Electroabsorption (Stark effect) spectroscopy of monomeric purine and pyrimidine bases

Chemical Physics 314 (2005) 309–316 www.elsevier.com/locate/chemphys

Electroabsorption (Stark effect) spectroscopy of monomeric purine and pyrimidine bases Rafał Luchowski, Stanisław Krawczyk

*

Institute of Physics, Maria Curie-Skłodowska University, Pl. M. Curie-Skodowskiej 1, 20-031 Lublin, Poland Received 15 December 2004; accepted 22 March 2005 Available online 26 April 2005

Abstract Electronic charge distribution in individual purine and pyrimidine bases and in their systems is an important parameter useful for characterization of their molecular interactions. This paper describes experiments devoted to determination of changes in dipole moments and polarizabilities of DNA bases which take place on electronic excitation, using the Stark effect in absorption spectra ˚3 of glassy solutions at low temperature (80–100 K). Dipole moment differences of 2–3 D and polarizability changes about 30–60 A were obtained for electronic transitions corresponding to the
260 nm absorption band. The comparison of experimental data on difference dipoles with the results of semiempirical calculations allows for assignment of their directions within molecular structures.
2005 Elsevier B.V. All rights reserved. Keywords: Purine; Pyrimidine; Nucleotide; DNA; Stark effect; Absorption spectroscopy

1. Introduction Electronic spectroscopy methods, particularly the measurement of electronic absorption and circular dichroism (CD) spectra, are widely used for characterization of nucleic acids and polynucleotides. The basic interpretation of these spectra has been given long ago [1–3] in terms of intermolecular interactions that lead to shifts and intensity changes of absorption spectra through the coupling of electronic transitions in the system of closely spaced base molecules as compared to spectra of separated monomers. Since that time, several papers considered the coupling of electronic states of bases by examining the possibility of electron exchange interactions [4] and formation of dense clusters of electronic energy levels [5–8]. In recent years, the extensive studies on electron transfer mechanisms in DNA and related molecular systems posed the question pertaining to the relative role of *

Corresponding author. Tel.: +4881 5376253; fax: +4881 5376191. E-mail address: [email protected] (S. Krawczyk).

0301-0104/$ - see front matter
2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chemphys.2005.03.012

coulombic (dispersive and electrostatic) interactions and exchange interactions [9,10] and, irrespective of the significance of the latter, indicated the need for new experimental data capable of characterizing the electronic charge distribution in individual bases and in their systems. Among the key photophysical parameters useful for the interpretation of spectral changes that occur on stacking of planar chromophores are the changes in permanent dipole moment and of molecular polarizability that occur on aggregation, as shown recently for the dimer of acridine orange [11]. The changes in the electronic structure take place also in stacked purine and pyrimidine bases in polynucleotides and in DNA. Besides, nucleic acids and polynucleotides present an interesting case of a self-organized molecular system with well-known spatial distribution of chromophore molecules, and for this reason are good model for testing and extending the applicability of Stark effect spectroscopy in its classical formulation [12,13] to systems of interacting molecules. There have been several experimental [14–18] and computational [18,19] studies aimed at the determination

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of the structure and the ground-state dipole moments of the monomeric bases, but less efforts concerning the excited states [18,20–22]. In this paper, we describe experiments using the electroabsorption (Stark effect) spectroscopy, a technique which offers insights into molecular structure useful in various fields of chemistry and biophysics [23–25]. In some respect, electroabsorption spectroscopy turns out to be a valuable and essentially a unique tool providing direct information about charge redistribution on electronic excitation and on its sensitivity to an externally applied electric field. Electronic transitions are examined here for the purine and pyrimidine bases used in the form of nucleosides and nucleotides frozen in glassy solvents at low temperature. Due to experimental difficulties, the measurements are limited to the lowest-energy absorption bands of bases known to result mainly from one p ! p* electronic transition in pyrimidines or two transitions in purines [26]. The electroabsorption spectroscopy of interacting bases in polynucleotides will be described separately.

