An approach to the interpretation of the vibrational spectra of 2′-deoxyinosine by means of DFT calculations

Journal of Molecular Structure 687 (2004) 7–15 www.elsevier.com/locate/molstruc

An approach to the interpretation of the vibrational spectra of 20 -deoxyinosine by means of DFT calculations M. de la Fuente*, R. Navarro Department of Ciencias y Te´cnicas Fisicoquı´micas, Facultad de Ciencias, Universidad Nacional de Educacio´n a Distancia, C/Senda del Rey, 9, Madrid 28040, Spain Received 3 September 2002; revised 15 July 2003; accepted 29 July 2003

Abstract The FT-IR and FT-Raman spectra of 20 -deoxyinosine (20 -dI) are reported. This modified nucleoside of the four natural DNA bases binds with weak hydrogen bonds to any of them, feature used in PCR technique or in hybridisation essays [Howard Huge Medical Institute]. The FT-IR and FT-Raman spectra of its synthesized trideuterated derivative (20 -dI-d3, deuteration at N1, and hydroxyl groups O30 and O50 ) have also been recorded. The observed isotopic shifts, the spectra of related molecules and theoretical calculations have been used to interpret the vibrational spectra of 20 -dI. The accurate knowledge of the vibrational spectra is an important prerequisite for understanding its structure, conformation and possible behaviour in physiological conditions. Therefore, different optimised structures have been obtained, including variable number of water molecules (none, 1 or 2) surrounding the nucleoside. They have been situated close to the carbonyl group. The wavenumbers of the normal modes of vibration for each one have been calculated using Density Functional Theory methods, with the Becke’s Three Parameter Hybrid Functional and the LYP Correlation Functional (B3LYP) model, using 6-31G** basis set. An assignment of FT-IR and Raman spectra is proposed. The influence of the water molecules in the theoretical results is shown. q 2003 Elsevier B.V. All rights reserved. Keywords: 20 -deoxyinosine; FT-Raman; FT-IR; Density functional theory calculations

1. Introduction 20 -Deoxyinosine (Fig. 1) is a nucleoside with important applications, such as being incorporated into oligonucleotides used in PCR, or being used as hybridisation probe for the detection or analysis of a target DNA strand containing ambiguities. These applications are due to its property of binding with weak hydrogen bonds to any of the four natural DNA bases [1]. In this paper it is proposed an assignment of its vibrational spectra, which is necessary for understanding its structure, conformation and possible auto- and hetero-association processes. On the other hand, these features wide our knowledge about the behaviour of the nucleoside under physiological conditions. The assignment of the vibrational spectra of 20 -dI is proposed in the light of information provides by theoretical calculations, the observed isotopic shifts in the spectra of its trideuterated derivative, 20 -dI-d3, * Corresponding author. Tel.: þ 34-91-3987207; fax: þ 34-91-3966697. E-mail address: [email protected] (M. de la Fuente). 0022-2860/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2003.07.009

and comparing 20 -dI spectra with the spectra of related molecules, such as inosine and 20 ,30 -dideoxyinosine. It has been shown the importance of consider explicit water molecules at the quantum mechanical calculations involving nitrogen bases [2 – 5]. In the present work, calculated vibrational modes for 20 -deoxyinosine in interactions with one and two water molecules have been analysed, in order to study the effect of these interactions on the vibrational spectra. The water molecules have been located close to carbonyl group since this is one of the worst theoretically predicted modes and it notably affects other vibrations of the base residues in the 1800 – 1500 cm21 region.

2. Experimental The 20 -deoxyinosine was purchased from Sigma Chemical Co. and was used as supplied. A isotopomer (deuteration at N1, and hydroxyl groups: O30 and O50 ) and a control sample were synthesized by dissolving the commercial product in 2H2O (Scharlau, 99.999%) and deionised H2O,

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M. de la Fuente, R. Navarro / Journal of Molecular Structure 687 (2004) 7–15

respectively. Both solid products were obtained by freezedrying.

