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Structural features of DNA interaction with caffeine and theophylline
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Journal of Molecular Structure 875 (2008) 392–399 www.elsevier.com/locate/molstruc
Structural features of DNA interaction with caffeine and theophylline Shohreh Nafisi
b
a,*
, Firouzeh Manouchehri a, Heidar-Ali Tajmir-Riahi b, Maryam Varavipour c
a Department of Chemistry, Azad University, Central Tehran Branch, Tehran 14169 63316, Iran Department of Chemistry-Biology, University of Quebec at Trois-Rivieres, C.P. 500, TR, Que., Canada G9A 5H7 c Department of Iirigation & Drainage Engineering, College of Aboreyhan, University of Tehran, Iran
Received 26 February 2007; received in revised form 9 May 2007; accepted 9 May 2007 Available online 17 May 2007
Abstract Caffeine and theophylline are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these xanthine derivatives with individual DNA at molecular level. The aim of this study was to examine the stability and structural features of calf-thymus DNA complexes with caffeine and theophylline in aqueous solution, using constant DNA concentration (6.25 mM) and various caffeine or theophylline/DNA(P) ratios of 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1. FTIR, UV–visible spectroscopic methods were used to determine the ligand external binding modes, the binding constant and the stability of caffeine, theophylline–DNA complexes in aqueous solution. Spectroscopic evidence showed that the complexation of caffeine and theophylline with DNA occurred via G-C and A-T and PO2 group with overall binding constants of K(caffeine–DNA) = 9.7 · 103 M1 and K(theophylline–DNA) = 1.7 · 104 M1. The affinity of ligand–DNA binding is in the order of theophylline > caffeine. A partial B to A-DNA transition occurs upon caffeine and theophylline complexation. 2007 Elsevier B.V. All rights reserved. Keywords: DNA; Caffeine; Theophylline; Binding constant; Binding mode; Conformation; Stability; Secondary structure; FTIR; UV–visible spectroscopy
1. Introduction Caffeine is the most widely consumed in the world. It is found in seeds, citrus fruits, olive oil, tea, cocoa beverages. Because of the vast consumption of caffeine, research has focused on its physiological effects [1]. Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione) and theophylline (3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione) bases (Scheme 1) are structurally related to nucleic acids components. Caffeine has a CH3 group at the N7 site which blocks coordination with N7 atom, while in theophylline the N7 site is accessible for ligand coordination. The presence of oxy and hydroxyl groups as well as double bonds in specific positions make them strong *
Corresponding author. Tel./fax: +98 21 22426554. E-mail address: drsnafi[email protected] (S. Nafisi).
0022-2860/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2007.05.010
antioxidants. Caffeine has significant abilities to scavenge highly reactive free radicals and excited states of oxygen and to protect crucial biological molecules against these species. Antioxidant ability of caffeine is similar to that of the established biological antioxidant glutathione and significantly much higher than that of ascorbic acid [2–7]. Caffeine is an efficient inhibitor of DNA repair and DNA damage-activated checkpoints. It inhibits retroviral transduction of dividing cells, most likely by blocking post integration repair. This effect may be mediated at least in part by a cellular target of caffeine. Caffeine belongs to a class of chemicals that strongly enhance the cytotoxic effect of ionizing radiation and other DNA damaging agents, at concentrations that are not otherwise toxic to cells [8–12]. However, it has been established that caffeine disrupts DNA damage-activated cell cycle checkpoints [13–22]. Caffeine and theophylline can modulate the binding of certain
S. Nafisi et al. / Journal of Molecular Structure 875 (2008) 392–399
O
H3C
O
H3C
N
N N
O
CH3
O
chemicals were of reagent grade and used without further purification.
N
N
N
H
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2.2. Preparation of stock solutions N
N
CH3
CH3
Caffeine
Theohphylline Scheme 1.
DNA damaging agents and reduce DNA directed toxicity [23,24]. The molecular mechanisms underlying the effects of caffeine and theophylline on DNA are still not fully understood. The bindings of caffeine and theophylline to nucleic acids are studied and were found to exhibit a dose dependent binding behaviour [23,24]. Investigations on the interaction of caffeine and theophylline with DNA suggests that intercalation is not the predominant mechanism [25]. The studies on the interaction of RNA and xanthine derivatives (caffeine, theophylline and theobromine) showed that G-C and A-U bases of RNA are the targets for caffeine and theophylline bindings so that the C@O and NH groups of the ligand and RNA mutually involved in H-bonding. Among the three xanthine derivatives, theophylline showed greater binding efficacy with RNA than theobromine and caffeine [23–26], while the binding positions of caffeine and theophylline on B-DNA have not been firmly resolved. It is possible that both the base sequence and the environment have an important influence on xanthine binding. Therefore, it was of interest to examine the bindings of caffeine and theophylline to DNA bases and the backbone phosphate group. We now report the results of FTIR and UV–visible spectroscopic analysis of DNA interaction with caffeine and theophylline in aqueous solution at physiological conditions, using constant DNA concentration and various ligand/DNA (phosphate) ratios of 1/80 to 1/1. The ligand binding site, the binding constant, and the effects of ligand complexation on the stability and conformation of DNA duplex are discussed here.
