Photochemical and Collision-Induced Cross-Linking in Stereochemically Distinct Scaffolds of Peptides and Nitrile Imines in Gas-Phase Ions.

Intramolecular cross-linking between peptides and nitrile-imine intermediates was studied in stereochemically distinct conjugates in which the reacting components were mounted on cis-1,2-cyclohexane and trans-1,4-cyclohexane scaffolds that we call 1,2-s-peptides and 1,4-s-peptides, respectively. The nitrile-imine intermediates were generated by N2 loss from 2,5-diaryltetrazole tags upon UV-photodissociation at 213 and 250 nm or by collision-induced dissociation, and further interrogated by CID and UVPD-MS3. Peptide fragment ion series originating from linear structures and macrocyclic cross-links were distinguished and used to quantify the cross-linking yields. The yields in MS2 varied between 27% for AAAG conjugates to 78% for GAAAK conjugates, depending on the peptide sequence. The CID-MS3 yields were in a 57-97% range, depending on the peptide sequence. Structures of 1,2-s-peptide and 1,4-s-peptide ions as well as several of their nitrile-imine intermediates and cross-links were investigated by high-resolution cyclic ion mobility in combination with Born-Oppenheimer molecular dynamics and density functional theory calculations. Matches between the experimental and calculated collision cross sections and ion relative Gibbs energies were used to assign peptide structures. Peptide conjugates C-terminated with Gly and Lys residues underwent cross-linking by the carboxyl group, as established by MS3 sequencing and corroborated by carboxyl blocking experiments that lowered the cross-linking yields. Peptide conjugates C-terminated with Arg also cross-linked via the side-chain guanidine group. A notable feature of the 1,4-s-peptide ions was the participation of low-energy twist-boat cyclohexane conformers that was enforced by strong hydrogen bonds between the peptide and nitrile imine.

Cyclic Ion Mobility of Isomeric New Psychoactive Substances Employing Characteristic Arrival Time Distribution Profiles and Adduct Separation.

Analysis of new psychoactive substances (NPS), which is essential for toxicological and forensic reasons, can be made complicated by the presence of isomers. Ion mobility has been used as a standalone technique or coupled to mass spectrometry to detect and identify NPS. However, isomer separation has so far chiefly relied on chromatography. Here we report on the determination of isomeric ratios using cyclic ion mobility-mass spectrometry without any chromatographic separation. Isomers were distinguished by mobility separation of lithium adducts. Alternatively, we used arrival time distribution (ATD) profiles that were characteristic of individual isomers and were acquired for protonated molecules or fragment ions. Both approaches provided comparable results. Calculations were used to determine the structures and collision cross sections of both protonated and lithiated isomers that accurately characterized their ion mobility properties. The applicability of ATD profiles to isomer differentiation was demonstrated using direct infusion and flow injection analysis with electrospray of solutions, as well as desorption electrospray of solid samples. Data processing was performed by applying multiple linear regression to the ATD profiles. Using the proposed ATD profile-based approach, the relationships between the determined and given content of isomers showed good linearity with coefficients of determination typically greater than 0.99. Flow injection analysis using an autosampler allowed us to rapidly determine isomeric ratios in a sample containing two isomeric pairs with a minor isomer of 10% (determined 9.3% of 3-MMC and 11.0% of 3-FMC in a mixture with buphedrone and 4-FMC). The proposed approach is not only useful for NPS, but also may be applicable to small isomeric molecules analyzed by ion mobility when complete separation of isomers is not achieved.

Nucleoside Cation Radicals: Generation, Radical-Induced Hydrogen Atom Migrations, and Ribose Ring Cleavage in the Gas Phase.

