Development of a novel certified reference material for the determination of polycyclic aromatic hydrocarbons (PAHs) in whey protein powder.

Matrix-based certified reference materials (CRMs) play a central role in the analysis of food contaminants for ensuring measurement accuracy and comparability, as they exhibit the same matrix effects during sample preparation and measurement as the food sample under investigation. However, the availability of such CRMs is still limited. This is also true for matrix CRMs containing polycyclic aromatic hydrocarbons (PAHs), for which maximum levels in food are set in the EU by the Commission Regulation (EU) 2023/915 and in Switzerland by the regulation SR 817.022.15. Therefore, a whey protein powder-based certified reference material (WP-CBR001) was developed according to the requirements of ISO 17034 and the recommendations of ISO Guide 35:2017 containing the four PAHs benz[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), and chrysene (Chr). We show that the choice of solvent is of crucial importance to extract the PAHs completely from this matrix. Only polar and protic solvents such as methanol or water provided access for complete extraction of the PAHs. In contrast, nonpolar and polar aprotic solvents, such as n-hexane or ethyl acetate, exhibited only very low extraction efficiencies below 20%. The certified mass fractions and expanded uncertainties (k = 2) were (3.17 ± 0.32) µg/kg BaA, (4.18 ± 0.48) µg/kg BaP, (4.73 ± 0.49) µg/kg BbF, and (2.85 ± 0.33) µg/kg Chr. These values were verified by an interlaboratory comparison study and by the gravimetric mass fractions obtained from production data.

Planarizing cytosine: The S1 state structure, vibrations, and nonradiative dynamics of jet-cooled 5,6-trimethylenecytosine.

We measure the S0 → S1 spectrum and time-resolved S1 state nonradiative dynamics of the "clamped" cytosine derivative 5,6-trimethylenecytosine (TMCyt) in a supersonic jet, using two-color resonant two-photon ionization (R2PI), UV/UV holeburning, and ns time-resolved pump/delayed ionization. The experiments are complemented with spin-component scaled second-order approximate coupled cluster (SCS-CC2), time-dependent density functional theory, and multi-state second-order perturbation-theory (MS-CASPT2) ab initio calculations. While the R2PI spectrum of cytosine breaks off ∼500 cm-1 above its 000 band, that of TMCyt extends up to +4400 cm-1 higher, with over a hundred resolved vibronic bands. Thus, clamping the cytosine C5-C6 bond allows us to explore the S1 state vibrations and S0 → S1 geometry changes in detail. The TMCyt S1 state out-of-plane vibrations ν1', ν3', and ν5' lie below 420 cm-1, and the in-plane ν11', ν12', and ν23' vibrational fundamentals appear at 450, 470, and 944 cm-1. S0 → S1 vibronic simulations based on SCS-CC2 calculations agree well with experiment if the calculated ν1', ν3', and ν5' frequencies are reduced by a factor of 2-3. MS-CASPT2 calculations predict that the ethylene-type S1 ⇝ S0 conical intersection (CI) increases from +366 cm-1 in cytosine to >6000 cm-1 in TMCyt, explaining the long lifetime and extended S0 → S1 spectrum. The lowest-energy S1 ⇝ S0 CI of TMCyt is the "amino out-of-plane" (OPX) intersection, calculated at +4190 cm-1. The experimental S1 ⇝ S0 internal conversion rate constant at the S1(v'=0) level is kIC=0.98-2.2⋅108 s-1, which is ∼10 times smaller than in 1-methylcytosine and cytosine. The S1(v'=0) level relaxes into the T1(3ππ*) state by intersystem crossing with kISC=0.41-1.6⋅108 s-1. The T1 state energy is measured to lie 24 580±560 cm-1 above the S0 state. The S1(v'=0) lifetime is τ=2.9 ns, resulting in an estimated fluorescence quantum yield of Φfl=24%. Intense two-color R2PI spectra of the TMCyt amino-enol tautomers appear above 36 000 cm-1. A sharp S1 ionization threshold is observed for amino-keto TMCyt, yielding an adiabatic ionization energy of 8.114±0.002 eV.

Intersystem crossing rates of S1 state keto-amino cytosine at low excess energy.

