Amantadine–DNA interaction as studied by classical and resonance Raman spectroscopy

Journal of Molecular Structure 478 (1999) 129–138

Amantadine–DNA interaction as studied by classical and resonance Raman spectroscopy J. Stanicˇova´ a, G. Fabriciova´ b, L. Chinsky c, V. Sˇutiak d, P. Misˇkovsky´ b,* a

Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine, Komenske´ho 73, 041 81 Kosˇice, Slovak Republic b Biophysics Department, Sˇafa´rik University, Jesenna´ 5, 041 54 Kosˇice, Slovak Republic c L.P.B.C. (CNRS URA 2056), Universite´ P. et M. Curie, Case 138, 4 Place Jussieu, 752 31 Paris Cedex 05, France d Department of Pharmacology, University of Veterinary Medicine, Komenske´ho 73, 041 81 Kosˇice, Slovak Republic Received 28 July 1998; received in revised form 6 October 1998; accepted 6 October 1998

Abstract The interaction of the antiviral agent amantadine with calf thymus DNA was studied by classical and UV-resonance Raman spectroscopy. It was found that: (i) the drug interacts with purine bases adenine and guanine via hydrogen bonds formation between N7 positions of purines and amino group of amantadine and (ii) the interaction leads to partial DNA structure change, which is demonstrated by a deformation of the hydrogen bonds of the A–T base pairs and by a partial deformation of the sugarphosphate backbone of DNA, which does not lead to the DNA conformation transition. 䉷 1999 Elsevier Science B.V. All rights reserved. Keywords: Amantadine; Calf thymus DNA; Raman spectroscopy

1. Introduction Amantadine (Fig. 1) is an antiviral agent that specifically inhibits influenza A virus replication at a micromolar concentration [1]. Higher concentrations of amantadine are required to inhibit viruses of influenza B, rubeola and other viruses [1]. The drug is used in the therapy, mainly in chemoprophylaxis, of influenza A [2,3]. Other important clinical applications of amantadine have been recently studied, ranging from viral infections e.g. herpes, herpes zoster neuralgia to granulomatis [4], from Parkinson’s disease [5–7], neuroleptic extrapyramidal movement disease [4], depression [8] to cocaine dependence [9]. In addition, an antimalarial activity of amantadine has recently * Corresponding author.

been proposed [10]. Some adverse effects of amantadine have also been observed [11]. The exact mode of action of amantadine in influenza virus replication remains unclear [12]. It blocks two steps of infection. Early inhibition by amantadine is related to inhibition of virus uncoating [13]. The molecular basis of this early inhibition by amantadine relies on the accumulation of the drug in endosomes, an event that leads to increased endosomal pH [13]. It was suggested that amantadine binds to the pore region of the channel formed by M2 protein [14]. The M2 protein present in virions would permit the flow of ions, including protons, from endosome into virus particle interior to disrupt macromolecular interactions, freeing the nucleocapsid from the M1 protein. This activity of M2 would be blocked by amantadine [13,14]. More recent experiments suggest

0022-2860/99/$ - see front matter 䉷 1999 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(98)00659-0

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gives the resonance enhancement of the purine and pyrimidine bases vibrational modes which leads to a simplification of very complicated complex spectra and allows us to identify the exact position of amantadine binding place in the DNA structure. As it is known, classical Raman spectroscopy (visible excitation) is widely used for the DNA conformation analysis [23,24]. We therefore applied this method to characterize the conformational form of DNA in the complex with amantadine.

2. Experimental 2.1. Sample preparation Calf thymus DNA and amantadine were purchased from Sigma and they were used without further purification. Fig. 1. Structure of amantadine.

that amantadine acts as an allosteric blocker that binds to another portion of M2 not directly involved in the pore region [15]. At low concentration amantadine blocks a very late step of influenza virus maturation; viruses are able to bud from the cell surface, but they are unable to pinch off from the cell [16]. Thus, a better understanding of the interaction of this potent drug with various possible cellular targets such as membranes, proteins, or nucleic acids is important to determine its basic mode of action on biological systems. To our knowledge no mechanism of antiviral activity, based on direct interaction with DNA or RNA, has been proposed, and no study of the amantadine–nucleic acids interaction has been published so far. In view of the biochemical and aforementioned pharmacological activities of amantadine, we studied the amantadine DNA interaction with the aim of determining the drug–DNA binding site. Vibrational spectroscopy (Raman and infrared absorption) is often used to characterize the nature of drug–nucleic acids interaction and to monitor the effects of various drugs on DNA or RNA structures [17–22]. Resonance Raman spectroscopy (RRS) with 257 nm excitation wavelength was used to investigate the character of amantadine interaction with calf thymus DNA. The excitation wavelength of 257 nm

