OH Radical-Induced Oxidation in Nucleosides and Nucleotides Unraveled by Tandem Mass Spectrometry and Infrared Multiple Photon Dissociation Spectroscopy.

OH⋅-induced oxidation products of DNA nucleosides and nucleotides have been structurally characterized by collision-induced dissociation tandem mass spectrometry (CID-MS2 ) and Infrared Multiple Photon Dissociation (IRMPD) spectroscopy. CID-MS2 results have shown that the addition of one oxygen atom occurs on the nucleobase moiety. The gas-phase geometries of +16 mass increment products of 2'-deoxyadenosine (dA(O)H+ ), 2'-deoxyadenosine 5'-monophosphate (dAMP(O)H+ ), 2'-deoxycytidine (dC(O)H+ ), and 2'-deoxycytidine 5'-monophosphate (dCMP(O)H+ ) are extensively investigated by IRMPD spectroscopy and quantum-chemical calculations. We show that a carbonyl group is formed at the C8 position after oxidation of 2'-deoxyadenosine and its monophosphate derivative. For 2'-deoxycytidine and its monophosphate derivative, the oxygen atom is added to the C5 position to form a C-OH group. IRMPD spectroscopy has been employed for the first time to provide direct structural information on oxidative lesions in DNA model systems.

Binding Motifs of Carboplatin and Oxaliplatin with Guanine: A Combined MS/MS, IRMPD, and Theoretical Study.

Complexes generated in the gas phase involving the purine nucleobase guanine bound to second and third generation platinum drugs, namely, carboplatin (CarboPt) and oxaliplatin (OxaliPt), were investigated by combining tandem mass spectrometry, collision-induced dissociation (CID), infrared multiple photon dissociation spectroscopy (IRMPD), and density functional theory (DFT) calculations. As the first step, a spectroscopic characterization of the protonated platinum drugs was accomplished. Protonation of both CarboPt and OxaliPt in the gas phase occurs on one of the two carbonyl groups of the cyclobutanedicarboxylate and oxalate ligand, respectively. Such protonation has been postulated by several theoretical studies as a key preliminary step in the hydrolysis of Pt drugs under acidic conditions. Subsequently, the protonated drugs react with guanine in solution to generate a complex of general formula [Pt drug + H + guanine]+, which was then mass-selected. CID experiments provided evidence of the presence of strong binding between guanine and platinum-based drugs within the complexes. The structures of the two complexes have also been examined by comparing the experimental IRMPD spectra recorded in two spectral regions with DFT-computed IR spectra. For each system, the IRMPD spectra agree with the vibrational spectra calculated for the global minimum structures, which present a monodentate complexation of Pt at the N7 position of canonical guanine. This binding scheme is therefore akin to that observed for cisplatin, while other coordination sites yield substantially less stable species. Interestingly, in the case of oxaliplatin, the IRMPD spectra are consistent with the presence of two isomeric forms very close in energy.

Applications of Infrared Multiple Photon Dissociation (IRMPD) to the Detection of Posttranslational Modifications.

Infrared multiple photon dissociation (IRMPD) spectroscopy allows for the derivation of the vibrational fingerprint of molecular ions under tandem mass spectrometry (MS/MS) conditions. It provides insight into the nature and localization of posttranslational modifications (PTMs) affecting single amino acids and peptides. IRMPD spectroscopy, which takes advantage of the high sensitivity and resolution of MS/MS, relies on a wavelength specific fragmentation process occurring on resonance with an IR active vibrational mode of the sampled species and is well suited to reveal the presence of a PTM and its impact in the molecular environment. IRMPD spectroscopy is clearly not a proteomics tool. It is rather a valuable source of information for fixed wavelength IRMPD exploited in dissociation protocols of peptides and proteins. Indeed, from the large variety of model PTM containing amino acids and peptides which have been characterized by IRMPD spectroscopy, specific signatures of PTMs such as phosphorylation or sulfonation can be derived. High throughput workflows relying on the selective fragmentation of modified peptides within a complex mixture have thus been proposed. Sequential fragmentations can be observed upon IR activation, which do not only give rise to rich fragmentation patterns but also overcome low mass cutoff limitations in ion trap mass analyzers. Laser-based vibrational spectroscopy of mass-selected ions holding various PTMs is an increasingly expanding field both in the variety of chemical issues coped with and in the technological advancements and implementations.