The amino-keto tautomer of supersonic jet-cooled cytosine undergoes intersystem crossing (ISC) from the v = 0 and low-lying vibronic levels of its S1((1)ππ(∗)) state. We investigate these ISC rates experimentally and theoretically as a function of S1 state vibrational excess energy Eexc. The S1 vibronic levels are pumped with a ∼5 ns UV laser, the S1 and triplet state ion signals are separated by prompt or delayed ionization with a second UV laser pulse. After correcting the raw ISC yields for the relative S1 and T1 ionization cross sections, we obtain energy dependent ISC quantum yields QISC (corr)=1%-5%. These are combined with previously measured vibronic state-specific decay rates, giving ISC rates kISC = 0.4-1.5 ⋅ 10(9) s(-1), the corresponding S1⇝S0 internal conversion (IC) rates are 30-100 times larger. Theoretical ISC rates are computed using SCS-CC2 methods, which predict rapid ISC from the S1; v = 0 state with kISC = 3 ⋅ 10(9) s(-1) to the T1((3)ππ(∗)) triplet state. The surprisingly high rate of this El Sayed-forbidden transition is caused by a substantial admixture of (1)nOπ(∗) character into the S1((1)ππ(∗)) wave function at its non-planar minimum geometry. The combination of experiment and theory implies that (1) below Eexc = 550 cm(-1) in the S1 state, S1⇝S0 internal conversion dominates the nonradiative decay with kIC ≥ 2 ⋅ 10(10) s(-1), (2) the calculated S1⇝T1 ((1)ππ(∗)⇝(3)ππ(∗)) ISC rate is in good agreement with experiment, (3) being El-Sayed forbidden, the S1⇝T1 ISC is moderately fast (kISC = 3 ⋅ 10(9) s(-1)), and not ultrafast, as claimed by other calculations, and (4) at Eexc ∼ 550 cm(-1) the IC rate increases by ∼50 times, probably by accessing the lowest conical intersection (the C5-twist CI) and thereby effectively switching off the ISC decay channels.

Low-lying excited states and nonradiative processes of 9-methyl-2-aminopurine.

The UV spectrum of the adenine analogue 9-methyl-2-aminopurine (9M-2AP) is investigated with one- and two-color resonant two-photon ionization spectroscopy at 0.3 and 0.05 cm(-1) resolution in a supersonic jet. The electronic origin at 32,252 cm(-1) exhibits methyl torsional subbands that originate from the 0A1'' (l = 0) and 1E(″) (l = ±1) torsional levels. These and further torsional bands that appear up to 00 (0)+230 cm(-1) allow to fit the threefold (V3) barriers of the torsional potentials as |V3''|=50 cm(-1) in the S0 and |V3'|=126 cm(-1) in the S1 state. Using the B3LYP density functional and correlated approximate second-order coupled cluster CC2 methods, the methyl orientation is calculated to be symmetric relative to the 2AP plane in both states, with barriers of V3''=20 cm(-1) and V3'=115 cm(-1). The 00 (0) rotational band contour is 75% in-plane (a/b) polarized, characteristic for a dominantly long-axis (1)ππ(*) excitation. The residual 25% c-axis polarization may indicate coupling of the (1)ππ(*) to the close-lying (1)nπ(*) state, calculated at 4.00 and 4.01 eV with the CC2 method. However, the CC2 calculated (1)nπ oscillator strength is only 6% of that of the (1)ππ(*) transition. The (1)ππ(*) vibronic spectrum is very complex, showing about 40 bands within the lowest 500 cm(-1). The methyl torsion and the low-frequency out-of-plane ν1' and ν2' vibrations are strongly coupled in the (1)ππ(*) state. This gives rise to many torsion-vibration combination bands built on out-of-plane fundamentals, which are without precedence in the (1)ππ(*) spectrum of 9H-2-aminopurine [S. Lobsiger, R. K. Sinha, M. Trachsel, and S. Leutwyler, J. Chem. Phys. 134, 114307 (2011)]. From the Lorentzian broadening needed to fit the 00 (0) contour of 9M-2AP, the (1)ππ(*) lifetime is τ ⩾ 120 ps, reflecting a rapid nonradiative transition.

Isomer- and species-selective infrared spectroscopy of jet-cooled 7H- and 9H-2-aminopurine and 2-aminopurine·H2O clusters.

