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An Effective Strategy for a Conformer-Selective Detection of Short-Lived Excited State Species: Application to the IR Spectroscopy of the N1H Keto Tautomer of Guanine Hiroya Asami, Munefumi Tokugawa, Yoshiaki Masaki, Shun-ichi Ishiuchi, Eric Gloaguen, Kohji Seio, Hiroyuki Saigusa, Masaaki Fujii, Mitsuo Sekine, and Michel Mons J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.6b01194 • Publication Date (Web): 18 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016

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The Journal of Physical Chemistry

1. INTRODUCTION Tautomerism in DNA bases is thought to play an important role since occurrence of rare tautomers can lead to a pairing mismatch and potentially to mutation in the genetic material.1-3 In this respect the DNA base guanine (Gua) exhibits four stable tautomers, corresponding to Htransfers between the N1 and O6 sites (Cf. Chart 1), on a one hand, and between the N7 and N9 sites, on the other hand; high level quantum chemistry methods predict these four forms to be nearly isoenergetic when isolated in the gas phase.4,5 The issue of the relative stability of these species has motivated many gas phase studies during the past decade, in order to provide an experimental counterpart to these theoretical predictions and characterize the corresponding species.6-17

Several groups have tried to observe selectively the most stable forms of Gua, in

particular the most stable keto tautomers, in the gas phase by using resonance-enhanced twophoton ionization (R2PI) with nanosecond lasers. However a careful analysis of the data eventually showed that only high energy species, one enol and two imino forms,8,17 were actually observed.617

The reason for the absence of the most stable forms in the spectra, in particular the keto forms,

was ascribed to the existence of an ultrashort lifetime of the S1 state of these species, caused by the existence of a conical intersection with the ground state at a geometry close to that of the S1 state minimum, corresponding to a distortion of the six-membered ring in the  excitation.18-25 The consequently ultrafast deactivation forbids S1 state detection with ns lasers. The use of shorter pulses could be used to solve this issue, but at the expense of conformational selectivity owing to the large bandwidth of these lasers. Conformer-selective gas phase detection of isolated Gua had thus to rely on other strategies: Choi and Miller26 found conclusive evidence for the four most stable tautomers (keto and enol with both 7H and 9H forms) from IR spectroscopy in He droplets. This result was later supported by Alonso et al. who detected them by gas-phase microwave

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spectroscopy27. However these methods are neither conformer-selective, nor directly sensitive to the mass, in contrast to IR/UV double resonance spectroscopy with a R2PI detection carried out in conjunction with mass spectrometry. In order to make possible R2PI ionization of the Gua system, we have relied to a convenient chromophore that absorbs in the near UV and whose S1 state is long-lived and high enough to be easily ionized through R2PI. By grafting a 2methylnaphthyl moiety on the N9 site of Gua, resulting in the 9-(2-naphthylmethyl) guanine, referred to as GNap in short (Chart 1), one also blocks the 7/9NH tautomerism, which leaves us with a model for the biologically relevant 9-substituted Gua derivative. The S1 state of naphthalene (Nap) is significantly red-shifted from that of Gua, which reduces the risk of mixing between electronic states of these UV chromophores.28 This makes possible fluorescence or ion detection from near UV excitation of a Gua-containing molecule, without being hampered by the short lifetime of the Gua excited states. Although the approach is similar to grafting a phenyl-containing group to the N- or C-terminals of peptides29-31, it differs by the introduction of a second chromophore, chosen for its specific properties (S1 lifetime, S1S0 and ionization energies). The naphthyl moiety UV spectroscopy is also expected to be slightly sensitive to the tautomeric nature of the Gua attached to it, both via through-bond or through-space interactions, making the GNap molecule a convenient model system from which tautomer-selective features of substituted Gua can be studied. In the present work, we employed the laser desorption technique, where nonvolatile molecules are successfully vaporized without extended degradation.32 Subsequent supersonic-jet cooling of molecular beam enables us to isolate low-energy tautomers under low-temperature conditions. The neutral molecules are ionized by nanosecond R2PI, and the structural assignment is determined from IR-UV double resonance spectroscopy aided by quantum chemistry calculations. Specifically, we here report the mass-selected R2PI observation of the N1H keto form


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of GNap isolated in the gas phase, as well as for the first time its mid-IR absorption spectrum in the region of the CO stretch, an emblematic vibration of the keto form. The complex IR spectrum obtained is ascribed to significant couplings among the vibrational modes of the Gua moiety, as identified from an anharmonic vibrational analysis.