Inter- and Intramolecular Proton Transfer in an Isolated (Cytosine-Guanine)H+ Pair: Direct Evidence from IRMPD Spectroscopy.

The collision-induced dissociation of the protonated cytosine-guanine pair was studied using tandem mass spectrometry (MS3) coupled to infrared multiple photon dissociation spectroscopy with the free electron laser at Orsay (CLIO) to determine the structure of the CH+ and GH+ ionic fragments. The results were rationalized with the help of electronic structure calculations at the density functional theory level with the B3LYP/6-311++G(3df,2p) method. Several tautomers of each fragment were identified for the first time, some of which were previously predicted by other authors. In addition, two unexpected and minor tautomers were also found: cytosine keto-imino [CKI(1,2,3,4)H+] and guanine keto-amino [GKA(1,3,7)H+]. These results highlight the importance of the DNA base tautomerization assisted by inter- and intramolecular proton or hydrogen transfer within the protonated pairs.

Binding Motifs in the Naked Complexes of Target Amino Acids with an Excerpt of Antitumor Active Biomolecule: An Ion Vibrational Spectroscopy Assay.

The structures of proton-bound complexes of 5,7-dimethoxy-4H-chromen-4-one (1) and basic amino acids (AAs), namely, histidine (His) and lysine (Lys), have been examined by means of mass spectrometry coupled with IR ion spectroscopy and quantum chemical calculations. This selection of systems is based on the fact that 1 represents a portion of glabrescione B, a natural small molecule of promising antitumor activity, while His and Lys are protein residues lining the cavity of the alleged receptor binding site. These species are thus a model of the bioactive adduct, although clearly the isolated state of the present study bears little resemblance to the complex biological environment. A common feature of [1+AA+H]+ complexes is the presence of a protonated AA bound to neutral 1, in spite of the fact that the gas-phase basicity of 1 is comparable to those of Lys and His. The carbonyl group of 1 acts as a powerful hydrogen-bond acceptor. Within [1+AA+H]+ the side-chain substituents (imidazole group for His and terminal amino group for Lys) present comparable basic properties to those of the α-amino group, taking part to a cooperative hydrogen-bond network. Structural assignment, relying on the comparative analysis of the infrared multiple photon dissociation (IRMPD) spectrum and calculated IR spectra for the candidate geometries, derives from an examination over two frequency ranges: 900-1800 and 2900-3700 cm-1 . Information gained from the latter one proved especially valuable, for example, pointing to the contribution of species characterized by an unperturbed carboxylic OH or imidazole NH stretching mode.

Molecular Properties of Bare and Microhydrated Vitamin B5-Calcium Complexes.

Pantothenic acid, also called vitamin B5, is an essential nutrient involved in several metabolic pathways. It shows a characteristic preference for interacting with Ca(II) ions, which are abundant in the extracellular media and act as secondary mediators in the activation of numerous biological functions. The bare deprotonated form of pantothenic acid, [panto-H]-, its complex with Ca(II) ion, [Ca(panto-H)]+, and singly charged micro-hydrated calcium pantothenate [Ca(panto-H)(H2O)]+ adduct have been obtained in the gas phase by electrospray ionization and assayed by mass spectrometry and IR multiple photon dissociation spectroscopy in the fingerprint spectral range. Quantum chemical calculations at the B3LYP(-D3) and MP2 levels of theory were performed to simulate geometries, thermochemical data, and linear absorption spectra of low-lying isomers, allowing us to assign the experimental absorptions to particular structural motifs. Pantothenate was found to exist in the gas phase as a single isomeric form showing deprotonation on the carboxylic moiety. On the contrary, free and monohydrated calcium complexes of deprotonated pantothenic acid both present at least two isomers participating in the gas-phase population, sharing the deprotonation of pantothenate on the carboxylic group and either a fourfold or fivefold coordination with calcium, thus justifying the strong affinity of pantothenate for the metal.