Nucleoside ions that were furnished on ribose with a 2'-O-acetyl radical group were generated in the gas phase by multistep collision-induced dissociation of precursor ions tagged with radical initiator groups, and their chemistry was investigated in the gas phase. 2'-O-Acetyladenosine cation radicals were found to undergo hydrogen transfer to the acetoxyl radical from the ribose ring positions that were elucidated using specific deuterium labeling of 1'-H, 2'-H, and 4'-H and in the N-H and O-H exchangeable positions, favoring 4'-H transfer. Ion structures and transition-state energies were calculated by a combination of Born-Oppenheimer molecular dynamics and density functional theory and used to obtain unimolecular rate constants for competitive hydrogen transfer and loss of the acetoxyl radical. Migrations to the acetoxyl radical of ribose hydrogens 1'-H, 2'-H, 3'-H, and 4'-H were all exothermic, but product formation was kinetically controlled. Both Rice-Ramsperger-Kassel-Marcus (RRKM) and transition-state theory (TST) calculations indicated preferential migration of 4'-H in a qualitative agreement with the deuterium labeling results. The hydrogen migrations displayed substantial isotope effects that along with quantum tunneling affected the relative rate constants and reaction branching ratios. UV-vis action spectroscopy indicated that the cation radicals from 2'-O-acetyladenosine consisted of a mixture of isomers. Radical-driven dissociations were also observed for protonated guanosine, cytosine, and thymidine conjugates. However, for those nucleoside ions and cation radicals, the dissociations were dominated by the loss of the nucleobase or formation of protonated nucleobase ions.

Competitive Radical Migrations and Ribose Ring Cleavage in Adenosine and 2'-Deoxyadenosine Cation Radicals.

We report a combined experimental and computational study of adenosine cation radicals that were protonated at adenine and furnished with a radical handle in the form of an acetoxyl radical, •CH2COO, that was attached to ribose 5'-O. Radicals were generated by collision-induced dissociation (CID) and characterized by tandem mass spectrometry and UV-vis photodissociation action spectroscopy. The acetoxyl radical was used to probe the kinetics of intramolecular hydrogen transfer from the ribose ring positions that were specifically labeled with deuterium at C1', C2', C3', C4', C5', and in the exchangeable hydroxyl groups. Hydrogen transfer was found to chiefly involve 3'-H with minor contributions by 5'-H and 2'-H, while 4'-H was nonreactive. The hydrogen transfer rates were affected by deuterium isotope effects. Hydrogen transfer triggered ribose ring cleavage by consecutive dissociations of the C4'-O and C1'-C2' bonds, resulting in expulsion of a C6H9O4 radical and forming a 9-formyladenine ion. Rice-Ramsperger-Kassel-Marcus (RRKM) and transition-state theory (TST) calculations of unimolecular constants were carried out using the effective CCSD(T)/6-311++G(3d,2p) and M06-2X/aug-cc-pVTZ potential energy surfaces for major isomerizations and dissociations. The kinetic analysis showed that hydrogen transfer to the acetoxyl radical was the rate-determining step, whereas the following ring-opening reactions in ribose radicals were fast. Using DFT-computed energies, a comparison was made between the thermochemistry of radical reactions in adenosine and 2'-deoxyadenosine cation radicals. The 2'-deoxyribose ring showed lower TS energies for both the rate-determining 3'-H transfer and ring cleavage reactions.

Nitrile Imines as Peptide and Oligonucleotide Photo-Cross-Linkers in Gas-Phase Ions.

Nitrile imines produced by photodissociation of 2,5-diaryltetrazoles undergo cross-linking reactions with amide groups in peptide-tetrazole (tet-peptide) conjugates and a tet-peptide-dinucleotide complex. Tetrazole photodissociation in gas-phase ions is efficient, achieving ca. 50% conversion with 2 laser pulses at 250 nm. The formation of cross-links was detected by CID-MS3 that showed structure-significant dissociations by loss of side-chain groups and internal peptide segments. The structure and composition of cross-linking products were established by a combination of UV-vis action spectroscopy and cyclic ion mobility mass spectrometry (c-IMS). The experimental absorption bands were found to match the bands calculated for vibronic absorption spectra of nitrile imines and cross-linked hydrazone isomers. The calculated collision cross sections (CCSth) for these ions were related to the matching experimental CCSexp from multipass c-IMS measurements. Loss of N2 from tet-peptide conjugates was calculated to be a mildly endothermic reaction with ΔH0 = 80 kJ mol-1 in the gas phase. The excess energy in the photolytically formed nitrile imine is thought to drive endothermic proton transfer, followed by exothermic cyclization to a sterically accessible peptide amide group. The exothermic nitrile imine reaction with peptide amides is promoted by proton transfer and may involve an initial [3 + 2] cycloaddition followed by cleavage of the oxadiazole intermediate. Nucleophilic groups, such as cysteine thiol, did not compete with the amide cyclization. Nitrile imine cross-linking to 2'-deoxycytidylguanosine was found to be >80% efficient and highly specific in targeting guanine. The further potential for exploring nitrile-imine cross-linking for biomolecular structure analysis is discussed.

