Excited-State Structure, Vibrations, and Nonradiative Relaxation of Jet

Feb 24, 2014 - The spectral region from 0 to 300 cm–1 is dominated by overtone and combination bands of the out-of-plane ν 1 ′ (boat), ν 2 ′ (...
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Excited-State Structure, Vibrations, and Nonradiative Relaxation of Jet-Cooled 5‑Fluorocytosine Simon Lobsiger, Maria A. Trachsel, Takuya Den, and Samuel Leutwyler* Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland S Supporting Information *

ABSTRACT: The S0 → S1 vibronic spectrum and S1 state nonradiative relaxation of jetcooled keto-amino 5-fluorocytosine (5FCyt) are investigated by two-color resonant twophoton ionization spectroscopy at 0.3 and 0.05 cm−1 resolution. The 000 rotational band contour is polarized in-plane, implying that the electronic transition is 1ππ*. The electronic transition dipole moment orientation and the changes of rotational constants agree closely with the SCS-CC2 calculated values for the 1ππ* (S1) transition of 5FCyt. The spectral region from 0 to 300 cm−1 is dominated by overtone and combination bands of the out-of-plane ν1′ (boat), ν2′ (butterfly), and ν3′ (HN−C6H twist) vibrations, implying that the pyrimidinone frame is distorted out-of-plane by the 1ππ* excitation, in agreement with SCS-CC2 calculations. The number of vibronic bands rises strongly around +350 cm−1; this is attributed to the 1ππ* state barrier to planarity that corresponds to the central maximum of the double-minimum out-of-plane vibrational potentials along the ν′1, ν′2, and ν′3 coordinates, which gives rise to a high density of vibronic excitations. At +1200 cm−1, rapid nonradiative relaxation (knr ≥ 1012 s−1) sets in, which we interpret as the height of the 1 ππ* state barrier in front of the lowest S1/S0 conical intersection. This barrier in 5FCyt is 3 times higher than that in cytosine. The lifetimes of the ν′ = 0, 2ν1′ , 2ν2′ , 2ν1′ + 2ν2′ , 4ν2′ , and 2ν1′ + 4ν2′ levels are determined from Lorentzian widths fitted to the rotational band contours and are τ ≥ 75 ps for ν′ = 0, decreasing to τ ≥ 55 ps at the 2ν1′ + 4ν2′ level at +234 cm−1. These gasphase lifetimes are twice those of S1 state cytosine and 10−100 times those of the other canonical nucleobases in the gas phase. On the other hand, the 5FCyt gas-phase lifetime is close to the 73 ps lifetime in room-temperature solvents. This lack of dependence on temperature and on the surrounding medium implies that the 5FCyt nonradiative relaxation from its S1 (1ππ*) state is essentially controlled by the same ∼1200 cm−1 barrier and conical intersection both in the gas phase and in solution.

1. INTRODUCTION In room-temperature aqueous solution, the five canonical nucleic acid bases exhibit excited-state lifetimes in the 0.5−5 ps range and fluorescence quantum yields Φfl of ∼10−4−10−5, reflecting correspondingly high nonradiative decay rates.1−4 In the gas phase, several femtosecond (fs) pump−probe studies of the cytosine nucleobase have yielded excited-state decay times in the τ = 0.5−2 ps range.5−7 However, recent measurements of lifetimes in the 1ππ* state of jet-cooled cytosine (Cyt) and 5methylcytosine (5MCyt) at low (0−500 cm−1) vibrational energies have revealed longer lifetimes in the ranges 25−45 and 30−60 ps, respectively.8,9 Thereby, vibronic spectroscopy9−13 and single vibronic level lifetime measurements8,9 may be used to probe the gas-phase excited-state structure, dynamics, and photochemical properties of cytosine and its derivatives. 5-Fluorocytosine (5FCyt) is an antitumor and antifungal prodrug.14 In suicide gene therapy, 5FCyt is used in combination with tumor-selective replicating retrovirus vectors that encode for cytosine deaminase.15,16 Within infected tumor cells, the cytosine deaminase converts 5FCyt to the highly cytotoxic metabolite 5-fluorouracil, leading to inhibition of tumor growth.17 © 2014 American Chemical Society