2. EXPERIMENTAL AND COMPUTATIONAL METHODS The laser-desorption/supersonic-jet cooling/R2PI or LIF apparatus used in this study has been described elsewhere.32-36 GNap was synthesized according to Scheme S1 in Supporting Information. 40 mg of GNap were mixed with a graphite matrix (5%) which absorbs the second harmonic output of a YAG laser. The plume of desorbed molecules was entrained into a supersonic expansion of a He-Ne (7:3) mixture and ionized by one-color R2PI using a frequency tunable UV laser. The resulting ions were analyzed by a reflectron time-of-flight mass spectrometer.36 Massselected UV spectra were recorded by probing the ion signal at a particular mass channel while scanning the UV frequency. The LIF spectra were obtained with a 30 nm bandwidth monochromator, centered on a detection wavelength of 330 nm. The corresponding IR spectra were obtained in the region 1600-1800 cm-1 and 3400-3600 cm-1 by IR-UV double resonance spectroscopy.37 The IR laser (LaserVision OPO) is sent 40 ns before the UV probe laser. The UV signals obtained from the jet population in presence and absence of the IR laser are recorded simultaneously for each laser pulse, according to a procedure described elsewhere.35,36 GNap structures were optimized at the B3LYP/6-311++G(d,p) level of theory. The harmonic vibrational frequencies and zero-point energy correction were also estimated at the same level. Thereafter single-point calculation at the MP4(SDQ)/6-311++G(d,p) level were carried out for the most stable structures, having an electronic energy below 10 kJ/mol. The calculated frequencies



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are scaled according to different factors (0.960 for NH2 and N1H, and 0.951 for enol-OH), chosen to fit the previous experimental data obtained in He droplets26 and in R2PI experiments7,13,16. Excitation energies are evaluated from single-point calculations at the TD-M06-2X/6311++G(d,p) level and ionization energies are calculated at the M06-2X/6-311++G(d,p) level of theory. Anharmonic vibrational calculations were carried out for 9MG: the naphthyl moiety was disregarded in order to reduce the computational costs. The potential energy surfaces consist of a full 51 dimensional anharmonic quartic force field, constructed by a multiresolution method38 based on a simple electronic structure at the B3LYP/6-311++G(d,p) level of theory. The vibrational energy levels and the anharmonic intensities were estimated using the second-order vibrational quasi-degenerate perturbation theory (VQDPT2), based on the vibrational selfconsistent field zeroth-order Hamiltonian.39 Details of these computational methods are described elsewhere.39-43

3. RESULTS AND DISCUSSION The structures of the low-energy tautomers of GNap relevant for the discussion are shown in Figure 1. The N1H keto form (left) is calculated to be the most stable structure, followed by the two O6H enol forms (at least ~ 6 kJ/mol higher at both 0 and 300 K), which differ by the orientation of their enol-OH group. It should be noticed that each of these tautomers is expected to occur as four different rotamers, due to the four possible orientations of the naphthyl moiety relative to Gua. This Gua/Nap rotamerism, which is described by the N9-C-C2’-C3’ dihedral angle (Chart 1), indeed gives rise to synperiplanar (0 ), synclinal (± 60°), anticlinal ((±120°), and antiperiplanar (180º) species.1 In addition, inversion of the amino group is also expected to occur in all these species. As shown in Figure S1, the calculated relative energies of these conformers