Covalent crosslinking in gas-phase biomolecular ions. An account and perspective.

Photochemical crosslinking in gas-phase ion complexes has been introduced as a method to study biomolecular structures and dynamics. Emphasis has been on carbene-based crosslinking induced by photodissociation of diazirine-tagged ions. The features that characterize gas-phase crosslinking include (1) complex formation in electrospray droplets that allows for library-type screening; (2) well defined stoichiometry of the complexes due to mass-selective isolation; (3) facile reaction monitoring and yield determination, and (4) post-crosslinking structure analysis by tandem mass spectrometry that has been combined with hydrogen-deuterium exchange, UV-vis action spectroscopy, and ion mobility measurements. In this account, examples are given of peptide-peptide, peptide-nucleotide, and peptide-ligand crosslinking that chiefly used carbene-based reactions. The pros and cons of gas-phase crosslinking are discussed. Nitrile-imine based crosslinking in gas-phase ions is introduced as a promising new approach to ion structure analysis that offers high efficiency and has the potential for wide ranging applications.

Do d(GCGAAGC) Cations Retain the Hairpin Structure in the Gas Phase? A Cyclic Ion Mobility Mass Spectrometry and Density Functional Theory Computational Study.

d(GCGAAGC) is the smallest oligonucleotide with a well-defined hairpin structure in solution. We report a study of multiply protonated d(GCGAAGC) and its sequence-scrambled isomers, d(CGAAGCG), d(GCGAACG), and d(CGGAAGC), that were produced by electrospray ionization with the goal of investigating their gas-phase structures and dissociations. Cyclic ion mobility measurements revealed that dications of d(GCGAAGC) as well as the scrambled-sequence ions were mixtures of protomers and/or conformers that had collision cross sections (CCS) within a 439-481 Å2 range. Multiple ion conformers were obtained by electrospray under native conditions as well as from aqueous methanol. Arrival time distribution profiles were characteristic of individual isomeric heptanucleotides. Extensive Born-Oppenheimer molecular dynamics (BOMD) and density functional theory (DFT) calculations of d(GCGAAGC)2+ isomers indicated that hairpin structures were high-energy isomers of more compact distorted conformers. Protonation caused a break up of the C2···G6 pair that was associated with the formation of strong hydrogen bonds in zwitterionic phosphate anion-nucleobase cation motifs that predominated in low energy ions. Multiple components were also obtained for d(GCGAAGC)3+ trications under native and denaturing electrospray conditions. The calculated trication structures showed disruption of the G···C pairs in low energy zwitterions. A hairpin trication was calculated to be a high energy isomer. d(GCGAAGC)4+ tetracations were produced and separated by c-IMS as two major isomers. All low energy d(GCGAAGC)4+ ions obtained by DFT geometry optimizations were zwitterions in which all five purine bases were protonated, and the ion charge was balanced by a phosphate anion. Tetracations of the scrambled sequences were each formed as one dominant isomer. The CCS calculated with the MobCal-MPI method were found to closely match experimental values. Collision-induced dissociation (CID) spectra of multiply charged heptanucleotides showed nucleobase loss and backbone cleavages occurring chiefly at the terminal nucleosides. Electron-transfer-CID tandem mass spectra were used to investigate dissociations of different charge and spin states of charge-reduced heptanucleotide cation radicals.