2. Materials and methods All experiments described below were performed using nucleosides, nucleotides and 9-ethyl adenine purchased from Sigma. The samples for low temperature measurements were prepared from nucleotide solutions in mixtures of water with glycerol or ethylene glycol. For nucleosides (except for cytosine) and 9-ethyl adenine, also 2-methyl tetrahydrofuran or mixtures of aliphatic ethers with isopropanol were used. Samples were also prepared in 10% polyvinyl alcohol (PVA) solution in water by drying for 2–5 h at 16–18 C to obtain a thin (35–50 lm) transparent foil which was clamped between electrodes after wetting with glycerol. Absorption and electroabsorption (Stark) spectra were recorded using UV light from a 450 W Xe lamp, which was dispersed by a single grating monochromator, linearly polarized with a Glan–Thompson airspaced polarizer and guided by quartz optics. Samples were frozen in a stream of cold N2 vapor and then transferred to a cryostat (Optistat, Oxford Instruments) equipped with quartz windows. Sample cuvettes were constructed from 1 mm thick quartz plates with evaporated conductive layers of SnO2 serving as electrodes. Multiple scanning and averaging of spectra was employed in order to improve the signal-to-noise ratio. Care was taken to obtain spectra free of distortions by keeping narrow slit widths and by applying corrections for scattered light if necessary. In most of experiments, absorption spectra were additionally measured after transferring the cryostat with the sample into the HP 8453 spectrometer. The electric field-induced change in absorbance, which results in a small modulation of the transmitted light intensity (DI), was induced by sinusoi-

dal voltage of
1000–2000 V r.m.s. DI was recorded at the second harmonic frequency to measure the quadratic Stark effect, and the absorbance change was calculated as DA = DI/(2.3 Æ I). The measurement of this very weak effect for DNA bases with broad and unstructured spectra requires appropriate light intensity, otherwise the shot noise of photons becomes a limiting factor, and this made our measurements unfeasible at wavenumbers higher than 42,000 cm1 (238 nm). The spectrum of absorbance changes, DA(m) = DI/ (2.3 Æ I), can be expressed as a linear combination of the absorption spectrum itself and of its first and second derivatives with respect to the wavenumber m [13,24,27] DA ¼ a0  A þ a1  m

dðA=mÞ d2 ðA=mÞ þ a2  m : dm dm2

ð1Þ

The coefficients a1 and a2 are functions of the differences in molecular polarizability tensor and in the permanent dipole moment, Da and Dl, upon electronic excitation: 
 ðfF Þ2 5 3 1 TrðDaÞ þ ð3cos2 v  1Þ pðDaÞp  TrðDaÞ a1 ¼ 2 2 15hc 2 n X 1 þ mi 10Aij Dlj þ ð3cos2 v  1Þ jmj2 i;j o  ð3Aji Dlj þ 3Ajj Dli  2Aij Dlj Þ ; ð2Þ 2

a2 ¼

ðfF Þ jDlj2 f5 þ ð3cos2 v  1Þð3cos2 d  1Þg: 30h2 c2

ð3Þ

In these formulas, v is the experimentally variable angle between the electric vector of light and the vector of applied electric field F, and p is a unit vector in the direction of the dipole transition moment m. In the following, the second term in Eq. (2) is neglected (see Section 3). Since the experimental data are insufficient to fully determine the difference polarizability tensor Da, the trace of the polarizability tensor, Tr(Da), was determined from the value of a1 for v equal to the magic angle 54.7. The first term in Eq. (1), which is associated with DA proportional to the absorption spectrum, reflects the electric field-induced change in the dipole transition moment mF ¼ m þ A  F þ 12F  B  F;

ð4Þ

where m is the transition moment without the electric field F. This field dependence of the transition moment leads to the complex expression [13,27,28] for the coefficient a0 in Eq. (1) ðfF Þ2 a0 ¼ 30m2

(

X ½10A2ij þ ð3cos2 v  1Þ  ð3Aii Ajj þ 3Aij Aji  2A2ij Þ i;j

) X 2 ½10Bijj þ ð3cos v  1Þ  ð3Bjij þ 3Bjji  2Bijj Þ : þ

ð5Þ

i;j

The electrooptical parameters reported here include the local field factor f which relates the macroscopic (mean)

R. Luchowski, S. Krawczyk / Chemical Physics 314 (2005) 309–316

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electric field F to the one actually acting on the pigment molecule. The values of jDlj and Da given below correspond to f Æ jDlj and f2 Æ Da, with the local field factor making them apparently larger by about 1.1–1.3 [24]. In addition to experiments, the ground- and excitedstate permanent dipoles and transition dipole moment vectors were calculated with the semiempirical AM1, INDO/S and ab initio Hartree-Fock methods with CI, all with program implementations in the HyperChem package [29].

