Influence of Solvent Polarity and DNA-Binding on Spectral Properties of Quaternary Benzo[c]phenanthridine Alkaloids

RESEARCH ARTICLE Influence of Solvent Polarity and DNABinding on Spectral Properties of Quaternary Benzo[c]phenanthridine Alkaloids Michal Rájecký 1 , Kristýna Šebrlová 2 , Filip Mravec 3 , Petr Táborský 1,4 * 1Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic, 2Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 3Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic, 4Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic *[email protected] Abstract Quaternary benzo[c]phenanthridine alkaloids are secondary metabolites of the plant families Papaveraceae,Rutaceae, and Ranunculaceae with anti-inflammatory, antifungal, antimicrobial and anticancer activities. Their spectral changes induced by the environment could be used to understand their interaction with biomolecules as well as for analytical purposes. Spectral shifts, quantum yield and changes in lifetime are presented for the free form of alkaloids in solvents of different polarity and for alkaloids bound to DNA. Quantum yields range from 0.098 to 0.345 for the alkanolamine form and are below 0.033 for the iminium form. Rise of fluorescence lifetimes (from 2–5nsto3–10 ns) and fluorescence intensity are observed after binding of the iminium form to the DNA for most studied alkaloids. The alkanolamine form does not bind to DNA. Acid-base equilibrium constant of macarpine is determined to be 8.2–8.3. Macarpine is found to have the highest increase of fluorescence upon DNA binding, even under unfavourable pH conditions. This is probably a result of its unique methoxy substitution at C 12 a characteristic not shared with other studied alkaloids. Association constant for macarpine-DNA interaction is 700000 M -1 . Introduction Quaternary benzo[c]phenanthridine alkaloids (QBAs) are secondary metabolites of some species of the plant families Papaveraceae,Rutaceae, and Ranunculaceae. The most abundant representatives are commercially available sanguinarine and chelerythrine. Isolations of other QBAs, namely sanguilutine, sanguirubine, chelilutine, chelirubine, and macarpine, were reported by Slavik et al.[1–3]. The source of these rare alkaloids has been plant material [4,5] even though the synthetic approach was published for macarpine [6]. QBAs are composed of a N-methylbenzo[c]phenanthridinium core with several methoxy or methylenedioxy substituents (Fig 1). Due to the reactive iminium bond, QBAs are susceptible PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 1/16 OPEN ACCESS Citation: Rájecký M, Šebrlová K, Mravec F, Táborský P (2015) Influence of Solvent Polarity and DNA-Binding on Spectral Properties of Quaternary Benzo[c]phenanthridine Alkaloids. PLoS ONE 10(6): e0129925. doi:10.1371/journal.pone.0129925 Academic Editor: Heidar-Ali Tajmir-Riahi, University of Quebec at Trois-Rivieres, CANADA Received: March 16, 2015 Accepted: May 14, 2015 Published: June 19, 2015 Copyright: © 2015 Rájecký et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funding was provided by the Ministry of Education, Youth and Sports of the Czech Republic (KONTAKT II LH12176). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. to nucleophilic addition on carbon C 6 [7]. Therefore, equilibrium between the iminium and alkanolamine forms is established in aqueous solution (Fig 1). The biological effects of QBAs that have been studied were mainly for sanguinarine and chelerythrine. Anti-inflammatory, antifungal, antimicrobial activities and possible anticancer effects of QBAs have been reported (reviewed in [8,9]). At the molecular level, interactions with various forms of DNA, proteins and enzymes have been reported for sanguinarine and chelerythrine [10–16]. The properties of other QBAs are less known and have been studied mainly at cellular level [17,18]. Their ability to quickly enter into cells has been used for cell staining [19]. While the fluorescence of sanguinarine and chelerythrine is well described [20–23], knowledge of the fluorescence properties of other QBAs is limited. Fluorescence spectra in aqueous solution and their change upon binding to DNA has previously been reported [24] and their potential use as probes in fluorescence microscopy and flow cytometry was proposed [19]. A detailed description of the fluorescence of iminium and alkanolamine forms of QBAs has not yet been