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belonging to the same Gua tautomer are found to be nearly isoenergetic within 2 kJ/mol, which implies that all rotatmers/conformers can coexist in the molecular beam. Coming back to the ketoenol tautomerism, the energetics of the GNap tautomers are found to correspond to those of 9HGua as obtained at the MP4 level of theory44-46, suggesting that GNap can be considered as a good model system for the Gua tautomerism. The mass-selected one-color R2PI (1C-R2PI) spectrum recorded on the parent mass (Figure 2b) exhibits a strong band observed at 31798 cm-1 that we assign to the origin band of the first UV transition of the naphthyl chromophore in a conformer of the GNap molecule. Figure 2a shows the laser induced fluorescence (LIF) spectrum in the same region. As in the R2PI spectrum, the origin band is observed at the same position. However, it is accompanied by two weak satellite bands, located 5 and 10 cm-1 to the red, which can be ascribed to either hot bands of a major conformer observed or to other weakly populated species, for example minor conformers. The weak peak observed at 31712 cm-1 is assigned to 2-methylnaphthalene (2-Nap), in fair agreement with previous data, owing to the precision of the measurements;28 this neutral molecule being also generated from the GNap sample during the desorption process. At higher energies, only a broad background is observed in the LIF spectrum (Figure S2) beyond 32000 cm-1, in contrast with expectations from a naphthyl chromophore, as compared to the weak but significant FranckCondon activity reported for 2-Nap.28 This could be assigned either to a mixing of electronic states of the naphthyl moiety with those of guanine, to a congestion effect involving the low frequency torsional modes of the molecule along the 9NGua-2CNap, or to the presence of GNap complexes or clusters in the expansion. Furthermore it is noted that the possibility of the charge transfer from guanine to naphthalene cannot be negligible, which would leave the LIF spectrum unresolved.



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The IR-UV double resonance spectrum in the NH/OH stretch region has been recorded using a 1C-R2PI detection, with the UV laser tuned on the main transition at 31798 cm-1 (Figure 3a). Composed of three bands observed at 3435, 3440, and 3539 cm-1, it strongly differs from that recorded with the same technique on 9-methylguanine7,13,16 (9MG, Figure 3b), whose three bands (3464, 3581, and 3589 cm-1) have been assigned to the symmetric and anti-symmetric NH2 (denoted sNH2 and aNH2, respectively) and enol-OH stretch vibrations of the enol-anti form. In the present case, the good agreement between experimental data and scaled calculated harmonic frequencies (Figure 3a) for the keto-antiperiplanar GNap form (Figure 1,left) enables us to assign confidently the observed spectrum to a keto tautomer of GNap. One can notice that the IR bands of the GNap spectrum present significant shoulders. As anticipated before, they can be interpreted by the presence of secondary conformers (rotamers, having a similar UV transition as the main form) since, as illustrated in Figure S3, the stretch modes of the Gua moiety are found by calculations to be sensitive neither to the naphthyl moiety orientation, nor to the amino group orientation. It should also be noted that, expectedly, the present experimental IR transitions also match well those reported for the N1H/N9H keto form of Gua in He droplets26 (3437.9, 3444.5 and 3544.5 cm-1; the N9H being found at 3506.9 cm-1), which confirms our detection of a keto form and establishes GNap as a good model system. Note in passing that the present scaled frequencies suggest an inverted assignment within the sNH2 / N1H doublet components compared to that proposed by Choi and Miller.26 In order to discuss this issue more quantitatively, anharmonic vibrational calculations have been performed using the vibrational quasi-degenerate perturbation theory (VDQPT2; See Figure S4 for details). As a result, taking anharmonicity into account in the GNap system leads to a permutation of the assignment within the doublet, suggesting that Choi and Miller’ assignment is likely to be the right one.