2. Methods and material 2.1. Materials Caffeine and theophylline were purchased from Merck Co. Highly polymerized type I calf-thymus DNA sodium salt (7% Na content) was purchased from Sigma Chemical Co., and deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. The absorbance at 260 and 280 nm was recorded, in order to check the protein content of DNA solution. The A260/A280 ratio was 1.80, showing that the DNA was sufficiently free of protein [27]. Other
Sodium–DNA (5 mg/ml) was dissolved in distilled water (pH 7) at 5 C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the calf-thymus DNA solution was determined spectrophotometrically at 260 nm using molar extinction coefficient 260 = 6600 cm1 M1 (expressed as molarity of phosphate groups) [28,29]. The UV absorbance at 260 nm of a diluted solution (1/250) of calf-thymus DNA used in our experiments was 0.661 (path length was 1 cm) and the final concentration of the DNA solution was 12.5 mM in DNA phosphate. The appropriate amounts of caffeine and theophylline (0.15–12.5 mM) were prepared in distilled water and added dropwise to DNA solution in order to attain the desired ligand/DNA(P) molar ratios (r) of 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1 with a final DNA(P) concentration of 6.25 mM (path length was 0.03 cm). The pH of the solutions was adjusted at 7.0 ± 0.2 using NaOH solution. 2.3. Absorption spectroscopy The absorption spectra were recorded on a LKB model 4054 UV–vis spectrometer, using various ligands concentrations (5 lM to 0.05 mM) and DNA concentration of 0.1 mM. 2.4. FTIR spectroscopic measurements Infrared spectra were recorded on a Jasco FTIR spectrometer equipped with a liquid-nitrogen-cooled HgCdTe (MCT) detector and a KBr beam splitter. The spectra of the caffeine and theophylline/DNA solutions were acquired using a cell assembled with AgBr windows. Spectra were collected after 2 h incubation of ligand with the DNA solution and were measured in triplicate (three individual samples of the same DNA and ligand concentrations). For each spectrum, 100 scans were collected at a resolution of 4 cm1. The water subtraction was carried out using NaCl (0.1 mol/L) solution at pH 6.5–7.5 as a reference. A good water subtraction was considered to be achieved when there was a flat baseline around 2200 cm1, where the water combination mode is located. This method yields a rough estimate of the subtraction scaling factor, but removes the spectral features of water in a satisfactory way [30]. The difference spectra [(DNA solution + ligand) (DNA solution)] were obtained using a sharp DNA band at 968 cm1 as internal reference. This band, which is due to sugar C–C and C–O stretching vibrations, exhibit no spectral change (shifting or intensity variations) upon caffeine and theophylline–DNA complexation, and it is cancelled out upon spectral subtraction [31]. The intensity ratios of bands due to several
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DNA-in-plane vibrations related to A-T, G-C base pairs and the PO2 stretching vibrations were measured with respect to the reference band at 968 cm1 (DNA) as a function of caffeine and theophylline concentration with an error of ±3%. Similar intensity variations have been used to determine the ligand binding to DNA bases and backbone phosphate groups [32–34]. The plots of the relative intensity (R) of several peaks of DNA-in-plane vibrations related to A-T, G-C base pairs and the PO2 stretching vibrations such as 1710 (guanine), 1662 (thymine), 1610 (adenine), 1492 (cytosine), and 1226 cm1 (PO2 groups), versus the caffeine and theophylline concentrations were obtained after peak normalization using, Ri = Ii/I968, where Ii is the intensity of absorption peak for pure DNA in the complex with i as ligand concentration, and I968 is the intensity of the 968 cm1 peak (internal reference). 3. Results and discussion Caffeine and theophylline showed intense IR bands in the region of 1680–1720 cm1, that were assigned to the C@O stretching vibrations. Other bands in the region of 1550–1666 cm1 were assigned to C@C and C@N stretching modes in purine ring system [35–37]. 3.1. FTIR spectra of caffeine–DNA complexes FTIR spectra of caffeine–DNA complexes between 1800 and 600 cm1 are presented in Fig. 1. The spectral changes (intensity and shifting) of several prominent DNA-in-plane vibrations at 1710 cm1 (G, T mainly G), 1662 cm1 (T, G, A and C, mainly T), 1610 cm1 (A, C, mainly A), 1492 cm1 (C, G mainly C) and 1226 cm1 (PO2 asymmetric stretch) and 1088 cm1 [30,38–41] were monitored at different caffeine–DNA molar ratios and the results are shown in Figs. 1 and 2a. At low caffeine concentration (r = 1/80), helix stabilization and conformational change occurred upon the interaction of caffeine with DNA. Evidence for this comes from the changes in intensity and shifting of the absorption bands in the region of 1800–1550 cm1 due to the in-plane DNA vibrational frequencies and 1200–1450 cm1 (phosphate vibrational frequencies). Major decrease in intensity of DNA vibrations was observed as a result of caffeine– phosphate interaction and biopolymer conformational change. Similar spectral changes were observed in the spectra of drug–DNA complexes in which loss of intensity of the DNA-in-plane vibrations attributed to partial helix stabilization, while the increase in intensity of DNA vibrations at high drug content attributed to some degree of helix destabilization [41–44]. The major caffeine–PO2 interaction (through H-bonding), resulted in the shifting of the PO2 asymmetric band at 1226 cm1 towards a higher frequency at 1236 cm1 and decrease in intensity by 66%. Similarly, the PO2 symmetric stretching band at 1088 shifted to 1092 cm1 and
Fig. 1. FTIR spectra and difference FTIR spectra [(DNA solution + caffeine) DNA solution] of the free calf-thymus DNA and caffeine complexes at different molar ratios in the region of 1800–600 cm1 in aqueous solution.
decreased in intensity by 75%. In addition to a major spectral shifting of the PO2 stretching at 1226 cm1, the relative intensities of the asymmetric (mas) and symmetric (ms) vibrations were altered upon caffeine interaction. The msPO2 (1088 cm1) and masPO2 (1226 cm1) ratios changed, with ms/mas decreasing from 1.95 (uncomplexed DNA) to 1.41 (caffeine–DNA complexes) [30]. At r = 1/80, major interaction of caffeine (via N-9 and C–O groups) was observed with A-T and G donor sites
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Fig. 2. Intensity ratio variations for several DNA vibrations at 1710 (G), 1662 (T), 1610 (A), 1492 (C) and 1226 cm1 (PO2 asymmetric stretch) and 1088 cm1 (PO2 symmetric stretch) as a function of caffeine (a) and theophylline (b) concentrations.