performed. Spectral changes are an important indicator of the microenvironment around a fluorescent probe. Sensitivity to solvent polarity could help to explain the mode of binding to biomacromolecules especially when no structure of such complexes is available. Additional information could be provided by fluorescence lifetimes. Therefore, fluorescence properties of both iminium and alkanolamine forms of QBAs (except chelilutine) in solutions of different polarity and upon binding to DNA are presented. Fig 1. Structure, numbering and acid-base equilibrium of studied QBAs. doi:10.1371/journal.pone.0129925.g001 Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 2/16 Materials and Methods Quaternary benzo[c]phenanthridine alkaloids and chemicals All studied alkaloids were isolated from plant material as published earlier [25] and were obtained as chloride salts of at least 93% purity. Stock solutions were prepared by dilution in MilliQ water (Millipore, USA) and were stored at room temperature in the dark. Trizma base, ethylenediaminetetraacetic acid disodium salt (EDTA), quinine, colloidal silica (Ludox), calf thymus DNA (ctDNA), and salmon testes DNA were purchased from Sigma-Aldrich, USA. All other chemicals were obtained from Lach-ner, Czech Republic. The ctDNA was dissolved in 10mM Tris–1mM EDTA buffer, pH 7; salmon testes DNA was dissolved in MilliQ water. The concentration of DNA was determined spectrophotometrically using the relationship that 1 absorbance unit at 260 nm corresponds to 50 μgml -1 (0.075 mM in base-pairs (bp)) of double-stranded DNA. Absorbance and fluorescence measurements Absorbance was measured on a Shimadzu UV-1601 spectrophotometer (Shimadzu, Japan). Steady-state fluorescence was measured on an Aminco Bowman Series-2 spectrofluorometer (SLM Aminco, USA). All measurements were performed at 25°C. If not stated otherwise, fluorescence spectra were corrected for lamp instabilities and non-ideal instrument detection. Validity of corrected spectra produced by correction factors supplied by the manufacturer of the spectrofluorometer was assessed by comparison with corrected spectra of fluorescent probes published by Lakowicz [26]. When necessary, spectra were converted to a wavenumber scale using Eq (1), where Idenotes intensity at a particular wavenumber (V ˜ ) or wavelength (λ), to take non-constant bandpass in wavenumber scale into account. Spectra in wavenumber scale were smoothed by a Savitzki-Golay filter using 2 nd order polynomial over 11 points. Ið~ nÞ¼IðlÞl2ð1Þ Acid-base behaviour of macarpine Acid-base equilibrium of a QBA (Fig 1) is defined by the equilibrium constant K ROH (Eq (2), also denoted as K R+ ) that is connected to a common acid-base equilibrium constant K (Eq (3), Fig 1) by a water ion product K ROH =KK W [27,28]. Negative logarithm of K ROH is analogous to pK a of common acids and will be marked pK ROH in this article. KROH ¼½Hþ½QOH ½Qþð2Þ K¼½QOH ½Qþ½OHð3Þ Samples were prepared by diluting macarpine stock solution to 3 μM by Britton-Robinson universal buffer with a pH between 3 and 12 and ionic strength 0.15 M [29]. Absorption and fluorescence spectra were recorded as described above. Measurements were performed 3 times and mean values of absorbance at 495 nm and fluorescence at 450 nm were fitted to Eq (4)[30] A¼A1þA210pHpKROH 1þ10pHpKROH ð4Þ where Ais the signal (absorbance or fluorescence) at a particular pH, A 1 is the signal of Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 3/16 iminium form, A 2 is signal of the alkanolamine form and pK ROH is the constant defined above. QtiPlot 0.9.8.9 was used for weighted fitting with variance used as a weight. Standard errors of best-fit parameters (SE) are reported in Table 1. Macarpin binding to DNA Measurements were performed in 0.05 M citrate buffer, pH 6.15, containing 0.122 M Na + .A set of samples with a constant macarpin concentration 1 × 10 -5 M and varying concentrations of salmon testes DNA were prepared. Absorption and fluorescence spectra were measured as stated above. Inner-filter effect was corrected using Eq (5)[31], where F corr is fluorescence corrected for inner-filter effect, F meas is measured fluorescence, A ex is absorbance at excitation wavelength, A em is absorbance at emission wavelength, and d ex and d em are dimensions of light path in excitation and emission direction, respectively. Fcorr ¼Fmeas 10ðAexdex=2þAemdem=2Þð5Þ DynaFit program [32] was used to analyse binding of macarpin to double-stranded DNA by observing corrected fluorescence at 625 nm as a function of the total DNA concentration and conditional association constant for 1:1 binding was obtained. Measurements were performed 3 times and standard deviations (SD) for small samples were calculated according to Dean and Dixon [33]. Fluorescence lifetimes QBAs in 3 × 10 -6 M concentration were prepared in 0.1M borate buffer, pH 9.45 (alkanolamine form) or in 0.1M acetate buffer, pH 3.95 (iminium form). For measurement of lifetimes of Table 1. Spectroscopic properties of alkanolamine (QOH) and iminium (Q + ) forms of QBAs. Fluorescence lifetime (ns) e pH 9.45 (QOH) pH 3.95 (Q + )S 1 !S 0 rate const. of QOH (10 7 s -1 ) Alkaloid pK ROHa QY QOH d QY Q +d no DNA ctDNA no DNA ctDNA k rS k nrS sanguinarine 8.05 0.210 0.033 3.2 f 3.2 2.4 i 2.4 (10.1) 6.56 24.69 chelerythrine 9.0 0.156 0.004 3.2 3.2 —3.1 (10.3) 4.88 26.38 sanguilutine 8.8 0.208 —3.5 3.5 —3.8 5.94 22.63 sanguirubine 7.9 0.233 —3.9 3.9 —2.9 5.97 19.67 chelirubine 7.7 0.345 —4.5 4.5 —3.1 7.67 14.56 macarpine 8.24 b (8.30 c ) 0.098 —3.9 3.3 g (4.6 h )—3.1 (5.1) 2.51 23.13 Where two results were obtained (pK ROH by two methods, two lifetimes), second value is indicated in brackets. a) all QBAs except macarpine ref. [30]. b) this work, spectrophotometry, n = 3, SE = 0.02 c) this work, spectrofluorometry, n = 3, SE = 0.06 d) n = 4, estimated RSD ca. 15% e) n = 3, SE 0.1 ns f) 3.3 ns (ref. [22]) g) 3.5 ns (610 nm) h) 5.8 ns (610 nm) i) 2.4 ns (ref. [22]) doi:10.1371/journal.pone.0129925.t001 Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 4/16 QBA-DNA complexes, ctDNA was added (DNA base pair:drug ratio 1.6:1). Measurements were performed on a Fluorocube (Horiba Jobin Yvon, France) with a 329 nm excitation LED diode (pulse width 1.2 ns) and emission monochromator set to 440 nm (alkanolamine form) or 610 nm (iminium form, 570 nm for chelerythrine). Instrument response function was obtained by measuring a solution of colloidal silica. When necessary, intensity was lowered by neutral filters. For each QBA three measurements were performed and lifetimes were globally fitted using DecayFit 1.3. Quality of a fit was considered by inspecting residuals and reduced χ 2 value around 1. SE are reported in Table 1. Quantum yields Quinine sulfate dissolved in 0.1M H 2 SO 4 and anthracene dissolved in ethanol for spectroscopy were used as standards. Alkanolamine forms of QBAs were measured in 0.01M borate buffer, pH 9.45. Iminium forms of sanguinarine and chelerythrine were measured in 0.01M acetate buffer, pH 3.95. Five to 8 solutions with absorbances at 322 nm between 0.01 and 0.05 were prepared for each alkaloid and standard. Their absorption and fluorescence spectra were measured using excitation at 322 nm and an emission range covering the whole emission spectrum of a particular QBA or standard. Inner-filter effect was corrected using Eq (5)[31]. The fluorescence spectra were integrated and plotted against corresponding absorbance at the excitation wavelength. Resulting slopes were used for quantum yield (QY) calculation according to Eq (6), where Fis fluorescence QY, slope is slope of integrated fluorescence vs. absorbance plot and ηis refractive index of the solvent. Subscripts QBA and ST denote QBA and standard, respectively. FQBA ¼FST slopeQBA slopeST Z2 QBA Z2 ST ð6Þ This is a version of a well-known Eq (7)[26] recognizing that when integrated fluorescence of a sample is plotted as a function of its absorbance at the excitation wavelength, the slope equals fluorescence-to-absorbance ratio. FQBA ¼FST FQBA FST AST AQBA Z2 QBA Z2 ST ð7Þ Standards were cross-correlated to find out if measurements give reliable QYs (0.54 for quinine, 0.27 for anthracene [34]). For each QBA, QY was calculated using both standards and the result was obtained by averaging these two calculated values. Each experiment was repeated 4 times and a relative standard deviation (RSD) of 15% was estimated, based on the error of fit and quality of instrument correction factors supplied with the spectrofluorometer. Fluorescence rate constants were calculated from measured lifetimes and QYs for the alkanolamine form of QBA according to Eqs (8)–(11)[35]: tS¼1 kS rþkS nr ð8Þ where τ S is fluorescence lifetime from singlet excited state, k rS is rate constant for radiative deactivation S 1 !S 0 with emission of