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Taking advantage of the possibility to record tautomer-selective spectroscopy, the spectrum of a keto tautomer in the region of the CO stretch (Figure 4a) is reported for the first time. The spectrum is composed of two asymmetric doublets, whose main bands, located at 1632 and 1763 cm-1, are accompanied by satellite bands in the blue. Calculated harmonic frequencies (Figure 4b) suggest to assign the main bands to C2-N2 and C=O stretching Gua vibrations respectively, illustrating the similarities between the C2-N2 and C=O bonds, in qualitative agreement with the partially double bond character of the former due to the conjugation with the 6-membered ring. Beyond this preliminary assignment, one remarks however that the weaker doublet bands can be accounted for, neither by harmonic vibrational calculations of any enol form, nor by the presence of other rotamers of the keto form (see their calculated spectra presented in Figure S5). This motivated us again to consider anharmonicity effects. Figure 4c shows the anharmonic vibrational spectrum in this region, where satellite bands are well accounted for by the coupling of the C2-N2 and C=O stretching Gua modes with combination modes (labelled by letters in Figure 4c), involving two or three modes of the Gua moiety, respectively, in particular in-plane NH bending modes (see mode details in Figure S6). The nice agreement of this synthetic spectrum with the experimentally observed doublets suggests therefore to assign these latter to Fermi-like resonances. Although such vibrational coupling was already observed in the NH/OH stretch region around 3200 cm-1 in other related H-bonded systems42,43, the present result demonstrates that resonances of the same type also occur in the CO stretch vibrational region for the bare molecule. Let us finally consider the issue of tautomer populations and relative abundances, in connection with the appearance of satellite bands in the LIF spectrum of GNap, not present in the R2PI spectrum (Figure 2) and tentatively assigned to either hot bands or to secondary tautomers. For the purpose of discussion, vertical excitation and ionization energies (IEs) have been calculated for



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the three GNap tautomers of Figure 1 (Table 1). Even if calculated absolute TD-DFT energies remain far from experimental data, qualitative conclusion can probably be inferred about the relative energies for the keto and enol forms. It turns out that the S1 vertical excitation values of the three tautomers are found to be very similar (within 50 cm-1), which supports the assignment of the satellite bands (Figure 2) to enol forms, which, in this case, would thus be minor, weakly populated, tautomers (assuming similar fluorescence efficiencies). Similarly, examination of IEs suggests that those of the enol forms are slightly higher than that of the keto form. Such a dependence of IE upon the tautomer is indeed expected, owing to the difference in intensity and direction of the dipole moments of the Gua tautomers , which should affect the interaction between the Gua dipole and the charged naphthyl moiety in the ion. Consequently the absence of enol forms in the UV spectrum of Figure 2 could be ascribed to a too weak two-photon energy in the R2PI experiment (7.885 eV; 63596 cm-1), below the IEs of these enol forms. This would also be consistent with the fact that experimental adiabatic IE of the bare 2-methylnaphthalene (7.955 eV; 64157 cm-1)47 is found to be in this energy range. In order to be able to detect enol forms by 1C-R2PI, we had thus to use a higher UV energy, which was chosen in the S2 excitation region of the naphthyl moiety (Figure S8). In the S2 transition48 region of naphthalene, the UV spectrum of GNap is composed of a series of broadened features superimposed on a broad background, which rules out conformation-resolved IR spectroscopy; however, as already noticed for the S1 state, the weak dependence of the calculated S2 excitation energy of GNap with the guanine moiety (see Table 1) suggests that all the tautomers should be excited and their IR spectral features therefore detected in the IR/UV double resonance spectrum (Figure 5). As a matter of fact, whereas the aNH2 stretch of the keto form are observed and unchanged (within the experimental errors) at 3538 cm-1, a broader barely resolved band is


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observed at 3434 cm-1 instead of the two other keto features. In addition, two new bands are present at 3582 and 3589 cm-1, which match well the anti-enol form of 9MG, shown in Figure 2, as well as the aNH2 and OH scaled harmonic frequencies of the GNap enol forms (Figure 5b). The same calculations show that the enol sNH2 stretches have also to be expected in the region of the broadened red-most doublet. Finally the experimental IR spectrum can be fairly reproduced by a simulated spectrum assuming a probing efficiency in the ratio 3 : 1 : 0.4 for the keto : enol-syn : enol-anti forms, illustrating the simultaneous R2PI detection of the several tautomers in this spectral region. This suggests that the weak bands detected in the LIF spectrum of Figure 2 could be ascribed to enol forms. IR/UV spectra with LIF detection, not available at the moment, would confirm this point. Determining the relative abundance of tautomers or conformers in a floppy molecule from intensity of spectral lines is in general a difficult and risky task, partly because of uncontrolled excited state lifetimes or two-photon ionization efficiency issues.49 In the present case the S1 spectroscopy suggests that the keto form is by far the most abundant species, in agreement with calculated energetics. The apparently different ratio resulting from a S2 state probe can be explained by a fortuitous sampling of the congested spectrum, leading to an uncontrolled probe of the tautomers populated.