3. Results and discussion All experiments were performed using nucleosides and nucleotides of respective bases. Besides of close similarity between these molecules and the chemical structures involving the purine and pyrimidine bases in DNA, this choice eliminates main sources of tautomerization and ensures sufficient solubility in a very limited set of solvents capable of forming good quality glasses at low temperature. We also consider the experimentally determined values of jDlj and Tr(Da) for all five nucleotides and 9-EA to be the characteristics of the bases as the chromophores irrespective of their substitution. This assumption supposes the similarity of the 9-ethyl and 9-ribose substitution as well as the noneffect of the phosphate group. The latter assumption is supported by the close spectral similarity of nucleosides and nucleotides [30]. 3.1. Thymidine and uridine Fig. 1 presents the absorption and Stark spectra of thymidine. Two vibrational progressions with frequencies of 750 and 1450 cm1 are clearly seen in the Stark spectrum making it rich in details. Two fitting curves calculated for the entire spectral range are shown, which result from either a two-component fit with only the first and second derivatives of the absorption spectrum or from a fit including an additional term proportional to the absorbance (cf. Eq. (1)). Their comparison shows the relevance of the component proportional to the absorption spectrum, which is not always the case in many dyes and pigments examined with this spectroscopic method. All our results were carefully tested in this respect and the zeroth-derivative term (i.e., proportional to the absorption spectrum) is consequently included if it once turned out to be necessary for a given transition. For uridine both the absorption and Stark spectra (not shown) are very similar to those of thymidine and are only shifted by 400 cm1 toward higher frequencies. All details in the Stark spectra of thymidine and uridine resulting from the vibrational structure of the electronic transition can be well fitted with similar sets of electrooptical parameters in both 2-MeTHF and in glyc-

Fig. 1. Spectra of 3.3 mM thymidine in 2-MeTHF at 80 K recorded with electric field intensity 274,000 V/cm (r.m.s.). Both spectra are normalized to unitary maximum absorbance, and the Stark spectrum additionally normalized so as to correspond to electric field intensity of 105 V/cm. (a) Absorption spectrum; (b) Stark spectrum (points) and the least squares fits: with only the first and second derivatives (dotted line), and including the third component proportional to the absorption spectrum (continuous line). Part (c) shows the composition of the three-component fit: the scaled absorbance (continuous line) and its first (dashed line) and second (dotted line) derivatives. The fits in part (b) were calculated for data in the whole spectral range shown in the figure.

erol/water, although the vibrational structure of the electronic transition is much weaker in the latter solvent (spectra not shown). The electrooptical parameters obtained are listed in Table 1. They are independent of whether the solvent is a hydrogen bond acceptor or donor, indicating small effect of hydrogen bonding on the electrooptical parameters of the bases. Generally, for T and U, the good quality of fits indicates the lack of aggregation effects and the presence of a single electronic transition in the whole spectral range examined. 3.2. Cytidine Cytosine appears to form concentration- and solventdependent aggregates most easily among the bases

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Table 1 Transition energies and electrooptical parameters of the DNA bases in nucleoside and nucleotide forms Base

Transition No.

