Measured Infrared Multiple Photon Dissociation Action Spectra Research Articles

Influence of the local environment on the intrinsic structures of gas-phase cytidine-5′-monophosphates

Gas-phase conformations of the deprotonated disodium cationized 2′-deoxycytidine-5′-monophosphate and cytidine-5′-monophosphate nucleotides, [pdCyd−H+2Na]+ and [pCyd−H+2Na]+, are studied by infrared multiple photon dissociation (IRMPD) action spectroscopy. Analysis of the experimental results is assisted by complimentary electronic structure calculations of low-energy conformers at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory. These calculations provide relative energetics and predicted IR spectra of the calculated conformers for comparison to the measured IRMPD action spectra in the IR fingerprint and hydrogen-stretching regions. Comparisons between the predicted IR and measured IRMPD spectra provide insight into the conformations accessed by [pdCyd−H+2Na]+ and [pCyd−H+2Na]+ during the experiments. Comparison of these calculations and spectroscopic analysis with those performed in previous studies for cytidine nucleotides representing those present in different local environments allows for elucidation of the impact of the local environment on the intrinsic structure of these nucleotides. Comparison of these results with similar studies of the cytidine nucleosides also helps reveal the impact of the phosphate moiety on structure. Although several conformers of both [pdCyd−H+2Na]+ and [pCyd−H+2Na]+ are observed experimentally, a common sodium cation binding mode is observed, highlighting the importance of the stabilization it provides to the cytidine nucleotides.

Impact of the 2'- and 3'-Sugar Hydroxyl Moieties on Gas-Phase Nucleoside Structure.

Modified nucleosides have been an important target for pharmaceutical development for the treatment of cancer, herpes simplex virus, and the human immunodeficiency virus (HIV). Amongst these nucleoside analogues, those based on 2',3'-dideoxyribose sugars are quite common, particularly in anti-HIV applications. The gas-phase structures of several protonated 2',3'-dideoxyribose nucleosides are examined in this work and compared with those of the analogous protonated DNA, RNA, and arabinose nucleosides to elucidate the influence of the 2'- and combined 2',3'-hydroxyl groups on intrinsic structure. Infrared multiple photon dissociation (IRMPD) action spectra are collected for the protonated 2',3'-dideoxy forms of adenosine, guanosine, cytidine, thymidine and uridine, [ddAdo+H]+, [ddGuo+H]+, [ddCyd+H]+, [ddThd+H]+, and [ddUrd+H]+, in the IR fingerprint and hydrogen-stretching regions. Molecular mechanics conformational searching followed by electronic structure calculations generates low-energy conformers of the protonated 2',3'-dideoxynucleosides and corresponding predicted linear IR spectra to facilitate interpretation of the measured IRMPD action spectra. These experimental IRMPD spectra and theoretical calculations indicate that the absence of the 2'- and 3'-hydroxyls largely preserves the protonation preferences of the canonical forms. The spectra and calculated structures indicate a slight preference for C3'-endo sugar puckering. The presence of the 3'- and further 2'-hydroxyl increases the available intramolecular hydrogen-bonding opportunities and shifts the sugar puckering modes for all nucleosides but the guanosine analogues to a slight preference for C2'-endo over C3'-endo. Graphical Abstract.

N3 and O2 Protonated Conformers of the Cytosine Mononucleotides Coexist in the Gas Phase.

The gas-phase conformations of the protonated forms of the DNA and RNA cytosine mononucleotides, [pdCyd+H]+ and [pCyd+H]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy over the IR fingerprint and hydrogen-stretching regions complemented by electronic structure calculations. The low-energy conformations of [pdCyd+H]+ and [pCyd+H]+ and their relative stabilities are computed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers allow the conformers present in the experiments to be determined. Similar to that found in previous IRMPD action spectroscopy studies of the protonated forms of the cytosine nucleosides, [dCyd+H]+ and [Cyd+H]+, both N3 and O2 protonated cytosine mononucleotides exhibiting an anti orientation of cytosine are found to coexist in the experimental population. The 2'-hydroxyl substituent does not significantly influence the most stable conformations of [pCyd+H]+ versus those of [pdCyd+H]+, as the IRMPD spectral profiles of [pdCyd+H]+ and [pCyd+H]+ are similar. However, the presence of the 2'-hydroxyl substituent does influence the relative intensities of the measured IRMPD bands. Comparisons to IRMPD spectroscopy studies of the deprotonated forms of the cytosine mononucleotides, [pdCyd-H]- and [pCyd-H]-, provide insight into the effects of protonation versus deprotonation on the conformational features of the nucleobase and sugar moieties. Likewise, comparisons to results of IRMPD spectroscopy studies of the protonated cytosine nucleosides provide insight into the influence of the phosphate moiety on structure. Comparison with previous ion mobility results shows the superiority of IRMPD spectroscopy for distinguishing various protonation sites. Graphical Abstract ᅟ.