of DNA duplex, such as adenine and guanine N-7 atoms as well as thymine O-2 atom. Evidence for this comes from the shifting of the guanine band at 1710–1699, thymine band at 1662–1672 cm1 and adenine band at 1610– 1608 cm1. As caffeine concentration increased (r = 1/40, and 1/20), the intensity of the vibrations increased and reached a maximum at r = 1/20 (Figs. 1 and 2a). The major intensity increases were attributed to a partial helix destabilization induced by the caffeine–DNA interaction. Similar increases in the intensities of DNA-in-plane vibrations were observed when the DNA duplex was destabilized upon cation interaction [45,46]. The increase in intensity was also accompanied by shifting of the guanine band at 1710–1699 cm1 (r = 1/40, and 1/20), thymine band at 1662–1668 cm1 (r = 1/40), adenine
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band at 1610–1608 cm1 (r = 1/40, 1/20), cytosine band at 1492–1489 cm1 (r = 1/40, and 1/20), PO2 (asymmetric) at 1226–1230 (r = 1/40) and 1236 cm1 (r = 1/20) and PO2 (symmetric) at 1088–1090 cm1 (r = 1/20). Thus, the spectral changes at r = 1/40, 1/20, can be related to major interaction of caffeine with mainly guanine N-7 atom (major groove), A-T base pair (minor groove) and backbone phosphate group. It should be noted that the C@O and NH groups of caffeine and DNA are effectively involved in Hbonding. In the difference spectra of caffeine–DNA (r = 1/20), positive features at 1697, 1684,1668, 1608, 1234 and 1092 cm1 are related to an increase in the intensity of G, T, A and asymmetric and symmetric phosphate vibrational frequencies as a result of caffeine bindings to the guanine N-7, adenine N-7, thymine O-2 and the backbone PO2 group. At high caffeine concentration (r = 1/10), some reduction in the intensity ratios of the bands at 1710 (G), 1662 (T), 1610 (A) and asymmetric and symmetric phosphate vibration at 1226 and 1088 cm1 were observed. The observed loss of intensity was attributed to DNA aggregation upon caffeine complexation. The band at 1492 cm1 (mainly cytosine) showed no major spectral changes upon caffeine interaction, which is indicative of minor participation of cytosine bases in caffeine complexation (Fig. 2a). Similar spectral changes was observed in the biogenic polyamine– and taxol–DNA complexes [31,47,48]. It should be noted that the absorption band with medium intensity at 1640 cm1 in the IR spectrum of the free DNA and at 1651–1653 cm1 for the complexes and in the difference spectra of ligand–DNA adducts are due to water deformation mode and they are not coming from DNA vibrations (Fig. 1) [48]. As concentration increased, r = 1/5, 1/2, 1/1 (Fig. 1), the DNA bases vibrations in the region of 1710–1610 cm1 were covered by caffeine C@O and C@N stretching bands at 1701 and 1655 cm1. Thus in the spectra of caffeine– DNA (Fig. 1, r = 1/5, 1/2, 1/1), the absorption bands at 1701, 1655, 1543, 1458, 1026 and 744 cm1 and in difference spectra, the bands at 1690, 1655, 1541, 1361 and 744 cm1 are related to caffeine vibrations and we could not draw a certain conclusion on the caffeine binding at high xanthine derivative content (Fig. 1). The UV results shows that caffeine binds externally to DNA (Fig. 5a). The increase in intensity of caffeine characteristic UV–vis band at 273 nm is due to major drug–DNA interaction at DNA surface, which does not limit the mobility of caffeine around DNA molecule (Fig. 5a) [49– 51]. 3.2. FTIR spectra of theophylline–DNA complexes Evidence related to theophylline–DNA complexation comes from the infrared spectroscopic results shown in Figs. 3 and 2b. At low theophylline concentration (r = 1/ 80), decreases in intensity of adenine, thymine and PO2
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Fig. 3. FTIR spectra and difference FTIR spectra [(DNA solution + theophylline) DNA solution] of the free calf-thymus DNA and theophylline complexes at different molar ratios in the region of 1800–600 cm1 in aqueous solution.
vibrations were observed as a result of theophylline–base and theophylline–PO2 interactions as well as biopolymer conformational change (Fig. 2b). The intensity of thymine (26%), adenine (53%), asymmetric and symmetric phosphate bands (16% and 44%) decreased. The loss of intensity of DNA-in-plane vibrations at 1662 (G), 1610 (A), 1226 (asy. PO2) and 1088 (sym. PO2) cm1 can be attributed to a partial helix stabilization as a result of ligand–phosphate binding. Minor interaction was observed between theophylline and G-C base pairs. Evidence for this comes from the small intensity increase of the G and C vibrations, the intensity of guanine band (7%) and cytosine band (20%) increased. The observed intensity changes were
accompanied by the shifting of the bases and phosphate bands. The guanine band at 1710 shifted to 1699 cm1, the cytosine band at 1492 shifted to 1489 cm1, the thymine band at 1662 shifted to 1657 cm1, the adenine band at 1610 shifted to 1608 cm1, the asymmetric and symmetric phosphate vibrations at 1226 and 1088 cm1 shifted to 1240 and 1092 cm1, respectively. In addition to major spectral shifting of PO2 stretching at 1226 cm1, the relative intensities of the asymmetric (mas) and symmetric (ms) vibrations were altered upon theophylline interaction. The msPO2 (1088 cm1) and masPO2 (1226 cm1) values changed, with the ms/mas decreasing from 1.95 (uncomplexed DNA) to 1.46 (theophylline–DNA complexes) [30]. As theophylline concentration increased (r = 1/40) (Figs. 3 and 2b), major interaction of ligand (via N-9 and C–O groups) with G (Guanine N-7), A–T base pairs and oxygen atom of PO2 group occurred. Evidence for this comes from major increase in intensity for these vibrations. The observed intensity changes were accompanied by shifting of the bands. The guanine band at 1710 shifted to 1699 cm1, the adenine band at 1610 shifted to 1608 cm1, the cytosine band at 1492 cm1 shifted to 1489 cm1, the asymmetric and symmetric phosphate vibrations