The infrared (IR) spectra of the supersonic-jet cooled 9H- and 7H-tautomers of 2-aminopurine (2AP) and of the 9H-2-aminopurine·H(2)O monohydrate clusters have been measured by mass- and species-selective IR-UV double resonance spectroscopy in the 3200-3900 cm(-1) region, covering the N-H and O-H stretching vibrations. The spectra are complemented by density functional (B3LYP and PW91) and by second-order Møller-Plesset (MP2) calculations of the electronic energies and vibrational frequenciesof the respective 2AP tautomers and clusters. The 9H- and 7H-2-aminopurine tautomers were definitively identified by the shifts of their NH and NH(2) symmetric and asymmetric stretching frequencies and by comparison to the B3LYP/TZVP calculated IR spectra. The H-bond topologies of the two previously observed 9H-2-aminopurine·H(2)O isomers (Sinha. R. K.; et al. J. Phys. Chem. A2011, 115, 6208) are definitively identified as the "sugar-edge" isomer A and the "trans-amino-bound" isomer B by comparing their IR spectra to the calculated frequencies and IR intensities of the cluster isomers A, B, C, and D, as well as to the IR spectrum of 9H-2AP. The sugar-edge isomer A involves N9-H···OH(2) and HOH···N3 hydrogen bonds and is predicted to be the most stable form. The amino-bound isomer B involves NH(2)···OH(2) and HOH···N1 hydrogen bonds and is calculated to lie 2.5 kJ/mol above isomer A. The H-bond topology of the "cis-amino-bound" isomer C is symmetrically related to isomer B, with a hydrogen bond to the N3 of the pyrimidine group. However, it is calculated to lie 7 kJ/mol above isomer A and indeed is not observed in the supersonic jet. Isomer D involves a single H-bond to the N7 position, is predicted to be 14 kJ/mol above A and is therefore not observed.

Intermolecular clamping by hydrogen bonds: 2-pyridone⋠NH3.

A combined spectroscopic and ab initio theoretical study of the doubly hydrogen-bonded complex of 2-pyridone (2PY) with NH(3) has been performed. The S(1)←S(0) spectrum extends up to ≈1200 cm(-1) above the 0(0) (0) band, close to twice the range observed for 2PY. The S(1) state nonradiative decay for vibrations above ≈300 cm(-1) in the NH(3) complex is dramatically slowed down relative to bare 2PY. Also, the Δv=2, 4,… overtone bands of the ν(1) ' and ν(2) ' out-of-plane vibrations that dominate the low-energy spectral region of 2PY are much weaker or missing for 2PY⋅NH3, which implies that the bridging (2PY)NH⋅⋅⋅NH(3) and H(2) NH⋅⋅⋅O=C H-bonds clamp the 2PY at a planar geometry in the S(1) state. The mass-resolved UV vibronic spectra of jet-cooled 2PY⋅NH(3) and its H/D mixed isotopomers are measured using two-color resonant two-photon ionization spectroscopy. The S(0) and S(1) equilibrium structures and normal-mode frequencies are calculated by density functional (B3LYP) and correlated ab initio methods (MP2 and approximate second-order coupled-cluster, CC2). The S(1)←S(0) vibronic assignments are based on configuration interaction singles (CIS) and CC2 calculations. A doubly H-bonded bridged structure of C(S) symmetry is predicted, in agreement with that of Held and Pratt [J. Am. Chem. Soc. 1993, 115, 9718]. While the B3LYP and MP2 calculated rotational constants are in very good agreement with experiment, the calculated H(2) NH⋅⋅⋅O=C H-bond distance is ≈0.7 Å shorter than that derived by Held and Pratt. On the other hand, this underlines their observation that ammonia can act as a strong H-bond donor when built into an H-bonded bridge. The CC2 calculations predict the H(2) NH⋅⋅⋅O distance to increase by 0.2 Å upon S(1)←S(0) electronic excitation, while the (2PY)NH⋅⋅⋅NH(3) H-bond remains nearly unchanged. Thus, the expansion of the doubly H-bonded bridge in the excited state is asymmetric and almost wholly due to the weakening of the interaction of ammonia with the keto acceptor group.

Vibronic spectra of jet-cooled 2-aminopurine·H2O clusters studied by UV resonant two-photon ionization spectroscopy and quantum chemical calculations.