Infrared-Assisted Synthesis of Prebiotic Glycine.

A novel approach has been developed to synthesize complex organic molecules (COMs) relevant to prebiotic chemistry, using infrared (IR) radiation to trigger the reaction. An original laboratory reactor working at low gas density and using IR irradiation was developed. In this way, glycine, the simplest brick of life, has been synthesized by assisting ion-molecule reaction with IR laser light. The ion-molecule complex constituted by acetic acid and hydroxylamine was formed in a mass spectrometer reactor and then irradiated with IR photons. As photoproducts, we obtained both glycine structures and some of its isomers. Anharmonic vibrational frequency calculations and fragmentation dynamics simulations allow for a better interpretation of the experimental data. This novel approach can be now extended to study other new synthetic pathways responsible for the formation of further COMs also with potential prebiotic relevance.

Effects of complexation with sulfuric acid on the photodissociation of protonated Cinchona alkaloids in the gas phase.

The effect of complexation with sulfuric acid on the photo-dissociation of protonated Cinchona alkaloids, namely cinchonidine (Cd), quinine (Qn) and quinidine (Qd), is studied by combining laser spectroscopy with quantum chemical calculations. The protonated complexes are structurally characterized in a room-temperature ion trap by means of infra-red multiple photon dissociation (IRMPD) spectroscopy in the fingerprint and the ν(XH) (X = C, N, O) stretch regions. Comparison with density functional theory calculations including dispersion (DFT-D) unambiguously shows that the complex consists of a doubly protonated Cinchona alkaloid strongly bound to a bisulfate HSO4- anion, which bridges the two protonated sites of the Cinchona alkaloid. UV excitation of the complex does not induce loss of specific photo fragments, in contrast to the protonated monomer or dimer, for which photo-specific fragments were observed. Indeed the UV-induced fragmentation pattern is identical to that observed in collision-induced dissociation experiments. Analysis of the nature of the first electronic transitions at the second order approximate coupled-cluster level (CC2) explains the difference in the behavior of the complex relative to the monomer or dimer towards UV excitation.

l-Cysteine Modified by S-Sulfation: Consequence on Fragmentation Processes Elucidated by Tandem Mass Spectrometry and Chemical Dynamics Simulations.

Low-energy collision-induced dissociation (CID) of deprotonated l-cysteine S-sulfate, [cysS-SO3]-, delivered in the gas phase by electrospray ionization, has been found to provide a means to form deprotonated l-cysteine sulfenic acid, which is a fleeting intermediate in biological media. The reaction mechanism underlying this process is the focus of the present contribution. At the same time, other novel species are formed, which were not observed in previous experiments. To understand fragmentation pathways of [cysS-SO3]-, reactive chemical dynamics simulations coupled with a novel algorithm for automatic determination of intermediates and transition states were performed. This approach has allowed the identification of the mechanisms involved and explained the experimental fragmentation pathways. Chemical dynamics simulations have shown that a roaming-like mechanism can be at the origin of l-cysteine sulfenic acid.

Vibrational signatures of curcumin's chelation in copper(II) complexes: An appraisal by IRMPD spectroscopy.

Curcumin (Cur) is a natural polyphenol with a wide spectrum of biological activities and appealing therapeutic potential. Herein, it has been delivered by electrospray ionization as gaseous protonated species, [Cur + H]+, and as a Cu(ii) complex, [Cu(Cur - H)]+, a promising antioxidant and radical scavenger. The gas phase structures were assayed by infrared multiple photon dissociation (IRMPD) spectroscopy in both the fingerprint (800-2000 cm-1) and hydrogen stretching (3100-3750 cm-1) ranges. Comparison between the experimental features and linear IR spectra of the lowest energy structures computed at the B3LYP/6-311+G(d,p) level reveals that bare [Cu(Cur - H)]+ exists in a fully planar and symmetric arrangement, where the metal interacts with the two oxygens of the syn-enolate functionality of deprotonated Cur and both OCH3 groups are engaged in H-bonding with the ortho OH. The effect of protonation on the energetic and geometric determinants of Cur has been explored as well, revealing that bare [Cur + H]+ may exist as a mixture of two close-lying isomers associated with the most stable binding motifs. The additional proton is bound to either the diketo or the keto-enol configuration of Cur, in a bent or nearly planar arrangement, respectively.

