometry of compound 3 was optimized using the B3LYP density functional method by implementing the 6-31G* basis set. All ground-state geometries were optimized at the B3LYP/6-31G* level. From the optimized B3LYP geometries CD spectra were generated using the TD-B3LYP functional, using dipole-length-computed rotational strengths. Antioxidant Assays. Ferric Reducing Antioxidant Power (FRAP) Assay. The antioxidant capacities of compounds were estimated according to the procedure described by Pulido et al.30 FRAP reagent (900 μL), prepared freshly and incubated at 37 °C, was mixed with 90 μL of distilled water and 30 μL of test sample or methanol (for the reagent blank). The test samples and reagent blank were incubated at 37 °C for 30 min in a water bath. The FRAP reagent contained 2.5 mL of 20 mmol/L TPTZ (2,4,6-tri(2-pyridyl)-1,3,5-triazine) solution in 40 mmol/L HCl plus 2.5 mL of 20 mmol/L FeCl3·6H2O and 25 mL of 0.3 mol/L acetate buffer (pH 3.6) as described by Siddhuraju and Becker.31 At the end of incubation, the absorbance was taken immediately at 593 nm, using a spectrophotometer. Methanolic solutions of known Fe(II) concentration, ranging from 100 to 2000 μmol/L (FeSO4·7H2O), were used for the preparation of the calibration curve. The parameter equivalent concentration (EC1) is defined as the concentration of antioxidant having a ferric-TPTZ reducing ability equivalent to that of 1 mmol/L FeSO4·7H2O. EC1 was calculated as the concentration of antioxidant giving an absorbance increase in the FRAP assay equivalent to the theoretical absorbance value of a 1 mmol/L concentration of Fe(II) solution, determined using the corresponding regression equation. Metal Chelating Assay. The chelation of ferrous ions was estimated by the method of Dinis et al.32 Briefly compounds and standard EDTA were added to a solution of 2 mmol/L FeCl2 (0.05 mL). The reaction was initiated by the addition of 5 mmol/L ferrozine (0.2 mL), and the mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm. Trolox Equivalent Antioxidant Capacity by Radical Cation (ABTS•+). The total antioxidant activity measured by the ABTS•+ radical cation decolorization assay involving preformed ABTS•+ radical cation.33 ABTS•+ was dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS•+) was produced by reacting ABTS•+ stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12−16 h before use. Prior to assay, the solution was diluted in ethanol (about 1:89 v/v) and equilibrated to 30 °C to give an absorbance at 734 nm of 0.700 ± 0.02 in a 1 cm cuvette. The concentration of sample that produced between 20% and 80% inhibitions of the blank absorbance was determined and adapted. After the addition of 1 mL of diluted ABTS•+ solution to the compounds or Trolox standards (final concentration 0−15 μM) in ethanol,
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ASSOCIATED CONTENT
S Supporting Information *
X-ray crystallographic data for compound 1, DNA binding, MTT assay procedures, as well as copies of the 1H, 13C, and 2D NMR spectra of compounds 1−3. This material is available free of charge via the Internet at http://pubs.acs.org. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 923598 (1). This can be obtained free of charge on application to Cambridge Crystallographic Data Centre, 2 Union Road, Cambridge CB2 1EZ, UK [fax: (+44) 1223-336033; e-mail:
[email protected]].
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AUTHOR INFORMATION
Corresponding Author
*Tel: +91-422-6611294. Fax: +91-422-6611394. E-mail: prof.
[email protected]. 7
dx.doi.org/10.1021/np400360h | J. Nat. Prod. 2014, 77, 2−8
Journal of Natural Products
Article
Notes
Harper, M. K.; Skalicky, J. J.; Mohammed, K. A.; Andjelic, C. D.; Barrows, L. R.; Ireland, C. M. J. Nat. Prod. 2006, 69, 1582−1586. (23) Stephens, P. J.; Pan, J. J.; Krohn, K. J. J. Org. Chem. 2007, 72, 7641−7649. (24) The intrinsic binding constant (Kb) of compound with CTDNA was determined from the following equation: [DNA]/(εa − εf) = [DNA]/(εa − εf) + 1/Kb(εa − εf), where [DNA] is the concentration of DNA in base pairs and the apparent absorption coefficient εa, εf, and εb correspond to Aobs/[compound], the extinction coefficient of the free compound, and the extinction coefficient of the compound when fully bound to DNA, respectively. The plot of [DNA]/(εa − εf) versus [DNA] gave a slope and intercept that are equal to 1/(εb − εf) and 1/Kb(εb − εf), respectively; Kb is the ratio of the slope to the intercept. (25) Quenching data were analyzed according to the following Stern−Volmer equation: I0/I = Kq[Q] + 1, where I0 is the emission intensity in the absence of a quencher, I is the emission intensity in the presence of a quencher, Kq is the quenching constant, and [Q] is the quencher concentration. The Kq value is obtained as a slope from the plot of I0/I versus [Q]. Further the apparent binding constant (Kapp) values were obtained for the compounds using the following equation: KEB[EB] = Kapp[compound] (where the compound concentration has the value at a 50% reduction of the fluorescence intensity of EB, KEB = 1.0 × 107 M−1 and [EB] = 5 μM). (26) (a) Haworth, I. S.; Elcock, A. H.; Freemann, J.; Rodger, A.; Richards, W. G. J. J. Biomol. Struct. Dyn. 1991, 9, 23−44. (b) Tysoe, S. A.; Morgan, R. J.; Baker, A. D.; Strekas, T. C. J. Phys. Chem. 1993, 97, 1707−1711. (27) The quenching constant (Kq) can be calculated using the plot of I0/I versus [Q]. If it is assumed that the binding of compounds with BSA occurs at equilibrium, the equilibrium binding constant (Kbin) can be analyzed according to the Scatchard equation: log[(I0 − I)/I] = log Kbin + n log [Q], from the plot of log(I0 − I)/I versus log [Q], the number of binding sites (n) and the binding constant (Kbin) have been obtained. (28) Raja, D. S.; Paramaguru, G.; Bhuvanesh, N. S. P.; Reibenspies, J. H.; Renganathan, R.; Natarajan, K. Dalton Trans. 2011, 40, 4548− 4559. (29) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzales, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 03, Revision A.11. 2; Gaussian, Inc.: Pittsburgh, PA, 2005. (30) Pulido, R.; Bravo, L.; Sauro-Calixto, F. J. Agric. Food Chem. 2000, 48, 3396−3402. (31) Siddhuraju, P.; Becker, K. Food Chem. 2006, 101, 10−19. (32) Dinis, T. C. P.; Madeira, V. M. C.; Almeida, L. M. Arch. Biochem. Biophys. 1994, 315, 161−169. (33) Kikuzaki, H.; Nakatani, N. J. Food Sci. 1993, 58, 1407−1410. (34) Siddhuraju, P.; Mohan, P. S.; Becker, K. Food Chem. 2002, 79, 61−67. (35) Klein, S. M.; Cohen, G.; Cederbaum, A. I. Biochemistry 1991, 20, 6006−6012. (36) Beauchamp, C.; Fridovich, I. Anal. Biochem. 1971, 44, 276−287. (37) Raja, D. S.; Bhuvanesh, N. S. P.; Natarajan, K. Inorg. Chem. 2011, 50, 12852−12866.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Dr. P. S. Mohan, Department of Chemistry, Bharathiar University, for isolation work and to K. Chandraprakash (Bharathiar University) for NMR spectral studies.
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