2.1.1. Resonance Raman spectroscopy Aqueous solution of the calf thymus DNA was prepared in 1 mg/ml concentration in pH 7 phosphate buffer. The mixed solution was prepared by adding 3 × 10 ⫺4 M amantadine solution to the DNA solution and it was gently stirred for 24 h at room temperature. The final drug/DNA(P) molar ratio was 1/10. 2.1.2. Classical Raman spectroscopy Aqueous solution of the calf thymus DNA was prepared in 30 mg/ml concentration in pH 7 phosphate buffer. The mixed solution was prepared by adding the drug solution in concentration 10 ⫺2 M to the DNA solution with constant stirring. The final drug/DNA(P) molar ratio was 1/10. Aqueous solutions of amantadine used for UV absorption and resonance Raman spectroscopy measurements were prepared in the same buffer in concentration of 10 ⫺3 M and for classical RS in concentration of 10 ⫺2 M. 2.2. Experimental procedures 2.2.1. Absorption spectroscopy The room temperature UV-Vis absorption spectrum of amantadine in a 1 cm cell was recorded with a Cecil CE 3040 spectrophotometer in the 200–800 nm wavelength range.

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Fig. 2. Resonance Raman spectrum of amantadine with excitation wavelength 257 nm. Inset figure: UV-Vis absorption spectrum of amantadine.

2.2.2. Resonance Raman spectroscopy Resonance Raman spectra of amantadine, calf thymus DNA and the DNA–amantadine complex were obtained in the 500–1800 cm ⫺1 range using the 257 nm excitation wavelength (the first harmonic of the 514 nm line delivered by a Lexel 95-4 cw Ar ⫹ laser) and a Jobin-Yvon Ramanor HG-2S double monochromator, operating in the second order of the gratings. The intensity of the sample illumination (1 mW) was controlled by monitoring the pathway, consisting of an external photomultiplier irradiated by split laser beam. The sample solution (1 ml) was placed in a quartz cell placed in a thermostated sample holder and maintained at 4⬚C. The sample was gently stirred with a little magnet to avoid an eventual thermal or photochemical damage. The water O–H

stretching band around 3450 cm ⫺1 was taken as an intensity standard for the spectra normalization. The spectra in this paper are the average of seven consecutive scans using 1 cm ⫺1 step, presented without buffer subtraction and smoothed by a fast Fourier transform method. The accuracy of the band position was ^ 2 cm ⫺1. 2.2.3. Classical Raman spectroscopy The classical Raman spectra were recorded at room temperature using the 514 nm/300 mW excitation wavelength and a Jobin-Yvon HRD1 double monochromator. The excitation wavelength, 514.5 nm, was provided by a Spectra Physics BeamLok 2060/65 argon ion laser. The phosphoionic band at 1094 cm ⫺1 was taken as an

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Fig. 3. Resonance Raman spectra of: (A) DNA–amantadine complex, (B) calf thymus DNA, (C) difference spectrum: (DNA–amantadine complex) ⫺ DNA. Excitation wavelength: 257 nm.

intensity standard for normalizing the spectra of DNA and the complex DNA–amantadine. The classical Raman spectra in this paper are the average of 16 consecutive scans recorded at 1 cm ⫺1 step, presented without buffer subtraction and smoothed by a fast Fourier transform method. The accuracy of the band position was ^ 2 cm ⫺1. The spectrum of amantadine was normalized on the water band at 1640 cm ⫺1.

3. Results and Discussion 3.1. Resonance Raman spectroscopy The resonance Raman spectrum of amantadine excited at 257 nm excitation wavelength is presented in Fig. 2. The inset figure plots the UV-Vis absorption spectrum of the amantadine. From this absorption spectrum it can be seen that the drug does not absorb

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Table 1 Raman frequencies (cm ⫺1) and assignments of calf thymus DNA and DNA–amantadine complex observed with excitation at 257 and 514 nm (abbreviations: s, strong; m, medium; w, weak; v, very; sh, shoulder; A, adenine; G, guanine; T, thymine; C, cytosine; amant., amantadine) 257 nm excitation DNA

DNA–amant.