Tracking Isomerizations of High-Energy Adenine Cation Radicals by UV-Vis Action Spectroscopy and Cyclic Ion Mobility Mass Spectrometry.

We report experimental and computational studies of protonated adenine C-8 σ-radicals that are presumed yet elusive reactive intermediates of oxidative damage to nucleic acids. The radicals were generated in the gas phase by the collision-induced dissociation of C-8-Br and C-8-I bonds in protonated 8-bromo- and 8-iodoadenine as well as by 8-bromo- and 8-iodo-9-methyladenine. Protonation by electrospray of 8-bromo- and 8-iodoadenine was shown by cyclic-ion mobility mass spectrometry (c-IMS) to form the N-1-H, N-9-H and N-3-H, N-7-H protomers in 85:15 and 81:19 ratios, respectively, in accordance with the equilibrium populations of these protomers in water-solvated ions that were calculated by density functional theory (DFT). Protonation of 8-halogenated 9-methyladenines yielded single N-1-H protomers, which was consistent with their thermodynamic stability. The radicals produced from the 8-bromo and 8-iodo adenine cations were characterized by UV-vis photodissociation action spectroscopy (UVPD) and c-IMS. UVPD revealed the formation of C-8 σ-radicals along with N-3-H, N-7-H-adenine π-radicals that arose as secondary products by hydrogen atom migrations. The isomers were identified by matching their action spectra against the calculated vibronic absorption spectra. Deuterium isotope effects were found to slow the isomerization and increase the population of C-8 σ-radicals. The adenine cation radicals were separated by c-IMS and identified by their collision cross sections, which were measured relative to the canonical N-9-H adenine cation radical that was cogenerated in situ as an internal standard. Ab initio CCSD(T)/CBS calculations of isomer energies showed that the adenine C-8 σ-radicals were local energy minima with relative energies at 76-79 kJ mol-1 above that of the canonical adenine cation radical. Rice-Ramsperger-Kassel-Marcus calculations of unimolecular rate constants for hydrogen and deuterium migrations resulting in exergonic isomerizations showed kinetic shifts of 10-17 kJ mol-1, stabilizing the C-8 σ-radicals. C-8 σ-radicals derived from N-1-protonated 9-methyladenine were also thermodynamically unstable and readily isomerized upon formation.

Carbene Cross-Linking in Gas-Phase Peptide Ion Scaffolds.

Scaffolds consisting of a peptide, a phthalate linker, and a 4,4-azipentyl group were synthesized and used to study intramolecular peptide-carbene cross-linking in gas-phase cations. Carbene intermediates were generated by UV-laser photodissociation at 355 nm of the diazirine ring in mass-selected ions, and the cross-linked products were detected and quantified by collision-induced dissociation tandem mass spectrometry (CID-MSn, n = 3-5). Peptide scaffolds containing Ala and Leu residues with a C-terminal Gly gave 21-26% yields of cross-linked products, while the presence of the Pro and His residues decreased the yields. Experiments using hydrogen-deuterium-hydrogen exchange, carboxyl group blocking, and analysis of CID-MSn spectra of reference synthetic products revealed that a significant fraction of cross-links involved the Gly amide and carboxyl groups. Interpretation of the cross-linking results was aided by Born-Oppenheimer molecular dynamics (BOMD) and density functional theory calculations that allowed us to establish the protonation sites and conformations of the precursor ions. Analysis of long (100 ps) BOMD trajectories was used to count close contacts between the incipient carbene and peptide atoms, and the counting statistics was correlated with the results of gas-phase cross-linking.

The DNA Radical Code. Resolution of Identity in Dissociations of Trinucleotide Codon Cation Radicals in the Gas Phase.

