Unlabeled and deuterium-labeled dimeric lignin model compounds with β-O-4 linkages were evaporated and ionized using negative ion mode electrospray ionization, transferred into a linear quadrupole ion trap, isolated, and subjected to collision-activated dissociation (CAD; MS2 experiments). The elemental compositions of the fragment ions were determined by using a high-resolution Orbitrap mass analyzer, and their structures were examined using further CAD experiments (MS n experiments wherein n = 2–5). Data analysis was facilitated by determining the fragmentation pathways for several deprotonated model compounds. The structures of the key fragment ions of several pathways were determined by comparison of the CAD mass spectra measured for undeuterated and deuterated analogues and for deprotonated authentic compounds. Some of the proposed reaction mechanisms were tested by examining additional deprotonated synthetic model compounds. Quantum chemical calculations were used to delineate the most likely reaction pathways and reaction mechanisms. This work provides basic information needed for the design of tandem mass spectrometry-based CAD sequencing strategies for mixtures of lignin degradation products.
Glucuronidation, a common phase II biotransformation reaction, is one of the major in vitro and in vivo metabolism pathways of xenobiotics. In this process, glucuronic acid is conjugated to a drug or a drug metabolite via a carboxylic acid, a hydroxy, or an amino group to form acyl-, O-, and/or N-glucuronide metabolites, respectively. This process is traditionally thought to be a detoxification pathway. However, some acyl-glucuronides react with biomolecules in vivo, which may result in immune-mediated idiosyncratic drug toxicity (IDT). In order to avoid this, one may attempt in early drug discovery to modify the lead compounds in such a manner that they then have a lower probability of forming reactive acyl-glucuronide metabolites. Because most drugs or drug candidates bear multiple functionalities, e.g., hydroxy, amino, and carboxylic acid groups, glucuronidation can occur at any of those. However, differentiation of isomeric acyl-, N-, and Oglucuronide derivatives of drugs is challenging. In this study, gas-phase ion−molecule reactions between deprotonated glucuronide metabolites and BF 3 followed by collision-activated dissociation (CAD) in a linear quadrupole ion trap mass spectrometer were demonstrated to enable the differentiation of acyl-, N-, and O-glucuronides. Only deprotonated Nglucuronides and deprotonated, migrated acyl-glucuronides form the two diagnostic product ions: a BF 3 adduct that has lost two HF molecules, [M − H + BF 3 − 2HF] − , and an adduct formed with two BF 3 molecules that has lost three HF molecules, [M − H + 2BF 3 − 3HF] − . These product ions were not observed for deprotonated O-glucuronides and unmigrated, deprotonated acyl-glucuronides. Upon CAD of the [M − H + 2BF 3 − 3HF] − product ion, a diagnostic fragment ion is formed via the loss of 2-fluoro-1,3,2-dioxaborale (MW of 88 Da) only in the case of deprotonated, migrated acyl-glucuronides. Therefore, this method can be used to unambiguously differentiate acyl-, N-, and O-glucuronides. Further, coupling this methodology with HPLC enables the differentiation of unmigrated 1-β-acyl-glucuronides from the isomeric acyl-glucuronides formed upon acyl migration. Quantum chemical calculations at the M06-2X/6-311++G(d,p) level of theory were employed to probe the mechanisms of the reactions of interest.
Examination of the reactions of σ-type quinoliniumbased triradicals with cyclohexane in the gas phase demonstrated that the radical site that is the least strongly coupled to the other two radical sites reacts first, independent of the intrinsic reactivity of this radical site, in contrast to related biradicals that first react at the most electron-deficient radical site. Abstraction of one or two H atoms and formation of an ion that formally corresponds to a combination of the ion and cyclohexane accompanied by elimination of a H atom ("addition-H") were observed. In all cases except one, the most reactive radical site of the triradicals is intrinsically less reactive than the other two radical sites. The product complex of the first H atom abstraction either dissociates to give the H-atom-abstraction product and the cyclohexyl radical or the more reactive radical site in the produced biradical abstracts a H atom from the cyclohexyl radical. The monoradical product sometimes adds to cyclohexene followed by elimination of a H atom, generating the "addition-H" products. Similar reaction efficiencies were measured for three of the triradicals as for relevant monoradicals. Surprisingly, the remaining three triradicals (all containing a meta-pyridyne moiety) reacted substantially faster than the relevant monoradicals. This is likely due to the exothermic generation of a meta-pyridyne analog that has enough energy to attain the dehydrocarbon atom separation common for H-atom-abstraction transition states of protonated meta-pyridynes.
The gaseous 2,6-didehydropyridinium cation and its derivatives transfer a proton to reagents for which the reaction for their singlet ground states is too endothermic to be observed. These reactions occur from the lowest-energy excited triplet states, which has not been observed (or reported) for other meta-benzyne analogues. Quantum chemical calculations indicate that the (excited) triplet states are stronger Brønsted acids than their (ground) singlet states, likely due to unfavorable three-center, four-electron interactions in the singlet-state conjugate bases. The cations have substantially smaller (calculated) singlet–triplet (S–T) splittings (ranging from ca. −11 to −17 kcal mol–1) than other related meta-benzyne analogues (e.g., −23.4 kcal mol–1 for the 3,5-isomer). This is rationalized by the destabilization of the singlet states (relative to the triplet states) by reduced (spatial) overlap of the nonbonding molecular orbitals due to the presence of the nitrogen atom between the radical sites (making the ring more rigid). Both the singlet and triplet states are believed to be generated upon formation of these biradicals via energetic collisions due to their small S–T splittings. It appears that once the triplet states are formed, the rate of proton transfer is faster than the rate of intersystem crossing unless the biradicals contain heavy atoms.
Substituted ureas correspond to a class of organic compounds commonly used in agricultural and chemical fields. However, distinguishing between different ureas and differentiating substituted ureas from other compounds with similar structures, such as amides, N-oxides, and carbamates, are challenging. In this paper, a four-stage tandem mass spectrometry method (MS 4 ) is introduced for this purpose. This method is based on gas-phase ion-molecule reactions of isolated, protonated analytes ([M + H] + ) with tris(dimethylamino)borane (TDMAB) (MS 2 ) followed by subjecting a diagnostic product ion to two steps of collisionactivated dissociation (CAD) (MS 3 and MS 4 ). All the analyte ions reacted with TDMAB to form a product ion [M + H + TDMAB − HN(CH 3 ) 2 ] + . The product ion formed for substituted ureas and amides eliminated another HN(CH 3 ) 2 molecule upon CAD to generate a fragment ion [M + H + TDMAB − 2HN(CH 3 ) 2 ] + , which was not observed for many other analytes, such as N-oxides, sulfoxides, and pyridines (studied previously). When the [M + H + TDMAB − 2HN(CH 3 ) 2 ] + fragment ion was subjected to CAD, different fragment ions were generated for ureas, amides, and carbamates. Fragment ions diagnostic for the ureas were formed via elimination of RNCO (R = hydrogen atom or a substituent), which enabled the differentiation of ureas from amides and carbamates. Furthermore, these fragment ions can be utilized to classify differently substituted ureas. Quantum chemical calculations were employed to explore the mechanisms of the reactions. The limit of detection for the diagnostic ion-molecule reaction product ion in HPLC/MS 2 experiments was found to range from 20 to 100 nM.
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