Manipulating Non-Dissociative Transformations of Gaseous Ion Ensembles Prior to Ion-Mobility Separation.

Molecules with multiple sites capable of accepting protons form ensembles of protomers. The manifested protomer ratios in such ensembles are influenced by many experimental conditions. In a Synapt G2 ion mobility (IM)-enabled mass spectrometry system, there are several physical locations where ion population changes can be manifested. Using APCI-generated protomers of aminonaphthalenes, we investigated its intramolecular proton transfers from the N-protomer to the C-protomer. This lossless transformation of the N-protomer to the thermodynamically favored C-protomer can take place in the ion source itself. Initially, we learned that the cone gas slows down the transformation to the C-protomer. Gaseous ions are then accelerated in the first vacuum region, where ions undergo collisional activation (heating), which facilitates the transformation to the C-protomer. Afterward, the ions are mass selected and transferred to the pre-IM (Trap)-collision cell, where ions can also be transformed to the thermodynamically favored protomers. Trap accumulated ions are then released to the IM separator via a helium-filled entry cell. The role of helium is to minimize ion activation and scattering taking place upon entry to the high-pressure T-Wave IM separator (TWIMS). The helium cell is known to increase the IM peak resolution. However, we found that significant changes occur depending on the presence or absence of helium. Without helium, source-generated protomers rapidly changed to a predominantly thermodynamically favorable ensemble protomers. Apparently, the introduction of helium into the precell induced a dramatic decrease in collisional "heating" effect, which effectively slowed down the conversion rate of the amino-protomer into the more favorable ring-protomer. The final message is that mobilograms should not be considered as direct real-time, or intrinsic, representations of the protomer ratios in the ion source.

Dependence of Collision-Induced Mass Spectra of Protonated Michler's Ketone on the Nature of LC-MS Mobile Phase.

Michler's ketone (MK) is a dimethylamino ketone that undergoes facile protonation under electrospray-ionization conditions to produce an ion of m/z 269. Initial LC-MS results showed that the collision-induced dissociation (CID) spectra of the m/z 269 ion depend heavily on the composition of the chromatographic mobile phase. Subsequent ion-mobility separation of the mass-selected m/z 269 ion revealed that protonated MK exists as two tautomeric forms. Moreover, the relative population of the two protomeric forms in the ion ensemble depends on the nature of the ambient molecules present in the atmospheric pressure ion source. For example, the ion-mobility arrival-time profile acquired from the mass-selected m/z 269 ion generated from an acetonitrile solution showed two peaks of near equal intensity. The peak with the shorter arrival time represented the O-protomer and that with the longer arrival time represented the N-protomer. However, when methanol or ammonia vapors were introduced to the ambient-pressure ion source, the intensity of the N-protomer peak decreased rapidly and that of the O-protomer signal soared until it became the dominant peak. When the introduction of methanol (or ammonia) vapors was stopped, the mobilogram signals gradually reverted back to their initial intensities. To rationalize this observation, we propose that the N-protomer of MK in the presence of methanol vapor undergoes transformation to the O-protomer by a Grotthuss-type mechanism via a methanol-based solvent bridge.

Evidence of Gas-Phase Attachment of Molecular Oxygen to Deprotonated Hydroquinone During Ion-Mobility Mass Spectrometry.