0.89 cm21 (RES ¼ 1.0, Hamming apodizing function). A DTGS/MIR detector, a Global source, and a KBr beamsplitter were used. The FT-Raman spectra of the polycrystalline 20 -deoxyinosine (Fig. 2) were recorded using the Raman accessory of the BOMEM DA3 interferometer. A krypton discharge lamp, pumped Nd3þ yag laser (Quantronix CW114) working at 1064 nm was the exciting source. Raman emission was collected at 1808 with backscattering geometry. A quartz beamsplitter and an IGA detector working at 77 K (cooled by liquid N2) were used to obtain the FT-Raman spectra. FT-Raman spectra was recorded working with the IGA detector at room temperature to obtain a better resolution in the 3500 –2000 cm21 interval. One thousand interferograms were coadded for each spectrum with a nominal resolution of 4 cm21 after Blackman – Harris apodization. The second derivatives of all the recorded spectra (Savitzky –Golay algorithm) [6] were calculated to resolve the bands containing more than one component.

2.1. FT-IR and FT-Raman spectra

2.2. Calculations

The FT-IR spectra were recorded in a Bomem-DA3 interferometer working under a vacuum (pressure # 133.3 Pa). The mid-IR spectra of polycrystalline 20 -dI and 20 -dI-d3 in KBr pellets (Fig. 2) were recorded by coadding 1000 interferograms with an effective apodized resolution(s) of

The quantum mechanical calculations were carried out using Titan [7]. The geometry of 20 -deoxyinosine was optimised with no restriction on different initial structures. MMFF optimised structures were used as initial geometries. Both, structure optimisation and vibrational analysis

Fig. 1. The 20 -deoxyinosine molecule. Molecular structure with conformational parameters.

Fig. 2. FT-IR (B) and FT-Raman (C) spectra (solid state) and second derivatives (2d2 A=dn2 ) (A and D) of 20 -deoxyinosine.

M. de la Fuente, R. Navarro / Journal of Molecular Structure 687 (2004) 7–15

calculations, were implemented by using Density Functional Theory (DFT) at the B3LYP level with the 6-31G** basis set. After each change of atomic coordinates, the energy of the fundamental state of the system was evaluated by self-consistent field using the Direct Inversion in the Iterative Subspace convergence scheme. The optimised geometries and force constants resulting from the analytical second derivatives of the energies were used to compute the wavenumbers corresponding to the normal modes. Local minima were verified by establishing that only real vibrational wavenumbers are predicted. The vibrational mode descriptions were made on the basis of calculated nuclear displacements associated with the vibrational wavenumbers. Only those degrees of freedom which dominate the vibrational form were used for assigning a normal vibration. The B3LYP method was chosen because different studies have shown that the B3LYP level of theory is the most promising quantum mechanical method for modelling biomolecules method containing the ribose ring and in the vibrational frequencies predictions of DNA bases [8].

3. Results and discussion Spectra of control sample (commercial product dissolved in deionised H2O) show that the dissolving process itself does not affect the vibrational spectra of this compound [9]. Two different optimised geometries of the 20 -dI have been taken into consideration, labeled as 20 -dI (A) and 20 dI (B). The main difference between these two molecules are the conformation of the deoxyribose ring: the former is south, while it is north the latter (Table 1) [10]. The calculated vibrational wavenumbers for the 20 -dI (B) approach better the experimental wavenumbers that