1250w 1336s 1379m,sh 1486vs 1578m

1254w 1338s 1379m,sh 1486vs 1582m

1611m 1649m

1646m

514 nm excitation DNA DNA–amant.

Assignment

Ref.

678m 792s

681m 791s

834m,sh 1094m 1234w 1257m 1339m 1376m 1493m 1579m

825m,sh 1095m 1235m 1258m 1342m 1376m 1494m 1584m

G(d N7C8N9 ⫹ d C5N7C8) C(N1R–C4N–C4C5C6) OPO symmetric stretch OPO asymmetric stretch PO2⫺ symmetric stretch T(-d C6H ⫹ C2N3) C(d C6H ⫹ C4N4 0 ) A(-N7C5 ⫹ C8N7) T(C6C5–C4O) G(d C8H–N9C8 ⫹ C8N7) G(-C4N3 ⫹ C5C4–N7C5) A(C5C4–C4N3) A(d NH2–C5C6 ⫹ C6N6 0 ) T(C5C4–C4O)

[23,25] [25] [23] [23] [23] [25,39] [25,39] [25,39] [25,39] [25,39] [25,39] [25,39] [25] [25]

in the spectral region of the laser excitation line and consequently the Raman spectrum of amantadine does not exhibit a resonance enhancement for this excitation. This phenomenon gives us an advantage for the DNA–amantadine complex study, as for the 257 nm excitation only the DNA base vibrations are enhanced and so, the amantadine vibrational modes do not participate in the Raman spectrum of the complex. This fact allows us to present also a high quality difference spectra which are not influenced by the drug spectrum contribution (Fig. 3). Fig. 3 shows the normalized resonance Raman spectra of calf thymus DNA (Fig. 3B), DNA–amantadine complex (Fig. 3A), and the difference spectrum (DNA–amantadine complex) ⫺ DNA (Fig. 3C). The excitation in the absorbance maximum of the DNA molecule leads to the resonance enhancement of the DNA bases, adenine and guanine in particular. Due to this resonance enhancement for the DNA vibrational modes, all observed spectral changes in going from the DNA to the complex spectrum can be associated with the structural changes of the DNA molecule caused by the interaction with amantadine. The observed Raman frequencies of calf thymus DNA and the DNA–amantadine complex and their assignments are listed in Table 1. Three intense bands at 1336, 1486, and 1578 cm ⫺1 can be seen in our calf thymus DNA resonance Raman spectrum (Fig. 3B). Fodor et al. [25] have measured

the resonance Raman spectra (RRS) of deoxyribonucleotides using excitation wavelengths 266, 240, 218, and 200 nm. RR spectrum of dAMP obtained with the 266 nm excitation, shows a strong band at 1339 cm ⫺1 and a medium intensity band at 1581 cm ⫺1. Likewise, RRS of dGMP contains a strong band at 1489 cm ⫺1 and a medium intensity band at 1578 cm ⫺1. Thus, in our DNA resonance Raman spectrum (Fig. 3B) we can attribute the band at 1336 cm ⫺1 to adenine vibrations, the band at 1486 cm ⫺1 to the guanine vibrations, and finally the band at 1578 cm ⫺1 to guanine and adenine vibrations (see also Table 1). In the same spectrum the cytosine band at 1250 cm ⫺1 and the thymine bands at 1379 cm ⫺1 (shoulder), and at 1649 cm ⫺1 (medium intensity band) can be seen (Fig. 3B). These bands were also observed in the study of dTMP and dGMP excitated at 266 nm by Fodor et al. [25]. The formation of the DNA–amantadine complex leads to a hyperchromic effect of the whole spectrum (Fig. 3). As it can be observed in the difference spectrum (Fig. 3C), the 1336 cm ⫺1 adenine and the 1486 cm ⫺1 guanine bands are enhanced and shifted in the complex spectrum in comparison to the DNA isolated molecule. As a consequence of this change, the bands at 1343 cm ⫺1 (for adenine) and 1490 cm ⫺1 (for guanine) are observed in the difference spectrum (Fig. 3C). The medium intensity increase and an upshift of the overlapped adenine–guanine band at