Gas-phase addition of dioxygen to certain ions is a well-known phenomenon in mass spectrometry. For this reaction to occur, the presence of a distonic radical site on the precursor ion is thought to be a prerequisite. Herein, we report that oxygen adduct formation can take place also with deprotonated hydroquinone, which in fact is an even-electron species without a radical site. When the product-ion spectrum of the m/z 109 ion, generated by electrospray ionization from a solution of hydroquinone in acetonitrile, was recorded under ion-mobility conditions, a new peak was observed at m/z 141. However, an analogous peak was not visible in the spectrum acquired under nonmobility conditions (i.e., without any gas introduced to the mobility cell). Presumably, traces of oxygen present in the collision gas instigate an ion-molecule reaction to produce an adduct of m/z 141, which upon activation results in CO and H2O loss to form a product ion of m/z 95. Isotope-labeling studies confirmed that one of the hydrogen atoms from the hydroxy group and another from the aromatic ring contribute to the water loss instigated from the m/z 141 adduct. Furthermore, computational methods indicated the three-dimensional structure of the ground-state deprotonated hydroquinone to be distinctly different from those of its 1,2- and 1,3-isomers. Calculations predicted that all atoms in the two m/z 109 ions generated from catechol and resorcinol lie on one plane. In contrast, the structure of the m/z 109 ion from hydroquinone was significantly different. Computations predicted that the hydrogen atom on the intact hydroxyl group of deprotonated hydroquinone protrudes out of plane from rest of the atoms. Consequently, the exposed OH group can interact with an incoming dioxygen molecule. Computations conducted at the CAM-B3LYP/6-311++g(2d,2p) level of theory detected a minimum energy crossing point (MECP) at -4.3 kJ mol-1 below the separated O2 + deprotonated hydroquinone triplet threshold. In contrast, similar calculations conducted for catechol and resorcinol yielded MECPs of +116.9 and +69.1 kJ mol-1, respectively, above the associated triplet thresholds. These results indicated that the curve crossing required to form singlet products upon reaction with triplet O2 is favorable in the case of hydroquinone and unfavorable in the cases of catechol and resorcinol. In practical terms, the selective oxygen addition appears to be a diagnostically useful reaction to differentiate hydroquinone from its ring isomers.

Fragmentation pathways of deprotonated ortho-hydroxybenzyl alcohol.

The ortho, meta, and para isomers of hydroxybenzyl alcohol can be unequivocally distinguished by the collision-induced dissociation mass spectra of their anions. The presence of a prominent peak at m/z 121 for an elimination of a dihydrogen molecule renders the ortho-isomer spectrum markedly different from those of its meta and para congeners. Investigations carried out with deuterium-labeled isotopologues of the ortho isomer verified that the labile hydrogen atom on the hydroxyl group and one of the benzylic hydrogen atoms are specifically removed in the formation of the m/z 121 ion. The ortho-isomer spectrum also showed a prominent peak at m/z 93. Experimental data indicated that the m/z 93 product ion originates either from a two-step H2 and CO elimination mechanism or from a direct loss of a HCHO molecule from the precursor anion. The intensity ratio of the m/z 93 and 94 peaks in the spectrum recorded from the m/z 124 ion generated from a sample of o-hydroxybenzyl alcohol dissolved in D2 O supported the notion that the direct HCHO loss is the more dominant pathway for the generation of the phenolate ion under low activation conditions. In contrast, the two-step mechanism becomes the more dominant pathway under high collisional activation conditions. The spectrum also showed a weak peak at m/z 105 for a water loss. Based on computational data, the m/z 105 ion generated in this way appears to be a composite generated from a common ion-neutral complex intermediate in which a hydroxyl anion is positioned equidistantly between one of the benzylic hydrogens and a nearby hydrogen atom of the benzene ring. Upon activation, the complex dissociates to form either a phenide or a quinone methide anion. The reaction forming a carbon dioxide adduct under ion-mobility conditions was used to support the proposed water-loss mechanism.

Dehydrogenation of 1-Phenylethanol Catalyzed by Nickel(II)diphosphine Complexes.

Catalytic efficacy of the nickel(II)-diphosphine systems in the dehydrogenation of 1-phenylethanol to acetophenone under acceptorless conditions was investigated. Steric and electronic factors of the phosphine ligands were found to play an important role in the catalysis, while the nature of the base used and the reaction conditions, viz. time, temperature, and stoichiometry, have also shown major influence. Based on the preliminary analysis, a homogeneous pathway, perhaps involving nickel hydride species, was proposed. Due to the gradual disintegration of the catalytic species, deterioration of catalytic activity was observed resulting into low to moderate conversions. Among the series of catalysts examined, the highest conversion of 52% was exhibited by the catalyst C4, dichloro(1,2-bis(diphenylphosphino)ethane)nickel(II) (5 mol%), when loaded with 50 mol% of sodium ethoxide in toluene at 120 °C.

Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.