Unimolecular and collision-induced fragmentation of protonated nitroarenes

The unimolecular fragmentation reactions of 28 protonated nitroarenes, occurring on the metastable ion time-scale, are reported. In addition, the collision-induced fragmentation of the same species have been studied at 10 eV and at 50 eV collision energy. When an OH, COOH or NH₂ substituent is ortho to the nitro function, the dominant fragmentation involves loss of H₂O, for both unimolecular and collision-induced reactions. When there is an electron-releasing substituent ortho or para to the litro group, loss of OH is the dominant fragmentation reaction both on the metastable ion time-scale and for ions activated by collision. When the electron-releasing substituent is meta to the nitro group, loss of NO₂ is the dominant low-energy unimolecular fragmentation reaction while loss of HNO₂ is the most important fragmentation for ions activated by 50 eV collisions. Elimination of NO from [MH]⁺ occurs to a significant extent in the unimolecular fragmentation of protonated nitrobenzene and those protenated nitrobenzenes containing electron- attracting substituents. In the collision-induced dissociation of these species loss of HNO₂ occurs at the expense of loss of NO. The results are consistent with protonation predominantly at the nitro group. The results are discussed in terms of the use of neutral loss scans in tandem mass spectrometry to monitor complex mixtures for nitroarenes.     

Analysis of protonated and alkali metal cationized aminoglycoside antibiotics by collision-activated dissociation and infrared multi-photon dissociation in the quadrupole ion trap

Nine aminoglycoside antibiotics were analyzed in two quadrupole ion trap mass spectrometers using electrospray ionization. Structural information was obtained via collision-activated dissociation (CAD) and infrared multi-photon dissociation (IRMPD) of the protonated species. Several of the compounds, having multiple basic sites, preferred the doubly protonated form while some existed in the singly charged state or were distributed between single and doubly protonated species, allowing comparison of the fragmentation patterns of the two charge states. In general, IRMPD is as efficient as CAD, produces more low-mass fragment ions, and is more universally applied owing to its low dependence on trapping, pressure and tuning conditions. Alkali metal complexation using Li(+) and Na(+) was probed as a means of producing different fragmentation patterns, but in most cases the resulting fragmentation patterns were simplified versions of those obtained for the protonated analogs.     

An unprecedented rearrangement in collision-induced mass spectrometric fragmentation of protonated benzylamines

The collision-induced dissociation (CID) mass spectra of several protonated benzylamines are described and mechanistically rationalized. Under collision-induced decomposition conditions, protonated dibenzylamine, for example, loses ammonia, thereby forming an ion of m/z 181. Deuterium labeling experiments confirmed that the additional proton transferred to the nitrogen atom during this loss of ammonia comes from the ortho positions of the phenyl rings and not from the benzylic methylene groups. A mechanism based on an initial elongation of a C--N bond at the charge center that eventually cleaves the C--N bond to form an ion/neutral complex of benzyl cation and benzylamine is proposed to rationalize the results. The complex then proceeds to dissociate in several different ways: (1) a direct dissociation to yield a benzyl cation observed at m/z 91; (2) an electrophilic attack by the benzyl cation within the complex on the phenyl ring of the benzylamine to remove a pair of electrons from the aromatic sextet to form an arenium ion, which either donates a ring proton (or deuteron when present) to the amino group forming a protonated amine, which undergoes a charge-driven heterolytic cleavage to eliminate ammonia (or benzylamine) forming a benzylbenzyl cation observed at m/z 181, or undergoes a charge-driven heterolytic cleavage to eliminate diphenylmethane and an immonium ion; and (3) a hydride abstraction from a methylene group of the neutral benzylamine to the benzylic cation to eliminate toluene and form a substituted immonium ion. Corresponding benzylamine and dibenzylamine losses observed in the spectra of protonated tribenzylamine and tetrabenzyl ammonium ion, respectively, indicate that the postulated mechanism can be widely applied. The postulated mechanisms enabled proper prediction of mass spectral fragments expected from protonated butenafine, an antifungal drug.     

Methyl group transfer upon gas phase decomposition of protonated methyl benzoate and similar compounds

Gas phase decompositions of protonated methyl benzoate and its conjugates have been studied by using electrospray ionization‐collision induced dissociation‐tandem mass spectrometry. Loss of CO₂ molecule, thus transfer of methyl group, has been observed. In order to better understand this process, the theoretical calculations have been performed. For methyl benzoate conjugates, it has been found that position of substituent affects the loss of CO₂ molecule, not the electron donor/withdrawing properties of the substituent. Therefore, electrospray ionization‐mass spectrometry in positive ion mode may be useful for differentiation of isomers of methyl benzoate conjugates.     