The intrinsic basicity of the phosphate backbone exceeds that of uracil and thymine residues: protonation of the phosphate moiety is preferred over the nucleobase for pdThd and pUrd.

The gas-phase conformations of the protonated forms of thymidine-5'-monophosphate and uridine-5'-monophosphate, [pdThd+H]+ and [pUrd+H]+, are investigated by infrared multiple photon dissociation (IRMPD) action spectroscopy and electronic structure calculations. The IRMPD action spectra of [pdThd+H]+ and [pUrd+H]+ are measured over the IR fingerprint and hydrogen-stretching regions using the FELIX free electron laser and an OPO/OPA laser system. Low-energy conformations of [pdThd+H]+ and [pUrd+H]+ and their relative stabilities are computed at the MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers indicate that the dominant conformers of [pdThd+H]+ and [pUrd+H]+ populated in the experiments are protonated at the phosphate oxo oxygen atom, with a syn nucleobase orientation that is stabilized by strong P[double bond, length as m-dash]OH+O2 and P-OHO4' hydrogen-bonding interactions, and C2'-endo sugar puckering. Minor abundance of conformers protonated at the O2 carbonyl of the nucleobase residue may also contribute for [pdThd+H]+, but do not appear to be important for [pUrd+H]+. Comparisons to previous IRMPD spectroscopy investigations of the protonated forms of thymidine and uridine, [dThd+H]+ and [Urd+H]+, and the deprotonated forms of pdThd and pUrd, [pdThd-H]- and [pUrd-H]-, provide insight into the effects of the phosphate moiety and protonation on the conformational features of the nucleobase and sugar moieties. Most interestingly, the thymine and uracil nucleobases remain in their canonical forms for [pdThd+H]+ and [pUrd+H]+, unlike [dThd+H]+ and [Urd+H]+, where protonation occurs on the nucleobases and induces tautomerization of the thymine and uracil residues.

Influence of Sodium Cationization versus Protonation on the Gas-Phase Conformations and Glycosidic Bond Stabilities of 2'-Deoxyadenosine and Adenosine.

The influence of noncovalent interactions with a sodium cation on the gas-phase structures and N-glycosidic bond stabilities of 2'-deoxyadenosine (dAdo) and adenosine (Ado), [dAdo+Na](+) and [Ado+Na](+), are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and energy-resolved collision-induced dissociation (ER-CID) experiments. ER-CID experiments are also performed on the protonated forms of these nucleosides, [dAdo+H](+) and [Ado+H](+), for comparison purposes. Complementary electronic structure calculations are performed to determine the structures and relative stabilities of the stable low-energy conformations of the sodium cationized nucleoside complexes and to predict their IR spectra. Comparison between the measured IRMPD action spectra and calculated IR spectra enables the conformations of the sodium cationized nucleosides present in the experiments to be elucidated. The influence of sodium cationization versus protonation on the structures and IR spectra is elucidated by comparison to IRMPD and theoretical results previously reported for the protonated forms of these nucleosides. The influence of sodium cationization versus protonation on the glycosidic bond stability of the adenine nucleosides is determined by comparison of the ER-CID behavior of these systems. All structures present in the experiments are found to involve tridentate binding of Na(+) to the N3, O4', and O5' atoms forming favorable 5- and 6-membered chelation rings, which requires that adenine rotate to a syn configuration. This mode of sodium cation binding results in moderate flexibility of the sugar moiety such that the sugar puckering of the conformations present varies between C2'-endo and O4'-endo. Sodium cationization is found to be less effective toward activating the N-glycosidic bond than protonation for both dAdo and Ado. Both the IRMPD yields and ER-CID behavior indicate that the 2'-hydroxyl substituent of Ado stabilizes the N-glycosidic bond relative to that of dAdo.