(0–0) (cm1)

jDljcalc (D)

dcalc ()

jDljexp (D)

dexp ()

˚ 3) Tr(Da) (A

U T C

I I I

35,600 35,300 34,700

1.54 1.39 2.04

24 5 53

3.26 ± 0.35 2.54 ± 0.12 1.65 ± 0.2

12 ± 2 12 ± 6 16 ± 3

70 ± 15 50 ± 5 26 ± 4

9.4 ± 1.4 6.6 ± 1.9 5.1 ± 1.4

A

I II

35,600 37,150

3.44 2.19

36 7

3.25 ± 0.3 2.1 ± 0.25

40 ± 10 29 ± 5

43 ± 14 36 ± 11

n.d. 4.5 ± 1.7

9-EA

I II

35,450 36,900

3.44 2.19

36 7

2.82 ± 0.15 1.8 ± 0.1


55
30


30 26 ± 3

n.d. 4.8 ± 0.5

G

I II

34,900 38,850

3.60 8.38

50 22

2.7 ± 0.15 1.85 ± 0.2

26 ± 2 18 ± 2

50 ± 7 26 ± 7

10.9 ± 1.2 4.8 ± 0.8

(a0/F2) · 1017 (cm2/V2)

The wavenumber of the (0–0) transition is given for samples in glycerol–water or ethylene glycol–water glasses (4:1, v/v), except for 9-ethyladenine in diethyl ether/tert-butyl ether/isopropanol (1:1:0.35, v/v). The quantity dcalc in the fifth column is the angle between the vector Dl from our AM1 calculations and the experimental transition moment direction taken from LD data in the literature (see text). Data marked as approximate were averaged from only two separate experiments or, in the case of angles, were obtained from poor angular dependence of fit coefficients.

studied, in both aqueous and organic glasses. The full set of CMP spectra are shown in Fig. 2 with additional absorption and Stark data for cytosine in organic solvents. In the latter cases, the spectra show more clear structure with a progression of 780 cm1 vibrational transitions. The positive feature at 34,000–34,500 cm1 is split into two components with intensity ratio dependent on solvent polarity so that the higher energy component is stronger in solvents of lower polarity. This suggests that the formation of stacked dimers or higher aggregates of H type held together by electrostatic interactions, with the transition energy shifted up by 400– 500 cm1, i.e., in the range predicted for interacting transition dipoles of two bases in van der Waals contact [6]. The large permanent dipole moment of cytosine chromophore in the ground state, 8 D from experiment [16,17], 8.1 D from ab initio [21] and 7.1 D from DFT calculations [17,19] (5.93 D from AM1 in this work), can be responsible for stacking interactions. Since cytidine in organic solvents used always appears to be partly aggregated, we estimated the electrooptical data only for CMP in aqueous glass (Fig. 2). Although, the positive Stark band at 34,000 cm1 may look to contain the monomer and dimer bands, its width is in fact even smaller (FWHM 850 cm1) than for analogous lowest energy (0–0) bands in TMP or UMP spectra (FWHM 1200 cm1). Also much better solubility of CMP in aqueous solvent (like other nucleotides) allows to ascribe the spectra to the monomeric form of CMP. This is indirectly supported by a single fit covering the spectral range considered. However, a satisfactory fit to the Stark spectrum is possible only in the spectral range below 37,000 cm1. The fits extended above 37,000 cm1 show systematic deviations from experimental data and this effect is illustrated with a dotted line in Fig. 2. This is in agreement with analyses of absorption [1,26] and linear dichroism (LD) spectra [31,32] of cytosine which indicate

Fig. 2. Normalized spectra of cytidine. Panel (a), absorption spectra of 9.6 mM CMP in H2O:glycerol = 1:4 (v/v) (solid line), cytosine in ethanol:ethyl-butyl ether:diethyl ether = 1.4:1:1 (dots) and cytosine in ethanol:methanol = 2:5 (dashes). (b) Stark spectra of CMP (circles, vertical scale at left), and cytosine in ethanol/ethers (dots) and cytosine in alcohols (dashes), both referring to the right vertical scale. Solid line is the least squares fit (line) calculated with experimental data in the range 32,000–37,000 cm1 but shown with the dotted line in a wider range. (c) Fit components: the zeroth (solid line), first (dashed) and second (dotted) derivatives of absorbance. Spectra normalized as in Fig. 1.

