Vibrational motions of bases of nucleic acids as revealed by neutron

J. Phys. Chem. 1993,97, 1074-1084

1074

Vibrational Motions of Bases of Nucleic Acids As Revealed by Neutron Inelastic Scattering and Resonance Raman Spectroscopy. 1. Adenine and Its Deuterated Species Z. Dbaouadi and M. Cbomi' Physique Theorique des Macromolecules Biologiques, UFR SantC- Medecine- Biologie Humaine, Universite Paris- Nord, 74 rue Marcel Cachin, 9301 2 Bobigny Cedex, France

J. C. Austin, R. B. Girling, and R. E. Hester Department of Chemistry, University of York, Heslington, York YO1 5DD, United Kingdom

P. Mojzes,+L. Chinsky, and P. Y. Turpin Laboratoire de Physique et Chimie Bimolikulaires, CNRS URA 198, Institut Curie et UniversitC Paris VI, 1 1 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France

C. Codombeau Laboratoire d'Etudes Dynamiques et Structurales de la Silectivite, CNRS URA 332, Universite Joseph Fourier, B. P. 53X, 38041 Grenoble Cedex, France H, Jobic Institut de Recherche sur la Catalyse, 2 avenue Albert Einstein, 69626 Villeurbanne Cedex, France

J. Tomkinson Rutherford Appleton Laboratory. Chilton, Didcot, Oxon OX1 1 OQX, United Kingdom Received: July 29, 1992; In Final Form: October 8. 1992

Neutron inelastic scattering (NIS) and ultraviolet resonance Raman scattering (RRS) experimental data have

been obtained from purine nucleic base samples, Le., adenine, C8-deuterated adenine, and a (N9, C6-amino)deuterated species. The two types of spectra have made it possible to carry out complete theoretical assignments of the vibrational modes of adenine and its derivatives. Normal mode wavenumbers as well as atomicdisplacements have been calculated by the Wilson GF method, using a valence force field and a non-redundant set of internal coordinates. In addition, the N I S intensities have been simulated by considering the fundamentals of the internal vibrational motions as well as their interaction with the lattice modes situated below 150 cm-1 in adenine and its deuterated species. The R R S intensities were simulated by using the bond-order changes induced by the electronic transition from the ground to the first excited state. It is clearly shown that the NIS spectra mainly arise from out-of-plane vibrations, while the ultraviolet R R S spectra are principally related to highwavenumber in-plane stretching motions (above 1000 cm-').

Introduction In this paper experimental and theoretical studies are presented in order to show how the simultaneous use of neutron inelastic scattering (NIS) and resonance Raman scattering (RRS) enables a better description of the nucleic acid base vibrational motions to be obtained than that available from vibrational frequency analysis alone. NIS and RRS are complementary approaches, with respect to the spectral regions they cover as well as the nature of the vibrational modes they can probe. NIS presents several advantages as compared with optical spectroscopicmethods for investigating molecular vibrations: (i) no selection rule has to be applied to the incident neutron during its interaction with the molecular vibrations, (ii) low-wavenumber vibrations give more intense bands than do high-wavenumber ones (this appears in the Debye-Waller factor involved in the expression of the NIS cross section, for details see Theoretical Procedure), and these band intensities are still considerably enhanced at low temperatures, and (iii) the inelastic hydrogen

* Author to whom correspondence should be addressed.

' Permanentaddress: lnstituteof Physicsof Charles University,Ke Karlovu

5, 12116 Prague 2, Czechoslovakia

0022-3654/58/2091- 1074504.00/0

atom cross section is much higher than that of other atoms; consequently, the vibrational modes in which hydrogen motions participate give NIS bands with much higher intensities than those in which they do not. In NIS spectra the modes mainly involving heavy atom motions are either not observed or are very weak compared with those involving hydrogen vibrations. This property of NIS is a very interesting one in relation to biological molecules which contain a large number of hydrogen atoms. However, a major disadvantage of neutron spectroscopy is in the low intensity of the inelastically scattered beam; thus, a considerable amount of sample is necessary to run NIS spectra. In contrast, the optical techniquesof vibrational analysisreveal those modes contributing to the variation of the molecular dipole moments (infrared absorption) or to thevariation of theelectronic polarizability (Raman scattering). The selection rules governing the optical spectroscopies are those imposed by the transition dipole moment (IR process) or the molecular polarizability derivatives (Raman process). As far as nucleic acid vibrational spectra are concerned, offresonance (or classical) Raman scattering as well as IRabsorption generally provide a satisfactory analysisof the high-wavenumber modes located above 500 cm-I. On the other hand resonance 0 1993 American Chemical Society