The photophysics of 5FCyt is of considerable interest because the efficient nonradiative relaxation of excited-state cytosine (Cyt) has been linked to the lowest-lying conical intersection involving the C5C6 double bond torsion (the C5-twist conical intersection).4,18−20 The influence of 5-fluoro substitution on this relaxation has been investigated in several femtosecond (fs) excited-state studies of 5FCyt. Using transient absorption measurements (263 nm pump/570 nm probe) in solution, Kohler and co-workers4,21 have showed that the H → F substitution increases the excited-state lifetime from 720 fs to 73 ± 4 ps in ethanol and dimethyl sulfoxide4 and to 88 ± 5 ps in water.21 Using fs pump−probe photoionization mass Received: January 14, 2014 Revised: February 20, 2014 Published: February 24, 2014 2973

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spectrometry in the gas phase, Ho et al. have observed two exponential decay components in the transients of 5FCyt: The dominant “slow” component depends on the excitation wavelength and decreases from 100 ps at 290 nm to 9.5 ps at 258 nm.7 The “fast” (τ ∼ 3 ps) component has smaller amplitude and does not depend on the excess vibrational energy; it was attributed to the intramolecular vibrational relaxation (IVR) from the Franck−Condon point (vertical excitation) to the 1ππ* state minimum.7 Blancafort et al. showed by CASSCF/CASPT2 calculations of the 1nπ* and 1ππ* potentials that the minimum energy paths connecting the minimum of the optically accessed 1ππ* state with the conical intersection responsible for internal conversion are very similar for Cyt and 5FCyt.4 They have therefore suggested that Cyt and 5FCyt decay nonradiatively by the same mechanisms and that the nearly 100-fold difference in lifetimes is due to subtle changes along the decay coordinate.4 Using configuration interaction singles (CIS) and CR-EOM-CCSD(T) excited-state electronic calculations, Lim and co-workers22,23 have investigated possible pathways for S1 → S0 internal conversion of electronically excited pyrimidine bases, including 5FCyt. They have proposed a deactivation channel over an outof-plane deformed excited state of biradical character.22,23 While previous experimental work on 5FCyt has focused on time-domain measurements,4,7,21 no gas-phase spectroscopy of 5FCyt has been performed. However, the knowledge of the vibronic spectrum and excited-state vibrations of 5FCyt are a key to understanding its excited-state behavior especially compared to other cytosines.8,9 Here, we investigate the 1ππ* excited state of jet-cooled 5FCyt using mass-selective two-color resonant two-photon ionization (2C-R2PI) and UV/UV holeburning spectroscopies. These reveal that the three lowest-frequency out-of-plane vibrations couple to the electronic excitation; the respective vibronic band intensities indicate extensive out-of-plane deformation. The in-plane orientation of the electronic transition dipole moment identifies the nature of the excited state as 1ππ*. The lifetimes of the vibrationless and of five low-lying vibronic levels are determined from the rotational contours. The resulting lifetimes as a function of excess vibrational energy are compared to those of Cyt, giving insight into the mechanisms that lead to nonradiative deactivation of cytosine derivatives. We complement the experimental study with correlated ab initio and density functional (DFT) calculations.

Figure 1. The six most stable tautomers/rotamers of 5-fluorocytosine.