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the presence of a next electronic state at about 230 nm (43,500 cm1) with the wing extending to 260 nm (38,400 cm1). The low-energy tail of this band contributes to the Stark spectrum at frequencies above 37,000 cm1 by adding a positive signal up to
39,500 cm1 and a negative feature at higher energies. 3.3. Adenosine The absorption band of purine bases is known to result from two electronic transitions [26], of which the one at lower energy is significantly less intense. Accordingly, their Stark spectra contain overlapping contributions from two transitions with potentially different sets of electrooptical parameters. In the case of adenine chromophore, this difficulty can be overcome by making comparison with reference data for 9-ethyladenine which can be used in organic solvents and thus shows better resolved spectra (cf. Fig. 3) with a clear vibrational struc-

313

ture. In the spectra of 9-EA, the lower energy (0–0) band of transition I is at 35,450 cm1, and there is also a weak feature in the absorption spectrum at about 36,200 cm1 indicating the vibrational excitation of
750 cm1. Vibrational quanta accompany the electronic transition II with the (0–0) origin at 36,900 cm1, for which a
700 cm1 vibration can be inferred from the second derivative. The lower energy transition I in the Stark spectrum of 9-EA can be well approximated in the range up to 36,200 cm1 with the combination of only the first and second derivatives of the absorption spectrum (Fig. 3(c)). Trial fits with three components have shown that the term proportional to the absorption spectrum itself is insignificant for transition I. However, a significant component proportional to the absorbance remains necessary in the higher energy transition II as shown in Fig. 3(c). Guided by the data for 9-EA, we retained the twocomponent fits also for transition I in AMP in the range from 34,000 to 35,800 cm1, and used three-component fits for transition II (data not shown). The data sets for A and 9-EA in Table 1 are similar within the limits of statistical scatter. 3.4. Guanosine In the spectra for GMP shown in Fig. 4, the two electronic transitions are separated well enough for the fits to provide reliable assignment of electrooptical parameters. Additionally, the two-component fit gets remarkably poorer if extended above 35,000 cm1, where the band overlap effects shall not be expected. We thus applied three-component fits to both transitions in Stark spectra of G. They also contain traces of vibrational structure very useful in guiding the fit composition. The fits for both transitions shown in Fig. 4(b) are of good quality, provided the component proportional to the absorbance is included. The values of jDlj and Tr(Da) for GMP quoted in Table 1 are based on fits in the ranges below and above 37,500 cm1. The boundary between the two regions with dominant contribution from one or the other transition becomes clear if the fits are continued beyond this limiting wavenumber. Fig. 4(b) shows also the fits to the Stark spectrum with only the first and second derivatives, indicating the importance of the change in absorption intensity (0th derivative). The neglect of the 0th derivative makes the estimates of Tr(Da) and jDlj for transition I lower by 20–25% and larger by 10–15%, respectively.

Fig. 3. Normalized spectral data for 1.3 mM 9-ethyladenine in tertbutyl ether:diethyl ether:isopropanol (1:1:0.35, v/v) at 80 K. (a) Absorption spectrum, (b) Stark spectrum, and (c) the fit components drawn below and above 36,200 cm1, according to the left and right vertical scales, respectively. Part (b) shows the two- and threecomponent fits in the higher energy range (dashed and solid lines, respectively), and the two-component fit including only the derivatives in the lower energy range. Fit components in part (c) denoted as in previous figure.

3.5. Directional properties For each electronic transition there are four possible directions for the vector Dl at the (experimentally determined) angle d with the transition moment. In principle, this ambiguity in experimental data can be removed and the directions of Dl for most transitions can be assigned

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The ground state dipole moments calculated with the INDO/S method were incompatible with the experimental and other theoretical data and this method was not used further. The ground state dipole moments calculated with AM1 for T (4.48 D, 136), U (4.59 D, 140), C (5.93 D, 80), A (2.30 D, 93) and G (7.09 D, 172) were found to be in very good agreement (differences less than 10%, for cytosine 16%) with those from the ab initio method for T, C, A, G [19], A, G [18,20] and U, C [21]. In the assignments given below, the angles are measured counterclockwise from the N1–C4 axis in pyrimidine bases and from the N3–C6 axis in purine bases (cf. Fig. 5), according to the accepted convention [1]. Both the values and directions