Vibrational Motions of Bases of Nucleic Acids

The Journal of Physical Chemistry, Vol. 97, No. 5, 1993 1075

a

&*k/

b

\

H

H

Adenine Figure 1. Chemical structure and atom numbering of adenine molecule. C

Raman spectra, obtained by exciting the nucleic bases in their ultraviolet electronic absorption bands, provide valuable information on the vibrationswhich mainly involve in-planestretching motions of the atoms (or angle deformations highly coupled to stretchings), the better resonance enhancement is generally observed for bands higher than 1000 cm-I. In addition, some of these vibrational modes are much more coupled than the others to a specific electronic transition, by tuning the laser wavelength in the ultraviolet region, one can preferentially detect the modes which are in resonance with the electronic transition in question. Conversely, the spectral analysis of these particular modes leads toa better understandingof the molecular geometry in this excited state.' Recently,the guanine NIS spectrum has been published2along with a normal coordinateanalysis and a first-order NIS simulation (considering only the fundamentals). Despite the extreme efficiency of the NIS process for analyzing out-of-plane modes (which are detected by the optical techniques with greater difficulty), it is not sufficient for studying all the in-plane modes, especially those located in the high-wavenumber region of the vibrationalspectra: above IOOOcm-', the NIS spectral resolution becomes poor and the intensities become low. In an earlier paper we also mentioned that the NIS spectra should be complemented by RRS spectra, if a reliable assignmentof the vibrational modes in both the high- and low-wavenumber regions is desired. On the basis of the above-mentioned criteria, we report here the RRS and NIS spectra of adenine (and its deuterated derivatives), obtained from aqueous solutions and from polycrystalline samples, respectively. The spectral features are assigned by using a valence force field, which enables both the vibrational frequencies and the spectral bandshapes to be simulated. The investigation described here follows those carried out in the past on the theoretical assignment of the in-plane3and out-of-plane4modes of adenine.

Experimental hocedure and Results

NIS Spectra. The adenine (A) (Figure 1) base sample was purchased from Sigma and used as supplied. Conversion to the (N9, C6-amino)-deuterated species (hereafter designated as A-d3) was achieved by repeated recrystallization from D2O (purchased from Goss Scientific, 99.9% pure) at ambient temperature. Replacement of the H-atom on C8 (giving the A-dl species) was effected by heating a D2O solution of adenine at 80 OC for 2 h and then recrystallizing from H20. Approximately 2-g samples of thedry polycrystalline powders were used for NIS spectroscopy. The NIS spectra of adenine and its deuterated derivatives were obtained with the time-focused crystal analyzer TFXA, on the ISIS pulsed neutron source at the Rutherford Appleton Laboratory, UK.5 They were recorded at a low temperature ( T = 15 K)in order to sharpen the fundamental bands by decreasing the Debye-Waller factor.6 Traces a, b, and c of Figure 2 show the NIS spectra of adenine, A-dl, and A-d3, respectively, in the spectral region below 1800 cm-I. We have recently shown that the guanine lattice modes are located below 150cm-l in the NIS spectrum:2 this was verified by a normal mode analysis of the internal modes. Here theadenine latticemodesgive rise tointense

I 0

I

I

I

I

4.50

900

1350

18M)

Wakenumtcr / cm.' Figure 2. NIS spectra of adenine (a), A-dl (b), and A-d3 (c) species, observed from a polycrystalline sample at T = 15 K, and RRS spectra of the same species (adenine (d), A-dl (e), and A-d3 (0)observed from aqueous solutions at room temperature with the 257-nm excitation wavelength.

and poorly resolved bands below 120 cm-I: this will also be confirmed in the next section by the normal mode analysis. RRSSpectra. The adenine water solubilityin l e 2M phosphate buffer, pH 7,at room temperature is sufficient (ca. 6 X 10-3 M) for resonance Raman measurements. The C8-deuterium-substituted molecule (A-dl) was obtained as above and redissolved in the aqueous phosphate buffer for Raman measurements. The A-d3 species was prepared simply by dissolving adenine in a deuterium phosphate buffer in D20, pD 7, at room temperature. Resonance Raman spectra of the various solutions of adenine and its deuterated species were recorded by using a 257-nm excitation wavelength, i.e., the first harmonic of the 514-nm line of a Lexel Ar+ C W laser obtained through an Inrad Corp. doubling system. The spectrometer was a Jobin-Yvon HGZS double monochromator (home-modified for UV transmission)equipped with two holographicgratings and a single-channel PM detector. The whole setup, the photon counting device, the data acquisition system, and the spectrum treatment procedure have been described extensively elsewhere.' Traces d, e, and f of Figure 2 show the RRS spectra of the native adenine. A-dl, and A-d3 species, respectively, in the spectral region above 400 cm-I. Below IO00 cm-I, the RRS information content is rather low (in contrast with the NIS spectra), apart from a medium intensity contribution in the 600-700-~m-~ range. The strong Raman peaks detected above 1000 cm-I, where the NIS spectra provide only broad and weak bands, confirm the complementaryaspects of the NIS and RRS techniques for the purpose of vibrational mode analysis of this nucleic base.