Sixty DNA trinucleotide cation radicals covering a large part of the genetic code alphabet were generated by electron transfer in the gas phase, and their chemistry was studied by collision-induced dissociation tandem mass spectrometry and theoretical calculations. The major dissociations involved loss of nucleobase molecules and radicals, backbone cleavage, and cross-ring fragmentations that depended on the nature and position of the nucleobases. Mass identity in dissociations of symmetrical trinucleotide cation radicals of the (XXX+2H)+• and (XYX+2H)+• type was resolved by specific 15N labeling. The specific features of trinucleotide cation radical dissociations involved the dominant formation of d2+ ions, hydrogen atom migrations accompanying the formation of (w2+H)+•, (w2+2H)+, and (d2+2H)+ sequence ions, and cross-ring cleavages in the 3'- and 5'-deoxyribose moieties that depended on the nucleobase type and its position in the ion. Born-Oppenheimer molecular dynamics (BOMD) and density functional theory calculations were used to obtain structures and energies of several cation-radical protomers and conformers for (AAA+2H)+•, (CCC+2H)+•, (GGG+2H)+•, (ACA+2H)+•, and (CAA+2H)+• that were representative of the different types of backbone dissociations. The ion electronic structure, protonation and radical sites, and hydrogen bonding were used to propose reaction mechanisms for the dissociations.

UV-vis spectroscopy of gas-phase ions.

Photodissociation action spectroscopy has made a great progress in expanding investigations of gas-phase ion structures. This review deals with aspects of gas-phase ion electronic excitations that result in wavelength-dependent dissociation and light emission via fluorescence, chiefly covering the ultraviolet and visible regions of the spectrum. The principles are briefly outlined and a few examples of instrumentation are presented. The main thrust of the review is to collect and selectively present applications of UV-vis action spectroscopy to studies of stable gas-phase ion structures and combinations of spectroscopy with ion mobility, collision-induced dissociation, and ion-ion reactions leading to the generation of reactive intermediates and electronic energy transfer.

Resolution of Identity in Gas-Phase Dissociations of Mono- and Diprotonated DNA Trinucleotide Codons by 15N-Labeling and Computational Structure Analysis.

Dissociations of DNA trinucleotide codons as gas-phase singly and doubly protonated ions were studied by tandem mass spectrometry using 15N-labeling to resolve identity in the nucleobase loss and backbone cleavages. The monocations showed different distributions of nucleobase loss from the 5'-, middle, and 3'-positions depending on the nucleobase, favoring cytosine over guanine, adenine, and thymine in an ensemble-averaged 62:27:11:<1 ratio. The distribution for the loss of the 5'-, middle, and 3'-nucleobase was 49:18:33, favoring the 5'-nucleobase, but also depending on its nature. The formation of sequence w2+ ions was unambiguously established for all codon mono- and dications. Structures of low-Gibbs-energy protomers and conformers of dAAA+, dGGG+, dCCC+, dTTT+, dACA+, and dATC+ were established by Born-Oppenheimer molecular dynamics and density functional theory calculations. Monocations containing guanine favored classical structures protonated at guanine N7. Structures containing adenine and cytosine produced classical nucleobase-protonated isomers as well as zwitterions in which two protonated bases were combined with a phosphate anion. Protonation at thymine was disfavored. Low threshold energies for nucleobase loss allowed extensive proton migration to occur prior to dissociation. Loss of the nucleobase from monocations was assisted by neighboring group participation in nucleophilic addition or proton abstraction, as well as allosteric proton migrations remote from the reaction center. The optimized structures of diprotonated isomers for dAAA2+ and dACA2+ revealed combinations of classical and zwitterionic structures. The threshold and transition-state energies for nucleobase-ion loss from dications were low, resulting in facile dissociations involving cytosine, guanine, and adenine.

Low-scale syringe-made synthesis of 15 N-labeled oligonucleotides.

Fast and reasonable low-scale (200 nmol) syringe-made synthesis of 15 N-labeled oligonucleotides representing DNA trinucleotide codons is communicated. All codons were prepared by solid-phase controlled pore glass synthesis column technique via the phosphoramidite method. Twenty-four labeled oligonucleotides covering the DNA genetic code alphabet were prepared using commercially available reagents and affordable equipment in a reasonably short period of time, with acceptable yields and purity for direct applications in mass spectrometry.