Strong intramolecular hydrogen bonding in protonated ß-methylaminoalanine: A vibrational spectroscopic and computational study.

The gas-phase structure of protonated ß-methylaminoalanine was investigated using infrared multiple photon dissociation spectroscopy in the C-H, N-H, O-H stretching region (2700-3800 cm-1) and the fingerprint region (1000-1900 cm-1). Calculations using density functional theory methods show that the lowest energy structures prefer protonation of the secondary amine. Formation of hydrogen bonds between the primary and secondary amine, and the secondary amine and carboxylic oxygen further stabilize the lowest energy structure. The infrared spectrum of the lowest energy structure originating with harmonic density functional theory has features that generally match the positions of the experimental spectra; however, the overall agreement with the experimental spectrum is poor. Molecular dynamics calculations were used to generate a gas-phase infrared spectrum. With these calculations a reasonable match with the experimental spectrum, especially in the high-energy region, was obtained. The results of the molecular dynamics simulation support the density functional theory calculations, with protonation of the secondary amine and the formation of a hydrogen bond between the protonated secondary amine and the primary amine. This work shows the importance of accounting for anharmonic effects in systems with very strong intramolecular hydrogen bonding.

Complexation of halide ions to tyrosine: role of non-covalent interactions evidenced by IRMPD spectroscopy.

The binding motifs in the halide adducts with tyrosine ([Tyr + X]-, X = Cl, Br, I) have been investigated and compared with the analogues with 3-nitrotyrosine (nitroTyr), a biomarker of protein nitration, in a solvent-free environment by mass-selected infrared multiple photon dissociation (IRMPD) spectroscopy over two IR frequency ranges, namely 950-1950 and 2800-3700 cm-1. Extensive quantum chemical calculations at B3LYP, B3LYP-D3 and MP2 levels of theory have been performed using the 6-311++G(d,p) basis set to determine the geometry, relative energy and vibrational properties of likely isomers and interpret the measured spectra. A diagnostic carbonyl stretching band at ∼1720 cm-1 from the intact carboxylic group characterizes the IRMPD spectra of both [Tyr + X]- and [nitroTyr + X]-, revealing that the canonical isomers (maintaining intact amino and carboxylic functions) are the prevalent structures. The spectroscopic evidence reveals the presence of multiple non-covalent forms. The halide complexes of tyrosine conform to a mixture of plane and phenol isomers. The contribution of phenol-bound isomers is sensitive to anion size, increasing from chloride to iodide, consistent with the decreasing basicity of the halide, with relative amounts depending on the relative energies of the respective structures. The stability of the most favorable phenol isomer with respect to the reference plane geometry is in fact 1.3, -2.1, -6.8 kJ mol-1, for X = Cl, Br, I, respectively. The change in π-acidity by ring nitration also stabilizes anion-π interactions yielding ring isomers for [nitroTyr + X]-, where the anion is placed above the face of the aromatic ring.

Undervalued N3 Coordination Revealed in the Cisplatin Complex with 2'-Deoxyadenosine-5'-monophosphate by a Combined IRMPD and Theoretical Study.

The complex obtained by the reaction of cisplatin and 2'-deoxyadenosine-5'-monophosphate (5'-dAMP) in water has been isolated and detected by electrospray ionization mass spectrometry. The so-formed cis-[PtCl(NH3)2(5'-dAMP)]+ complex has been studied in detail by infrared multiple photon dissociation (IRMPD) spectroscopy in two spectral ranges, namely, 700-1900 and 2800-3800 cm-1, backed by quantum-chemical calculations at the B3LYP/LACV3P/6-311G** level of theory. In agreement with the computational results, the vibrational spectroscopic characterization of cis-[PtCl(NH3)2(5'-dAMP)]+ shows that the sampled ionic population comprises two major isomers, differentiated in the X-H stretching region by their distinct fragmentation patterns. One of these species presents coordination of the platinum moiety at the N3 position of adenine, whereas in the second one, platinum is bound at the N1 position of adenine. IRMPD kinetics have allowed an estimation of their relative proportions. Surprisingly, the most abundant component of cis-[PtCl(NH3)2(5'-dAMP)]+ is the N3 isomer, although it is slightly less stable than the other potential isomers in the gas phase. In contrast, the lowest-energy species, namely, the one showing cisplatin binding to the N7 position of adenine, seems to be the one less represented in the sampled ionic population. These findings suggest that the reaction of cisplatin with 5'-dAMP is governed by the kinetics of the process occurring in solution rather than by the thermodynamic factors.