Theoretical Procedure

A. Background. NZS Spectra. The NIS spectra arise from an inelastic interaction between neutrons and the external (lattice)

1076 The Journal of Physical Chemistry, Vol. 97,No.5, 1993

TABLE I: Valence Force Constants for Adenine In-Plane Modes'

Dhaouadi et al.

TABLE 11: Valence Force Constants for Adenine Out-of-Plane Modes'

In-Plane Force Constants KN9-H KN9-C8 KNI=C6 KC2-H KN748 KC2=N3 KC544 KCS-C6 K(C8-H) K(N6-H) K(N7-CS) KN9-c4 KC6-N6 KNl-c2 KN3-c4 HN9C4N3 H(NC2-H) N9C8. C4N9 C 4 , C-Np c = c , c-cp C-C, C=Np NlC6, N1C2 C-C, CNip CN, CNip C6N6, C6C5 C8N7, C2N3 C8N7, C4C5 C2N3. C4C5 N 1 C6N6, N 1 C6CS CSC6N6, N1C6C5 C5C6N6, N7C5C6 N-C, DCHp C = C , DRp C-N, DRp C-C, DRp C=N3, DRp C6N6, DRp C-N, DRip C-C, DRip H-N9, DRi C=N, DCHp N7C5, N7C5C6 N7C5, C5N7C8

Diagonal 4.35 HN7C8N9 5.92 HN342N1 6.2 H(N6C6C5) 5.13 H(N7C5C6) 7 H(N7-CSC4) 6.88 H(NlC6C5) 6.4 H(C4C5C6) 5.73 H(NC8-H) 5.41 H(N6C6N1) 5.86 H(C-N6-H) 5.51 H(H-N-H) 5.92 HC8N9C4 6.2 HC6N 1C2 6.21 HC-N9-H 6.4 HCSN748 1 HC4N342 0.52 HN9C445 Stretchstretch 0.2 NlC6, N3C2 0.35 C8N7, C5N7 0.44 C8N7, C8N9 0.65 C8N7, C4N9 0.641 N3C2, NlC2 -0.15 N3C2, N3C4 0.2 C6N6, N 1C6 0.75 C5C4, C4N9 -0.17 N 1C6, N7C8 0.15 N9C4, C6C5 -0.15 Bend-Bend 0.39 C6N6H, NlC6N6 -0.1 C6N6H, C5C6N6 -0.45 Stretch-Bend 0.54 C4N9, C4N9C8 0.75 C4N9. C4N9C5 0.701 C8N7, C8N7C5 0.2 C8N7, N7C8N9 0.75 C5C4, C5C4N3 0.901 C5C4, C5C4N9 0.55 NlC6, N1C6N6 -0.3 NlC6, NlC6C5 -0.03 NlC6, C6NlC2 0.35 N9C8, N9C8H 0.75 N9C4, HN9C4 0.75 N9C8, N9C8C4

Out-of-Plane Force Constants 1.39 1.53 1.44 1.35 1.23 1.29 1

0.425 1.28 0.415 0.452 1.4 1.998 0.31 1.68 1.9 1.6 0.15 0.9 0.9 -0.71 0.5 0.25 0.801 0.15 0.22 0.15

Diagonal 0.362 TO C=N (im) 0.33 TOC=C 0.43 TO C-N (py) 0.044 TO C=N (py) 0.3 TO C-C (py) 0.55 TOC6-N6 Wag-Wag W(NH2). W(C6N6) -0.03 W(N9-H), W(C2-H) W(NH2'), W(C6N6) -0.062 W(C6N6), W(C2-H) 0.02 W(C8-H), W(C2-H) Wag-Torsion W(N9-H), TO(N9C8) -0.05 W(C2-H), TO(C2Nl) W(N9-H), TO(C4N9) 0.22 W(C2-H), TO(C2N3) W(C8-H), TO(N9C8) 0.12 W(C6N6). TO(C5C6) W(C8-H), TO(C8N7) -0.245 W(C6N6), TO(NlC6) Torsion-Torsion TO(C4N9), TO(N9C8) 0.195 TO(C6N6), TO(NlC6) TO(C2N3), TO(N3C4) TO(C4N9), TO(C8N7) -0.05 TO(C4C5), TO(N3C4) -0.12 TO(C2NI). TO(C2N3) TO(N9C8), TO(N7C5) 0.05 TO(C2NI), TO(C6NI) TO(C8N7), TO(C4C5) 0.12 TO(C5C6), TO(NlC6) TO(N9C8), TO(C4C5) -0.05 TO(C5C6), TO(C4CS) TO(C4N9), TO(N7C5) -0.14 TO(NlC6), TO(C2N3) TO(N9C8), TO(C8N7) 0.035 TO(NlC6), TO(C4C5) TO(N7C5), TO(C4CS) 0.1 TO(C2N3), TO(C4C5) TO(C4C5), TO(C4N9) 0.22 TO(N3C4), TO(C5C6) W(C8-H) W(N9-H) W(C6-N6) W(NH2) W(CZ-H2) TO C-N (im)