For understanding the major- and minor-groove hydration patterns of DNAs and RNAs, it is important to understand the local solvation of individual nucleobases at the molecular level. We have investigated the 2-aminopurine·H(2)O monohydrate by two-color resonant two-photon ionization and UV/UV hole-burning spectroscopies, which reveal two isomers, denoted A and B. The electronic spectral shift δν of the S(1) ← S(0) transition relative to bare 9H-2-aminopurine (9H-2AP) is small for isomer A (-70 cm(-1)), while that of isomer B is much larger (δν = -889 cm(-1)). B3LYP geometry optimizations with the TZVP basis set predict four cluster isomers, of which three are doubly H-bonded, with H(2)O acting as an acceptor to a N-H or -NH2 group and as a donor to either of the pyrimidine N sites. The "sugar-edge" isomer A is calculated to be the most stable form with binding energy D(e) = 56.4 kJ/mol. Isomers B and C are H-bonded between the -NH2 group and pyrimidine moieties and are 2.5 and 6.9 kJ/mol less stable, respectively. Time-dependent (TD) B3LYP/TZVP calculations predict the adiabatic energies of the lowest (1)ππ* states of A and B in excellent agreement with the observed 0(0)(0) bands; also, the relative intensities of the A and B origin bands agree well with the calculated S(0) state relative energies. This allows unequivocal identification of the isomers. The R2PI spectra of 9H-2AP and of isomer A exhibit intense low-frequency out-of-plane overtone and combination bands, which is interpreted as a coupling of the optically excited (1)ππ* state to the lower-lying (1)nπ* dark state. In contrast, these overtone and combination bands are much weaker for isomer B, implying that the (1)ππ* state of B is planar and decoupled from the (1)nπ* state. These observations agree with the calculations, which predict the (1)nπ* above the (1)ππ* state for isomer B but below the (1)ππ* for both 9H-2AP and isomer A.

S0 and S1 state structure, methyl torsional barrier heights, and fast intersystem crossing dynamics of 5-methyl-2-hydroxypyrimidine.

We report the analysis of the S1<--S0 rotational band contours of jet-cooled 5-methyl-2-hydroxypyrimidine (5M2HP), the enol form of deoxythymine. Unlike thymine, which exhibits a structureless spectrum, the vibronic spectrum of 5M2HP is well structured, allowing us to determine the rotational constants and the methyl group torsional barriers in the S0 and S1 states. The 0(0)(0), 6a(0)(1), 6b(0)(1), and 14(0)(1) band contours were measured at 900 MHz (0.03 cm(-1)) resolution using mass-specific two-color resonant two-photon ionization (2C-R2PI) spectroscopy. All four bands are polarized perpendicular to the pyrimidine plane (>90% c type), identifying the S1<--S0 excitation of 5M2HP as a 1nπ* transition. All contours exhibit two methyl rotor subbands that arise from the lowest 5-methyl torsional states 0A" and 1E". The S0 and S1 state torsional barriers were extracted from fits to the torsional subbands. The 3-fold barriers are V3" = 13 cm(-1) and V3' = 51 cm(-1); the 6-fold barrier contributions V6" and V6' are in the range of 2-3 cm(-1) and are positive in both states. The changes of A, B, and C rotational constants upon S1 <--S0 excitation were extracted from the contours and reflect an "anti-quinoidal" distortion. The 0(0)(0) contour can only be simulated if a 3 GHz Lorentzian line shape is included, which implies that the S1(1nπ*) lifetime is ~55 ps. For the 6a(0)(1) and 6b(0)(1) bands, the Lorentzian component increases to 5.5 GHz, reflecting a lifetime decrease to ~30 ps. The short lifetimes are consistent with the absence of fluorescence from the 1nπ* state. Combining these measurements with the previous observation of efficient intersystem crossing (ISC) from the S1 state to a long-lived T1 (3nπ*) state that lies ~2200 cm(-1) below [S. Lobsiger, S. et al. Phys. Chem. Chem. Phys. 2010, 12, 5032] implies that the broadening arises from fast intersystem crossing with k(ISC) ≈ 2 × 10(10) s(-1). In comparison to 5-methylpyrimidine, the ISC rate is enhanced by at least 10 000 by the additional hydroxy group in position 2.

Low-lying excited states and nonradiative processes of the adenine analogues 7H- and 9H-2-aminopurine.