agreement with the calculations of Ho et al., who used the G3MP2B3 method.7 The 2b tautomerwhich is not at the focus of this workis clearly the dominant species in the supersonic jet together with the cis amino-enol tautomer 2a, which is calculated at ∼0.8 kcal/mol higher energy. The aminoketo tautomer 1 is predicted 2.2−3.8 kcal/mol higher, and the imino-keto-trans 3a is 2.5−5.0 kcal/mol higher. The relative energies of the imino-keto-cis 3b and the amino-keto 4 tautomers lie at 6−9 and 10−12 kcal/mol, respectively, so 3b and 4 should be present in very low concentrations in the molecular beam. The imino-enol tautomers and rotamers are higher by 13 kcal/mol or more; their B3LYP/TZVP relative energies are given in the Supporting Information. 2.2. Excited States. Table 2 shows the TD-B3LYP/TZVP, CC2/aug-cc-pVTZ, and SCS-CC2/aug-cc-pVDZ calculated adiabatic and vertical transition energies for the amino-keto 1, which is of central interest for this work, as well as for the most stable amino-enol 2b tautomer. The TD-B3LYP and SCS-CC2 calculated adiabatic transitions to the lowest 1ππ* state lie within 50−80 cm−1 of the experimentally observed 000 transition (see below); the CC2 calculated transition energy is 1500 cm−1 lower. By comparison to the amino-keto tautomer 1, the TDB3LYP calculated lowest 1ππ* adiabatic transition energies of the amino-enol 2a and 2b and imino-keto 3a and 3b tautomers are shifted by 6400−7900 cm−1 to higher frequencies; see Table 2 and Table 2S in the Supporting Information. Hence, they do not contribute to the absorption in the 30000−32500 cm−1 region investigated here. Although the CC2 calculated adiabatic transition energy of the amino-keto-N3H 4 tautomer is similar to that of tautomer 1, its relative energy is too high and its abundance too low to render it observable in the supersonic jet. The two lowest-energy singlet electronic transitions of 1 are dominated by the HOMO-1 → LUMO and HOMO → LUMO orbital excitations and correspond to 1nπ* and 1ππ* states, respectively; see Figure 2. Vertically, the 1nπ* state is 2000−

2. COMPUTATIONAL METHODS AND RESULTS The electronic ground state of 5FCyt was optimized with the spin-component scaled (SCS) resolution-of-identity (RI) Møller−Plesset (MP2) method using the aug-cc-pVTZ basis set. Ground- and excited-state calculations were also performed with the approximate second-order coupled-cluster method (CC2) and with its spin-component scaled variant SCS-CC2 using the aug-cc-pVDZ and aug-cc-pVTZ basis sets. Additional ground- and excited-state calculations were performed with the B3LYP density functional and the TZVP basis set. Excited-state normal-mode frequencies were calculated by the numerical differentiation of analytical gradients. The calculations were performed using Turbomole 6.3.24,25 2.1. Electronic Ground State. 5-Fluorocytosine exists in 14 different tautomer/rotamer forms, the six most stable of which are shown in Figure 1. Their relative energies are given in Table 1. With all correlated methods (MP2, SCS-MP2, and CC2), the trans amino-enol 2b tautomer is the most stable, in 2974

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Table 1. Calculated Ground-State Energies of the Six Lowest-Energy 5-Fluorocytosine Tautomers/Rotamers (in kcal/mol), Relative to the Amino-enol-trans 2b Tautomer tautomer

B3LYP/TZVP

CC2/aug-cc-pVTZ

MP2/aug-cc-pVTZ

SCS MP2/aug-cc-pVTZ

amino-keto-N1H 1 amino-enol-cis 2a amino-enol-trans 2b imino-keto-trans 3a imino-keto-cis 3b amino-keto-N3H 4

0.52 0.85 0.00 2.07 5.95 7.97

2.14 0.78 0.00 4.09 7.88 10.66

3.75 0.77 0.00 4.96 8.78 12.64

2.47 0.78 0.00 2.50 6.23 11.62

Table 2. Time-Dependent B3LYP, CC2, and SCS-CC2 Calculated Vertical and Adiabatic Transition Energies (in cm−1) and Oscillator Strengths fel of the Keto-Amino 1 and Amino-Enol 2b Tautomers of 5-Fluorocytosine B3LYP/TZVP tautomer state

adiab.

fel

nπ* ππ* 3 ππ* 3 nπ* 3 ππ*

38379 36414 25730 32796 36264

28993 30726 23084

0.00034 0.052

nπ* ππ* 3 ππ* 3 nπ* 3 ππ*

39722 40295 30161 33637 36231

34962 38351 25900

0.0046 0.11

1 1

1 1

a

vertical

CC2/aug-cc-pVTZ a

vertical

adiab.

Keto-Amino 1 38584 25173 35654 29100 28519 36976 42238 Amino-Enol 2b

SCS-CC2/aug-cc-pVDZ fel

a

0.00066 0.060

vertical

adiab.

fela

Exp.

42155 36509 28729 37334 40876

31304 30592

0.0037 0.062

30643

Vertical excitation from S0 equilibrium geometry, length representation.