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calculated ones for the 20 -dI (A) (Tables 2– 4). Other two different local minima were verified for 20 -dI þ water complexes: 20 -dI þ H2O and 20 -dI þ 2H2O. These optimised geometries were reached from 20 -dI (B) as initial structure, adding the water molecules. In order to analyse the effect of carbonyl group –water interaction, since this is one of the worst theoretically predicted modes, a first water molecule interacting with the carbonyl group have been included. The location of the second water molecule is basically the result of the global minimum reached in the optimization of the monohydrated form. As can be seen in Fig. 3c, the water molecule located firtsly in the proximities of carbonyl group, after the optimization is situated between group CyO and N1 –H. Therefore, a second global minimum was reached from a initial geometry with a second water molecule close to N1 –H group, being this another step covering the donor/accetor sites of the pirimidine ring. The bigger structure which was become optimised (20 -dI þ 2H2O) has 36 atoms. Studies adding more molecules of water are being carried out. Fig. 2 shows FT-IR and FT-Raman of the 20 -deoxyinosine. The optimised DFT geometries are shown in Fig. 3, detailing the geometrical features for each one in Table 1, in accordance with Ref. [10]. The main results of the vibrational analysis are shown in Tables 2– 4 and is discussed by spectral regions below. 3.1. 3600 –2500 cm21 spectral region CH, OH, and NH stretching modes are expected in this region, where Raman spectrum is not solved. The IR bands due to N –H and O – H stretching vibrations will be clearly identified due to their shift under deuteration (Fig. 4). O30 – H stretching mode could be assigned to the IR band

Table 1 Geometrical features of DFT B3LYP/6-31G** optimised geometries of 20 -deoxyinosine Parameters

20 -dI (A)

20 -dI (B)

20 -dI þ H2O

20 -dI þ 2H2O

Exocyclic torsion angles (8) g (O50 – C50 –C40 –C30 or O50 –C50 –C40 –O40 ) x (O40 – C10 –N9–C4 or O40 –C10 –N9–C8) d (C50 –C40 –C30 –O30 )

50.17/269.53 (gg) 2128.89/50.59 (anti) 139.13

46.70/272.42 (gg) 2150.51/30.68 (anti) 86.61

46.50/271.34 (gg) 2153.63/28.15 (anti) 87.65

46.76/272.34 (gg) 2150.83/31.24 (anti) 86.18

Endocyclic torsion angles (8) n0 (C40 –O40 –C10 –C20 ) n1 (O40 –C10 –C20 –C30 ) n2 (C10 –C20 –C30 –C40 ) n3 (C20 –C30 –C40 –O40 ) n4 (C30 –C40 –O40 –C10 )

214.89 28.85 231.05 23.06 25.27

4.77 225.84 35.21 233.25 18.26

3.71 225.43 35.66 233.91 19.49

4.46 225.74 35.32 233.56 18.66

Pa (8) nmax b (8) Sugar pucker

170.81 31.45 2 T3 (C20 -endo; south)

10.91 35.86 3 T2 (C30 -endo; north)

12.46 36.52 3 T2 (C30 -endo; north)

11.44 36.04 3 T2 (C30 -endo; north)

a b

gg ¼ gauche– gauche. tgP ¼ ððn4 þ n1 Þ 2 ðn3 þ n0 ÞÞ=2n2 ðsinð36Þ þ sinð72ÞÞ: nmax ¼ n2 =cos P:

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M. de la Fuente, R. Navarro / Journal of Molecular Structure 687 (2004) 7–15

Table 2 Observed (FT-IR and FT-Raman) and calculated (DFT B3LYP/6-31G**) wavenumbers for 20 -dI molecule in 3500–2000 cm21 region: tentative assignment for vibrational bands Vibrational modes (fundamental contributions)

n(O –H) anti. W1 n(O –H) anti. W2 n(O50 H) n(O30 H) n(O –H) anti. W1 n(OH) W1 o´ W1 y W2 n(N1H)

DFT n (cm21) 20 -dI (A)

DFT n (cm21) 20 -dI (B)

3900 3815

3840 3837

3606

3594

n(OH) W1,W2 þ n(N1H) n(N1H) n(C8H) n(C2H) n(C20 H2) anti. n(C30 H) n(C20 H2) anti. n(C10 H) n(C20 H2) symm. n(C20 H2) symm. þ n(C10 H) þ n(C30 H) n(C30 H) þ n(C20 H00 ) n(C40 H) þ n(C10 H) n(C50 H2) anti. n(C10 H) þ n(C40 H) n(C50 H2) anti. n(C40 H) þ n(C50 H2) symm. in-phase

3847 3847 3845 3569

DFT n (cm21) 20 -dI·2H2O

IR n (cm21)

Tentative assignment

3453w (2894)

n(O30 H)