0.6 1 0.585 0.257 0.43 0.455 0.0935 0.03 -0.01

0.115 -0.06 0.14 0.1 1 -0.01 -0.04 0.065 0.02 0.05

-0.08 0.09 -0.08 -0.1

0.195

Symbols: W = waggingforceconstant,TO =torsional forceconstant. All of these force constants are in mdyn.A. a

0.25 0.2

0.9 0.9 0.58 0.58 0.3 0.3 0.78 6.2 0.4 0.76 0.27 0.8

a Symbols: K = stretching force constant (mdyn/A), H = bending force constant (mdyn-A),DCH = CH bending internal coordinate, DR = ring bending internal coordinate, p (i) = pyrimidine (imidazole) ring. Str bend interaction force constants are in mdyn.

and internal (molecular) vibrational modes of a polycrystalline sample of the molecule in question. Here the lattice modes of adenine are located below 150 cm-I, while the molecular modes are situated above this wavenumber. This fact considerably simplifies the simulation of the couplings between internal and external modes. This kind of interaction gives rise to the socalled "phonon wings" (broad side bands arising from the additive and subtractive combinations of the lattice modes with the molecular fundamentals). In a first-order NIS process, the incident neutron interacts with only one molecular vibrational quantum. In a second- or higher-order process, two or more vibrational quanta also contribute. The fundamental physical laws governing NIS are the energy and momentum conservation between the incident and scattered neutrons and the molecular vibrations. In NIS t h e ~ r y , ~the < ~neutron -~ scattering differential cross section (Le., the probability that an incident neutron be scattered by a nucleus in a given direction with a known energy change) can be expressed as follows for an incoherent and inelastic process,

where 6 is the Dirac delta function, W Ais the Ath vibrational mode angular wavenumber (A = 1, 2, ..., 3N-6), nA = 0, f l , *2, designates the number of vibrational quanta adsorbed (minus sign) or emitted (plus sign) during the scattering process, fl is a solidangle, k~ and T a r e the Boltzmann constant and the absolute temperature, respectively. K = Kr- Ki is the wavevector associated with the momentum transfer, where Ki and Kf are the incident and final wave vectors of the neutron, S(K,w) is the so-called partial reduced scattering function. At low temperature, S(K,o) in the nth order interaction can be evaluated by the following expression,I0J I

...

where b~ is the scattering length, m~ is the mass of the atom L, eLAis the normalized Ath normal mode eigenvector for the atom L, exp(-2W~) is the so-called Debye-Waller factor, WLbeing expressed by

W, =

E(h)(K.c,")2 A

4mLwA

coth

("-)

2k0T

(3)

RRS Spectra. The nucleic acid bases give rise to resonance Raman enhancements when they are excited with incident laser radiation coincident with their ultraviolet absorption band. In the first-order Stokes Raman process, a simplified theory has been developed in the case of conjugated molecules and totally

Vibrational Motions of Bases of Nucleic Acids

The Journal of Physical Chemistry, Vol. 97, No. 5, 1993 1077

TABLE III: Comparison between Experimental and Calculated Wavenumbers (cm-I) for the Adenine In-Plane Modes’ wavenumber/cm-I IR

R

RRS NIS

3295 3215 3125 3038 2790 1672 1675 1604 1612 1484 1465 1416 1364 1330

calc

3310 3204 3163 3075 2817 1680 1653 1601

1597 1598 1588 1482 1483 1471 1473 1462 1467 1418 1412 1370 1366 1358 1331 1329 1328

1308 1307

1300

1251 1248 1248 1234 1253 1155 1164 1168 1153 1125 1126 1129 1090 1068 1022 1023 1032 1015 942 949 940 940 912 898 934 72 1

722

722

620 540 337

620 535 330

620

876 721 529

878 709 665 604 545 310

assignments (PED/%) N6-H2 asym. st. (100) N6-H2 sym. st. (100) C8-H (99) C2-H (99) N9-H (99) H-N-H (23); C6-N6 (23); C 6 - C 5 (12); C6N6H (1 1); C5=C4 (10) C 6 4 5 (24); N 7 4 5 (10); C4-N9 (10); C4-N3 (9); N3C4C5 (7); N7C5C4 (6); H-N-H (6); C6C5C4 ( 5 ) ; N9C4C5 ( 5 ) ( 2 5 4 4 (28); C4-N3 (18); H-N-H (15) C4-N9 (16); C6-C5 (13); C8=N7 (12); N7