at 1226 and 1088 cm1 shifted to 1240 and 1090 cm1, respectively. It should be noted that the cytosine band at 1492 cm1 exhibited minor shifting while its relative intensity did not change significantly at different theophylline concentrations. This can be due to minor interaction of cytosine and theophylline. In the difference spectra of theophylline–DNA (r = 1/ 40), the presence of several positive derivative features at 1697, 1685, 1664, 1489, 1240, and 1092 cm1 are related to the intensity increase of DNA-in-plane vibrations as a result of major ligand–DNA interaction (Fig. 3). At r = 1/20, some reduction in the intensity ratios of the bands at 1710 (G), 1662 (T), 1610 (A), 1492 (C), 1226 (asy. PO2) and 1088 cm1 (sym. PO2) were observed (Fig. 2b). The loss of intensity ratios were probably due to aggregation of DNA complexes. At higher concentrations (r = 1/10), minor increase in intensities of bases and phosphate bands was accompanied by the shifting of the guanine band at 1710–1697 cm1, adenine band at 1610–1606 cm1, no shifting was observed for thymine, but a new band emerged at 1670 cm1. The asymmetric PO2 vibration at 1226 shifted to 1238 cm1. The observed spectral changes can be attributed to continued interaction of theophylline with DNA bases and the alterations of DNA conformation (will be discussed further on). At r = 1/5, the intensity of all bands decreased that can be related to DNA aggregation upon theophylline interaction (Fig. 2b). At higher contents of theophylline (r = 1/2, 1/1) (Fig. 3), the DNA base vibrations in the region of 1720–1610 cm1 was covered by theophylline C@O stretching band at 1714 and C@C and C@N stretching modes at 1666–1650 cm1
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and thus, we could not draw a certain conclusion on the nature of theophylline–DNA binding at high ligand concentrations. In the spectra of theophylline–DNA complexes (Fig. 3, r = 1/2, 1/1), the absorption bands at 1703, 1666, 1606, 1489, 1188 and 742 cm1 and the positive features in the difference spectra at 1714, 1666, 1489, 1188, 980, 926 and 742 cm1 are coming from theophylline vibrational frequencies and not DNA vibrations. The UV results shows that theophylline binds externally to DNA (Fig. 5b). The increase in intensity of theophylline characteristic UV–vis band at 278 nm is due to major drug–DNA interaction at DNA surface, which does not limit the mobility of theophylline around DNA molecule (Fig. 5a) [49–51]. 3.3. DNA conformation A partial B to A-DNA transition occurred upon caffeine and theophylline complexation. Evidence for this comes
Fig. 5. UV–visible spectra characteristics of caffeine, theophylline and their DNA adducts for caffeine at 273 nm (a) and theophylline at 278 nm (b), with final DNA concentration of 0.1 mM and caffeine and theophylline of 0.05 mM.
from the shift of DNA marker infrared bands for the guanine at 1710 (G) to 1699–1701 cm1, the PO2 stretching vibration at 1226 to 1230–1240 cm1 in the spectra of caffeine and theophylline–DNA complexes (Figs. 1 and 3). When a complete B to A transition occurs, the DNA marker bands such as 836 cm1 appears at about 820810 cm1, while the PO2 stretching vibration at 1222 shifts towards a higher frequency at 1230–1240 cm1 and the guanine band at 1710 appears at 1700 cm1 [39,40,52–56]. Thus, the spectral changes observed for the DNA marker bands are due to a partial B to A transition upon caffeine and theophylline interactions (Figs. 1 and 3). It should be mentioned that the shift of the sugar band from 1055 to 1065–1070 cm1 can be related to some degree of DNA denaturation. 3.4. Absorption spectra of caffeine, theophylline–DNA complexes
Fig. 4. The plot of 1/(A A0) vs. 1/L for DNA and their ligand complexes where A0 is the initial absorption of DNA (260 nm) and A is the recorded absorption at different caffeine and theophylline concentrations (L).
The calculation of the overall binding constants were carried out using UV spectroscopy as reported [57,58]. If the equilibrium for each ligand and DNA complex was established as: ligand þ DNA () ligand : DNA K ¼ ½ligand : DNA=½ligand½DNA
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The double reciprocal plot of 1/[A A0] vs 1/[ligand] is linear and the association binding constant (K) is calculated from the ratio of the intercept on the vertical coordinate axis to the slope [44,59–61] (Fig. 4). Concentrations of complexed ligand were determined by subtracting absorbance of free DNA at 260 nm from those of the complexed DNA. Concentration of the free ligand was determined by subtraction of complexed ligand from total ligand used for the experiment. Our data of 1/[ligand complexed] almost proportionally increased as a function of 1/[free ligand] (Fig. 4), and thus the overall binding constants are estimated to be K(caffeine–DNA) = 9.7 · 103 M1 for caffeine–DNA and K(theophylline–DNA) = 1.7 · 104 M1 for theophylline–DNA. A larger K value is estimated for theophylline–DNA, because it provides greater accessibility for DNA binding rather than caffeine. The overall binding constants of the caffeine–DNA and theophylline/DNA complexes are consistent with those of the corresponding xanthine-mononucleotide binding that show weak ligand– DNA interaction [37,38]. 4. Conclusions On the basis of our spectroscopic results the following remarks can be made. (i) The order of stability of adduct formation was theophylline > caffeine. (ii) In caffeine–DNA complexes, the helix destabilization occurred at r = 1/20, while in theophylline–DNA complexes, helix destabilization occurred at r = 1/ 40, because theophylline induces more perturbations of DNA duplex. (iii) The interaction of DNA with caffeine and theophylline was established through H-bonding. (iv) The complexation occurred with the G-C and A-T bases and PO2 group (through H-bonding). (v) A partial B to A-DNA transition occurred at high concentrations of caffeine and theophylline.