3303

3405sh,b (2890) 3312m (2849)

n(O50 H) n(N1H) þ n(OH) W n(OH) n(OH)

3268 3203 3145

3222sh,b (2802) 3190w,b (2817) 3149w 3132w–m 3115w

3099

3095w

3077 3076 3063

3073vw,b

3869 3862 3841 3837 3426

3273

3277 3200

3272 3194 3146

3145 3139

3283 3215 3164 3137

3097 3081

3107 3076 3061

3059 3053

3079 3065

3051

3044vw,b

3013

3048

3011

2990

3027

2989

3002vw 2986vw 2959vw

3052 3021

n(C50 H2) symm. þ n(C40 H) out-of-phase n(C50 H2) symm.

DFT n (cm21) 20 -dI·H2O

2935

2940w 2925vw 2914vw 2904vw 2856w 2810vw,b (2675) 2666vw (2704) 2628vw (2690)

9 > > > > > > > > > > > > > > > > > > > > > > > > > > = > > > > > > > > > > > > > > > > > > > > > > > > > > ; )

n(CH)

n(OH)

Observed isotopic shifts for 20 -dI-d3 are in parentheses; vs, very strong; s, strong; m, medium; w, weak; vw, very weak; b, broad; sh, shoulder; n; stretching; symm., symmetric; anti., antisymmetric; W, water molecule.

appearing at 3453 cm21, by comparing the spectra of 20 -dI, Inosine and ddI [11]: while the two former present a hydroxyl group at C30 position (and a IR band at 3433 and 3453 cm21, respectively), the latter, ddI, is deoxylated at this position as unique difference (Fig. 5). In this case, a IR band is not observable at these wavenumbers. The vibrational bands between 3150 and 2850 cm21 that are not affected by deuteration, might be related with the different C – H stretching modes of the molecule (Fig. 4). Theoretical vibrational wavenumbers in this range are higher than the experimental values, especially those due to OH and NH stretchings. This is explained taken into account the possible hydrogen bonding in which these groups are involved in solid phase. In order to approach the vibrational calculations to the observed vibrational spectra in condesed phase, it is necessary to consider explicit water molecules, especially

surrounding the polar groups [2]. In this work one or two H2O molecules have been considered, close to base polar groups (CyO and N – H). Coupling of stretching modes of these groups with water motions has been evidenced. Thus, it has been possible to assign, the observed IR band at 3312 cm21 to the N1H stretch coupled with OH stretch of the two water molecules, calculated at 3303 cm21 in the 20 -dI þ 2H2O (Table 2). This assignment is in agreement with expected wavenumber for N – H stretchings of related molecules [12 – 14] and with deuteration effects. 3.2. 1750 – 600 cm21 spectral region Between 1750 and 1650 cm21 four IR bands appear at 1719, 1708, 1703 and 1688 cm21. The two former are not

1504 1497

Observed isotopic shifts for 20 -dI-d3 are in parentheses; (d.a.) ¼ deuteration affected, without state it accurately; vs, very strong; s, strong; m, medium; w, weak; vw, very weak; b, broad; sh, shoulder; n; stretching; d; in-plane bending; W, water molecule; sciss., scissoring.

1506m (25) 1506w (28) 1505

1521vw 1538 1521

1533 1510 1496 1520 1528

50 CH2 sciss. d(N1H) 20 CH2 sciss.

1553 1555

d(N1H) þ d(C8H) þ n(C4N9) n(CyC) þ n(CyN)

1557

; CH2 sciss. (sugar)

n(CyC) þ n(CyN) þ d(N–H) þ d(C– H)

> ; 9 = 1576w 1555s (25) 1546s 1579

1653 1603 1650 1591 1651 1597

d(HOH) W1 n(C2N3) þ d(C2H) n(C4C5) þ n(C4N3)

1666 1637 1591

1855

n(C6yO) þ d(N1H) d(HOH) W1 þ W2 d(HOH) W2 þ W1

1849

1821

1795 1707 1691

1719vs (d.a.) 1708vs (d.a.) 1703vs (d.a.) 1688w,sh (d.a.) 1671vw,sh (d.a.) 1664vw 1592vw 1582vw 1577vw 1554w (26)