5500 cm−1 above the 1ππ* state; see Table 2. Both the TDB3LYP and CC2 methods predict the 1nπ* state minimum to lie below the 1ππ* minimum, by 1733 cm−1 (TD-B3LYP) and by 3927 cm−1 (CC2). In contrast, the SCS-CC2 method predicts the 1nπ* minimum 710 cm−1 above the 1ππ* minimum. The 1nπ* oscillator strength is calculated to be ∼100 times smaller than that of the 1ππ* transition. Figure 3 shows an overlay of the calculated S0 and 1ππ* state geometries calculated by the SCS-CC2 method. Both the SCSCC2 and CC2 methods predict the pyrimidinone moiety to be planar in the S0 state, with the −NH2 group bent slightly out of the ring plane. For the 1ππ* excited state, both methods predict a stronger pyramidalization of the −NH2 group, an out-of-plane bend of the C6 hydrogen, and an in-plane angle deformation of the six-membered ring. The N1−C2−N3 and C5−C4−N(H2) angles increase by ∼9 and ∼7°, while the other ring angles decrease by 4−5°. The calculated out-of-plane geometry changes are much larger for the SCS-CC2 method; this point will be taken up when comparing the simulated and experimental Franck−Condon intensity patterns. Figure 4 compares the side views of the CC2 and SCS-CC2 geometries that emphasize the out-of-plane deformations. Tables with Cartesian coordinates are given in the Supporting Information. The CC2, B3LYP, and SCS-CC2 calculated 1ππ* state normal modes and frequencies are given in Table 3. For the inplane framework vibrations, we follow the Wilson nomenclature for benzene derivatives. The eight lowest energy vibrations of 5FCyt are out-of-plane modes. The lowest frequency inplane vibration is closely related to the 6a mode of benzene derivatives and is calculated at 458 cm−1 (SCS-CC2), 465 cm−1 (CC2), and 486 cm−1 (B3LYP). The next in-plane vibration is similar to the 6b vibration of benzene derivatives and lies at 478

cm−1 (SCS-CC2), 475 cm−1 (CC2), or 520 cm−1(B3LYP). Generally, the CC2 and SCS-CC2 calculated frequencies are close together and lower than the TD-B3LYP frequencies. The main exception to this is the NH2 inversion mode, which is predicted 200 cm−1 lower by the TD-B3LYP method.

3. EXPERIMENTAL METHODS 5-Fluorocytosine (Sigma, >99% purity) was used without further purification. The molecular-beam/mass spectrometer experimental setup has been previously described.26 Neon carrier gas (≥99.995% purity) at ∼1.8 bar backing pressure was passed through a pulsed nozzle (0.4 mm diameter) containing the 5FCyt heated to 215 °C. Mass-selected two-color R2PI spectra in the 30000−32300 cm−1 range were measured by crossing the skimmed supersonic jet with unfocused UV excitation and ionization laser beams that are spatially and temporally overlapped in the ion source of a linear time-offlight (TOF) mass spectrometer. The mass spectra were recorded with a LeCroy LT374 digitizer, averaged over 32 laser shots, and transferred to a PC. S0 → S1 excitation was performed with the frequencydoubled output of a Radiant Dyes NarrowScan D-R dye laser (300 μJ/pulse) with a ∼0.038 cm−1 bandwidth in the visible, as measured with a HighFinesse Ångstrom WS6 wavemeter. After frequency doubling, the UV bandwidth is expected to be 0.053 cm−1 (1600 MHz). The wavelength was calibrated by measuring the DCM dye laser fundamental frequency with the WS6 Wavemeter. The high-resolution scans were measured at 200 μJ/pulse. The ionization light at 226 nm was generated by an Ekspla NT342B optical parametric oscillator (OPO) (∼90 μJ/pulse, ∼8 cm−1 bandwidth). For UV/UV holeburning and depletion experiments, the frequency-doubled output of a 2975

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Figure 3. SCS-CC2/aug-cc-pVDZ calculated geometry changes of 5fluorocytosine upon 1ππ* excitation; the excited-state geometry is in color. Only bond length changes ≥0.04 Å and angle changes ≥4° are indicated.

Figure 2. Frontier molecular orbitals of 5-fluorocytosine (SCS-CC2/ aug-cc-pVTZ calculation).

second dye laser with pulse energies of ∼1 mJ was used. The burn laser was set 200 ns before the UV excitation and ionization lasers to efficiently deplete the ground state.