Protonation induces base rotation of purine nucleotides pdGuo and pGuo.

Infrared multiple photon dissociation (IRMPD) action spectra of the protonated forms of 2'-deoxyguanosine-5'-monophosphate and guanosine-5'-monophosphate, [pdGuo+H](+) and [pGuo+H](+), are measured over the IR fingerprint and hydrogen-stretching regions using the FELIX free electron laser and an OPO/OPA laser system. Electronic structure calculations are performed to generate low-energy conformations of [pdGuo+H](+) and [pGuo+H](+) and determine their relative stabilities at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparative analyses of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers are performed to determine the most favorable site of protonation and the conformers present in the experiments. These comparisons and the computed energetics find that N7 protonation is considerably preferred over O6 and N3, and the N7 protonated ground-state conformers of [pdGuo+H](+) and [pGuo+H](+) are populated in the experiments. The 2'-hydroxyl substituent does not significantly impact the stable low-energy conformers of [pdGuo+H](+)vs. those of [pGuo+H](+). The effect of the 2'-hydroxyl substituent is primarily reflected in the relative intensities of the measured IRMPD bands, as the IRMPD profiles of [pdGuo+H](+) and [pGuo+H](+) are quite similar. Comparisons to previous IRMPD spectroscopy investigations of the protonated forms of the guanine nucleosides, [dGuo+H](+) and [Guo+H](+), and deprotonated forms of the guanine nucleotides, [pdGuo-H](-) and [pGuo-H](-), provide insight into the effects of the phosphate moiety and protonation on the conformational features of the nucleobase and sugar moieties. Protonation is found to induce base rotation of the guanine residue to an anti orientation vs. the syn orientation found for the deprotonated forms of the guanine nucleotides.

Infrared multiple photon dissociation action spectroscopy of sodium cationized halouracils: Effects of sodium cationization and halogenation on gas-phase conformation

The gas-phase structures of sodium cationized complexes of 5- and 6-halo-substituted uracils are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. The halouracils examined in this investigation include: 5-flourouracil, 5-chlorouracil, 5-bromouracil, 5-iodouracil, and 6-chlorouracil. Experimental IRMPD action spectra of the sodium cationized halouracil complexes are measured using a 4.7T Fourier transform ion cyclotron resonance mass spectrometer coupled to the FELIX free electron laser (FEL). Irradiation of the mass selected sodium cationized halouracil complexes by the FEL was carried out over the range of frequencies extending from 950 to 1900cm−1. Theoretical linear IR spectra predicted for the stable low-energy conformations of the sodium cationized halouracils, calculated at B3LYP/6-31G(d) level of theory, are compared with the measured IRMPD action spectra to identify the structures accessed in the experiments. Relative stabilities of the low-energy conformations are determined from single-point energy calculations performed at the B3LYP/6-311+G(2d,2p) level of theory. The evolution of IRMPD spectral features as a function of the size (F, Cl, Br, and I) and position (5 versus 6) of the halogen substituent are examined to elucidate the effects of the halogen substituent and noncovalent interactions with sodium cations on the structure of the nucleobase. Present results are compared with results from energy-resolved collision-induced dissociation and IRMPD action spectroscopy studies previously reported for the protonated and sodium cationized forms of uracil, and halo-, methyl-, and thioketo-substituted uracils. The present results suggest that only a single conformer is accessed for all of the 5-halouracil complexes, whereas multiple conformers are accessed for the Na+(6ClU) complex. In all cases, the experimental IRMPD action spectra confirm that the sodium cation binds to the O4 carbonyl oxygen atom of the canonical diketo tautomer in the ground-state conformers, and gains additional stabilization via chelation interactions with the halogen substituent in the complexes to the 5-halouracils as predicted by theory.