We have investigated the UV vibronic spectra and excited-state nonradiative processes of the 7H- and 9H-tautomers of jet-cooled 2-aminopurine (2AP) and of the 9H-2AP-d(4) and -d(5) isotopomers, using two-color resonant two-photon ionization spectroscopy at 0.3 and 0.045 cm(-1) resolution. The S(1) ← S(0) transition of 7H-2AP was observed for the first time. It lies ∼1600 cm(-1) below that of 9H-2AP, is ∼1000 times weaker and exhibits only in-plane vibronic excitations. In contrast, the S(1) ← S(0) spectra of 9H-2AP, 9H-2AP-d(4), and 9H-2AP-d(5) show numerous low-frequency bands that can be systematically assigned to overtone and combinations of the out-of-plane vibrations ν(1)', ν(2)', and ν(3)'. The intensity of these out-of-plane bands reflects an out-of-plane deformation in the (1)ππ∗(L(a)) state. Approximate second-order coupled-cluster theory also predicts that 2-aminopurine undergoes a "butterfly" deformation in its lowest (1)ππ∗ state. The rotational contours of the 9H-2AP, 9H-2AP-d(4), and 9H-2AP-d(5) 0(0)(0) bands and of eight vibronic bands of 9H-2AP up to 0(0)(0) + 600 cm(-1) exhibit 75%-80% in-plane (a∕b) polarization, which is characteristic for a (1)ππ∗ excitation. A 20%-25% c-axis (perpendicular) transition dipole moment component may indicate coupling of the (1)ππ∗ bright state to the close-lying (1)nπ∗ dark state. However, no (1)nπ∗ vibronic bands were detected below or up to 500 cm(-1) above the (1)ππ∗ 0(0)(0) band. Following (1)ππ∗ excitation, 9H-2AP undergoes a rapid nonradiative transition to a lower-lying long-lived state with a lifetime ≥5 µs. The ionization potential of 9H-2AP was measured via the (1)ππ∗ state (IP = 8.020 eV) and the long-lived state (IP > 9.10 eV). The difference shows that the long-lived state lies ≥1.08 eV below the (1)ππ∗ state. Time-dependent B3LYP calculations predict the (3)ππ∗ (T(1)) state 1.12 eV below the (1)ππ∗ state, but place the (1)nπ∗ (S(1)) state close to the (1)ππ∗ state, implying that the long-lived state is the lowest triplet (T(1)) and not the (1)nπ∗ state.

Supersonic jet UV spectrum and nonradiative processes of the thymine analogue 5-methyl-2-hydroxypyrimidine.

We investigate the infrared and electronic absorption spectra and the excited-state nonradiative processes of supersonic jet-cooled 5-methyl-2-hydroxypyrimidine (5M2HP), the enol form of deoxythymine, using two-color resonant two-photon ionization (R2PI) and infrared-UV depletion spectroscopies. Unlike uracil and thymine, which exhibit structureless electronic absorption spectra, the vibronic spectrum of 5M2HP is structured with narrow vibronic bands, allowing for the first time to probe the excited state of a thymine analogue. The S(0) state infrared depletion spectrum shows an O-H and no N-H stretch band, identifying the spectrum as that of the enol tautomer. The S(1)<--S(0) electronic transition is (1)npi*, as evidenced by the rotational contour of the 0 band. Vibronic excitations of the in-plane benzene-type vibrations nu'(6a), nu'(6b), nu'(14) and nu'(15) are observed, while none are observed for the out-of plane fundamental excitations, implying that the (1)npi* excited state of 5M2HP has a planar pyrimidine frame. From 1200 to 3600 cm(-1) the vibronic bands become steadily broader, signaling a coupling to a lower-lying electronic state that increases with increasing energy. At approximately 3600 cm(-1) above the origin, the R2PI spectrum broadens completely, indicating that the two states are strongly mixed. Delayed ionization measurements show that the coupled electronic state has a >5 micros lifetime. No fluorescence has been observed from the (1)npi* state, implying relaxation to the lower-lying long-lived state is very efficient. Separate ionization potentials are measured for the (1)npi* state (9.178 eV) and for the long-lived state (approximately 9.46 eV), hence the latter lies approximately 2200 cm(-1) below the (1)npi* state. Time-dependent B3LYP calculations of the excited states of 5M2HP indeed predict the S(1) state to be (1)npi* with a planar hydroxypyrimidine moiety. The T(1) ((3)pipi*) state is calculated to lie 3000 cm(-1) below the S(1) state, in excellent agreement with the experiment.

Locating Cytosine Conical Intersections by Laser Experiments and Ab Initio Calculations.