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134 ⫺1

⫺1

1578 cm (1586 cm in the difference spectrum) are provoked by the complex formation. Some spectral changes caused by the complex formation can also be seen in the region at about 1650 cm ⫺1, where the thymine vibrations are presented. The spectral variation in this region leads to the doublet appearing at 1642 and 1662 cm ⫺1 in the difference spectrum (Fig. 3C). This is associated with the influence of the amantadine molecule on the DNA structure at the level of thymine residues. From the point of view of the previous analysis of the spectra, we can conclude that the amantadine interacts preferentially with adenine and guanine molecules. The low intensity doublet appearing at 1642 and 1662 cm ⫺1 in the difference spectrum indicates that the thymine molecule which forms two hydrogen bonds with adenine in the DNA structure participates in this interaction. An interesting question is: what part of the DNA molecule and consequently what part of the adenine, guanine, and thymine molecules are included in this interaction? The strong band at 1339 cm ⫺1 is mainly caused by the N7C5 and C8N7 adenine groups vibrations with 39% of the N7C5 group participation [25]. Thus, the intensity increase and the upshift of the adenine band from 1336 cm ⫺1 to 1338 cm ⫺1 (1343 cm ⫺1 band in the difference spectrum) are caused mainly by the amantadine interaction with the N7 position of adenine, which is accessible for amantadine in the major groove of the DNA structure (Fig. 3). The localization of the amantadine molecule in the major groove of DNA can influence the NH2 deformation vibration of the adenine molecule. This is the reason why the complex formation leads to the appearance of the 1611 cm ⫺1 band (Fig. 3A) which is attributed to the adenine d NH2–C5C6–C6N6 0 vibrations with 73% of the dNH2 group vibrations contribution [25]. The complex formation also stimulates the downshift of the 1649 cm ⫺1 thymine band to the final position at 1646 cm ⫺1 (Fig. 3). This band corresponds to the C5C4–C4O vibrations of thymine groups with 37% contribution of the C4O group [25,26]. It means that the presence of the amantadine molecule in this position could change the DNA structure via deformation of the hydrogen bond between NH2 group of adenine and the C4O group of thymine molecule. This structure deformation leads to a

change of the thymine band position in the RR spectrum of the complex (Fig. 3A). The strong band located at 1486 cm ⫺1 in the resonance Raman DNA spectrum (Fig. 3) corresponds to the d C8H⫺N9C8⫹C8N7 guanine vibration [21,25,27,28]. The intensity increase of this band in the process of complex formation is associated with its upshift clearly seen in the difference spectrum (band at 1490 cm ⫺1) (Fig. 3C). Thus, the interaction of amantadine with DNA in the region of the G–C pairs occurs at the N7 site of the guanine, but it is probably shifted to the external side of the imidazole moiety of the guanine because the PED distribution for this band indicates only 21% for the C8N7 bond and 40% and 32% for d C8H and N9C8 bonds, respectively [25]. The band at 1578 cm ⫺1 is attributed to the adenine C5C4–C4N3 internal vibrations and guanine C4N3– C5C4–N7C5 internal vibrations [25,26,28] and the reason why the 257 nm excitation the guanine contribution in this band is stronger that the adenine one [25]. The intensity and the position variation of this band during the complex formation are mainly caused by amantadine interaction with guanine in which the N7 guanine position is also included. Thus, from the resonance Raman data it is clear that amantadine interacts with N7 positions of adenine and guanine and that this interaction leads a partial deformation of the hydrogen bonds between adenine and thymine bases. An interesting question is: which part of the amantadine molecule is implicated in this interaction? With regards to the amantadine structure (Fig. 1) and the nature of the RR spectra any type of intercalation of amantadine to the DNA structure can be excluded. As the N7 positions of guanine and adenine are proton-acceptor sites of the purine rings, it seems very probable that amantadine interacts with guanine and adenine via a hydrogen bond formation between the protons of its amino group and the N7 positions of the purines. This conclusion is suported by our previous study of the other DNA-hydrogen bonded agent hypericin, where similar spectral features have been observed in the resonance Raman spectra of the complex [19,29]. An effect of hydrogen bonding on the adenine ring vibrations, in solvents with varied hydrogen bonding properties, was recently presented by Fujimoto et al. [30]. In this study the upshift of the 1330 cm ⫺1 adenine band has

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Fig. 4. Classical Raman spectra of: (A) DNA–amantadine complex, (B) calf thymus DNA, (C) amantadine. Excitation wavelength: 514 nm.

been interpreted as being caused by a strong hydrogen bond formation with the adenine proton-acceptor sites (N1, N3 and N7). The N7C5 group contribution represents 25% in this vibration [25] and N1 nitrogen forms a hydrogen bond with thymine molecule in the DNA structure. Thus for our RR spectra it seems reasonable to interpret the observed spectral variation in going from the DNA to the complex spectrum as being a consequence of the hydrogen bond formation between the N7 position of adenine and the NH2 group of amantadine. We propose the same model of interaction for the guanine molecule.