4. RESULTS AND DISCUSSION 4.1. Resonant Two-Photon Ionization, UV/UV Holeburning, and Depletion Spectra of 5FCyt. Figure 5a shows the vibrationally resolved 2C-R2PI spectrum of jet-cooled 5FCyt between 30430 and 32240 cm−1. The electronic origin lies at 30643 cm−1 and has only 30% of the intensity of the strongest vibronic band, signaling a marked geometry change between the ground and excited states. The origin is shifted by δν = −1192 cm−1 relative to the 000 band of keto-amino Cyt (31835 cm−1).9,11,13 The CAS-PT2 calculations of Blancafort et al. predict the 000 band of 5FCyt at 29360 cm−1 and red-shifted by δν = −807 cm−1 relative to Cyt (calculated at the same theoretical level).4 Both the absolute and red-shift wavenumbers are in good agreement with our experiment. The spectral shift of 5FCyt relative to Cyt in the gas phase is similar to the experimental red-shift of the absorption spectrum of

Figure 4. Side views of (a) CC2 and (b) SCS-CC2 calculated 1ππ* geometries of 5-fluorocytosine. Note the much larger out-of-plane excited-state distortions with the SCS-CC2 method.

5FCyt relative to Cyt in aqueous solution, which is δν = −1200 cm−1.4 Since both Cyt and 5FCyt in aqueous solution are dominantly (>85%) in the keto-amino form, this also supports 2976

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Table 3. Calculated Normal Modes and Frequencies (in cm−1) of the S1 (1ππ*) Excited State of 5-Fluorocytosine ν1 ν2 ν3 ν4 ν5 ν6 ν7 ν8 ν9 ν10 ν11 ν12 ν13 ν14 ν15 ν16 ν17 ν18 ν19 ν20 ν21 ν22 ν23 ν24 ν25 ν26 ν27 ν28 ν29 ν30 ν31 ν32 ν33

irrepa

descriptionb

CC2/aug-cc-pVDZ

B3LYP/TZVP

SCS-CC2/aug-cc-pVDZ

Exp.c

a″ a″ a″ a″ a″ a″ a″ a″ a′ a′ a″ a′ a″ a″ a″ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′ a′

boat butterfly HN−C6 twist γC6H/γC6/γasNH2 γN7Ha γC6H/γasNH2 γasNH2/δC2O γasNH2/γC4/γC5 6a 6b γN1H 3 γC4/γC5/γN1H NH2 inversion γN1/γN1H/γC2/γN3/NH2 inversion δN1C6C5/δC2O 1/νC2O βasNH2/δN1H/δC6H/νC2O βasNH2/δC6H βasNH2/δN1C2N3 βasNH2/δN1H/δC6H βasNH2/νC5F/δC6H βasNH2/δC6H 15/δN7Hb νN1C6/δN1H νC5C6/δN1H/δC6H βsNH2/νC2N3 νN1C6/νC4NH2/νC2N3 βsNH2/νC4C5/νC2N3 νC6H νsNH2 νN1H νasNH2

131.8 139.7 245.8 260.1 286.9 325.6 344.6 395.3 464.7 475.2 519.4 560.8 563.5 647.4 673.0 725.7 742.6 927.8 960.4 1022.3 1147.9 1189.8 1215.0 1316.8 1388.3 1458.8 1588.2 1593.6 1652.0 3271.6 3507.4 3584.7 3637.6

152.0 142.9 271.8 307.0 343.6 372.9 387.8 437.6 486.2 519.8 570.5 602.5 616.4 447.0 744.7 783.0 774.8 1044.1 971.9 1141.0 1200.8 1229.0 1261.8 1357.8 1436.2 1516.1 1623.2 1550.0 1673.9 3269.4 3576.0 3625.7 3698.7

107.0 147.1 218.8 238.2 279.4 299.4 328.0 367.6 458.2 478.2 406.8 536.4 573.6 646.5 674.0 744.9 749.1 802.4 961.2 1001.3 1155.4 1185.6 1213.4 1302.0 1371.2 1468.8 1610.1 1569.4 1634.9 3266.5 3511.4 3606.9 3634.6

(49) (85) (267)

461 492 567

835 981

a

Irreducible representation in the Cs(M) point group. bBased on CC2/aug-cc-pVDZ eigenvectors; ν = stretching vibration; δ = in-plane bending vibration; γ = out-of-plane bending vibration; βs = scissoring vibration; βas = in-plane rocking vibration; γs = out-of-plane wagging vibration; γas = torsion vibration. cOut-of-plane fundamentals (in parentheses) estimated as half the overtone frequency.