Fig. 4. Normalized absorption and Stark spectra of GMP (6 mM) in H2O:ethylene glycol (1:4, v/v) at temperature 100 K. (a) Absorption, (b) Stark spectrum (points) with fitting curves based on derivatives only (thin lines) and including the absorption intensity changes (thick lines), (c) fit components denoted as in previous figures.

by comparing the experimental data with the results of quantum chemical computations. However, making the proper choice of a computational method capable of providing reliable dipole transition moments and permanent dipole moment vectors in the ground and excited states poses some difficulty. We tested two semiempirical methods, INDO/S (spectroscopically calibrated [33]) and AM1 (parameterized so as to reproduce well the structure, thermodynamics, redox and dipole moment properties [34]), and the ab initio method (however, only with the small basis set 3-21G). The calculations were performed using the ground state structures of 1-Me pyrimidines and 9-Me purines optimized using AM1. Excited state calculations with AM1 and INDO/S were performed with the full set of singly excited configurations (SCI) resulting from valence orbitals – about 900 single-determinant configurations for pyrimidines and 1300 for purines. With the ab initio method, the limited SCI expansion included similar numbers of occupied and unoccupied orbitals as in AM1 calculations.

Fig. 5. Molecular structures of DNA bases with sketched directions of the transition moment dipoles (lines) and permanent dipole moment differences (arrows). Experimental data are shown with continuous lines and calculated with dashed lines. For each base, the scheme on the left shows the experimental transition dipole(s) from the literature LD data (solid line), the calculated transition dipole moment (dashed line) and calculated Dl (dashed arrow). The structure on the right shows the transition moment and the vector of permanent dipole difference between the ground and excited state directed so as to fulfil the computational predictions and in agreement with the experimental angles (d) in Table 1.

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of the dipole moments from our ab initio calculations were also very similar to the above AM1 values for C, T, U and A; only for G, a 25 difference in orientation was noted. More significant discrepancies were noted for transition moments and excited state dipole orientations when comparing our values with transition moments from the literature. They indicate that AM1 results are more compatible with the experimental data. For all bases, AM1 gives the lowest energy transition moment orientations close to those determined experimentally (vide infra), however, with unacceptable discrepancies for the second electronic transitions in A and G. The p–p* transition energies obtained with AM1 were very accurate for 1-methylthymine and 1methyluracil, and too low by a factor of 0.84 ± 0.06 for 1-methylcytosine and the two transitions in both 9methyladenine and 9-methylguanine. We thus present the assignments of experimental electrochromic data based on AM1 results, but it should be emphasized that, especially for all three pyrimidines, identical interpretations of our experimental data emerge from the use of ab initio results. The assignments of data obtained from Stark spectra are depicted in Fig. 5, which includes also the transition moment orientations from LD measurements of other authors [31,35] (summarized in [36]). These data are in good agreement with other LD studies for adenine [32,37,38], guanine [32], cytosine [32,39], thymine [32] and uracil [40]. It can be seen in Fig. 5 that for T, U, C and for the lower energy transitions in A and G the computed directions of the transition moments (shown with the dashed line in the left part of each figure) are in reasonable agreement with the experimental data from LD (continuous lines). Also the values of jDlj calculated with AM1 (see Table 1) remain in reasonable agreement with the experimental data for CMP, AMP and 9-EA (transitions I and II), and for GMP (transition I). For TMP and UMP, the calculated jDlj
s are significantly smaller than their experimental values, and for the higher energy transition II in GMP the calculated value of 8 D was unacceptably large. We take the prevailing conformity of calculated and experimental data as a support for assigning the directions of Dl at the angle d to the experimentally determined [35,36] transition moment, and aligned on the side indicated by the relation between the calculated Dl and the calculated transition moment. The assignments of the difference dipoles Dl with respect to transition dipole moments are depicted in Fig. 5 in the right part of each picture corresponding to a given base. The data in Table 1, columns 5 and 7, provide comparison of the angles between the calculated Dl and the experimental transition moment with the angles determined from the Stark spectra. The directions of Dl for pyrimidine bases are all similar and indicate the electron donating character of nitrogen atoms towards the ring