Dehydration versus deamination of N-terminal glutamine in collision-induced dissociation of protonated peptides

Some of the most prominent "neutral losses" in peptide ion fragmentation are the loss of ammonia and water from N-terminal glutamine. These processes are studied by electrospray ionization mass spectrometry in singly- and doubly-protonated peptide ions undergoing collision-induced dissociation in a triple quadrupole and in an ion trap instrument. For this study, four sets of peptides were synthesized: (1) QLLLPLLLK and similar peptides with K replaced by R, H, or L, and Q replaced by a number of amino acids, (2) QLnK (n = 0, 1, 3, 5, 7, 9, 11), (3) QLnR (n = 0, 1, 3, 5, 7, 9), and (4) QLn (n = 1, 2, 3, 4, 8). The results for QLLLPLLLK and QLLLPLLLR show that the singly protonated ions undergo loss of ammonia and to a smaller extent loss of water, whereas the doubly protonated ions undergo predominant loss of water. The fast fragmentation next to P (forming the y5 ion) occurs to a larger extent than the neutral losses from the singly protonated ions but much less than the water loss from the doubly protonated ions. The results from these and other peptides show that, in general, when N-terminal glutamine peptides have no "mobile protons", that is, the number of charges on the peptide is no greater than the number of basic amino acids (K, R, H), deamination is the predominant neutral loss fragmentation, but when mobile protons are present the predominant process is the loss of water. Both of these processes are faster than backbone fragmentation at the proline. These results are rationalized on the basis of resonance stabilization of the two types of five-membered ring products that would be formed in the neutral loss processes; the singly protonated ion yields the more stable neutral pyrrolidinone ring whereas the doubly protonated ion yields the protonated aminopyrroline ring (see Schemes). The generality of these trends is confirmed by analyzing an MS/MS spectra library of peptides derived from tryptic digests of yeast. In the absence of mobile protons, glutamine deamination is the most rapid neutral loss process. For peptides with mobile protons, dehydration from glutamine is far more rapid than from any other amino acid. Most strikingly, end terminal glutamine is by far the most labile source of neutral loss in excess-proton peptides, but not highly exceptional when mobile protons are not available. In addition, rates of deamination are faster in lysine versus arginine C-terminus peptides and 20 times faster in positively charged than negatively charged peptides, demonstrating that these formal neutral loss reactions are not "neutral reactions" but depend on charge state and stability.     

Microhydration of protonated nucleic acid bases and protonated nucleosides in the gas phase

Thermochemical data, ΔH _n ^o , ΔS _n ^o , and ΔG _n ^o , for the hydration of protonated nucleic acid bases and protonated nucleosides have been experimentally studied by equilibrium measurements using an electrospray high-pressure mass spectrometer equipped with a pulsed ion-beam reaction chamber. For protonated nucleobases the hydration enthalpies were found to be similar for all studied systems and varied between 12.4–13.1 kcal/mol for the first and 11.2–11.5 kcal/mol for the second water molecule. While for protonated nucleosides the water binding enthalpies (11.7–13.3 kcal/mol) are very close to those for protonated nucleobases, the entropy values are “more negative.” The structural and energetic aspects of hydrated ions are discussed in conjunction with the available theoretical data.     

Collision induced dissociation of protonated N-nitrosodimethylamine by ion trap mass spectrometry: Ultimate carcinogens in gas phase

Collision induced dissociation of protonated N-nitrosodimethylamine (NDMA) and isotopically labeled N-nitrosodimethyl-d6-amine (NDMA-d6) was investigated by sequential ion trap mass spectrometry to establish mechanisms of gas phase reactions leading to intriguing products of this potent carcinogen. The fragmentation of (NDMA + H⁺) occurs via two dissociation pathways. In the alkylation pathway, homolytic cleavage of the N–O bond of N-dimethyl, N′-hydroxydiazenium ion generates N-dimethyldiazenium distonic ion which reacts further by a CH₃ radical loss to form methanediazonium ion. Both methanediazonium ion and its precursor are involved in ion/molecule reactions. Methanediazonium ion showed to be capable of methylating water and methanol molecules in the gas phase of the ion trap and N-dimethyldiazenium distonic ion showed to abstract a hydrogen atom from a solvent molecule. In the denitrosation pathway, a tautomerization of N-dimethyl, N′-hydroxydiazenium ion to N-nitrosodimethylammonium intermediate ion results in radical cleavage of the N–N bond of the intermediate ion to form N-dimethylaminium radical cation which reacts further through α-cleavage to generate N-methylmethylenimmonium ion. Although the reactions of NDMA in the gas phase are different to those for enzymatic conversion of NDMA in biological systems, each activation method generates the same products. We will show that collision induced dissociation of N-nitrosodiethylamine (NDEA) and N-nitrosodipropylamine (NDPA) is also a feasible approach to gain information on formation, stability, and reactivity of alkylating agents originating from NDEA and NDPA. Investigating such biologically relevant, but highly reactive intermediates in the condensed phase is hampered by the short life-times of these transient species.     