1591vw

9 Pyrimidine > =

n(C6yO) þ d(N1– H) þ d(HOH) water

> > ;

1702m (24) 1689m (210)

9 > > =

Tentative assignment Raman n (cm21) IR n (cm21) DFT n (cm21) 20 -dI·2H2O DFT n (cm21) 20 -dI·H2O DFT n (cm21) 20 -dI (B) DFT n (cm21) 20 -dI (A) Vibrational modes (fundamental contributions)

Table 3 Observed (FT-IR and FT-Raman) and calculated (DFT B3LYP/6-31G**) wavenumbers for 20 -dI molecule in 1800–1500 cm21 region: tentative assignment for vibrational bands

M. de la Fuente, R. Navarro / Journal of Molecular Structure 687 (2004) 7–15

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observable in Raman spectra. All of them are sensitive to the deuteration, the two former almost completely disappearing in deuterated derivative FT-IR spectrum (Fig. 6). In this region the CyO stretching mode is expected. However, the isotopic shift indicates that other atomic motions are involved, such as N1 – H and H – O –H of water molecules deformations. This is supported by theoretical calculations, when water molecules are involving close to the N –H and CyO groups. As it can be seen in Table 3, the calculated wavenumber for 20 -dI þ 2H2O are much closer to experimental values that the other vibrational mode predicted in the rest of calculations. This is also in agreement with theoretical calculations of the vibrational spectra of a related molecule (uracil base) including water molecules surrounding it [2]. Spectral features within the range 1670 –1550 cm21 seem to belong to base vibrations. Between 1550 and 1500 cm21 CH2 scissoring motions are predicted (Table 3). A tentative assignment of the principal observed vibrational bands in the 1500 –600 cm21 region is proposed in Table 4. In this sense, it would be important to mention that some vibrational modes features in the 1400 – 1300 cm21 ranges seem to reflect the conformation of the 20 -deoxyinosine. As it can be observed in Table 4, three vibrational modes are predicted at 1380, 1327 and 1300 cm21 for the 20 -dI (A) molecule (C20 -endo conformation). These vibrational modes are calculated at approximately 1368, 1320 and 1311 cm21 for the 20 -dI molecules with C30 -endo sugar puckering (20 -dI (B), 20 -dI þ H2O and 20 -dI þ 2H2O). These vibrational modes, involving sugar vibrations, are related with the three intense Raman bands that appear at 1364, 1323 and 1309 cm21 in the spectrum of 20 -dI in solid phase. Similar results have been previously reported for other related molecules, such as guanosine derivatives. Thus, three conformation sensitive Raman lines were found in 1400 –1300 cm21 region. These are: 1369– 65, 1333 –27 and 1317 –1306 cm21 characteristic for C20 – C30 -endo sugar puckering, respectively [15]. Further vibrational studies in relation to different conformations would be necessary to establish more accurately vibrational modes-structure correlations. A very strong Raman band at 724 cm21 is found, which is distinctive for all purines (with the exception of guanine derivatives) [13]. In accordance with DFT calculations, it can be attributable to ring stretching vibration of the base skeleton (ring breathing mode), as it was expected [13].

4. Conclusions A tentative assignment of the vibrational spectra of 20 -dI has been proposed on the basis of DFT calculations. The results of the calculations were in reasonable agreement with the observed wavenumbers in most of the cases, although this does not happen at a much higher wavenumbers (3600 –1650 cm21). Theoretical calculations trends to

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Table 4 Principal observed (FT-IR and FT-Raman) and calculated (DFT B3LYP/6-31G**) wavenumbers for 20 -dI molecule in 1500– 600 cm21 region: tentative assignment for this vibrational bands DFT n (cm21) 20 -dI (A)

DFT n (cm21) 20 -dI (B)

n(CN) þ d(NH) þ d(CH) þ 50 CH2 wagg. þ d(O0 H) þ d(C0 H)