315

in the electronic excited state. However, there are also significant discrepancies for the electronic transition in C and for transition I in G. The directional assignments for Dl given in Fig. 5 bear some level of uncertainty associated with the choice of one of the two possible directions ascribable to a given transition moment on the basis of LD data [31,35,36]. We checked also the alternative transition moment orientations that were considered in the original analyses of LD but rejected by their authors on grounds of substituent effects [31] or by comparison with computational results [35]. The alternative transition moments for T (at +51) and U (at +7) lead to the values of d = 76 and 41, respectively. For transitions I (+66) and II (+19) in A the corresponding values of d would be 6 and 69. A comparison with experimental values of d in Table 1 indicates that the assumption of corresponding transition moments is unreliable. The alternative transition moment in C (at 46) gives d = 10, in better agreement with experiment (d = 16) than the transition moment orientation preferred in the LD data. Also for G, whether using the preferred orientation for transition moments (Fig. 5) or the alternative ones (+4 (I) and 88 (II) and thus d = 65 and d = 39, respectively), the values of d poorly correlated with experimental data result, especially for transition I. In the case of cytidine, the discrepancy between the transition moment orientation emerging from LD and that required to fit the Dl direction in our electroabsorption data can be tentatively explained by the possibility of aggregation as mentioned above, that leads to imprecise experimental value of d. This discrepancy, however, similar to what is observed for G, can be due as well by the effect of transition moment rotation induced by the external perturbation, first invoked to explain the differences between the calculated transition moments and those from LD measurements for crystals of 9-ethylguanine [41]. This effect has been shown later to be operative also in amorphous environment and, being due to the reaction field, has been found sensitive to the size of the cavity filled by the chromophore [22]. In the case considered here, both C and G possess the largest ground state dipole moments, 6–8 and 7 D, respectively, compared with
4.5 D for T, U and 2.3 D for A (vide supra). Taking into account the enhancement of the reaction field by the smaller size of the cavity in the case of C, we indicate the possibility of transition moment rotation by the strong reaction field in G and possibly also in C. 3.6. Transition intensity changes Almost all electronic transitions examined in this work exhibit the dependence of transition intensity on the electric field. This property is related to molecule
s electronic structure in a very complex way [12,13,27]. It has found an approximate interpretation only in the

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case of electronic transitions with significant charge transfer character [25,42,43], where the values of a0/F2 (cf. Eq. (5)) are larger by more than an order of magnitude than those for DNA bases. The relatively small changes in transition intensity contribute significantly to Stark spectra of bases mainly because the other two components, which are related to derivatives of weakly structured absorption spectra, are weak. Since there are no indications which electronic states mix with the lowest excited states in DNA bases in the electric field (which can be based, e.g., on molecular symmetry [27]), the exact origin of field-induced transition intensity modulation cannot be reliably indicated. The values of the modulation rates, a0/F2, which are quoted in Table 1 for v = 90, remain negative also at v = 54.7, when the angle-dependent terms in Eq. (5) vanish. This suggests that the field dependence of transition moments originates mainly from the second term in Eq. (5) stemming from transition hyperpolarizability B, since the first term related to transition polarizability A can be only positive for magic angle v. As indicated by Eq. (2), the polarizability difference may contain a contribution from the transition polarizability A.

[3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]

3.7. Conclusion [21]

The application of low temperature absorption and Stark spectroscopy to the chromophores of nucleic acid bases provided spectra with sufficiently resolved structure to produce unique fits of good quality for making estimates of permanent dipole and polarizability changes on electronic excitation. Their values were found in approximate agreement with the results of semiempirical calculations for the lowest excited states of all bases, leaving the comparison of data for the second allowed transition in G and A for more advanced computational study. The parameters of base monomers obtained here are among the basic experimentally available characteristics to be compared with those of interacting bases in single- and double-stranded polynucleotides, which can be expected to reflect the electronic structure modulation by intra- and interstrand interactions.

[22] [23]

[24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]

Acknowledgment This work was supported by research funds of Maria Curie-Skłodowska University.

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