3.2. Classical Raman spectroscopy in the visible range From the point of view of the previous findings, a study of amantadine influence on the DNA conformation is interesting. It can be supposed that the amantadine action on the DNA bases will influence the DNA sugar-phosphate backbone structure. Therefore, it is important to make a conformational analysis of the DNA–amantadine complex by classical Raman spectroscopy for getting a better understanding of the influence of amantadine on the DNA molecule.

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Fig. 4 shows the normalized classical Raman spectra of calf thymus DNA (Fig. 4B), DNA– amantadine complex (Fig. 4A) and free amantadine molecule (Fig. 4C). The spectra were normalized using the phosphoionic band at 1094 cm ⫺1 as an intensity standard. The intensity of this band does not change for different DNA conformations [22,23]. The 1 cm ⫺1 wavelength shift of this line observed in going from the DNA to the complex spectrum is not significant as the accuracy of the band positions identification is 2 cm ⫺1. Thus, the same position of this band in both spectra excludes an eventual possibility of significant amantadine interaction with the PO2 group of DNA which could be proposed for such type of molecules. A minor intensity variation of this band in going from the DNA to the complex spectrum (Fig. 4) is caused by a low intensity amantadine band observed at 1090 cm ⫺1 (Fig. 4C). As a result of the drug contribution in the complex spectra, a high quality complex ⫺ DNA difference spectrum was not possible to obtain and so it is not presented here. The positions and intensities of Raman bands for the DNA molecule (Fig. 4B) correspond to the B-form of DNA conformation and they are in good agreement with those published by other authors [23,24,31–35]. The conformation sensitive Raman marker bands linked with phosphate group vibrations observed at 834 and 792 cm ⫺1 [23,32,36,37] are the most intense in our spectrum (Fig. 4B) and thus it enables us to make the conformational analysis of the DNA in the complex with amantadine. The band at 792 cm ⫺1 (Fig. 4) is a result of overlap of the cytosine vibration seen at 784 cm ⫺1 and the symmetric phosphodiesteric OPO group vibration observed at 790 cm ⫺1[23,38,39]. The low intensity amantadine band at 781 cm ⫺1 (Fig. 4C) contributes to the intensity at the low frequency side of the 792 cm ⫺1 band in the complex spectrum (Fig. 4A). This symmetric phosphodiesteric vibration is sensitive to the DNA conformational change. A shift to 748 cm ⫺1 frequency for Z-form of DNA conformation was observed by Benevides and Thomas in the study of the B–Z transition of poly(dG-dC)poly(dG-dC) and DNA [23]. In our spectra no spectral shift and intensity change for this band is observed. So it does not reflect an eventual DNA conformation change provoked by the interaction with amantadine.

The asymmetric stretching vibration of the phosphodiesteric bond OPO is represented by the bond at 834 cm ⫺1 [23]. In our DNA Raman spectrum this band is observed as a shoulder of the 792 cm ⫺1 band (Fig. 4B) and indicates the B-conformation form of the calf thymus DNA [23,40]. The structural transition from the B to Z and A conformation forms of DNA is characterized by the downshift of this line, to 810 cm ⫺1 in the Z form [23] and to 807 ^ 3 cm ⫺1 in the A-form spectrum [23]. In our spectrum, the formation of the DNA–amantadine complex leads to the downshift of this band from 834 cm ⫺1 to 825 cm ⫺1 (Fig. 4). Thus, this downshift is not sufficient to associate it with a conformation change of the DNA, but it seems to be reasonable to associate this downshift with a partial deformation of the sugar-phosphate DNA backbone. Other important DNA conformation marker is the 682 cm ⫺1 guanine band which includes the breathing vibration of the guanine ring coupled to the sugar vibrations [23]. The position of this line in going from the B to Z and A DNA forms shifts to 625 cm ⫺1 and 665 cm ⫺1, respectively and reflects the sugar conformation change from the C2 0 -endo/ anti in the B-form to the C3 0 -endo/syn and C3 0 endo/anti in the Z and A-forms, respectively [23,41]. In our DNA Raman spectrum this guanine band is observed at 678 cm ⫺1 (Fig. 4B). In going from the DNA to the complex spectrum one can observe an intensity decrease and an upshift of about 3 cm ⫺1 of this band (Fig. 4A). As the accuracy of the band position is ^ 2 cm ⫺1 in our spectra, we can associate both these bands with the guanine vibration superposed by the low intensity thymine band at 675 cm ⫺1 [42] and with low intensity amantadine band observed at 674 cm ⫺1 for the complex spectrum (Fig. 4). With regard to our results obtained by RR spectroscopy, we can propose that the intensity decrease and a weak upshift of this band observed for the complex spectrum is caused by amantadine interaction with guanine, but the observed variation in position and intensity of this band does not reflect an eventual conformation transition of the DNA molecule. The base vibrations observed in the high frequency classical Raman spectral region are not very sensitive to the DNA conformation, but a spectral variation in this region can support our interpretation of the