The decay mechanism of S0 → S1 excited cytosine (Cyt) and the effect of substitution are studied combining jet-cooled spectroscopy (nanosecond resonant two-photon ionization (R2PI) and picosecond lifetime measurements) with CASPT2//CASSCF computations for eight derivatives. For Cyt and five derivatives substituted at N1, C5, and C6, rapid internal conversion sets in at 250-1200 cm-1 above the 000 bands. The break-off in the spectra correlates with the calculated barriers toward the "C5-C6 twist" conical intersection, which unambiguously establishes the decay mechanism at low S1 state vibrational energies. The barriers increase with substituents that stabilize the charge shifts at C4, C5, and C6 following (1ππ*) excitation. The R2PI spectra of the clamped derivatives 5,6-trimethyleneCyt (TMCyt) and 1-methyl-TMCyt (1M-TMCyt), which decay along an N3 out-of-plane coordinate, extend up to +3500 and +4500 cm-1.

Accurate determination of the structure of cyclooctatetraene by femtosecond rotational coherence spectroscopy and ab initio calculations.

We combine femtosecond time-resolved rotational coherence spectroscopy with high-level ab initio theory to obtain accurate structural information for the nonpolar antiaromatic molecule 1,3,5,7-cyclooctatetraene (C8H8, COT) and its perdeuterated isotopomer COT-d8 (C8D8). We measure the rotational B0 and centrifugal distortion constants D(J), D(JK) of the v = 0 states of COT and COT-d8 to high accuracy, e.g. B0 (COT) = 2710.329(56) MHz, as well as B(v) for the v = 1 states nu6, nu11, nu17, nu22, and nu41/nu42 of COT. The experimental rotational constants are compared to those obtained from calculations at the coupled-cluster with single, double, and perturbative triples [CCSD(T)] level. The latter also take into account vibrational averaging effects of the ground and vibrationally excited states. Combining the experimental and calculated rotational constants with the calculated equilibrium bond lengths and angles allows us to determine accurate equilibrium structure parameters, e.g., r(e) (C-C) = 147.0 +/- 0.05 pm, r(e) (C=C) = 133.7 +/- 0.1 pm, and r(e) (C-H) = 107.9 +/- 0.1 pm. The equilibrium C-C and C=C bond lengths of COT are compared to those of 1,3-butadiene. The expected effect of decreased pi-electron delocalization due to the twisting of adjacent C=C double bonds in COT relative to butadiene is observed for the C-C bonds but not for the C=C bonds.

Gas-Phase Cytosine and Cytosine-N1-Derivatives Have 0.1-1 ns Lifetimes Near the S1 State Minimum.

Ultraviolet radiative damage to DNA is inefficient because of the ultrafast S1 ⇝ S0 internal conversion of its nucleobases. Using picosecond pump-ionization delay measurements, we find that the S1((1)ππ*) state vibrationless lifetime of gas-phase keto-amino cytosine (Cyt) is τ = 730 ps or ∼ 700 times longer than that measured by femtosecond pump-probe ionization at higher vibrational excess energy, Eexc. N1-Alkylation increases the S1 lifetime up to τ = 1030 ps for N1-ethyl-Cyt but decreases it to 100 ps for N1-isopropyl-Cyt. Increasing the vibrational energy to Eexc = 300-550 cm(-1) decreases the lifetimes to 20-30 ps. The nonradiative dynamics of S1 cytosine is not solely a property of the amino-pyrimidinone chromophore but is strongly influenced by the N1-substituent. Correlated excited-state calculations predict that the gap between the S2((1)nOπ*) and S1((1)ππ*) states decreases along the series of N1-derivatives, thereby influencing the S1 state lifetime.

Concerted hydrogen-bond breaking by quantum tunneling in the water hexamer prism.

The nature of the intermolecular forces between water molecules is the same in small hydrogen-bonded clusters as in the bulk. The rotational spectra of the clusters therefore give insight into the intermolecular forces present in liquid water and ice. The water hexamer is the smallest water cluster to support low-energy structures with branched three-dimensional hydrogen-bond networks, rather than cyclic two-dimensional topologies. Here we report measurements of splitting patterns in rotational transitions of the water hexamer prism, and we used quantum simulations to show that they result from geared and antigeared rotations of a pair of water molecules. Unlike previously reported tunneling motions in water clusters, the geared motion involves the concerted breaking of two hydrogen bonds. Similar types of motion may be feasible in interfacial and confined water.

Molecular Structure and Chirality Detection by Fourier Transform Microwave Spectroscopy.