Experimental and Computational Investigation of Salophen-Zn Gas Phase Complexes with Cations: A Source of Possible Interference in Anionic Recognition.

We explore the possibility that protonated molecular ions might be an unexpected source of interference in the recognition process of anions and neutral species by Zn-salophen receptors. Zn-salophen complexes are known to bind anions and neutral molecules in solution. We present here evidence (from computational work and IRMPD spectroscopy) that these complexes can also be the binding site for protonated pyridine or quinuclidine. The resulting binding pattern does not involve the Zn ion, but one of the oxygen atoms directly attached to it. The resulting complex therefore turns out to have a positive charge adjacent to the Zn-salophen binding site. This finding seems to point to the existence of an interfering factor in the quantification of the experimental data about the association constant.

Does the Residues Chirality Modify the Conformation of a Cyclo-Dipeptide? Vibrational Spectroscopy of Protonated Cyclo-diphenylalanine in the Gas Phase.

The structure of a protonated diketopiperazine dipeptide, cyclo-diphenylalanine, is studied by means of infrared multiple photon dissociation spectroscopy combined with quantum chemical calculations. Protonation exclusively occurs on the oxygen site and, in the most stable conformer, results to an intramolecular OH···π interaction, accompanied by a CH···π interaction. Higher-energy conformers with free OH and NH···π interactions are observed as well, due to kinetic trapping. Optimization of the intramolecular interactions involving the aromatic ring dictates the geometry of the benzyl substituents. Changing the chirality of one of the residues has consequences on the CH···π interaction, which is of CαH···π nature for LD, while LL shows a CßH···π interaction. Higher-energy conformers also display some differences in the nature of the intramolecular interactions.

Amino Acid Oxidation: A Combined Study of Cysteine Oxo Forms by IRMPD Spectroscopy and Simulations.

The redox activity of cysteine sulfur allows numerous post-translational protein modifications involved in the oxidative regulation of metabolism, in metal binding, and in signal transduction. A combined approach based on infrared multiple photon dissociation spectroscopy at the Centre Laser Infrarouge d'Orsay (CLIO) free electron laser facility, calculations of IR frequencies, and finite temperature ab initio molecular dynamics simulations has been employed to characterize the gas-phase structures of deprotonated cysteine sulfenic, sulfinic, and sulfonic acids, [cysSOx ]- (x=1, 2, 3, representing the number of S-bound oxygen atoms), which are key intermediates in the redox-switching chemistry of proteins. The ions show different structural motifs owing to preferential binding of the proton to either the carboxylate or sulfur-containing group. Due to the decreasing basicity of the sulfenic, sulfinic, and sulfonic terminals, the proton bound to SO- in [cysSO]- migrates to the carboxylate in [cysSO3 ]- , whereas it turns out to be shared in [cysSO2 ]- . Evidence is gathered that a mixture of close-lying low-energy conformers is sampled for each cysteine oxo form in a Paul ion trap at room temperature.

Chirality-dependent structuration of protonated or sodiated polyphenylalanines: IRMPD and ion mobility studies.

Ion mobility experiments are combined with Infra-Red Multiple Photon Dissociation (IRMPD) spectroscopy and quantum chemical calculations for assessing the role of chirality in the structure of protonated and sodiated di- or tetra-peptides. Sodiated systems show a strong chirality dependence of the competition between Na(+)O and Na(+)π interactions. Chirality effects are more subtle in protonated systems and manifest themselves by differences in the secondary interactions such hydrogen bonds between neutral groups or those involving the aromatic rings.