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Journal of Molecular Structure 875 (2008) 392–399 www.elsevier.com/locate/molstruc
Structural features of DNA interaction with caffeine and theophylline Shohreh Nafisi
b
a,*
, Firouzeh Manouchehri a, Heidar-Ali Tajmir-Riahi b, Maryam Varavipour c
a Department of Chemistry, Azad University, Central Tehran Branch, Tehran 14169 63316, Iran Department of Chemistry-Biology, University of Quebec at Trois-Rivieres, C.P. 500, TR, Que., Canada G9A 5H7 c Department of Iirigation & Drainage Engineering, College of Aboreyhan, University of Tehran, Iran
Received 26 February 2007; received in revised form 9 May 2007; accepted 9 May 2007 Available online 17 May 2007
Abstract Caffeine and theophylline are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these xanthine derivatives with individual DNA at molecular level. The aim of this study was to examine the stability and structural features of calf-thymus DNA complexes with caffeine and theophylline in aqueous solution, using constant DNA concentration (6.25 mM) and various caffeine or theophylline/DNA(P) ratios of 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1. FTIR, UV–visible spectroscopic methods were used to determine the ligand external binding modes, the binding constant and the stability of caffeine, theophylline–DNA complexes in aqueous solution. Spectroscopic evidence showed that the complexation of caffeine and theophylline with DNA occurred via G-C and A-T and PO2 group with overall binding constants of K(caffeine–DNA) = 9.7 · 103 M1 and K(theophylline–DNA) = 1.7 · 104 M1. The affinity of ligand–DNA binding is in the order of theophylline > caffeine. A partial B to A-DNA transition occurs upon caffeine and theophylline complexation. 2007 Elsevier B.V. All rights reserved. Keywords: DNA; Caffeine; Theophylline; Binding constant; Binding mode; Conformation; Stability; Secondary structure; FTIR; UV–visible spectroscopy
1. Introduction Caffeine is the most widely consumed in the world. It is found in seeds, citrus fruits, olive oil, tea, cocoa beverages. Because of the vast consumption of caffeine, research has focused on its physiological effects [1]. Caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione) and theophylline (3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione) bases (Scheme 1) are structurally related to nucleic acids components. Caffeine has a CH3 group at the N7 site which blocks coordination with N7 atom, while in theophylline the N7 site is accessible for ligand coordination. The presence of oxy and hydroxyl groups as well as double bonds in specific positions make them strong *
Corresponding author. Tel./fax: +98 21 22426554. E-mail address: drsnafi[email protected] (S. Nafisi).
0022-2860/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2007.05.010
antioxidants. Caffeine has significant abilities to scavenge highly reactive free radicals and excited states of oxygen and to protect crucial biological molecules against these species. Antioxidant ability of caffeine is similar to that of the established biological antioxidant glutathione and significantly much higher than that of ascorbic acid [2–7]. Caffeine is an efficient inhibitor of DNA repair and DNA damage-activated checkpoints. It inhibits retroviral transduction of dividing cells, most likely by blocking post integration repair. This effect may be mediated at least in part by a cellular target of caffeine. Caffeine belongs to a class of chemicals that strongly enhance the cytotoxic effect of ionizing radiation and other DNA damaging agents, at concentrations that are not otherwise toxic to cells [8–12]. However, it has been established that caffeine disrupts DNA damage-activated cell cycle checkpoints [13–22]. Caffeine and theophylline can modulate the binding of certain
S. Nafisi et al. / Journal of Molecular Structure 875 (2008) 392–399
O
H3C
O
H3C
N
N N
O
CH3
O
chemicals were of reagent grade and used without further purification.
N
N
N
H
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2.2. Preparation of stock solutions N
N
CH3
CH3
Caffeine
Theohphylline Scheme 1.
DNA damaging agents and reduce DNA directed toxicity [23,24]. The molecular mechanisms underlying the effects of caffeine and theophylline on DNA are still not fully understood. The bindings of caffeine and theophylline to nucleic acids are studied and were found to exhibit a dose dependent binding behaviour [23,24]. Investigations on the interaction of caffeine and theophylline with DNA suggests that intercalation is not the predominant mechanism [25]. The studies on the interaction of RNA and xanthine derivatives (caffeine, theophylline and theobromine) showed that G-C and A-U bases of RNA are the targets for caffeine and theophylline bindings so that the C@O and NH groups of the ligand and RNA mutually involved in H-bonding. Among the three xanthine derivatives, theophylline showed greater binding efficacy with RNA than theobromine and caffeine [23–26], while the binding positions of caffeine and theophylline on B-DNA have not been firmly resolved. It is possible that both the base sequence and the environment have an important influence on xanthine binding. Therefore, it was of interest to examine the bindings of caffeine and theophylline to DNA bases and the backbone phosphate group. We now report the results of FTIR and UV–visible spectroscopic analysis of DNA interaction with caffeine and theophylline in aqueous solution at physiological conditions, using constant DNA concentration and various ligand/DNA (phosphate) ratios of 1/80 to 1/1. The ligand binding site, the binding constant, and the effects of ligand complexation on the stability and conformation of DNA duplex are discussed here.