Synthesis and characterization of some derivatives of lupinine and aminolupinine. Collision induced dissociation mass spectrometry studies of protonated molecules

Some ester, amide and thioether derivatives of lupinine, aminolupinine and bromolupinine were synthesized and characterized in order to search biologically active compounds. The protonated molecules were studied by tandem mass spectrometry using collision induced dissociation (CID) technique in order to find out how different structural features and functional groups in different quinolizidine derivatives influence fragmentation behavior. Some theoretical calculations were also made to clarify the conformations of neutral and protonated molecules, to reveal the fragmentation routes and the product ions obtained. The functional groups clearly directed the fragmentations although the typical fragments for lupinine were still observed in all the cases. Theoretical calculations were in agreement with observed fragmentations and greatly helped interpretation of the CID spectra.     

McLafferty-type rearrangement in the collision-induced dissociation of Li+, Na+ and Ag+ cationized esters of N-acetylated peptides

In this study we investigated the multi-stage collision-induced dissociation (CID) of N-terminally acetylated di-, tri- and tetrapeptides in the form of C-terminal ethyl, n-propyl, isopropyl, n-butyl and tert-butyl esters and cationized by the attachment of Li(+), Na(+) and Ag(+). While methyl ester versions of the metal cationized peptides primarily eliminate H(2)O following collisional activation and dissociation, the ethyl, propyl and butyl ester versions of the peptides exhibit a dissociation pathway consistent with gamma-hydrogen transfer to the C-terminal carbonyl group, with associated elimination of an alkene, in a McLafferty-type rearrangement. The rearrangement leaves a metal cationized, free-acid form of the peptide, as confirmed by comparing the multi-stage CID of rearrangement products generated from peptide esters with the CID of corresponding metal cationized free-acid peptides. The transfer of a gamma-hydrogen in the rearrangement reaction was confirmed by investigating the CID of ethyl esters for which the terminal methyl group was labeled with deuterium. We found that the rearrangement product was significantly more abundant, relative to other product ions, when derived from isopropyl and tert-butyl esters than from ethyl, n-propyl or n-butyl ester analogues.     

Theoretical study of alpha/beta-alanine and their protonated/alkali metal cationized complexes

Density functional theory has been employed to model the structure and the relative stabilities of alpha/beta-alanine conformers and their protonated and alkali metal cationized complexes. In general, we find that the behavior of the beta-alanine (beta-Ala) system is quite similar to that of alpha-alanine (alpha-Ala). However, the presence of the methylene group (-CH2-) at the beta position in beta-Ala leads to a few key differences. First, the intramolecular hydrogen bonding patterns are different between free alpha- and beta-Ala. Second, the stability of zwitterionic species (in either the free ligand or alkali metal cationized complexes) is often enhanced in beta-Ala. Third, the preferred mode of alkali metal cation (M+) binding may also differ in alpha- and beta-Ala. Natural energy decomposition analysis has been applied here to gain further insight into the effects of the ligand, cation size, and mode of binding on the nature of interaction in these M+-Ala complexes.     

Doubly protonated benzene in the gas phase

Structural aspects and the unimolecular fragmentations of doubly protonated benzene are studied by means of tandem-mass spectrometry. The corresponding dications are generated by electron ionization (EI) of 1,3- and 1,4-cyclohexadienes, respectively. It is suggested that EI of 1,3-cyclohexadiene leads to the singlet state of doubly protonated benzene, whereas EI of 1,4-cyclohexadiene yields a mixture of singlet and triplet states. Unimolecular fragmentation of doubly protonated benzene exclusively proceeds via dehydrogenation leading to the benzene dication. The proton affinities (PAs) of protonated benzene amount to PA(C(6)H(7)(+))(meta) = 1.9 +/- 0.3 eV for protonation taking place at the meta-position, PA(C(6)H(7)(+))(ortho) = 1.5 +/- 0.2 eV, and PA(C(6)H(7)(+))(para) = 0.9 +/- 0.2 eV, respectively. Various facets of the experiments are compared with density functional theory calculations and generally good agreement is found.     