Infrared multiple photon dissociation action spectroscopy of alkali metal cation–cyclen complexes: Effects of alkali metal cation size on gas-phase conformation

The gas-phase structures of alkali metal cationized complexes of cyclen (1,4,7,10-tetraazacyclododecane) are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and electronic structure theory calculations. The measured IRMPD action spectra of four M+(cyclen) complexes are compared to IR spectra predicted for the stable low-lying conformers of these complexes calculated at the B3LYP/def2-TZVPPD level of theory to identify the structures accessed in the experiments. The IRMPD yields of the M+(cyclen) complexes investigated increase as the size of the alkali metal cation increases, in accordance with the decrease in the strength of alkali metal cation binding. The IRMPD spectrum of the Na+(cyclen) complex is relatively simple, and the features observed are retained for all of the other alkali metal cation–cyclen complexes. New spectral features begin to appear for K+(cyclen) and become very obvious for the Rb+(cyclen) and Cs+(cyclen) complexes. The IRMPD action spectra for the complexes of cyclen to K+, Rb+, and Cs+ are well reproduced by the calculated spectra predicted for the most stable conformations computed. Overall comparisons suggest that only the ground-state conformations of the M+(cyclen) complexes were accessed in the experiments for the complexes to Na+ and K+, whereas evidence for a very small population of the first excited conformers is observed for the complexes to Rb+ and Cs+.

Infrared Multiple Photon Dissociation Action Spectroscopy of Deprotonated DNA Mononucleotides: Gas-Phase Conformations and Energetics

The gas phase structures of the deprotonated 2'-deoxymononucleotides including 2'-deoxyadenosine-5'-monophosphate (dA5'p), 2'-deoxycytidine-5'-monophosphate (dC5'p), 2'-deoxyguanosine-5'-monophosphate (dG5'p), and thymidine-5'-monophosphate (T5'p) are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. The measured IRMPD action spectra of all four deprotonated DNA mononucleotides exhibit unique spectral features in the region extending from ~600 to 1800 cm(-1) such that they can be readily differentiated from one another. The measured IRMPD action spectra are compared to the linear IR spectra calculated at the B3LYP/6-311+G(d,p) level of theory to determine the conformations of these species accessed in the experiments. On the basis of these comparisons and the computed energetic information, the most stable conformations of the deprotonated forms of dA5'p, dC5'p, and T5'p are conformers where the ribose moiety adopts a C3' endo conformation and the nucleobase is in an anti conformation. By contrast, the most stable conformations of the deprotonated form of dG5'p are conformers where the ribose adapts a C3' endo conformation and the nucleobase is in a syn conformation. In addition to the ground-state conformers, several stable low-energy excited conformers that differ slightly in the orientation of the phosphate ester moiety were also accessed in the experiments.

Protonation Preferentially Stabilizes Minor Tautomers of the Halouracils: IRMPD Action Spectroscopy and Theoretical Studies

Tautomerization induced by protonation of halouracils may increase their efficacy as anti-cancer drugs by altering their reactivity and hydrogen bonding characteristics, potentially inducing errors during DNA and RNA replication. The gas-phase structures of protonated complexes of five halouracils, including 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 5-iodouracil, and 6-chlorouracil are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. IRMPD action spectra were measured for each complex in the IR fingerprint region extending from ~1000 to 1900 cm(-1) using the free electron laser (FELIX). Correlations are made between the measured IRMPD action spectra and the linear IR spectra for the stable low-energy tautomeric conformations computed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-31G* level of theory. Absence of an intense band(s) in the IRMPD spectrum arising from the carbonyl stretch(es) that are expected to appear near 1825 cm(-1) provides evidence that protonation induces tautomerization and preferentially stabilizes alternative, noncanonical tautomers of these halouracils where both keto functionalities are converted to hydroxyl groups upon binding of a proton. The weak, but measurable absorption, which does occur for these systems near 1835 cm(-1) suggests that in addition to the ground-state conformer, very minor populations of excited, low-energy conformers that contain keto functionalities are also present in these experiments.

Infrared Multiple Photon Dissociation Action Spectroscopy and Theoretical Studies of Triethyl Phosphate Complexes: Effects of Protonation and Sodium Cationization on Structure

The gas-phase structures of protonated and sodium cationized complexes of triethyl phosphate, [TEP + H](+) and [TEP + Na](+), are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy using tunable IR radiation generated by a free electron laser, a Fourier transform ion cyclotron resonance mass spectrometer with an electrospray ionization source, and theoretical electronic structure calculations. Measured IRMPD action spectra are compared to linear IR spectra calculated at the B3LYP/6-31 G(d,p) level of theory to identify the structures accessed in the experimental studies. For comparison, theoretical studies of neutral TEP are also performed. Sodium cationization and protonation produce changes in the central phosphate geometry, including an increase in the alkoxy ∠OPO bond angle and shortening of the alkoxy P-O bond. Changes associated with protonation are more pronounced than those produced by sodium cationization.