1489 1470 1463

1487 1467 1459 1454

d(C10 H) þ base (d(N1H) þ · · ·) or only base d(C40 H) þ d(C30 H) þ d(O30 H) Imid. ring breath. d(N1H) þ d(C2H) þ n(N7C5) Sugar: d(C0 H) Base þ sugar: d(CH) þ d(C0 H)

Sugar: d(C0 H) þ d(O0 H) Sugar: d(O0 –H) þ d(C0 –H)

DFT n (cm21) 20 -dI·H2O

1479 1465 1460

1449 1433 1406 1395 1380 1361 1346

1443 1425 1403 1393 1368 1361 1349

1443 1424 1405 1383 1367 1357 1340

1327 1300 1211

1322 1311 1218

1316 1311 1218

DFT n (cm21) 20 -dI·2H2O

IR n (cm21)

Raman n (cm21)

Tentative assignment

Base þ sugar vib.

1470 1467 1459

1481vw 1467vw

1481m–s

1447

1449vw 1441vw 1423w

1450s

1427 1417 1395 1369 1362 1351 1323 1311

1398vw 1366w 1351vw 1347w 1338vw 1323vw 1309w 1227w

1420sh 1415vs 1397vs 1364m 1346m 1335m 1323s 1309vs

d(C10 H) þ base vib. Sugar vib. Imid. vib. Base vib. Sugar vib. Base þ sugar vib.

Sugar vib.

1217 Def. skel. base (in-plane)

n(O0 C0 ) d(CH) oop Def. base oop d(O0 C0 C0 ) d(N1–H) oop Base rings breath. Base def. oop Base rings breath. Base def. oop

1207m 1126 1081 944 822 761 752 734

688 661 659

Def. skel. base þ sugar (in-plane) 639 620

1127 1083 944 927 821 787 754 732

1119 1081 979 940 839a 785

705 688 663

732 716 706 678 664 (W)

658

658

617 600

624 611

1195 1125 1087 955 932 830 790 781 740 727 713 681

661 627 618 591

1128w 1089vs 942m

1129w 1088w 942m

843w 789w 778vw 725vw,sh 719vw 706m 695vw 691vw

842vs 791w 778w 724vs 719vs 706w

671vw 643w–m 614w 610w

671m 645vw

Sugar vib. Base: d(CH) oop Def. base oop Sugar: d(O0 C0 C0 ) d(N1–H) oop Base rings breath. Base def. oop Base rings breath. Base def. oop

690w

Base þ sugar vib. (in-plane)

vs, very strong; s, strong; m, medium; w, weak; vw, very weak; b, broad; sh, shoulder; n; stretching; d; in-plane bending; def., deformation; oop, out-of-plane; imid., imidazol ring; breath., breathing; wagg., wagging; skel., skeleton; vib., vibration. a Include motion of water molecule: d(OH).

M. de la Fuente, R. Navarro / Journal of Molecular Structure 687 (2004) 7–15

Vibrational modes (fundamental contributions)

M. de la Fuente, R. Navarro / Journal of Molecular Structure 687 (2004) 7–15

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Fig. 3. DFT B3LYP/6-31G** optimised geometries of 20 -deoxyinosine: (A) 20 -dI (C20 -endo); (B) 20 -dI (C30 -endo); (C) 20 -dI þ H2O; (D) 20 -dI þ 2H2O.

overestimate stretching wavenumbers, especially for fundamentals of polar groups. The inclusion of water molecules interacting with these groups (CyO and N –H) approximates the calculated wavenumbers to the experimental ones from the spectra in solid phase. In view of this, further studies will be necessary to approach much more the rest of the vibrational modes, specially including water molecules close to the nitrogen atoms of the base residues and

the hydroxyl groups of sugar moiety. It will be interesting to analyse the DFT theoretical vibrational spectra of 20 -dI and its full first hydration shell model [3 –5], but the analysis of all of the vibrational modes obtained from all of the optimised configurations involving different number of explicit water molecules will be necessary to reach information about the hydration effect on the vibrational modes of 20 -dI, as it has been seen previously [2].

