Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase Mattanjah S

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7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase Mattanjah S. de Vries

7.1 Introduction

In his book The Double Helix, James Watson describes an early proposal for DNA structure in which equal bases pair with each other. He writes: ‘‘My scheme was torn to shreds the following noon. Against me was the awkward chemical fact that I had chosen the wrong tautomeric forms for guanine and thymine.’’ This fact was pointed out to him by the American crystallographer Jerry Donahue who ‘‘ ... protested that the idea would not work. The tautomeric forms I had copied out of Davidson’s book were, in Jerry’s opinion, incorrectly assigned. My immediate retort that several other texts also pictured guanine and thymine in the enol form cut no ice with Jerry. Happily he let out that for years organic chemists have been arbitrarily favoring particular tautomeric forms over the alternatives on only the ﬂimsiest of grounds’’ [1]. Without the correct keto form of these bases, Watson and Crick could not have arrived at their successful structure of DNA. In fact, for each of the bases, the keto tautomer is the form normally occurring in DNA. The rare imino and enol forms can lead to alternate base-pair combinations and thus cause mutations in replication. This type of mutation is called a tautomeric shift [2, 3]. Possible mismatches include iminoC–A, enolT–G, C–iminoA, and T–enolG. Tautomerization can occur in DNA by single or double proton transfer. In solution equilibrium, generally the keto form dominates, complicating efforts to study the properties of the individual forms. One approach to studying tautomeric properties is double-resonant gas-phase spectroscopy, as this technique offers isomeric selectivity. This chapter summarizes the results from such investigations.

7.2 Techniques

As molecules can exist in tautomeric equilibria, most experimental techniques need to address mixtures of tautomer populations. Double-resonant techniques in

Tautomerism: Methods and Theories, First Edition. Edited by Liudmil Antonov. © 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. 178 7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase

the gas phase offer opportunities to perform isomer-selective spectroscopy. This capability makes it possible to obtain detailed spectroscopy of individual tautomers. The first challenge is to form molecular beams with nucleobases that are thermally labile and have low vapor pressures. One solution is the combination of laser desorption and jet cooling. The process of neutral laser desorption is very poorly understood. The general idea is that fragmentation is minimized when the timescale for heating is shortened. The rates required to desorb molecules without fragmentation are of the order of 1011 Ks−1, which corresponds to a 1000 K temperature jump in a typical 10-ns laser pulse. Entraining the desorbed molecules in a supersonic expansion to achieve jet cooling offers three advantages: cooling (i) improves spectroscopic resolution, (ii) reduces fragmentation, and (iii) provides a pathway for cluster formation. Such combination requires careful optimization of the geometry of the apparatus [4–7]. By analyzing rotational profiles, Meijer et al. showed in the case of anthracene, entrained in helium, that a rotational temperature ◦ of less than 15 K is attainable. Commonly, the sample is moved to expose a fresh part of the sample in subsequent shots by a translating bar [6], a rotating rod [8], or a rotating wheel [9]. For ions, generally introduced by a electrospray, even better cooling can be achieved in a liquid- helium-cooled 22-pole trap [10]. The lowest temperatures are achieved in liquid helium droplets into which nucleobases have been introduced in a pickup source [11]. In principle, any molecular beam or gas- phase technique can be combined with such sources but the most relevant ones for investigating tautomer properties are forms of spectroscopy, particularly resonant two-photon ionization (R2PI), laser-induced fluorescence (LIF), hole-burning or double-resonant spectroscopy, microwave spectroscopy, infrared (IR) absorption, and combinations of these with any spectrometry and electron spectroscopy. The combination of R2PI and hole-burning makes it possible to obtain spectroscopy of selected individual tautomers. The resonant absorption of a primary photon is recorded by the detection of a photo-ion formed by successive absorption of a second photon [12]. Ions lend themselves to very sensitive detection and to mass selection. These properties provide several advantages: (i) good signal to noise, (ii) cluster selection, and (iii) potentially additional information from fragmentation. Similar to LIF, the technique is blind to short-lived dark states: if the excited-state lifetime is significantly shorter than the laser pulse, absorption of the second photon becomes very weak. Another problem occurs when the ionization potential is more than twice the resonant excitation energy. In that case, a two-color scheme may be required. For spectral hole-burning, the ionization step is preceded by a pulse from a tunable IR laser. This pulse resonantly alters a specific population in the beam, which is probed as a decrease in the ion signal [13–18]. This technique produces ground-state IR absorption spectra of optically selected as well as mass-selected molecules. The OH and NH stretching frequencies are especially highly specific for different tautomeric forms as well as for hydrogen-bonded structures. Fourier transform microwave spectroscopy can provide highly specific tautomeric data as well as conformational analysis derived from rotational spectra (Figure 7.1). Alonso and coworkers [19–21] have combined this approach with laser desorption 7.3 Guanine 179