2. Methods and material 2.1. Materials Caffeine and theophylline were purchased from Merck Co. Highly polymerized type I calf-thymus DNA sodium salt (7% Na content) was purchased from Sigma Chemical Co., and deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. The absorbance at 260 and 280 nm was recorded, in order to check the protein content of DNA solution. The A260/A280 ratio was 1.80, showing that the DNA was sufficiently free of protein [27]. Other
Sodium–DNA (5 mg/ml) was dissolved in distilled water (pH 7) at 5 C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the calf-thymus DNA solution was determined spectrophotometrically at 260 nm using molar extinction coefficient 260 = 6600 cm1 M1 (expressed as molarity of phosphate groups) [28,29]. The UV absorbance at 260 nm of a diluted solution (1/250) of calf-thymus DNA used in our experiments was 0.661 (path length was 1 cm) and the final concentration of the DNA solution was 12.5 mM in DNA phosphate. The appropriate amounts of caffeine and theophylline (0.15–12.5 mM) were prepared in distilled water and added dropwise to DNA solution in order to attain the desired ligand/DNA(P) molar ratios (r) of 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1 with a final DNA(P) concentration of 6.25 mM (path length was 0.03 cm). The pH of the solutions was adjusted at 7.0 ± 0.2 using NaOH solution. 2.3. Absorption spectroscopy The absorption spectra were recorded on a LKB model 4054 UV–vis spectrometer, using various ligands concentrations (5 lM to 0.05 mM) and DNA concentration of 0.1 mM. 2.4. FTIR spectroscopic measurements Infrared spectra were recorded on a Jasco FTIR spectrometer equipped with a liquid-nitrogen-cooled HgCdTe (MCT) detector and a KBr beam splitter. The spectra of the caffeine and theophylline/DNA solutions were acquired using a cell assembled with AgBr windows. Spectra were collected after 2 h incubation of ligand with the DNA solution and were measured in triplicate (three individual samples of the same DNA and ligand concentrations). For each spectrum, 100 scans were collected at a resolution of 4 cm1. The water subtraction was carried out using NaCl (0.1 mol/L) solution at pH 6.5–7.5 as a reference. A good water subtraction was considered to be achieved when there was a flat baseline around 2200 cm1, where the water combination mode is located. This method yields a rough estimate of the subtraction scaling factor, but removes the spectral features of water in a satisfactory way [30]. The difference spectra [(DNA solution + ligand) (DNA solution)] were obtained using a sharp DNA band at 968 cm1 as internal reference. This band, which is due to sugar C–C and C–O stretching vibrations, exhibit no spectral change (shifting or intensity variations) upon caffeine and theophylline–DNA complexation, and it is cancelled out upon spectral subtraction [31]. The intensity ratios of bands due to several
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DNA-in-plane vibrations related to A-T, G-C base pairs and the PO2 stretching vibrations were measured with respect to the reference band at 968 cm1 (DNA) as a function of caffeine and theophylline concentration with an error of ±3%. Similar intensity variations have been used to determine the ligand binding to DNA bases and backbone phosphate groups [32–34]. The plots of the relative intensity (R) of several peaks of DNA-in-plane vibrations related to A-T, G-C base pairs and the PO2 stretching vibrations such as 1710 (guanine), 1662 (thymine), 1610 (adenine), 1492 (cytosine), and 1226 cm1 (PO2 groups), versus the caffeine and theophylline concentrations were obtained after peak normalization using, Ri = Ii/I968, where Ii is the intensity of absorption peak for pure DNA in the complex with i as ligand concentration, and I968 is the intensity of the 968 cm1 peak (internal reference). 3. Results and discussion Caffeine and theophylline showed intense IR bands in the region of 1680–1720 cm1, that were assigned to the C@O stretching vibrations. Other bands in the region of 1550–1666 cm1 were assigned to C@C and C@N stretching modes in purine ring system [35–37]. 3.1. FTIR spectra of caffeine–DNA complexes FTIR spectra of caffeine–DNA complexes between 1800 and 600 cm1 are presented in Fig. 1. The spectral changes (intensity and shifting) of several prominent DNA-in-plane vibrations at 1710 cm1 (G, T mainly G), 1662 cm1 (T, G, A and C, mainly T), 1610 cm1 (A, C, mainly A), 1492 cm1 (C, G mainly C) and 1226 cm1 (PO2 asymmetric stretch) and 1088 cm1 [30,38–41] were monitored at different caffeine–DNA molar ratios and the results are shown in Figs. 1 and 2a. At low caffeine concentration (r = 1/80), helix stabilization and conformational change occurred upon the interaction of caffeine with DNA. Evidence for this comes from the changes in intensity and shifting of the absorption bands in the region of 1800–1550 cm1 due to the in-plane DNA vibrational frequencies and 1200–1450 cm1 (phosphate vibrational frequencies). Major decrease in intensity of DNA vibrations was observed as a result of caffeine– phosphate interaction and biopolymer conformational change. Similar spectral changes were observed in the spectra of drug–DNA complexes in which loss of intensity of the DNA-in-plane vibrations attributed to partial helix stabilization, while the increase in intensity of DNA vibrations at high drug content attributed to some degree of helix destabilization [41–44]. The major caffeine–PO2 interaction (through H-bonding), resulted in the shifting of the PO2 asymmetric band at 1226 cm1 towards a higher frequency at 1236 cm1 and decrease in intensity by 66%. Similarly, the PO2 symmetric stretching band at 1088 shifted to 1092 cm1 and
Fig. 1. FTIR spectra and difference FTIR spectra [(DNA solution + caffeine) DNA solution] of the free calf-thymus DNA and caffeine complexes at different molar ratios in the region of 1800–600 cm1 in aqueous solution.
decreased in intensity by 75%. In addition to a major spectral shifting of the PO2 stretching at 1226 cm1, the relative intensities of the asymmetric (mas) and symmetric (ms) vibrations were altered upon caffeine interaction. The msPO2 (1088 cm1) and masPO2 (1226 cm1) ratios changed, with ms/mas decreasing from 1.95 (uncomplexed DNA) to 1.41 (caffeine–DNA complexes) [30]. At r = 1/80, major interaction of caffeine (via N-9 and C–O groups) was observed with A-T and G donor sites
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Fig. 2. Intensity ratio variations for several DNA vibrations at 1710 (G), 1662 (T), 1610 (A), 1492 (C) and 1226 cm1 (PO2 asymmetric stretch) and 1088 cm1 (PO2 symmetric stretch) as a function of caffeine (a) and theophylline (b) concentrations.