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DNA–amantadine complex RR spectrum. The bands at 1339 and 1579 cm ⫺1 observed in the classical Raman spectrum of DNA (Fig. 4B) correspond to the enhanced purines vibrations observed at 1336 and 1578 cm ⫺1 in the DNA RR spectrum (Fig. 3B). These modes are not enhanced with 514 nm excitation wavelength and thus they are less intensive markers of interaction as that observed in RRS (Fig. 3). The most significant change in this region is the upshift and a relative intensity decrease of the overlapped adenine and guanine band at 1579 cm ⫺1 to the 1584 cm ⫺1 in the complex spectrum (Fig. 4). The band at 1339 cm ⫺1 which corresponds mainly to the adenine N7C5 and C8N7 groups vibrations [25] is shifted at 1342 cm ⫺1 owing to the complex formation which provokes also a low relative intensity decrease of this band (Fig. 4). Both these changes correspond to the previous conclusions obtained in RRS about drug binding to the purine bases adenine and guanine with preferential interaction with the N7 position of purines. The most intense change in the classical Raman spectra in this region caused by the complex formation is the intensity increase of the 1234 cm ⫺1 thymine band. This band is associated with the -d C6H and C2N3 group vibrations [25] and so the intensity increase of this band in the complex spectrum could reflect the influence of amantadine which interacts with the N7 position of adenine on the hydrogen bonds linked adenine and thymine in the N1…N3H position. This change seems to be rather clearly presented even if at the low frequency side of this band a contribution of the 1210 cm ⫺1 amantadine band can be seen. Thus the spectral changes between DNA and the DNA–amantadine complex Raman spectra observed with 514 nm excitation wavelength indicates a partial change of the DNA sugar-phosphate backbone structure caused by the interaction of amantadine with the N7 positions of adenine and guanine molecules. This partial structure deformation does not lead to a conformation transition of the DNA molecule.

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structure no absorption maximum in UV-Vis range can be seen. Therefore, resonance Raman spectroscopy, with a 257 nm excitation wavelength, was used for the determining the drug–DNA binding site. Classical Raman spectroscopy was mainly used for the DNA conformation determination in the complex with amantadine. The observed spectral changes as a result of the complex formation lead us to following conclusions: 1. The interaction of amantadine with calf thymus DNA in aqueous solution is realized by the drug binding to the purine bases adenine and guanine. The evidence for this conclusion are the spectral variations of the bands at 1336, 1486, and 1578 cm ⫺1 observed in the resonance Raman spectra (Fig. 3) and confirmed by features of the classical Raman spectrum in the base vibration region (Fig. 4). 2. Amantadine interacts with N7 position of purines via hydrogen bonds formation between the N7 position of purines and the NH2 group of the drug (bands at 1336, 1486 and 1578 cm ⫺1, and the appearance of 1611 cm ⫺1 band in the resonance Raman spectrum (Fig. 3)). Characteristic of the adenine–amantadine complex is a close placement of the amantadine with regard to the NH2 group of the adenine molecule. This interaction leads to the DNA structure deformation at the side of the adenine–thymine pairs, because the NH2 group of adenine molecule forms the hydrogen bond between adenine and thymine (variation of the bands at 1336, 1611, and 1649 cm ⫺1 in the resonance Raman spectra as shown in Fig. 3). 3. The amantadine interaction with guanine and adenine influences the DNA sugar-phosphate backbone chain structure (downshift of the 834 cm ⫺1 band as shown in Fig. 4), but this interaction does not change the DNA conformation.

Acknowledgements 4. Conclusion The antiviral agent, amantadine, has an unusual molecular structure and as a consequence of this

This work was supported by a grant of Slovak Ministry of Education, No 1/3258/96 and No 1/ 4387/96.

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