Lexes (D) DNA-Caffeine complexes, where A0 = absorbance (260 nm) of free DNA, [theophylline or theobromine or caffeine] = concentration of the respective drug. doi:10.1371/journal.pone.0050019.gMethylxanthines Binding with DNAFigure 3. FTIR spectra of free methylxanthines in the region of 1400?00 cm21. doi:10.1371/journal.pone.0050019.gIt is Cucurbitacin I observed that the carbonyl (C = O) vibration frequency (uC = O) of both drug (theophylline = 1718, 1666 cm21; caffeine = 1699.8, 1658.7 cm21) as well as DNA (1694.4 cm21) disappeared and a new vibration band with change in the intensity appeared around 1702.7 and 1693.35 cm21, in DNAtheophylline and DNA-caffeine Licochalcone A site complexes respectively. This indicates that the C = O and NH groups of drug and DNA are effectively involved in H-bonding interaction. However, we noticed only minor change in the C = O frequency of theobromine (1691.7 cm21) (Table 1). The H-bonding interaction of methylxanthines with DNA bases (G-C/A-T) is substantiated by the major spectral changes of DNA in-plane vibrations in the region of 1707?400 cm21 [41,42]. The band at 1694.4 cm21 (G, T) related to mainly guanine shifted to 1702.7, 1691.72 and 1693 cm21 in DNA-theophylline, DNAtheobromine and DNA-caffeine complexes respectively. The changes were also observed in the band at 1658 cm21 (T, G, C) mainly for thymine [41,42], cytosine band at 1484.2 cm21 (C, G) and for adenine at 1600 cm21 upon drug complexation. The shifting in the vibrational frequency of bases due to drug complexation clearly indicates that methylxanthines are able to interact with A-T and G-C bases where the NH and C = O ofFigure 4. FTIR spectra of free DNA and DNA-methylxanthines complexes obtained with repeated scanning between 1400?400 cm21. doi:10.1371/journal.pone.0050019.gDNA and drugs are mutually involved in H-bonding. This gains the support from the studies of Nafisi et.al, that caffeine and theophylline complexations with DNA are established through the hydrogen bonding 1676428 interaction [17]. Also splitting/shifting was observed in the vibrational intensities of assymetric (uas) and symmetric (us) PO22 band of free DNA at 1238.9, 1099 cm21 respectively. In DNA-theophylline complexes 24272870 the PO22 band of free DNA at 1238.9 cm21 exhibited splitting and shifting into 1250 and 1240.9 cm21. Similarly in DNAtheobromine and DNA-caffeine complexes the PO22 band exhibited shifting into 1223.83, 1205 cm21 and 1238.42, 1210 cm21 respectively. Whereas the usPO22 band of free DNA at 1099 showed shifting into 1090, 1070.95 and 1098 in DNAtheophylline, DNA-theobromine and DNA-caffeine complexes respectively (Table 1). The changes observed in the vibrational frequency of PO22 band are attributed to the possible interaction of methylxanthines with DNA bases as well as to the phosphate groups. At the same time only minor perturbations were observed in the IR marker bands of free DNA, sugar-phosphate stretch (890 cm21) and phosphodiester mode (828 cm21) upon drugMethylxanthines Binding with DNATable 1. The vibrational frequencies of C = O, NH and PO22 (FTIR, KBr cm21) bands of free DNA, free drugs and DNA-drug complexes.Free Drugs (cm21) X1 NH C=O PO22 (uas) PO22 (us) 3350?900 1694.4 1238.9 1099 3121 1718, 1666.8 — — X2 3113 1691.7 — — X3 3111 3600?600 3650?650 1691.7 1223.83, 1205 1070.95 3450?700 1693.35 1238.42, 1210Functional Groups Free DNA (cm21)DNA-X1 (cm21)DNA-X2 (cm21)DNA-X3 (cm21)1699.8, 1658.7 1702.7 — — 1250, 1240.9X1 = theophylline, X2 = theobromine