The R2PI spectrum exhibits two bands below the origin, at 000 −152 cm−1 and at 000 −93 cm−1. The −152 cm−1 band is absent in the UV/UV depletion and holeburning spectra, and we assign it to the 211 sequence band; see below. The band at −93 cm−1 is present also in the UV/UV depletion and holeburning spectra. It does not arise from complexation with the Ne carrier gas; R2PI spectra in Ar expansions also show the −93 cm−1 transition. Fragmentation of a H2O cluster can be ruled out, since the 5FCyt·(H2O)n+ mass channels in the R2PI experiments did not reveal any ion signal at the respective wavelength, and coexpansion of 5FCyt with water did not increase the signal intensity in the 5FCyt R2PI spectrum. However, UV holeburning spectra with the burn laser set at the 000 band did not exhibit a band at −93 cm−1. Hence, the −93 cm−1 band is assigned as a hot band that originates from a ground-state level with a vibronic transition that coincides with the band at 30728 cm−1 used for detection; see below. 4.2. Rotational Band Contours. In the S0 state, 5fluorocytosine is an effectively planar asymmetric rotor with the a/b axes in the pyrimidinone plane, drawn in red in Figure 6a; the c-axis is perpendicular to this plane. Figure 6b shows the analogous structure and inertial axes for the closely related 5methylcytosine (5MCyt).8 Figure 7 shows the experimental 000

that the jet spectrum of 5FCyt is that of the keto-amino tautomer. Above the 000 band, about 10 further intense and sharp bands are observed up to 315 cm−1. The density of vibronic excitations increases very strongly at ∼320 cm−1 above the origin, with well-resolved bands being intermingled with dense “clumps”. The relative intensity of the clumps decreases toward higher frequencies. Altogether, about 30 sharp and narrow bands are observed up to about 1200 cm−1 above the 000 band, where the spectrum breaks off. Figure 5b shows the corresponding UV/UV depletion spectrum with the detection laser fixed at the intense +85 cm−1 band (30728 cm−1). Up to ∼1000 cm−1 above the 000 band, the UV/UV depletion spectrum also reproduces the R2PI spectrum. From about 1000 to 1200 cm−1, the bands in the UV/UV depletion spectrum become increasingly broad, while the bands in the R2PI spectrum become weak and finally disappear completely. Beyond +1500 cm−1, the UV depletion spectrum reveals further vibronic bands that will be discussed in subsection 4.3. UV/UV holeburning spectra were also measured. These reproduce the 2C-R2PI spectrum in detail and thereby show that all observed bands originate from the same ground-state level as the 30728 cm−1 transition. 2977

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Figure 5. (a) Two-color resonant two-photon ionization spectrum and (b) UV/UV depletion spectrum of keto-amino 5-fluorocytosine; the scale is relative to the 000 band maximum at 30643 cm−1. In the UV/UV depletion spectrum (b), the detection laser was set to the intense 2ν′2 vibronic band at 000 + 85 cm−1 (30730 cm−1).

band contour for 5FCyt in gray. The double-wing a/b-hybrid shape is characteristic for an in-plane ππ* electronic transition. There is no sign of a central peak that would indicate c-type (and thus 1nπ*) polarization.27,28 Contour simulations were performed using the PGOPHER29 program with a rigid-rotor Hamiltonian, shown as the black traces in Figure 7. The SCS-CC2 A″, B″, and C″ constants given in Table 4 were fixed and the calculated changes A′ − A″, B′ − B″, and C′ − C″ used to initiate the fitting routine. The fitted B′ and C′ constants differed by less than the ±30 MHz fit uncertainty error from the SCS-CC2 starting values; hence, the SCS-CC2 calculated values were retained; see Table 4. Analogous fits with the TD-B3LYP calculated constants and differences lead to the same fit results. A Gaussian laser line shape with a fwhm of ΔGauss = 1.60 GHz was employed; see section 3. We varied the rotational temperature Trot between 2 and 8 K and determined Trot = 2.1 ± 0.6 K from the six bands listed in Table 4. This value is in good agreement with the Trot = 2.5−3 K determined for other pyrimidines and purines.9,26,28,30 The electronic transition dipole moment (TDM) components from the TD-B3LYP calculation in Table 4 were used as initial values for the fit, giving |μa|2:|μb|2 = 4:96. We could not fit a significant c-axis TDM contribution; the upper limit is |μc|2 < 5%. Since the electronic TDM lies fully (or dominantly) within the pyrimidinone plane, the electronic transition must be ππ*. Note that the experiment yields the unsigned angle between the TDM and the inertial axes, allowing two different projections.8,9,28,30 We prefer the orientation given in Figure 6a as a