Radical Cascade Dissociation Pathways to Unusual Nucleobase Cation Radicals.

We report unusual dissociations of protonated RNA nucleosides tagged with radical initiator groups at ribose 5'-O and furnished with a 2',3'-O-isopropylidene protecting group. The ions undergo collision-induced radical cascade dissociations starting at the radical initiator that break down the dioxolane ring and trigger the formation of nucleobase cations and cation radicals. The adenine cation radical that was formed by radical cascade dissociations was identified by MS5 UV-vis photodissociation action spectroscopy to be a higher-energy N-3-H tautomer of the canonical ionized nucleobase. The guanine cation radical was formed by radical cascade dissociations as the N-7-H tautomer. In contrast to adenosine and guanosine, radical cascade dissociations of the tagged ribocytidine ion produced protonated cytosine, whereas tagged ribothymidine showed yet different dissociations resulting in predominant thymine loss. Reaction mechanisms were suggested for the cascade dissociations that were based on Born-Oppenheimer molecular dynamics and density functional theory calculations that were used to map the relevant parts of the potential energy surfaces for adenosine, guanosine, and cytidine radical ions. The reported radical cascade dissociations represent a new, nonredox approach to nucleobase and nucleoside cation radicals that has the potential of being expanded to the generation of various oligonucleotide cation radicals.

Noncanonical Isomers of Nucleoside Cation Radicals: An Ab Initio Study of the Dark Matter of DNA Ionization.

Cation radicals of DNA nucleosides, 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine, can exist in standard canonical forms or as noncanonical isomers in which the charge is introduced by protonation of the nucleobase, whereas the radical predominantly resides in the deoxyribose moiety. Density functional theory as well as correlated ab initio calculations with coupled clusters (CCSD(T)) that were extrapolated to the complete basis set limit showed that noncanonical nucleoside ion isomers were thermodynamically more stable than their canonical forms in both the gas phase and as water-solvated ions. This indicated the possibility of exothermic conversion of canonical to noncanonical forms. The noncanonical isomers were calculated to have very low adiabatic ion-electron recombination energies (REad) for the lowest-energy isomers 2'-deoxy-(N-3H)adenos-1'-yl (4.74 eV), 2'-deoxy-(N-7H)guanos-1'-yl (4.66 eV), 2'-deoxy-(N-3H)cytid-1'-yl (5.12 eV), and 2'-deoxy-5-methylene-(O-2H)uridine (5.24 eV). These were substantially lower than the REad value calculated for the canonical 2'-deoxyadenosine, 2'-deoxy guanosine, 2'-deoxy cytidine, and 2'-deoxy thymidine cation radicals, which were 7.82, 7.46, 8.14, and 8.20 eV, respectively, for the lowest-energy ion conformers of each type. Charge and spin distributions in noncovalent cation-radical dA⊂dT and dG⊂dC nucleoside pairs and dAT, dCT, dTC, and dGC dinucleotides were analyzed to elucidate the electronic structure of the cation radicals. Born-Oppenheimer molecular dynamics trajectory calculations of the dinucleotides and nucleoside pairs indicated rapid exothermic proton transfer from noncanonical T+· to A in both dAT+· and dA⊂dT+·, leading to charge and radical separation. Noncanonical T+· in dCT+· and dTC+· initiated rapid proton transfer to cytosine, whereas the canonical dCT+· dinucleotide ion retained the cation radical structure without isomerization. No spontaneous proton transfer was found in dGC+· and dG⊂dC+· containing canonical neutral and noncanonical ionized deoxycytidine.

Tritium hydrogen-isotope exchange with electron-poor tertiary benzenesulfonamide moiety; application in late-stage labeling of T0901317.