(a) (b) (c)

S0 R2PI UV–UV IR–UV

Figure 7.1 Schematic diagram of spectroscopic techniques based on two-photon ionization and used in gas-phase experiments. Dashed arrows indicate tuned laser pulses; solid arrows indicate fixed wavelength laser pulses. In schemes (b) and (c), these are used as burn and probe lasers, respectively. jet cooling and applied the combination to the study of conformations of a number of amino acids. Their approach features a molecular beam along the axis of a large microwave cavity (55-cm mirrors) which monitors emission following a short microwave excitation pulse. Rotational analysis permits determination of all conformations present in the beam. For example, this technique identified six conformations for aspartic acid [20]. It is not clear at this point what maximum size will be amenable to this type of analysis. Helium droplets spectroscopy is usually performed in the IR. Resonant absorption by specific vibrational modes implies heating, resulting in helium atoms boiling off the droplets. By recording this effect in a mass spectrometer or with a bolometer, IR absorption spectra can be obtained. However, in this approach tautomeric selection is not possible. Progress in experimental capabilities is bolstered by the simultaneous progress in computational capabilities, creating a great synergy between experiment and theory. While quantum chemical calculations guide interpretation of spectra and help model structures and dynamics, at the same time gas-phase experimental data help calibrate algorithms, force fields, and functionals [22].

7.3 Guanine

The ﬁrst gas-phase spectroscopy of nucleobases was reported by Levy and coworkers in 1988 [23]. They measured the electronic spectra of the pyrimidine bases uracil and thymine in a supersonic molecular beam both by LIF and REMPI. The observed spectra were very broad and featureless, in spite of efﬁcient jet cooling. 180 7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase

OO O O O

1 6 5 N7 N N N N N 8 N N N N 2 4 N9 N N N N N N3 N N N N N N N N Watson–Crick Keto-N7H Keto-N9H trans-Enol-N9H cis-Enol-N9H trans-Enol-N7H 0 184 203 310 1189

O O O O O

N N N N N N N N N N

N N N N N N N N N N N N N N N

Keto-N3H-N7H Keto-imino-down-N7H Keto-imino-up-N7H cis-Enol-N7H Keto-imino-up-N9H 2073 2438 2464 3646 5128

O O O O O

N N N N N N N N N N

N N N N N N N N N N N N N N N

Keto-imino-down-N9H trans-Enol-imino-up-N7H Keto-N3H-N9H trans-Enol-imino-down-N7H trans-Enol-imino-up-N9H 5729 6523 6539 8084 6523

Figure 7.2 Fifteen of the lowest energy tautomers of guanine. Relative energies are in wavenumbers. From Ref. [27]. Solidly shaded structures are observed in gas-phase nanosecond R2PI experiments. Hashed structures have subpicosecond excited-state lifetimes. The rectangle marks the structures observed in helium droplets. Keto-N9H is the Watson–Crick structure. 7.3 Guanine 181