Photofragmentation mechanisms in protonated chiral cinchona alkaloids.

The photo-stability of protonated cinchona alkaloids is studied in the gas phase by a multi-technique approach. A multi-coincidence technique is used to demonstrate that the dissociation is a direct process. Two dissociation channels are observed. They result from the C8-C9 cleavage, accompanied or not by hydrogen migration. The branching ratio between the two photo-fragments is different for the two pseudo-enantiomers quinine and quinidine. Mass spectrometry experiments coupling UV photo-dissociation of the reactants and structural characterization of the ionic photo-products by Infra-Red Multiple Photo-Dissociation (IRMPD) spectroscopy provide unambiguous information on their structure. In addition, quantum chemical calculations allow proposing a reactive scheme and discussing it in terms of the ground-state geometry of the reactant.

Structure of protonated thymidine characterized by infrared multiple photon dissociation and quantum calculations.

RATIONALE: Many fundamental studies are motivated by the probable relationship between the presence of rare enol tautomers of nucleobases and point mutation developing during nucleic acid replication. The evaluation of the tautomeric behaviour of nucleobases is therefore of fundamental importance. This can be probed in the gas phase by combining action spectroscopy and mass spectrometry. METHODS: Experimental Infrared Multiple Photon Dissociation spectra in the fingerprint region of electrospray-generated and subsequently selected ions were recorded at the CLIO free electron laser (FEL) facility, by coupling the FEL to a quadrupole ion trap, and compared to calculated harmonic vibrational infrared spectra of the different low-lying isomers computed at the B3LYP/6-31++G(d,p) level. Relative energies were refined using the extended basis set 6-311++G(3df,2p). RESULTS: The Density Functional Theory (DFT) study shows that, as for protonated thymine, the global energy minimum of protonated thymidine corresponds to an enol tautomer, whose infrared absorption spectrum is found to be in very good agreement with the experimental IRMPD spectrum. A very weak IRMPD signal observed at ~1780 cm(-1) is very likely the signature of an oxo tautomer. Consequently, as for thymine, protonated thymidine generated by electrospray corresponds to a mixture of at least two tautomeric forms. CONCLUSIONS: Tautomerization can be characterized by IRMPD spectroscopy. Interestingly, the dominant enolic tautomeric form(s) presently observed cannot be directly generated from the most stable neutral tautomer of the thymine residue.

Interaction of cisplatin with 5'-dGMP: a combined IRMPD and theoretical study.

IR multiple photon dissociation (IRMPD) spectroscopy of cis-[Pt(NH3)2(5'-dGMP-H)](+) and cis-[PtCl(NH3)2(5'-dGMP)](+) ions (where 5'-dGMP is 2'-deoxyguanosine-5'-monophosphate), generated in the gas phase by electrospray ionization, was performed in two spectral regions, namely, 700-1900 cm(-1) and 2800-3800 cm(-1). For structural assignment, experimental IRMPD spectra were compared to IR spectra computed at the B3LYP/LACV3P/6-311G** level of theory. In agreement with computational results, the vibrational spectroscopic characterization of the cis-[Pt(NH3)2(5'-dGMP-H)](+) ion points to macrochelate species resulting from the simultaneous interaction of the metal with both the N7 atom of the guanine residue and an O atom of the phosphate group, structures that bear features in common with those characterized in solution by NMR spectroscopy. Concerning the cis-[PtCl(NH3)2(5'-dGMP)](+) ion, our study points to a monodentate complex involving exclusively the N7 position of guanine, as observed in solution. Also this species exhibits a compact form due to the formation of two hydrogen bonds involving the same ammonia ligand. For both complexes, IRMPD experiments show that a strong intramolecular hydrogen bond is established between one ammonia hydrogen and the carbonyl group of guanine. The strength of this particular interaction can be qualitatively estimated by looking at the redshift of the CO vibration with respect to an unperturbed C═O stretching mode in the fingerprint region. This point is also highlighted in the X-H (X = N, O) stretch region, by the shift of the N-H stretch frequency as a function of the number of hydrogen bonds involving the ammonia ligand.