4. CONCLUSIONS In conclusion, we report a successful strategy which enables spectroscopists to detect efficiently, with mass- and conformer-selectivity, natural chromophoric biomolecules having ultrashort-lived excited states that forbids fluorescence- and/or photoionization-based detections with ns lasers. The present work consists in grafting to the molecule of interest an additional chromophore having



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a long-lived, low-lying excited state together with a low-lying ionization potential. By using a naphthyl UV chromophore, we have demonstrated that a R2PI mass-selective detection of a molecule having a short-lived excited state, such as guanine, can nevertheless be carried out in a tautomer-selective way. The IR spectrum of the keto form of this 9-methylguanine derivative has thus been obtained by IR/UV double resonance. The C=O stretch region is reported for the first time and provides evidence for an extensive vibrational coupling within the vibrational manifold of the Gua moiety. Beyond these spectroscopic results, the presently developed strategy opens up a route to nanosecond investigations on molecules of biological interest exhibiting ultrashort-lived excited states, such as DNA bases, their complexes, e.g. hydrates, or peptides containing an aromatic residue, for which short-lived excited states have also been suspected.50,51

ASSOCIATED CONTENT Supporting Information Details of synthesis, structures, relative energies, LIF spectrum, calculated IR spectra, analyses of anharmonic vibrational calculations, and experimental spectra (S2 region) of GNap. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author


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* Tel: +81-3-3986-0221 ; fax: +81-3-5992-1029 ; e-mail: [email protected] * Tel: +81-45-924-5706 ; fax: +81-3-924-5772 ; e-mail: [email protected] Present Address ¶H.A.: Department of Chemistry, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Masato Koizumi (Material Analysis Suzukake-dai Center, Technical Department, Tokyo Institute of Technology) for ESI-MS measurements and Benjamin Tardivel (LIDyL) for technical assistance.

Funding Sources



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This work was supported by the Grant-in-Aid (25002918) from JSPS, and by “Investissements d’Avenir” LABEX PALM (ANR-10-LABX-0039-PALM).

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(33) Chin, W.; Mons, M.; Dimicoli, I.; Piuzzi, F.; Tardivel, B.; Elhanine, M. Tautomer Contributions to the Near UV Spectrum of Guanine: Towards a Refined Picture for the Spectroscopy of Purine Molecules. Eur. Phys. J. D 2002, 20, 347-355. (34) Chin, W.; Dognon, J. P.; Piuzzi, F.; Tardivel, B.; Dimicoli, I.; Mons, M. Intrinsic Folding of Small Peptide Chains: Spectroscopic Evidence for the Formation of -turns in the Gas Phase. J. Am. Chem. Soc. 2005, 127, 707–712. (35) Biswal, H. S.; Loquais, Y.; Tardivel, B.; Gloaguen, E.; Mons, M. Isolated Monohydrates of a Model Peptide Chain: Effect of a First Water Molecule on the Secondary Structure of a Capped Phenylalanine. J. Am. Chem. Soc. 2011, 133, 3931-3942. (36) Gloaguen, E.; Valdes, H.; Pagliarulo, F.; Pollet, R.; Tardivel, B.; Hobza, P.; Piuzzi, F.; Mons, M. Experimental and Theoretical Investigation of the Aromatic−Aromatic Interaction in Isolated Capped Dipeptides. J. Phys. Chem. A 2010, 114, 2973−2982. (37) Page, R. H.; Shen, Y. R.; Lee, Y. T. "Infrared-Ultraviolet Double-Resonance Studies of Benzene Molecules in a Supersonic Beam", Journal of Chemical Physics, 1988, 88, 5362-5376. (38) Yagi K.; Hirata, S.; Hirao, K. Multiresolution Potential Energy Surfaces for Vibrational State Calculations. Theor. Chem. Acc. 2007, 118, 681- 691. (39) Yagi, K.; Hirata, S.; Hirao, K. Vibrational Quasi-Degenerate Perturbation Theory: Applications to Fermi Resonance in CO2, H2CO, and C6H6. Phys. Chem. Chem. Phys. 2008, 10, 1781-1788. (40) Ishiuchi, S.; Yamada, K.; Chakraborty, S.; Yagi, K.; Fujii, M. Gas-Phase Spectroscopy and Anharmonic Vibrational Analysis of the 3-Residue Peptide Z-Pro-Leu-Gly-NH2 by the Laser Desorption Supersonic Jet Technique. Chem. Phys. 2013, 419, 145-152.