The authors speculated about photo-tautomerization as a possible explanation for such a spectrum. The absence of useful vibronic features discouraged the pursuit of gas-phase spectroscopy of nucleobases for a decade until Nir et al. [24] published a well-resolved R2PI spectrum of guanine. This ﬁnding was followed by similar spectra of adenine and cytosine [25, 26]. These initial spectra contained contributions of all the tautomers present in the beam, complicating interpretation. Such an R2PI spectrum could potentially consist of overlapping spectra of a number of tautomers. Figure 7.2 shows the 15 lowest energy tautomer structures of guanine with their energies given in wave numbers as computed by Seefeld et al. (B3LYP/TZVPP//RIMP2/cc-pVQZ, ZPE corrected) [27]. The second most stable form, keto-N9H at 184 cm−1, is the form in Watson–Crick base-pairing in DNA. In solution the keto forms dominate. In matrix-isolation IR observation of C=O stretch, frequencies points to keto forms but too many lines are present in the spectra to be able to exclude other tautomers as well [28]. The same problem manifests itself in resonance-enhanced multiphoton ionization (REMPI or R2PI) spectroscopy, where a dense spectrum is observed that potentially consists of contributions of multiple tautomers [29]. These contributions can be separated by UV–UV hole-burning, revealing the presence of four different isomers in the gas-phase jet-cooled conditions of these experiments [30]. Three of these tautomer-selective REMPI spectra appear in Figure 7.3. Further identiﬁcation is possible by IR–UV hole burning and by tautomeric blocking with methyl derivatives [30–32]. The spectra of species A (with the red-most origin at

32870 +405 +1046

A B C

32500 33000 33500 34000 34500 cm–1

Figure 7.3 Tautomer selective REMPI spectra of guanine obtained by UV-UV hole burning. Bottom trace: REMPI spectrum. Upper traces: UV-UV spectra obtained with the probe laser tuned to transitions of different tautomers, indicated as A, B, or C respectively. 182 7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase

32 870 cm−1) and species D (+1991 cm−1) correlate with enol tautomers, as clearly diagnosed by the characteristic OH stretching frequency in IR–UV spectra. However, species B and C (at +405 and +1046 cm−1, respectively) were not so straightforward to assign. Initially, their IR–UV hole-burning spectra appeared to present a reasonable match with the lowest energy species, keto-N7H and keto- N9H tautomers. 1-Methylguanine yielded the corresponding IR–UV spectrum, consistent with this assignment. Which of these two spectra correlated with the N7H and N9H tautomer could not be determined. At first this appeared to be a logical assignment, with four of the five lowest energy species appearing in the molecular beam. Curiously, 9-methylguanine yielded only the corresponding enol spectrum, which determined that the D spectrum corresponded to one of the two enol-N9H forms, A and D, but also raised the question why no spectrum was observed corresponding to the keto-N9H form [33]. On the other hand, 1-methylguanine produced spectra paralleling the B and C spectra. After considerable confusion, the puzzle was finally resolved following new data from experiments in helium droplets [34, 35]. In these experiments, room-temperature guanine vapor is entrained in helium droplets and IR absorption spectra are measured. Orientation of the molecules in an electric field and angular distributions relative to the light vector yield additional information about relative orientations of the various modes. The authors observed 16 vibrational bands for the guanine tautomers in the 3400–3650 cm−1 region of the spectrum. Although in these experiments isomer selection is not available, comparison with ab initio calculation allowed a complete assignment of four tautomers: keto-N7H, keto-N9H, trans-enol-N9H, and cis-enol-N9H in the ratio 1 : 0.8 : 0.4 : 0.3. The keto IR spectra differed slightly from those that had been assigned as in the gas-phase IR–UV spectra, leading to a number of important conclusions. First, the IR–UV data did not correspond to the lowest energy keto species, but rather to the higher energy imino tautomers [35]. This new assignment was conclusively confirmed by Seefeld et al., [27] who measured the imino stretch frequencies at 1690 cm−1. An important implication of these finding is that apparently the keto form does exist in the gas phase and is the lowest energy form, as predicted computationally, but goes unobserved in REMPI hole-burning measurements. There is a difference in the cooling rate in both experiments, with the helium droplet effecting very rapid ‘‘flash’’ cooling. One would therefore expect the helium droplet to reflect a room-temperature population distribution that is frozen into the droplet. The experience with jet entrainment is that, while the population distribution may not be strictly thermodynamic, it roughly corresponds to the abundances at the source temperature and certainly the most stable species are the most abundant. Therefore the failure to observe the keto tautomers in the REMPI experiments is curious and suggests that they may be present in the molecular beam but are not detected. This conclusion is beautifully supported by the subsequent findings of Alonso et al. [36] who used microwave spectroscopy to identify tautomers in a laser desorption source. Just as in the helium droplet experiments, these measurements are true absorption data and not affected by the nature of the electronic excited state. These authors identified the same two keto 7.3 Guanine 183