The multifunctional radioligand [3 H]T0901317 ([3 H]1) has been employed as a powerful autoradiographic tool to target several receptors, such as liver X, farnesoid X, and retinoic acid-related orphan receptor alpha and gamma subtypes at nanomolar concentrations. Although [3 H]1 is commercially available and its synthesis via tritiodebromination has been reported, the market price of this radioligand and the laborious synthesis of corresponding bromo-intermediate potentially preclude its widespread use in biochemical, pharmacological, and pathological studies in research lab settings. We exploit recent reports on hydrogen-isotope exchange (HIE) reactions in tertiary benzenesulfonamides where the sulfonamide represents an ortho-directing group that facilitates CH activation in the presence of homogenous iridium(I) catalysts. Herein, we report a time- and cost-efficient method for the tritium late-stage labeling of compound 1-a remarkably electron-poor substrate owing to the tertiary trifluoroethylsulfonamide moiety. Under a straightforward HIE condition using a commercially available Kerr-type NHC Ir(I) complex, [(cod)Ir (NHC)Cl], the reaction with 1 afforded a specific activity of 10.8 Ci/mmol. Additionally, alternative HIE conditions using the heterogeneous catalyst of Ir-black provided sufficient 0.72 D-enrichment of 1 but unexpectedly failed while repeating with tritium gas.

Charge-Tagged Nucleosides in the Gas Phase: UV-Vis Action Spectroscopy and Structures of Cytidine Cations, Dications, and Cation Radicals.

Cytidine ribonucleosides were furnished at O5' with fixed-charge 6-trimethylammoniumhexan-1-aminecarbonyl tags and studied by UV-vis photodissociation action spectroscopy in the gas phase to probe isolated nucleobase chromophores in their neutral, protonated, and hydrogen-adduct radical forms. The action spectrum of the doubly charged cytidine conjugate showed bands at 310 and 270 nm that were assigned to the N3- and O2-protonated cytosine tautomers formed by electrospray, respectively. In contrast, cytidine conjugates coordinated to dibenzo-18-crown-6-ether (DBCE) in a noncovalent complex were found to strongly favor protonation at N3, forming a single-ion tautomer. This allowed us to form cytidine N3-H radicals by electron transfer dissociation of the complex and study their action spectra. Cytidine radicals showed only very weak absorption in the visible region of the spectrum for dipole-disallowed transitions to the low (A and B) excited states. The main bands were observed at 360, 300, and 250 nm that were assigned with the help of theoretical vibronic spectra obtained by time-dependent density functional theory calculations of multiple (>300) radical vibrational configurations. Collision-induced dissociations of cytidine radicals proceeded by major cleavage of the N1-C1' glycosidic bond leading to loss of cytosine and competitive loss of N3-hydrogen atom. These dissociations were characterized by calculations of transition-state structures and energies using combined Born-Oppenheimer molecular dynamics and DFT calculations. Overall, cytidine radicals were found to be kinetically and thermodynamically more stable than previously reported analogous adenosine and guanosine radicals.

Flying DNA Cation Radicals in the Gas Phase: Generation and Action Spectroscopy of Canonical and Noncanonical Nucleobase Forms.

Gas-phase chemistry of cation radicals related to ionized nucleic acids has enjoyed significant recent progress thanks to the development of new methods for cation radical generation, ion spectroscopy, and reactivity studies. Oxidative methods based on intramolecular electron transfer in transition-metal complexes have been used to generate nucleobase and nucleoside cation radicals. Reductive methods relying on intermolecular electron transfer in gas-phase ion-ion reactions have been utilized to generate a number of di- and tetranucleotide cation radicals, as well as charge-tagged nucleoside radicals. The generated cation radicals have been studied by infrared and UV-visible action spectroscopy and ab initio and density functional theory calculations, providing optimized structures, harmonic frequencies, and excited-state analysis. This has led to the discovery of stable noncanonical nucleobase cation radicals of unusual electronic properties and extremely low ion-electron recombination energies. Intramolecular proton-transfer reactions in cation radical oligonucleotides and Watson-Crick nucleoside pairs have been studied experimentally, and their mechanisms have been elucidated by theory. Whereas the range of applications of the oxidative methods is currently limited to nucleobases and readily oxidizable guanosine, the reductive methods can be scaled up to generate large oligonucleotide cation radicals including double-strand DNA. Challenges in the experimental and computational approach to DNA cation radicals are discussed.