of DNA duplex, such as adenine and guanine N-7 atoms as well as thymine O-2 atom. Evidence for this comes from the shifting of the guanine band at 1710–1699, thymine band at 1662–1672 cm1 and adenine band at 1610– 1608 cm1. As caffeine concentration increased (r = 1/40, and 1/20), the intensity of the vibrations increased and reached a maximum at r = 1/20 (Figs. 1 and 2a). The major intensity increases were attributed to a partial helix destabilization induced by the caffeine–DNA interaction. Similar increases in the intensities of DNA-in-plane vibrations were observed when the DNA duplex was destabilized upon cation interaction [45,46]. The increase in intensity was also accompanied by shifting of the guanine band at 1710–1699 cm1 (r = 1/40, and 1/20), thymine band at 1662–1668 cm1 (r = 1/40), adenine
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band at 1610–1608 cm1 (r = 1/40, 1/20), cytosine band at 1492–1489 cm1 (r = 1/40, and 1/20), PO2 (asymmetric) at 1226–1230 (r = 1/40) and 1236 cm1 (r = 1/20) and PO2 (symmetric) at 1088–1090 cm1 (r = 1/20). Thus, the spectral changes at r = 1/40, 1/20, can be related to major interaction of caffeine with mainly guanine N-7 atom (major groove), A-T base pair (minor groove) and backbone phosphate group. It should be noted that the C@O and NH groups of caffeine and DNA are effectively involved in Hbonding. In the difference spectra of caffeine–DNA (r = 1/20), positive features at 1697, 1684,1668, 1608, 1234 and 1092 cm1 are related to an increase in the intensity of G, T, A and asymmetric and symmetric phosphate vibrational frequencies as a result of caffeine bindings to the guanine N-7, adenine N-7, thymine O-2 and the backbone PO2 group. At high caffeine concentration (r = 1/10), some reduction in the intensity ratios of the bands at 1710 (G), 1662 (T), 1610 (A) and asymmetric and symmetric phosphate vibration at 1226 and 1088 cm1 were observed. The observed loss of intensity was attributed to DNA aggregation upon caffeine complexation. The band at 1492 cm1 (mainly cytosine) showed no major spectral changes upon caffeine interaction, which is indicative of minor participation of cytosine bases in caffeine complexation (Fig. 2a). Similar spectral changes was observed in the biogenic polyamine– and taxol–DNA complexes [31,47,48]. It should be noted that the absorption band with medium intensity at 1640 cm1 in the IR spectrum of the free DNA and at 1651–1653 cm1 for the complexes and in the difference spectra of ligand–DNA adducts are due to water deformation mode and they are not coming from DNA vibrations (Fig. 1) [48]. As concentration increased, r = 1/5, 1/2, 1/1 (Fig. 1), the DNA bases vibrations in the region of 1710–1610 cm1 were covered by caffeine C@O and C@N stretching bands at 1701 and 1655 cm1. Thus in the spectra of caffeine– DNA (Fig. 1, r = 1/5, 1/2, 1/1), the absorption bands at 1701, 1655, 1543, 1458, 1026 and 744 cm1 and in difference spectra, the bands at 1690, 1655, 1541, 1361 and 744 cm1 are related to caffeine vibrations and we could not draw a certain conclusion on the caffeine binding at high xanthine derivative content (Fig. 1). The UV results shows that caffeine binds externally to DNA (Fig. 5a). The increase in intensity of caffeine characteristic UV–vis band at 273 nm is due to major drug–DNA interaction at DNA surface, which does not limit the mobility of caffeine around DNA molecule (Fig. 5a) [49– 51]. 3.2. FTIR spectra of theophylline–DNA complexes Evidence related to theophylline–DNA complexation comes from the infrared spectroscopic results shown in Figs. 3 and 2b. At low theophylline concentration (r = 1/ 80), decreases in intensity of adenine, thymine and PO2
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Fig. 3. FTIR spectra and difference FTIR spectra [(DNA solution + theophylline) DNA solution] of the free calf-thymus DNA and theophylline complexes at different molar ratios in the region of 1800–600 cm1 in aqueous solution.
vibrations were observed as a result of theophylline–base and theophylline–PO2 interactions as well as biopolymer conformational change (Fig. 2b). The intensity of thymine (26%), adenine (53%), asymmetric and symmetric phosphate bands (16% and 44%) decreased. The loss of intensity of DNA-in-plane vibrations at 1662 (G), 1610 (A), 1226 (asy. PO2) and 1088 (sym. PO2) cm1 can be attributed to a partial helix stabilization as a result of ligand–phosphate binding. Minor interaction was observed between theophylline and G-C base pairs. Evidence for this comes from the small intensity increase of the G and C vibrations, the intensity of guanine band (7%) and cytosine band (20%) increased. The observed intensity changes were
accompanied by the shifting of the bases and phosphate bands. The guanine band at 1710 shifted to 1699 cm1, the cytosine band at 1492 shifted to 1489 cm1, the thymine band at 1662 shifted to 1657 cm1, the adenine band at 1610 shifted to 1608 cm1, the asymmetric and symmetric phosphate vibrations at 1226 and 1088 cm1 shifted to 1240 and 1092 cm1, respectively. In addition to major spectral shifting of PO2 stretching at 1226 cm1, the relative intensities of the asymmetric (mas) and symmetric (ms) vibrations were altered upon theophylline interaction. The msPO2 (1088 cm1) and masPO2 (1226 cm1) values changed, with the ms/mas decreasing from 1.95 (uncomplexed DNA) to 1.46 (theophylline–DNA complexes) [30]. As theophylline concentration increased (r = 1/40) (Figs. 3 and 2b), major interaction of ligand (via N-9 and C–O groups) with G (Guanine N-7), A–T base pairs and oxygen atom of PO2 group occurred. Evidence for this comes from major increase in intensity for these vibrations. The observed intensity changes were accompanied by shifting of the bands. The guanine band at 1710 shifted to 1699 cm1, the adenine band at 1610 shifted to 1608 cm1, the cytosine band at 1492 cm1 shifted to 1489 cm1, the asymmetric and symmetric phosphate vibrations at 1226 and 1088 cm1 