and X3 = caffein.Lexes (D) DNA-Caffeine complexes, where A0 = absorbance (260 nm) of free DNA, [theophylline or theobromine or caffeine] = concentration of the respective drug. doi:10.1371/journal.pone.0050019.gMethylxanthines Binding with DNAFigure 3. FTIR spectra of free methylxanthines in the region of 1400?00 cm21. doi:10.1371/journal.pone.0050019.gIt is observed that the carbonyl (C = O) vibration frequency (uC = O) of both drug (theophylline = 1718, 1666 cm21; caffeine = 1699.8, 1658.7 cm21) as well as DNA (1694.4 cm21) disappeared and a new vibration band with change in the intensity appeared around 1702.7 and 1693.35 cm21, in DNAtheophylline and DNA-caffeine complexes respectively. This indicates that the C = O and NH groups of drug and DNA are effectively involved in H-bonding interaction. However, we noticed only minor change in the C = O frequency of theobromine (1691.7 cm21) (Table 1). The H-bonding interaction of methylxanthines with DNA bases (G-C/A-T) is substantiated by the major spectral changes of DNA in-plane vibrations in the region of 1707?400 cm21 [41,42]. The band at 1694.4 cm21 (G, T) related to mainly guanine shifted to 1702.7, 1691.72 and 1693 cm21 in DNA-theophylline, DNAtheobromine and DNA-caffeine complexes respectively. The changes were also observed in the band at 1658 cm21 (T, G, C) mainly for thymine [41,42], cytosine band at 1484.2 cm21 (C, G) and for adenine at 1600 cm21 upon drug complexation. The shifting in the vibrational frequency of bases due to drug complexation clearly indicates that methylxanthines are able to interact with A-T and G-C bases where the NH and C = O ofFigure 4. FTIR spectra of free DNA and DNA-methylxanthines complexes obtained with repeated scanning between 1400?400 cm21. doi:10.1371/journal.pone.0050019.gDNA and drugs are mutually involved in H-bonding. This gains the support from the studies of Nafisi et.al, that caffeine and theophylline complexations with DNA are established through the hydrogen bonding 1676428 interaction [17]. Also splitting/shifting was observed in the vibrational intensities of assymetric (uas) and symmetric (us) PO22 band of free DNA at 1238.9, 1099 cm21 respectively. In DNA-theophylline complexes 24272870 the PO22 band of free DNA at 1238.9 cm21 exhibited splitting and shifting into 1250 and 1240.9 cm21. Similarly in DNAtheobromine and DNA-caffeine complexes the PO22 band exhibited shifting into 1223.83, 1205 cm21 and 1238.42, 1210 cm21 respectively. Whereas the usPO22 band of free DNA at 1099 showed shifting into 1090, 1070.95 and 1098 in DNAtheophylline, DNA-theobromine and DNA-caffeine complexes respectively (Table 1). The changes observed in the vibrational frequency of PO22 band are attributed to the possible interaction of methylxanthines with DNA bases as well as to the phosphate groups. At the same time only minor perturbations were observed in the IR marker bands of free DNA, sugar-phosphate stretch (890 cm21) and phosphodiester mode (828 cm21) upon drugMethylxanthines Binding with DNATable 1. The vibrational frequencies of C = O, NH and PO22 (FTIR, KBr cm21) bands of free DNA, free drugs and DNA-drug complexes.Free Drugs (cm21) X1 NH C=O PO22 (uas) PO22 (us) 3350?900 1694.4 1238.9 1099 3121 1718, 1666.8 — — X2 3113 1691.7 — — X3 3111 3600?600 3650?650 1691.7 1223.83, 1205 1070.95 3450?700 1693.35 1238.42, 1210Functional Groups Free DNA (cm21)DNA-X1 (cm21)DNA-X2 (cm21)DNA-X3 (cm21)1699.8, 1658.7 1702.7 — — 1250, 1240.9X1 = theophylline, X2 = theobromine and X3 = caffein.