Possible conformational change within the desolvated and cationized sBBI/trypsin non-covalent complex during the collision-induced dissociation process

Electrospray ionization mass spectrometry (ESI-MS) has become an analytical technique widely used for the investigation of non-covalent protein-protein and protein-ligand complexes due to the soft desolvation conditions that preserve the stoichiometry of the interacting partners. Dissociation studies of solvated or desolvated complexes (in the source and in the collision cell, respectively) allow access to information on protein conformation and localization of the metal ions involved in protein structure stabilization and biological activity. The complex of bovine trypsin and small soybean Bowman-Birk inhibitor (sBBI) was studied by ESI-MS to determine changes occurring within the complex during its transfer from droplets to the gas phase independently of the ion polarity. Under collision-induced dissociation (CID) conditions, unexpected binding of the Ca(2+) ion (cofactor of native trypsin) to the inhibitor molecule was observed within the desolvated sBBI/trypsin/Ca(2+) complex (with a 1:1:1 stoichiometry). This formal gas-phase migration of the calcium ion from trypsin to the inhibitor may be related to conformational rearrangements in the solvent-free and likely collapsed complex. However, under conditions leading to the increase in complex charge state, the appearance of the cationized trypsin molecule was detected during complex dissociation, thus reflecting different pathways of the evolution of complex conformation.     

Theoretical and experimental studies of cationized uracil complexes in the gas phase

Cationized uracil clusters were generated in the gas phase by electrospray ionization (ESI). Mass spectrometry experiments showed that with particular experimental conditions, decameric uracil clusters are magic number clusters. MS/MS experiments demonstrated that the structure of these decameric uracil clusters depends substantially on the size and the charge of the cation. On the basis of the ab initio and density functional theory (DFT) quantum chemistry calculations, structures for these decameric clusters were proposed. These structures are in agreement with the experimental mass spectra of modified nucleobases. Theoretical calculations showed that complexes experimentally observed using ESI-MS techniques, are not naturally the most stable in the gas phase.     

Fragmentation studies and electrospray ionization mass spectrometry of lapachol: protonated,deprotonated and cationized species

Electrospray ionization mass spectrometric analysis of lapachol (2‐hydroxy‐3‐(3‐methyl‐2‐butenyl)‐1,4‐naphthoquinone) was accomplished in order to elucidate the gas‐phase dissociation reactions of this important biologically active natural product. The occurrence of protonated and cationized species in the positive mode and of deprotonated species in the negative mode was explored by means of collision‐induced dissociation (CID) experiments. For the protonated molecule, the H₂O and C₄H₈ losses occur by two competitive channels. For the deprotonated molecule, the even‐electron rule is not conserved, and the radicalar species are eliminated by formation of distonic anions. The fragmentation mechanism for each ion was suggested on the basis of computational thermochemistry. Atomic charges, relative energies, and frontier orbitals were employed aiming at a better understanding of the gas‐phase reactivity of lapachol. Potential energy surfaces for fragmentation reactions were obtained by the B3LYP/6‐31+G(d,p) model. Copyright © 2010 John Wiley & Sons, Ltd.     

Studies of rearrangement reactions of protonated and lithium cationized 2-pyrimidinyloxy-N-arylbenzylamine derivatives by MALDI-FT-ICR mass spectrometry

The rearrangement reactions of protonated and lithium-cationized 2-pyrimidinyloxy-N-arylbenzylamine derivatives were studied by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and infrared multiphoton dissociation mass spectrometry (IRMPD). Our results show that three kinds of rearrangement reactions occur in IRMPD processes. First, nearly all protonated 2-pyrimidinyloxy-N-arylbenzylamine derivatives undergo Pathway A to form the K ion series. It is proposed that this rearrangement (migration of a substituted benzyl group) proceeds by way of a gas-phase intramolecular S(N)2 reaction. Second, a gas phase intramolecular S(N)Ar type rearrangement mechanism is proposed to explain the formation of the F ion series from protonated and lithium-cationized 5 (or 6). This skeletal rearrangement reaction competes with the S(N)2 reaction of the Pathway A, which produces the K ion series, in IRMPD of protonated 5 and 6. Third, the formation pathway of the W ion series is explained by a gas phase Cope type rearrangement mechanism.