We describe a three-wave mixing experiment using time-separated microwave pulses to detect the enantiomer-specific emission signal of the chiral molecule using Fourier transform microwave (FTMW) spectroscopy. A chirped-pulse FTMW spectrometer operating in the 2-8 GHz frequency range is used to determine the heavy-atom substitution structure of solketal (2,2-dimethyl-1,3-dioxolan-4-yl-methanol) through analysis of the singly substituted (13)C and (18)O isotopologue rotational spectra in natural abundance. A second set of microwave horn antennas is added to the instrument design to permit three-wave mixing experiments where an enantiomer-specific phase of the signal is observed. Using samples of R-, S-, and racemic solketal, the properties of the three-wave mixing experiment are presented, including the measurement of the corresponding nutation curves to demonstrate the optimal pulse sequence.

Excited-state structure, vibrations, and nonradiative relaxation of jet-cooled 5-fluorocytosine.

The S0 → S1 vibronic spectrum and S1 state nonradiative relaxation of jet-cooled keto-amino 5-fluorocytosine (5FCyt) are investigated by two-color resonant two-photon ionization spectroscopy at 0.3 and 0.05 cm(­1) resolution. The 0(0)(0) rotational band contour is polarized in-plane, implying that the electronic transition is (1)ππ*. The electronic transition dipole moment orientation and the changes of rotational constants agree closely with the SCS-CC2 calculated values for the (1)ππ* (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' (HN­C6H twist) vibrations, implying that the pyrimidinone frame is distorted out-of-plane by the (1)ππ* excitation, in agreement with SCS-CC2 calculations. The number of vibronic bands rises strongly around +350 cm(­1); this is attributed to the (1)ππ* state barrier to planarity that corresponds to the central maximum of the double-minimum out-of-plane vibrational potentials along the ν1', ν2', and ν3' coordinates, which gives rise to a high density of vibronic excitations. At +1200 cm(­1), rapid nonradiative relaxation (k(nr) ≥ 10(12) 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 gas-phase lifetimes are twice those of S1 state cytosine and 10­100 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 ((1)ππ*) state is essentially controlled by the same ~1200 cm(­1) barrier and conical intersection both in the gas phase and in solution.

Switching on the fluorescence of 2-aminopurine by site-selective microhydration.

2-Aminopurine (2 AP) is a fluorescent isomer of adenine and has a fluorescence lifetime of ~11 ns in water. It is widely used in biochemical settings as a site-specific fluorescent probe of DNA and RNA structure and base-flipping and -folding. These assays assume that 2 AP is intrinsically strongly fluorescent. Here, we show this not to be the case, observing that gas-phase, jet-cooled 2-aminopurine and 9-methyl-2-aminopurine have very short fluorescence lifetimes (156 ps and 210 ps, respectively); they are, to all intents and purposes, non-fluorescent. We find that the lifetime of 2-aminopurine increases dramatically when it is part of a hydrate cluster, 2 AP · (H2O)n, where n = 1-3. Not only does it depend on the presence of water molecules, it also depends on the specific hydrogen-bonding site to which they attach and on the number of H2O molecules at that site. We selectively microhydrate 2-aminopurine at its sugar-edge, cis-amino or trans-amino sites and see that its fluorescence lifetime increases by 4, 50 and 95 times (to 14.5 ns), respectively.

Building up water-wire clusters: isomer-selective ultraviolet and infrared spectra of jet-cooled 2-aminopurine (H2O)n, n = 2 and 3.

2-Aminopurine (2AP) is an adenine analogue with a high fluorescence quantum yield in water solution, which renders it a useful real-time probe of DNA structure. We report the ultraviolet (UV) and infrared (IR) spectra of size-selected and jet-cooled 9H-2AP·(H2O)n clusters with n = 2 and 3. Mass- and species-specific UV/UV holeburning spectroscopy allows to separate the UV spectra of four cluster isomers in the 31,200­33,000 cm(­1) spectral region with electronic band origins at 31339, 31450, 31891, and 32163 cm(­1). Using IR/UV depletion spectroscopy in combination with B3LYP calculated harmonic vibrational frequencies, the H-bonding topologies of two isomers of the n = 2 and of two isomers of the n = 3 cluster are identified. One n = 2 isomer (denoted 2A) forms a water dimer chain between the N9H and N3 atoms at the sugar-edge site, the other isomer (denoted 2D) binds one H2O at the sugar-edge site and the other at the trans-amino site between the N1 atom and the NH2 group. For 2-aminopurine·(H2O)3, one isomer (denoted 3A) forms an H-bonded water wire at the sugar-edge site, while isomer 3B accommodates two H2O molecules at the sugar-edge and one at the trans-amino site. The approximate second-order coupled cluster (CC2) method predicts the adiabatic S1 ← S0 transitions of 9H-2-aminopurine and six water cluster isomers with n = 1­3 in very good agreement with the experimental 0(0)(0) frequencies, with differences of <0.6%. The stabilization of the S1(ππ*) state of 2-aminopurine by water clusters is highly regiospecific: Isomers with one or two H2O molecules H-bonded in the trans-amino position induce large spectra red shifts, corresponding to 1ππ* state stabilization of 10­12 kJ/mol, while water-wire cluster solvation at the sugar-edge leads to much smaller stabilization. The evolution of the IR spectra of the water-wire clusters with n = 1­3 that are H-bonded to the sugar-edge site is discussed. Qualitatively different regions (denoted I to IV) can be attributed to the different free and H-bonded OH, NH, NH2 and OH···OH water-wire stretch vibrations.