shifted to 1240 and 1090 cm1, respectively. It should be noted that the cytosine band at 1492 cm1 exhibited minor shifting while its relative intensity did not change significantly at different theophylline concentrations. This can be due to minor interaction of cytosine and theophylline. In the difference spectra of theophylline–DNA (r = 1/ 40), the presence of several positive derivative features at 1697, 1685, 1664, 1489, 1240, and 1092 cm1 are related to the intensity increase of DNA-in-plane vibrations as a result of major ligand–DNA interaction (Fig. 3). At r = 1/20, some reduction in the intensity ratios of the bands at 1710 (G), 1662 (T), 1610 (A), 1492 (C), 1226 (asy. PO2) and 1088 cm1 (sym. PO2) were observed (Fig. 2b). The loss of intensity ratios were probably due to aggregation of DNA complexes. At higher concentrations (r = 1/10), minor increase in intensities of bases and phosphate bands was accompanied by the shifting of the guanine band at 1710–1697 cm1, adenine band at 1610–1606 cm1, no shifting was observed for thymine, but a new band emerged at 1670 cm1. The asymmetric PO2 vibration at 1226 shifted to 1238 cm1. The observed spectral changes can be attributed to continued interaction of theophylline with DNA bases and the alterations of DNA conformation (will be discussed further on). At r = 1/5, the intensity of all bands decreased that can be related to DNA aggregation upon theophylline interaction (Fig. 2b). At higher contents of theophylline (r = 1/2, 1/1) (Fig. 3), the DNA base vibrations in the region of 1720–1610 cm1 was covered by theophylline C@O stretching band at 1714 and C@C and C@N stretching modes at 1666–1650 cm1
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and thus, we could not draw a certain conclusion on the nature of theophylline–DNA binding at high ligand concentrations. In the spectra of theophylline–DNA complexes (Fig. 3, r = 1/2, 1/1), the absorption bands at 1703, 1666, 1606, 1489, 1188 and 742 cm1 and the positive features in the difference spectra at 1714, 1666, 1489, 1188, 980, 926 and 742 cm1 are coming from theophylline vibrational frequencies and not DNA vibrations. The UV results shows that theophylline binds externally to DNA (Fig. 5b). The increase in intensity of theophylline characteristic UV–vis band at 278 nm is due to major drug–DNA interaction at DNA surface, which does not limit the mobility of theophylline around DNA molecule (Fig. 5a) [49–51]. 3.3. DNA conformation A partial B to A-DNA transition occurred upon caffeine and theophylline complexation. Evidence for this comes
Fig. 5. UV–visible spectra characteristics of caffeine, theophylline and their DNA adducts for caffeine at 273 nm (a) and theophylline at 278 nm (b), with final DNA concentration of 0.1 mM and caffeine and theophylline of 0.05 mM.
from the shift of DNA marker infrared bands for the guanine at 1710 (G) to 1699–1701 cm1, the PO2 stretching vibration at 1226 to 1230–1240 cm1 in the spectra of caffeine and theophylline–DNA complexes (Figs. 1 and 3). When a complete B to A transition occurs, the DNA marker bands such as 836 cm1 appears at about 820810 cm1, while the PO2 stretching vibration at 1222 shifts towards a higher frequency at 1230–1240 cm1 and the guanine band at 1710 appears at 1700 cm1 [39,40,52–56]. Thus, the spectral changes observed for the DNA marker bands are due to a partial B to A transition upon caffeine and theophylline interactions (Figs. 1 and 3). It should be mentioned that the shift of the sugar band from 1055 to 1065–1070 cm1 can be related to some degree of DNA denaturation. 3.4. Absorption spectra of caffeine, theophylline–DNA complexes
Fig. 4. The plot of 1/(A A0) vs. 1/L for DNA and their ligand complexes where A0 is the initial absorption of DNA (260 nm) and A is the recorded absorption at different caffeine and theophylline concentrations (L).
The calculation of the overall binding constants were carried out using UV spectroscopy as reported [57,58]. If the equilibrium for each ligand and DNA complex was established as: ligand þ DNA () ligand : DNA K ¼ ½ligand : DNA=½ligand½DNA
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The double reciprocal plot of 1/[A A0] vs 1/[ligand] is linear and the association binding constant (K) is calculated from the ratio of the intercept on the vertical coordinate axis to the slope [44,59–61] (Fig. 4). Concentrations of complexed ligand were determined by subtracting absorbance of free DNA at 260 nm from those of the complexed DNA. Concentration of the free ligand was determined by subtraction of complexed ligand from total ligand used for the experiment. Our data of 1/[ligand complexed] almost proportionally increased as a function of 1/[free ligand] (Fig. 4), and thus the overall binding constants are estimated to be K(caffeine–DNA) = 9.7 · 103 M1 for caffeine–DNA and K(theophylline–DNA) = 1.7 · 104 M1 for theophylline–DNA. A larger K value is estimated for theophylline–DNA, because it provides greater accessibility for DNA binding rather than caffeine. The overall binding constants of the caffeine–DNA and theophylline/DNA complexes are consistent with those of the corresponding xanthine-mononucleotide binding that show weak ligand– DNA interaction [37,38]. 4. Conclusions On the basis of our spectroscopic results the following remarks can be made. (i) The order of stability of adduct formation was theophylline > caffeine. (ii) In caffeine–DNA complexes, the helix destabilization occurred at r = 1/20, while in theophylline–DNA complexes, helix destabilization occurred at r = 1/ 40, because theophylline induces more perturbations of DNA duplex. (iii) The interaction of DNA with caffeine and theophylline was established through H-bonding. (iv) The complexation occurred with the G-C and A-T bases and PO2 group (through H-bonding). (v) A partial B to A-DNA transition occurred at high concentrations of caffeine and theophylline.
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