REMPI [μs] Reaction Internal (< ns) conversion [fs] Fluorescence (ns) Reaction [ps] S 1 E Internal conversion (< ps) Fluorescnce [ns]

q1 S0

Figure 7.4 Schematic representation of different competing decay processes and their timescales from the electronically excited state. Diagrams in the form of a Jablonski diagram (left) and in the form of a two dimensional potential energy diagram (right) as a function of two nuclear coordinates. and two enol tautomers as did Choi et al. in helium. The less stable higher energy imino tautomers do not appear in the microwave experiments, because of their lower abundance and lower sensitivity compared to REMPI detection. To explain this observation, another difference between the two measurements needs to be considered: With the helium droplets, the spectra represent direct IR absorption, while in REMPI experiments the IR spectrum is obtained indirectly as it is detected by an action spectrum via the S1 electronically excited state. If the lifetime of that excited state is short with respect to the laser pulse, two-photon ionization may not take place and the technique becomes blind to such a case. With the typical 10-ns laser pulses used in these experiments, the method may fail to detect species with electronic excited states in the picosecond or faster timescale. This explanation is borne out completely by quantum computational modeling. Following absorption, the resulting electronic excitation can initiate inter or intramolecular chemical reactions. Figure 7.4 shows a schematic Jabloski diagram, depicting the major possible competing pathways. The electronic energy can be radiated away by ﬂuorescence; this process occurs typically at a rate (∼109 s−1) which is generally too slow to compete with an excited-state reaction. Alternatively, the electronic energy can be converted to heat by internal conversion to the ground state; heat can subsequently be safely dissipated to the environment. When internal conversion is fast enough to prevent photochemical reactions from taking place, the molecule will have a very short excited-state lifetime and be stable against UV photodamage [37]. The key is the occurrence of conical intersections that connect the excited-state potential energy surface, reached by photon absorption, to the ground-state energy surface. The dramatic lifetime differences between tautomers of nucleobases appear to be due to variations in the excited-state potential surfaces that restrict or slow access to these conical intersections [38]. We can rationalize these differences as illustrated schematically in Figure 7.4b. Conical intersections are the crossings of multidimensional potential surfaces. Therefore these features 184 7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase

can occur only in regions of the potential energy landscape that represent a deformation of the molecular frame from the ground-state equilibrium geometry. For keto guanine, three types of conical intersections have been identiﬁed on

the S1/S0 crossing seam. Two of them feature structures with a strongly puckered pyrimidine ring, with the largest puckering at the C2 atom with the NH2 group distorted from the plane [34, 39–44]. In the third structure, the pyrimidine ring is puckered at the C6 atom, with the oxygen atom distorted from the plane of the ring. [34,

39, 40, 44] An S1 minimum of nπ* character was localized in the same energy region as well [39, 42]. The calculated character of the potential energy surface suggests two possible relaxation mechanisms according to which (i) the molecule relaxes directly to the ethylenic conical intersection using a reaction path of ππ* character, or (ii) it

ﬁrst relaxes to the S1 minimum with nπ* character. These direct and indirect mechanisms provide an interpretation of the experimentally observed relaxation times of 0.15 and 0.36 ps, respectively [39]. An alternative suggested that the relaxation pathway proceeds via a conical intersection with stretched NH bonds [45]. Barbatti et al. [44] performed dynamics simulation studies at the multireference conﬁguration interaction with singles (MR-CIS) level. At the ground-state mini-