Infrared multiple photon dissociation action spectroscopy of sodiated uracil and thiouracils: Effects of thioketo-substitution on gas-phase conformation

The gas phase structures of sodium cationized complexes of uracil and five thiouracils including 2-thiouracil (2SU), 5-methyl-2-thiouracil (5Me2SU), 6-methyl-2-thiouracil (6Me2SU), 4-thiouracil (4SU), and 2,4-dithiouracil (24dSU) are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. The IRMPD spectra of all six sodium cationized complexes exhibit both characteristic and unique spectral features over the range from ∼1000 to 1900 cm −1 such that the complexes are easily differentiated. The intense band at ∼1800 cm −1 in the IRMPD action spectrum of Na +(U) indicates that, as expected, a free carbonyl group is present in this complex. Absence of an intense band at ∼1800 cm −1 in the IRMPD action spectra for Na +(2SU), Na +(5Me2SU), Na +(6Me2SU), and Na +(4SU) complexes suggests that either sodium cationization preferentially stabilizes a minor tautomer of the nucleobase, or that the sodium cation binds to the keto group in these complexes, such that no free carbonyl stretch is observed. Measured IRMPD action spectra are compared to linear IR spectra calculated at the B3LYP/6-31G(d) level of theory to identify the structures accessed in the experimental studies. Based on these comparisons and the energetic predictions from theory, sodium cations preferentially bind at the 4-keto position of the canonical 2,4-diketo or 2-thioketo-4-keto tautomer in the Na +(U), Na +(2SU), Na +(5Me2SU), and Na +(6Me2SU) complexes. In contrast, sodium cationization results in preferential stabilization of a minor tautomer of the nucleobase in the Na +(4SU) and Na +(24dSU) complexes, where proton transfer from the N3H group to the 4-thioketo group, facilitates strong binding of the sodium cation via chelation with the O2(S2) and N3 atoms.

Infrared multiple photon dissociation action spectroscopy of protonated uracil and thiouracils: Effects of thioketo-substitution on gas-phase conformation

The gas-phase structures of protonated complexes of uracil and five thiouracils including 2-thiouracil (2SU), 5-methyl-2-thiouracil (5Me2SU), 6-methyl-2-thiouracil (6Me2SU), 4-thiouracil (4SU), and 2,4-dithiouracil (24dSU) are examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. IRMPD action spectra of all six protonated complexes exhibit both similar and distinct spectral features over the range of ∼1000–1900 cm −1, such that the complexes are easily differentiated by their IRMPD action spectra. Absence of the carbonyl stretch at ∼1825 cm −1 in the IRMPD spectra for the H +(U), H +(2SU), H +(5Me2SU), and H +(6Me2SU) complexes suggests that the binding of a proton preferentially stabilizes alternative tautomers of the nucleobases in these complexes such that no free carbonyl stretch is observed. In contrast, the intense band at ∼1825 cm −1 in the IRMPD action spectrum of H +(4SU) indicates that a free carbonyl group is still present in this complex. Measured IRMPD action spectra are compared to linear IR spectra calculated at the B3LYP/6-31G(d) level of theory to identify the structures accessed in the experimental studies. On the basis of these comparisons and energetics derived from the calculations, protonation results in preferential stabilization of a minor tautomer of the nucleobase in the H +(U), H +(2SU), H +(5Me2SU), H +(6Me2SU), and H +(24dSU) complexes, where both keto (thioketo) groups are converted to hydroxy (sulfhydryl) groups by proton binding and proton transfer from the neighboring N3H group. In contrast, the proton preferentially binds at the 4-thioketo position to the canonical keto–thioketo tautomer in the H +(4SU) complex. Additional band(s) are present in the IRMPD action spectra of the H +(U) and H +(4SU) complexes that suggest that a small population of excited low-energy conformers are also accessed in those systems.