Fig. 4. FT-IR spectra of 20 -dI (A) and 20 -dI-d3 (B) showing isotopic shift of the vibrational bands, which N1– H, O30 –H and O50 – H groups are implicated in.

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M. de la Fuente, R. Navarro / Journal of Molecular Structure 687 (2004) 7–15

Fig. 5. FT-IR spectra of inosine (A), 20 -dI (B) and 20 ,30 -ddI (C) between 3600 and 2800 cm21. Tentative assignment of OH and NH stretchings.

Fig. 6. Spectral region from 1800 to 1600 cm21 for the vibrational spectra of 20 -dI molecule. (A) 20 -dI FT-Raman spectrum, (B) 20 -dI-d3 FT-Raman spectrum, (C) 20 -dI-d3 FT-IR spectrum, (D) second derivative of 20 -dI-d3 FT-IR spectrum, (E) 20 -dI FT-IR spectrum, (F) Second derivative of 20 -dI.

M. de la Fuente, R. Navarro / Journal of Molecular Structure 687 (2004) 7–15

The observed isotopic shifts in the spectra of its trideuterated derivative, 20 -dI-d3, has allowed to identify the IR bands due to stretch motions of OH, NH and CH groups. Besides that, the vibrational band corresponding to the O30 –H stretching has been assigned by comparing the IR spectrum of 20 -dI with the spectra of inosine and 20 ,30 dideoxyinosine. A conformational sensitive Raman lines have been proposed in accordance to DFT calculations. These results reveal that the analysed polycrystalline 20 -deoxyinosine exhibits C30 -endo sugar puckering. Acknowledgements The authors wish to thank the Vicerrectorado de Investigacio´n de la Universidad Nacional de Educacio´n a Distancia (Spain) for financial support and for the postdoctoral grant which made this work possible. References [1] Howard Huge Medical Institute, http://bpf.med.harvard.edu/Pages/ techs/O/IDT/Internal_Modifications.html

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[2] M.P. Gaigeot, M. Ghomi, J. Phys. Chem. B 105 (2001) 5007. [3] O.V. Shishkin, L. Gorb, J. Leszczynski, J. Phys. Chem. B 104 (2000) 5357. [4] N.U. Zhanpeisov, J. Leszczynski, Struct. Chem. 12 (2001) 121. [5] O.V. Shishkin, L. Gorb, J. Leszczynski, Int. J. Mol. Sci. 1 (2000) 17. [6] A. Savitzky, M.J.E. Golay, Anal. Chem. 36 (1964) 1627. [7] Titan, Ver 1, Wavefunction, Inc., Irvine CA and Schro¨dinger, Inc, Portland OR, 1999. [8] A. Pelmenschikov, D.M. Hovorun, O.V. Shishkin, J. Leszczynski, J. Chem. Phys. 113 (2000) 5986. [9] M. de la Fuente, R. Navarro, A. Hernanz, Nuc. Nus. Nucleic Acids 21 (2002) 495. [10] W. Saenger, Principles of Nucleic Acid Structure, Springer, New York, 1984. [11] M. de la Fuente, R. Navarro, unpublished manuscript. [12] M. Tsuboi, S. Takahashi, I. Harada, in: J. Duchesne (Ed.), Physicochemical Properties of Nucleic Acids: Structural studies, Academic Press, London, 1973 (Chapter 11). [13] K.A. Hartman, R.C. Lord, G.J. Thomas Jr., in: J. Duchesne (Ed.), Physico-chemical Properties of Nucleic Acids: Structural Studies on Nucleic Acids and Other Biopolymers, Infrared and Raman Spectra of Nucleic Acids-Vibrations in the Base-Residues, Academic Press, London, 1973 (Chapter 10). [14] M. Tsuboi, Y. Takeuchi, E. Kawashima, Y. Ishido, M. Aida, Spectrochim. Acta 55A (1999) 1887. [15] Y. Nishimura, M. Tsuboi, T. Sato, K. Aoki, J. Mol. Struct. 146 (1986) 123.