mum geometry, the S1 state of ππ* character is almost isoenergetic with the S2 state of nπ* character. Thus, the trajectories are initially spread between the S1 → and S2 states; however, there is an almost instantaneous S2 S1 relaxation on the timescale of about 20 fs [44]. In the subsequent relaxation into the ground state, the vast majority of the population (95%) follows the direct ππ* pathway toward ethylenic conical intersections. The rest of the population decays into the ground state via the nπ* oop-O conical intersection. The predicted relaxation time is of the order of 0.22 ps. The ππ* relaxation mechanism was suggested also by a semiempirical OM2 method [40]. Remarkably, the other tautomers have lifetimes that are several orders of magnitude larger. According to calculations by Marian, ultrafast internal conversion depends dramatically on the tautomeric form [34]. The coordinates producing the low-energy conical intersections are the out-of-plane distortions at the C2 or C6 atoms of the purine ring. Ultrafast relaxation pathways via these conical intersections are available for the biologically relevant form of guanine, keto-N9H, for its 7H tautomer keto-N7H, and for the trans-enol-N9H form. These are the three lowest energy forms, shown as hashed in Figure 7.2, and are not observed in the REMPI experiments. The resulting short – subpicosecond – excited-state lifetime renders REMPI blind for these species. In the next higher energy forms, on the other hand, there are no accessible conical intersections, resulting in long excited-state lifetimes of the order of nanoseconds, which can be detected by REMPI. Four out of the next ﬁve lowest energy tautomers indeed appear in the REMPI experiments, indicated is solidly shaded in Figure 7.2. It is quite remarkable that the trans-enol-N9H and the cis-enol-N9H forms, which are very close in energy and only differ by the –OH rotamer orientation, appear to differ orders of magnitude in excited-state lifetime. Figure 7.5 shows Boltzmann distributions at two temperatures, based on the relative energies shown in Figure 7.2. At room temperature, diagonally hashed bars, the four lowest energy tautomeric forms would be populated, consistent 7.3 Guanine 185

300 K 1250 K Relative abundance

-N7H -N9H -N9H -N9H -N7H -N7H -N7H -N7H -N7H -N9H -up -up Keto Keto -Enol -Enol -Enol -N3H -Enol cis imino cis imino Keto - - trans trans -imino-down Keto Keto Keto Tautomer

Figure 7.5 Boltzmann distributions of guanine tautomer populations for two temperatures. Diagonally hashed bars indicate tautomers observed in helium droplets. Horizontally hashed bars indicate tautomers observed in laser desorption REMPI experiments. with the ﬁnding in helium droplets. In those experiments, the room-temperature vapor pressure distribution is expected to be frozen virtually instantaneously into the droplet. The reported relative abundances are consistent with such an equilibrium distribution. The higher than expected keto-N9H population might suggest a lower energy than calculated. The gray and horizontally hashed bars ◦ represent the equilibrium distribution at 1250 K. This is a rough estimate for the temperature reached in the laser desorption pulse. Subsequent cooling takes place in the supersonic expansion, but that is not an equilibrium process. The ﬁnal distribution is hard to predict but, as the cooling takes places in small steps in successive collisions, it is likely that the system gets trapped in local minima as the internal temperature is lowered, and barriers for tautomerization can no longer be overcome. This picture would lead to population distributions that could be similar to the initial one. Horizontally hashed bars indicate the tautomers observed in the laser desorption REMPI experiments. The three lowest energy forms, in gray, are too short-lived to be observed. Beyond the eighth form, the population becomes too small. The failure to observe to keto-N3H-N7H could suggest either a different UV absorption wavelength or another case of short excited-state lifetime. Nir et al. [46, 47] have shown by means of double-resonance laser spectroscopy and ab initio calculations that, in the investigated wavelength range around 290 nm, guanosine (Gs), 2-deoxyguanosine, and 3-deoxyguanosine each exhibit only the spectrum of one stable isomer, namely, the cis-enol form, which is stabilized by a strong intramolecular sugar(5-OH)···enolguanine(3-N) hydrogen bond. With the N9 position blocked by the sugar moiety, the other three forms of guanine 186 7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase

seen in the REMPI experiments cannot occur in the guanosine. The lower energy keto forms do not appear, presumably because in the nucleoside the excited-state dynamics are similar to those in the nucleobase cases. Clusters of guanine with one water molecule show the same trend. Crews et al. [48, 49] identiﬁed three structures based on IR–UV hole burning. Two of those correspond to the enol form, at least one of which is the cis-enol because the water bridges the –OH hydrogen and the N7. The third isomer was incorrectly interpreted as a keto form, based on comparison with the bare guanine spectra, which were also initially mis-assigned. Upon closer inspection, the third cluster appears to involve a N7H keto-imino tautomer. This could be either the up or down form or a combination of both, as their spectra might overlap. These ﬁndings are consistent with those of Chin et al. [32] on the monohydrate of 9-methylguanine, who found only the cis-enol form. Once again, the conclusion is that the three lowest energy tautomer forms are missing in the REMPI experiments, indicating a short excited-state lifetime, even as a monohydrate. It should be noted that in solution the keto form is the preferred tautomer. Saigusa et al. [50] performed double-resonance spectroscopy of the hydrated clusters of guanosine and 9-methylguanine. In interpreting the IR spectra, it is not possible to exclude the keto hydrates as the characteristic modes, OH for enol or N1H for keto, are masked when the water hydrogen-bonds to either of those sites (Figure 7.6).

o o N N (a) N N N NH a 2 Water NH 2s N9H free -OH

o N N (b) N N N OH o

o o N N N (c) N N

N N N N N (d)

N N o N N N N1H N N N-imino N N N (e,f)

(cm–1)3200 3400 3600 3800

Figure 7.6 IR–UV hole-burning spectra schematic only. Asterisks denote peaks in of guanine with (a–c) and without (d–f) (b) that also show up in (a), probably due to water. Corresponding modes for correspond- overlap in the probe spectra. Adapted from ing structures with and without water are [48]. indicated by dotted lines. Structures are 7.5 Cytosine 187

Excitation energies and ionization potentials are also tautomer-dependent properties [51]. This property can be seen in the observation of different photoionization curves for guanine resulting from different types of molecular beams, presumably reﬂecting different tautomer distributions [52].

7.4 Adenine

The tautomeric landscape of adenine is somewhat less varied than in the case of guanine because of the absence of the oxygen. Plutzer¨ and Kleinermanns reported IR–UV double-resonance and observed two tautomers [53, 54]. Both tautomers are of the amino form, with the 9H form most abundant and the 7H form with only a small presence. This ﬁnding is consistent with the microwave measurements by Brown et al. [55]. At the conditions of jet cooling, the imino form appears to be absent, although in the gas phase at elevated temperature the IR spectra seem to comprise multiple tautomers, including imino. The analysis is somewhat complicated by the fact that the UV spectra contain contributions to two excited states, of ππ*andnπ* character, respectively [56, 57]. Adenine is 6-aminopurine and the comparison with its isomer 2-aminopurine is interesting because the excited-state lifetimes differ by orders of magnitude [58].

7.5 Cytosine

As shown in Figure 7.7, cytosine has three major tautomeric forms which have different properties: enol, keto, and keto-imine. In the gas phase, the keto-imine is the most unstable and the enol tautomer is slightly more stable than the keto form by about 0.03 eV [59–61]. The keto form is the biologically important one, with Watson–Crick base-pairing in DNA, and predominant in solution. In matrix isolation, Szczesniak et al. [62] observed both keto and enol forms, with higher abundances for the latter and small contributions from the imino form. Brown et al. [63] have obtained the rotational constants for all three tautomeric forms by microwave spectroscopy. Schiedt et al. [64] also identiﬁed the existence of keto and enol tautomers of neutral cytosine in the gas phase by anion spectroscopy. While the energy difference between the enol and keto forms is very small, some of their other properties, such as their vertical excitation energies, are very different. Nir et al. [26, 65] reported REMPI and hole-burning spectra and concluded that the keto and enol tautomers have band origins that differ by a remarkable 0.5 eV at 314 and 278 nm, respectively. It should be noted that one of the consequences of this large difference in excited-state energy is that absorption below 278 nm involves all tautomers while at longer wavelengths only the keto tautomer can be excited. Many femtosecond pump–probe experiments are performed at 267 nm, thus probing 188 7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase