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Figure Captions Chart 1. Structure, with atom numbering, for the keto form of GNap.

Figure 1. Lowest energy antiperiplanar rotamers of the GNap molecule, for the three most stable tautomeric forms of the Gua moiety (see text for Gua/Nap rotamerism). The structures were optimized at the B3LYP/6-311++G(d,p) level, and the energetics were obtained from single-point calculations at the MP4(SDQ)/6-311++G(d,p) level. The relative enthalpies at 0 K (EH) and Gibbs energies at 300 K (EG), given between brackets in kJ/mol, were estimated from zero point vibrational energies and thermal corrections, respectively, obtained from harmonic vibrational frequencies at the B3LYP/6-311++G(d,p) level.

Figure 2. UV spectrum of GNap recorded using (a) LIF and (b) mass-selected (m/z= 291; parent mass) R2PI. The main band at 31798 cm-1 is assigned to the S1 state origin band of naphthyl moiety in GNap. The whole LIF spectrum is shown in Figure S2.

Figure 3. OH/NH stretch IR spectroscopy of (a) GNap and (b) 9MG, as obtained from IR-UV double resonance. For the GNap molecule the UV probe laser was set at 31798 cm-1 (main band Figure 2) and for 9MG the origin band of the main conformation observed was used16. Calculated IR spectra are obtained from scaled harmonic frequencies (B3LYP/6-311++G(d,p) level), with scaling factors taken to match the experimental bands of 9MG: 0.960, for NH2 and N1H, and 0.951 enol-OH. The spectrum b), reproduced from Ref. 16 red-shifted by 3 cm-1 to account for a systematic calibration shift in this paper. The enol structure of 9MG that accounts for the spectrum b),7,13,16 is also displayed in the lower panel.



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Figure 4. a) IR-UV double resonance spectrum of GNap in the C=O stretch vibrational region compared with b) scaled harmonic frequencies and c) anharmonic frequencies. The harmonic frequencies are evaluated at the B3LYP/6-311++G(d,p) level and scaled by 0.987. The anharmonic frequencies are evaluated at the VQDPT2 level, where the most intense combination tones are labelled with letters. The blue spectrum is a convolution of the calculated lines using a 7 cm-1 FWHM Lorentzian shape.

Figure 5. Comparison between (a) IR/UV double resonance spectrum of GNap, with a probe set at 36036 cm-1, in the S2 excitation region of the naphthyl group in which conformation-selectivity is no longer ensured (see details in the text) and (b) the synthetic spectrum based on scaled harmonic frequencies, in which the weight of the contributions of the enol forms is adjusted to reproduce the experimental IR relative intensities of the keto and enol-OH forms. The colors indicate the respective tautomer bands (keto: red, enol-syn: black, enol-anti: blue). The calculated frequencies are evaluated at the B3LYP/6-311++G(d,p) level scaled by 0.960 in NH2 and N1H, and 0.951 in enol-OH. The dotted contour is the convolution of the calculated lines with a Lorentz function having a full-width at half-maximum of 3 cm-1. The resulting apparent abundance ratio is keto : enol-syn : enol-anti = 3 : 1 : 0.4.


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Table 1. Theoretical vertical S1 and S2 excitation and ionization energies (naphthyl moiety) of the three tautomers of GNap (antiperiplanar rotamers of Figure1). Excitation energies are evaluated at the TD-M06-2X/6-311++G(d,p) level, and ionization energies at the M06-2X/6-311++G(d,p) level. Excitation Energy (S1)

Excitation Energy (S2)

Ionization Energy

keto

4.602

4.755

8.028

enol-syn

4.600

4.748

8.054

enol-anti

4.596

4.725

8.082

(eV)



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Chart 1.


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Figure 1.



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Figure 2.


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Figure 3.



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Figure 4.


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Figure 5.



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TOC GRAPHICS


Effective Strategy for Conformer-Selective ... - ACS Publications

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