fluorescence, k nrS is overall nonradiative rate constant Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 5/16 (internal conversion plus intersystem crossing) and F F is fluorescence QY. FF¼kS r kS rþkS nr ð9Þ kS r¼FF tS ð10Þ kS nr ¼1FF tS ð11Þ Effect of solvent polarity Small amounts of stock solutions of QBAs were transferred into Eppendorf tubes or glass volumetric flasks and were evaporated. The solvent (benzene, diethyl ether, methanol, ethanol, octanol, 0.01M borate buffer, pH 9.45) was added to prepare 3 × 10 -6 M solutions. Stokes shifts were determined from absorption (excitation in the case of benzene and diethyl ether) and fluorescence spectra plotted in wavenumber scale using Eq (1). Solvent polarity effect on QBA fluorescence was assessed by Lippert–Mataga plot of Stokes shift against orientation polarizability [35]. Orientation polarizability is defined as (ε r -1)/(2ε r +1)-(η 2 –1)/(2 η 2 +1). Published values of ε r (relative permittivity) and η(refractive index) were used [36]. Results Acid-base properties Fig 2 shows absorption and emission spectra of iminium and alkanolamine forms of macarpine and their change with changing pH. It could be seen that the iminium form is generally more absorbing, especially around 500 nm (20000 cm -1 ), but its emission is quenched. The alkanolamine form does not absorb above ca. 400 nm (25000 cm -1 ), but intensely fluoresces at 450 nm (ca. 22220 cm -1 ). In a highly alkaline solution fluorescence starts to decrease and was therefore omitted from fitting. Table 1 shows pK ROH values found by spectrophotometry and spectrofluorometry. Both methods give similar results. It is possible to align the QBAs by increasing pK ROH using published values [30] as follows: chelirubine <sanguirubine <sanguinarine < macarpine <chelilutine <sanguilutine <chelerythrine. Fluorescence lifetime Fluorescence lifetime of QBAs at pH 9.45 (mainly alkanolamine form present) is between 3 and 5 ns (Table 1). Previously reported lifetime for sanguinarine (3.3 ns) [22] is in good agreement with the found value of 3.2 ns. As can be seen from Table 1, addition of ctDNA does not change fluorescence lifetimes of the alkanolamine form (except macarpine). Fluorescence of iminium form of QBA is quenched except for sanguinarine and chelerythrine, but only the sanguinarine lifetime could be reliably measured. The measured value of 2.4 ns is the same as that reported previously [22,37]. The addition of DNA causes a decrease of sanguinarine steady-state fluorescence. The lifetime of DNA-bound sanguinarine increases to 10.1 ns with another component of 2.4 ns attributable to its free form in solution (S1 Fig). Again, lifetime of sanguinarine-DNA complex is the same as reported earlier [37]. Steady-state fluorescence of other QBAs increases in the presence of DNA. Their fluorescence decays are monoexponential with lifetimes of alkaloid-DNA complexes around 3 ns. The exceptions are Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 6/16 chelerythrine and macarpine. Chelerythrine, whose free form is fluorescent, shows double exponential decay similar to sanguinarine. Therefore, it can be argued that a longer lifetime belongs to a complex with DNA and shorter lifetime belongs to free form in solution. In the case of macarpine a double exponential is necessary to fit the decay, but obtained lifetimes are harder to interpret because lifetime of the free form is not measurable. Using the same assumption as for chelerythrine we could assign a shorter lifetime to free macarpine and a longer lifetime to macarpine-DNA complex. Fluorescence quantum yields To achieve minimum spectral overlap of iminium and alkanolamine forms of QBA, pH 3.95 and 9.45 were used in this study. Only QYs of sanguinarine and chelerythrine iminium form could be determined reliably due to the quenched fluorescence of other alkaloids. As can be seen from the values of pK ROH in Table 1, a higher pH would be better to measure the pure alkanolamine form of chelerythrine and sanguilutine. This was not possible due to a decrease of fluorescence above a pH of around 9.5–10 (Fig 2). Table 1 lists QYs of all alkanolamine forms of QBA and of sanguinarine and chelerythrine iminium forms. Iminium forms of other QBAs have too low a fluorescence for QY determination. The iminium form of sanguinarine has an order of magnitude lower QY than the alkanolamine form. For chelerythrine an even