N 4 3 5 N 2 6

O 1N

Keto

N N

O N O N

Enol Enol

N N

O N O N

Keto-imino Keto-imino

Figure 7.7 Tautomeric forms of cytosine.

multiple tautomers simultaneously. Kosma et al. [66] reported excited-state dynamics by probing with different excitation wavelengths in a range from 260 to 290 nm, which allowed them to distinguish between keto-only versus mixed populations. They conclude that the deactivation pathways are quite different for the different tautomers. Three time constants are found for keto-cytosine and two for the others. Theoretical treatments in the literature had focused on modeling the latter, while the new results point to the importance of tautomeric distinction in these analyses. For the keto form, the two faster channels, with femtosecond and picosecond lifetimes, are consistent with fast but not barrierless excited-state relaxation via conical intersections between the 1ππ* and ground state [59]. The third channel with a lifetime of hundreds of picoseconds at 290 nm is proposed to involve excited-state tautomerization to a low-lying 1nπ* state of the keto-imino tautomer [67]. The authors propose that in the condensed phase this channel would be quenched by rapid back hydrogen transfer, catalyzed by hydrogen-bond water molecules [68, 69]. Consequently, this pathway would not alter the inherent photostability.

7.6 Uracil and Thymine

When Brady et al. [23] reported the ﬁrst electronic spectra of uracil and thymine in a molecular beam, both of these nucleobases exhibited a broad spectrum in the 7.7 Base Pairs 189

UV with an onset around 36 000 cm−1. No spectroscopic detail could be extracted from these broad spectra, and certainly tautomeric information was limited for a long time to bulk measurements. Microwave measurements of uracil in a heated cell had suggested the diketo form as the most abundant [70]. Brown et al. [71] had reported the first microwave measurements in a seeded molecular beam and also concluded that the diketo form was predominant. Viant et al. [72] reported the first rotationally resolved gas-phase IR spectra of uracil. This work employed a slit nozzle, an IR diode laser, and a multipass arrangement to obtain high-resolution IR absorption spectra = = of the out-of-phase ν6(C2 O, C4 O) stretching vibration. The rotational analysis unambiguously assigned the species to the diketo tautomer. Brown et al. [73] also observed the diketo form of thymine in a seeded molecular beam, based on the hyperfine structure in the 144,10 –133,11 transition.

7.7 Base Pairs

Upon base-pairing, tautomeric stabilities may change dramatically, as certain tautomers can be stabilized by hydrogen-bonding structures, relative to others. Figure 7.8 shows the example of cytosine, for which the monomer keto and enol forms are within 240 cm−1 of each other while the lowest energy dimer involving an enol tautomer is 1343 cm−1 above the lowest energy keto–keto dimer [65]. The lowest energy enol–enol dimer is at 2127 cm−1. For GG, Nir et al. [74] calculated that most keto–keto dimers are consider- ably more stable than dimers with even one enol tautomer. The lowest energy keto–enol cluster was found at 2646 cm−1 above the lowest energy keto–keto cluster. Six keto–keto clusters were found to be more stable than the most stable keto–enol cluster. The authors observed two GG structures in IR–UV hole-burning experiments, identiﬁed as the second and third most stable keto–keto structures. Failure to observe the lowest energy structure could be due to a short excited- state lifetime, just as in the keto monomer case. It should be noted that this = ··· ··· = structure, (keto-N9H)2, is fully symmetric, with C O NH/NH O C hydrogen bonds, forming C2h symmetry. Consequently, the allowed S0 –S2 transition could be outside the investigated spectral range due to strong exciton splitting. An illustration of the importance of the tautomeric form for base-pairing properties can be found in the case of GC base-pairing. There are about 50 ways to form GC base pairs by hydrogen bonding. The one structure that is prevalent in DNA (the Watson–Crick structure) has a subpicosecond excited-state lifetime, evi- denced by a broad UV spectrum, while other structural arrangements of the same base-pair have sharp spectra, consistent with much longer excited-state lifetimes [75]. Figure 7.9 the shows IR–UV spectra for three structural isomers of GC base pairs. The stick spectra are density functional theory (DFT) calculated vibrational frequencies for the structures shown in the insets. Structure A is the Watson–Crick structure, while structure C is almost the same structure but with the cytosine 190 7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase

1 4 3 5 2 6 1

Keto–keto (0) Keto–keto (309) Keto–keto (899) Keto–enol-cis (1343)

Keto–enol-cis (1869) Enol-cis-enol-cis (2127) Enol-cis-enol-cis (2490) Keto–enol-trans (2885)

Keto–enol-trans (3043) Keto–enol-trans (3207) Enol-cis-enol-trans (3669) Enol-trans-enol-trans (4501)

Figure 7.8 The eight lowest energy structures of hydrogen-bonded cytosine dimers. Relative energies are in parenthesis in cm−1 accordingto[65].

in the enol form instead of the keto form. This subtle tautomeric difference has a dramatic effect on the photophysical properties. With the enol cytosine, the base-pair exhibits a sharp UV spectrum, while in the keto form the excited state has a lifetime too short to permit REMPI detection. The spectrum in panel A was obtained for a methylcytosine-ethylguanine cluster. With this tautomeric blocking, neither structure B nor C is possible and this made it possible to detect the faint broad UV spectrum of structure A. To explain the effect, the right column shows the calculated potential curves from Sobolewski and Domcke [76]. The reaction coordinate is hydrogen motion, N1-H, indicated with a circle in structure A. A

charge-transfer state (CT in red) connects the S1 (green) and S0 state (blue) by two conical intersections in A but not in B and C. The difference in tautomeric form between the A and the C case induces a subtle change in the excited-state potential

energy landscape. The CT state is just slightly higher in energy relative to S0 and S1, 7.8 Outlook 191

A 6.0 S2 N1-H 5.0 N9-H 4.0 NH2(a) CT NH (s) 2 3.0 O–H 2.0 Energy (eV)

1.0 S1

0.0 B

0.5 1.0 N1-H 2800 3200 3600 4000 (cm–1)

Figure 7.9 Left column shows three struc- potential curves. From [76]. A charge-transfer tures observed for isolated GC base pairs, state (CT) connects the S1 and S0 states by as identiﬁed by IR–UV spectroscopy. Stick two conical intersections in A but not in B spectra are DFT-calculated vibrational fre- and C. The reaction coordinate is middle quencies. Structure A is the Watson–Crick hydrogen motion, N1-H, indicated with a cir- structure. Right column shows calculated cle in structure A. creating both a barrier for reaching the ﬁrst conical intersection and avoiding the second conical intersection altogether. Thus a small change in potential energies upon tautomerization results in a difference of orders of magnitude in excited-state lifetime.

7.8 Outlook

Studies of nucleobases isolated in the gas phase have made it possible to investigate properties of individual tautomers. Such studies have revealed surprisingly large differences in properties such as excitation energies, ionization potentials, and excited-state dynamics between individual tautomers. This success in mapping intrinsic properties in single bases now raises new questions about the precise role of tautomeric variations in intermolecular structure involving neighboring 192 7 Tautomer-Selective Spectroscopy of Nucleobases, Isolated in the Gas Phase

molecules. Those intermolecular interactions also depend strongly on structure, which is especially critical because the conformational landscape is large in ﬂexible DNA and RNA strands. However, solution-phase data do not provide detailed insights into the precise role of structure variations, so further gas-phase studies are needed to obtain data for individually selected structural isomers in stacked and hydrogen-bonded clusters of nucleobases and their derivatives. Cluster studies can also detail the role of the solvent by investigating microsolvation effects in clusters with water. Work so far already indicates that base-pairing and hydrogen bonding can affect the tautomeric equilibria and the corresponding excited-state properties in important ways. One can hope that the capabilities of both theory and experiment to study molecular systems of increasing size in isolation in the gas phase will lead to a level of understanding that will allow extrapolation of individual molecular properties to conditions in solution and in the macromolecule.

Acknowledgments

This material is based upon work supported by the National Science Foundation under CHE-1301305 and by NASA under Grant No. NNX12AG77G.

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