black double-headed arrow, because (1) it lies closer to the TDB3LYP calculated TDM angle (blue double-headed arrow), (2) it nearly coincides with the SCS-CC2 calculated TDM angle (not shown), and (3) it is consistent with the experimental 1 ππ* TDM of 5-methylcytosine shown in Figure 6b.8 The excited-state lifetime τ of 5FCyt contributes a Lorentzian line shape of width ΔLorentz = 1/2πτ to every rovibronic line, which has to be convoluted with the laser line width ΔGauss = 1600 MHz. Figure 7 shows the effect on the 000 contour of varying ΔLorentz from 900 MHz (corresponding to τ = 177 ps) to 53 GHz (τ = 3 ps). The shortest lifetime for which simulation and experiment still agree is τ = 75 ps, corresponding to ΔLorentz = 2.1 GHz; see Figure 7c and Table 4. Given our laser resolution and the S/N ratio, we can exclude that τ < 60 ps. The uncertainty toward longer lifetimes is considerably larger, since the τ = 106 ps contour in Figure 7b is still compatible with the measurement and the simulation in Figure 7a shows a central dip that is only marginally too deep. From this, we estimate an upper lifetime limit of 110 ps. Figure 8 compares the experimental contours of the 000 band to those of the five intense bands at +49, +85, +138, +183, and +234 cm−1 measured at the same resolution. The vibrational assignments are discussed below. The contour simulations of the five vibronic bands show that they exhibit the same a/btype in-plane polarization as the 000 band. Hence, these five transitions lead to totally symmetric (a′) levels of the 1ππ* state. With increasing excess vibrational energy, the lifetimes as determined from the Lorentzian widths lie between τ ∼ 75 and 55 ps, with a slow decrease toward higher vibrational excess 2978

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Figure 6. B3LYP/TZVP calculated (blue) and experimental (black) transition dipole moment vectors of the lowest 1ππ* transition of (a) 5-fluorocytosine and (b) 5-methylcytosine. The in-plane a and b inertial axes are drawn in red. Figure 7. Two-color R2PI rotational 000 band contours of supersonically cooled 5-fluorocytosine (in gray) with ionization at 226 nm. In black are the simulated asymmetric rotor contours for an a/b polarized (ππ*) transition, with the contour simulation parameters as listed in Table 4. Six simulations are shown with excited-state lifetimes τ decreasing from (a) 177 ps to (f) 3 ps, which results in Lorentzian line width contributions ranging from (a) 900 MHz to (f) 54 GHz.

energy. The contours of three weak bands at −93, +120, and +131 cm−1 are also a/b in-plane polarized; these are shown in the Supporting Information. 4.3. Vibronic Band Assignments. Since the lowestfrequency in-plane mode ν′9 is predicted at 458 cm−1 (Table 3), the bands at lower frequency must arise from out-of-plane vibrations. The 1ππ* state potentials along the ν1′ , ν2′ , and ν3′ out-of-plane vibrational coordinates have a symmetric doubleminimum shape and a barrier at Q′1 = Q′2 = Q′3 = 0 that is the same along all three coordinates. The SCS-CC2 calculated 1ππ* state barrier (energy of the planar Cs structure relative to the 1 ππ* minimum) is 797 cm−1. If the ring is fixed in-plane and the amino group allowed to relax, the barrier decreases to 264 cm−1. The double-minimum potentials lead to tunneling splittings for all three out-of-plane vibrations, necessitating treatment in the molecular symmetry (MS) group Cs(M). Outof-plane vibronic excitations from the S0 ν″ = 0 level are only allowed to the v′ = 2, 4, ... (a′) overtones or to combination levels of a′ symmetry. When comparing experimental and calculated frequencies, note that the normal-mode calculations are performed at one of the two symmetry-equivalent C1 minima and do not take the out-of-plane tunneling into account; thus, the calculated fundamental frequencies must be compared to the experimental v′ = 2 values. Figure 9a shows the experimental 2C-R2PI spectrum of 5FCyt and gives vibronic assignments based on the SCS-CC2 calculation (see Table 3) and the PGOPHER simulation in Figure 9b. The frequencies and intensities of the bands at +49 and +85 cm−1 indicate that they cannot belong to the same progression, and we assign them as the overtones 120 and 220 of the out-of-plane ν1′ (boat) and ν2′ (butterfly) vibrations. The CC2, TD-B3LYP, and SCS-CC2 frequencies in Table 3 are 2−