Excited-state structure and dynamics of keto-amino cytosine: the 1ππ* state is nonplanar and its radiationless decay is not ultrafast.

We have measured the mass- and tautomer-specific S0 → S1 vibronic spectra and S1 state lifetimes of the keto­amino tautomer of cytosine cooled in supersonic jets, using two-color resonant two-photon ionization (R2PI) spectroscopy at 0.05 cm(­1) resolution. The rotational contours of the 0(0)(0) band and nine vibronic bands up to +437 cm(­1) are polarized in the pyrimidinone plane, proving that the electronic excitation is to a 1ππ* state. All vibronic excitations up to +437 cm(­1) are overtone and combination bands of the low-frequency out-of-plane ν1' (butterfly), ν2' (boat), and ν3' (H­N­C6­H twist) vibrations. UV vibronic spectrum simulations based on approximate second-order coupled-cluster (CC2) calculations of the ground and 1ππ* states are in good agreement with the experimental R2PI spectrum, but only if the calculated ν1' and ν2' frequencies are reduced by a factor of 4 and anharmonicity is included. Together with the high intensity of the ν1' and ν2' overtone vibronic excitations, this implies that the 1ππ* potential energy surface is much softer and much more anharmonic in the out-of-plane directions than predicted by the CC2 method. The 1ππ* state lifetime is determined from the Lorentzian line broadening necessary to reproduce the rotational band contours: at the 0(0)(0) band it is τ = 44 ps, remains at τ = 35­45 ps up to +205 cm(­1), and decreases to 25­30 ps up to +437 cm(­1). These lifetimes are 20­40 times longer than the 0.5­1.5 ps lifetimes previously measured with femtosecond pump­probe techniques at higher vibrational energies (1500­3800 cm(­1)). Thus, the nonradiative relaxation rate of keto­amino cytosine close to the 1ππ* state minimum is k(nr) 2.5 × 10(10) s(­1), much smaller than at higher energies. An additional nonradiative decay channel opens at +500 cm(­1) excess energy. Since high overtone bands of ν1' and ν2' are observed in the R2PI spectrum but only a single weak 2ν3' band, we propose that ν3' is a promoting mode for nonradiative decay, consistent with the observation that the ν3' normal-mode eigenvector points toward the "C6-puckered" conical intersection geometry.

The Adiabatic Ionization Energy and Triplet T1 Energy of Jet-Cooled Keto-Amino Cytosine.

Gas-phase cytosine exists in five different tautomer/rotamer forms 1, 2a, 2b, 3a, and 3b. We determine the threshold ionization energy (IE) of the keto-amino tautomer 1 as 8.73 ± 0.02 eV, using resonant two-photon ionization mass spectrometry in a supersonic molecular beam via the (1)ππ* excited state. This is the first IE threshold measurement for the biologically relevant tautomer 1. The IE of the thermal gas-phase mixture of cytosine has been measured as 8.60 ± 0.05 eV by Kostko et al. using single-photon VUV photoionization [Phys. Chem. Chem. Phys., 2010, 12, 2860]. Given the tautomer distribution and ionization energies calculated in that work, our determination of the keto-amino tautomer IE implies that the IE measured by Kostko et al. is dominated by the enol-amino tautomers 2a and 2b. Upon excitation of keto-amino cytosine to its (1)ππ* state, relaxation occurs to a lower-lying long-lived state. The IE threshold measured via this state places its energy about 0.69 eV below the (1)ππ* state, in good agreement with the triplet T1 energy of keto-amino cytosine calculated by several high-level ab initio methods. The identification of keto-amino cytosine T1 is the basis for characterizing the intersystem crossing rates into and the photochemical reactions of this long-lived state.