larger difference of two orders of magnitude is observed. Fig 2. Acid-base properties of 3 μM macarpine. Dependence of absorbance at 495 nm (black) and fluorescence at 450 nm (red) on pH, n = 3. Mean values ±SD and fits to Eq (4) are shown. Inset: Absorption (black) and emission (red) spectra of iminium (—) and alkanolamine (...) form. Ordinates are the same as for bigger figure. doi:10.1371/journal.pone.0129925.g002 Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 7/16 De-excitation rate constants Using measured fluorescence lifetimes and QYs, radiative and non-radiative rate constants of S 1 !S 0 transition of the alkanolamine form of QBAs were calculated using Eqs (10) and (11). All radiative constants are of the order 10 7 s -1 ; non-radiative constants are of the order 10 8 s -1 implying that radiative de-excitation is unfavourable. QBAs could be ordered according to k rS from lowest to highest rate constant as macarpine <chelerythrine < sanguilutine <sanguirubine <sanguinarine <chelirubine. Effect of solvent polarity Stokes shifts in various solvents were analysed by Lippert–Mataga plot. In all studied solvents (benzene, diethyl ether, octanol, methanol, ethanol, 0.1M borate buffer, pH 9.45), spectra of QBAs resemble that of the alkanolamine form with a peak around 430–450 nm (ca. 22000– 23000 cm -1 ,S3 Fig). There is a decrease in the Stokes shift between benzene and diethyl ether for all studied QBAs (Fig 3). An interesting effect was observed for spectra in hydrogen-bonding solvents. While methanol, ethanol and octanol decreased Stokes shift in comparison with benzene, borate buffer, pH 9.45 caused an increase. Binding to DNA Absorption spectra of QBA at pH 5 (Fig 4) show isosbestic points after addition of ctDNA confirming interaction of all studied alkaloids with double-stranded DNA. All QBAs except sanguinarine show increased fluorescence at ca. 16500 cm -1 (ca. 600 nm) in the presence of ctDNA. Additional fluorescence peaks of chelirubine and sanguirubine at ca. 23000 cm -1 (ca. 435 nm) belong to the alkanolamine form (Fig 4). Another experiment at pH 3.95 (S2 Fig) Fig 3. Lippert–Mataga plot of QBAs. Black–macarpine, red–sanguilutine, blue–chelirubine, green– sanguinarine, gold–sanguirubine, violet–chelerythrine; 1 –benzene, 2 –diethyl ether, 3 –octanol, 4 –ethanol, 5–methanol, 6–0.01M borate buffer, pH 9.45. Samples in borate buffer (dashed box) excluded from fitting. doi:10.1371/journal.pone.0129925.g003 Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 8/16 shows that QBAs in iminium form bind DNA. Absorbance spectra of alkanolamine forms of QBA at pH 9.45 slightly shift to the spectra of iminium-DNA complex in samples with DNA (S2 Fig). The largest shift was observed for macarpine. On the basis of previous experiments, macarpine was selected for further study of DNA interaction. Association constant in a citrate buffer, pH 6.15 with physiologically relevant Na + concentration (122 mM) was measured (Fig 5). Buffer pH was selected to prefer the iminium form of macarpine and stable B-form of DNA. Data were fitted to a 1:1 association constant of 7×10 5 M -1 (SD 2 × 10 5 M -1 ). According to the energy of iminium ground state (Fig 4) it is possible to order QBAs from lowest to highest: chelirubine ~ sanguirubine <sanguinarine ~ sanguilutine <chelerythrine (both for free and DNA-bound forms). Similarly, energy of the lowest singlet excited state could be ordered as chelirubine ~ sanguirubine ~ sanguilutine <sanguinarine <chelerythrine Fig 4. Absorbance and fluorescence spectra of QBAs in absence and presence of ctDNA. QBAs (3 μM in 20mM acetate buffer, 200 mM NaCl, 2mM EDTA, pH 5) in absence (—) and presence (...) of ctDNA (DNA base pair-to-drug ratio 15.9:1). a-f–absorption spectra, g-l–emission spectra; a, g– sanguinarine, b, h–chelerythrine, c, i–chelirubine, d, j–sanguilutine, e, k–sanguirubine, f, l–macarpine. Note that intensities are in arbitrary units due to conversion to wavenumber scale using Eq (1). doi:10.1371/journal.pone.0129925.g004 Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 9/16 44. Gregorova J, Babica J, Marek R, Paulova H, Taborska E, Dostal J. Extractions of isoquinoline alkaloids with butanol and octanol. Fitoterapia. 2010; 81: 565–568. doi: 10.1016/j.fitote.2010.01.020 PMID: 20117181 Spectral Properties of Benzo[c]phenanthridine Alkaloids PLOS ONE | DOI:10.1371/journal.pone.0129925 June 19, 2015 16 / 16