3 times larger than experiment for ν1′ and about 50% larger for ν′2. This indicates that the corresponding potentials are very anharmonic, in agreement with the anharmonicities of both progressions; see below. The ν1′ (boat) and ν2′ normal-mode eigenvectors are shown in Figure 10. Note that the character of these two vibrations is exchanged relative to those of cytosine.9 The PGOPHER vibronic band simulation29 in Figure 9b is based on the SCS-CC2 calculated S0 and 1ππ* state geometries, normal-mode frequencies, and l matrices and is plotted downward for easier comparison to the R2PI spectrum in part a. The band assignments of the ν′1 and ν′2 progressions are shown above Figure 9a, and the assignments of less intense bands are given in Figure 9b. The ν1′ and ν2′ harmonic frequencies in the PGOPHER simulation were scaled and diagonal anharmonicities adjusted to give the 102 and 104 overtones at +49 and +120 cm−1 and the 220 and 240 overtones at +85 and +183 cm−1. With these adjustments, the bands at +138, +234, and +287 cm−1 are predicted by the PGOPHER simulation to be the overtone or combination bands 120220, 120240, and 260, respectively. The next transition that could not be assigned as an overtone or combination of ν1′ and/or ν2′ is a medium weak band at +267 cm−1; see Figure 5a. We assign it to the first overtone of the ν′3 out-of-plane vibration, although the predicted intensity of this band in the PGOPHER simulation is much lower than experimentally observed; see Figure 9b. 2979

dx.doi.org/10.1021/jp500410s | J. Phys. Chem. B 2014, 118, 2973−2984

The Journal of Physical Chemistry B

Article

Table 4. S0 and 1ππ* State SCS-CC2 and B3LYP Calculated Rotational Constants and Transition Dipole Moments of 5Fluorocytosine, Compared to Fitted Values from Contour Simulations of the 000, +49 cm−1, +85 cm−1, +138 cm−1, +183 cm−1, and +234 cm−1 Bands

a

parameter

SCS-CC2

B3LYP

A″ (MHz) B″ (MHz) C″ (MHz) A′ − A″ (MHz) B′ − B″ (MHz) C′ − C″ (MHz) |μa|2:|μb|2:|μc|2 a ΔLorentz (GHz)

3135 1384 961 −122 6 −1 2:83:15

3190 1414 980 −72 −2 −5 1:99:0

000 band

120

220

120 + 220

240

120 + 240

−110(70) (6) (−1) 4(6):96(9):0(5) 2.1

−110(80) b b b 2.1

−126(80) b b b 3.6

−110(80) b b b 2.7

−110(80) b b b 4.0

−110(80) b b b 3.0

Sum of transition dipole moment components normalized to 100%; estimated uncertainty in parentheses. bValues taken over from the 000 band.

Figure 9. (a) Two-color resonant two-photon ionization spectrum of jet-cooled 5-fluorocytosine, with assignments for the ν1′ and ν2 bands; the scale is relative to the 000 band maximum at 30643 cm−1. (b) PGOPHER simulation of the vibronic spectrum (plotted in the negative direction) based on the SCS-CC2/aVDZ ground- and 1ππ*state calculations.

excited-state v′1 = 1 level is the upper component of the v′ = 0 tunnel doublet, denoted 0− in high-barrier notation. The 0+/0− tunneling splitting is expected to be