Characterization of the Retro-[3+2] Reaction of Protonated 2,3′-Bisindolylmethanes by Electrospray Ionization–Tandem Mass Spectrometry

Twelve 2,3′-bisindolylmethanes with various substituents were investigated using electrospray ionization quadrupole time-of-flight tandem mass spectrometry in positive ion mode. A retro-[3+2] reaction was observed in the collision-induced dissociation spectra of protonated 2,3′-bisindolylmethanes for the first time. The mechanism of retro-[3+2] reaction was concerted or stepwise. For the concerted pathway, carbon–carbon bonds of a protonated compound simultaneously cracked and the m/z 208 ion ([C₁₅H₁₀D₂N]⁺) was observed with hydrogen–deuterium exchange labeling. The stepwise pathway goes through 1,3-hydrogen migration twice and the m/z 208 ion ([C₁₅H₁₀D₂N]⁺) and m/z 207 ion ([C₁₅H₁₁DN]⁺) were detected with deuterium labeling. In the deuterium-labeled tandem mass spectrum for one compound, only the peak at m/z 208 was present at high abundance, suggesting that the concerted pathway is more likely. In addition, the substituents have no obvious trends on the ratios of the product intensity to the base intensity, further supporting the concerted pathway.     

Novel Copolymers of 4-Fluorostyrene. 1. Alkyl Ring-Substituted 2-Phenyl-1,1-dicyanoethylenes

Novel copolymers of trisubstituted ethylene monomers, alkyl ring-substituted 2-phenyl-1,1-dicyanoethylenes, RC₆H₄CH = C(CN)₂ (where R is 2-methyl, 3-methyl, 4-methyl, 4-ethyl, 4-i-propyl, 4-butyl, 4-i-butyl, and 4-t-butyl) and 4-fluorostyrene were prepared at equimolar monomer feed composition by solution copolymerization in the presence of a radical initiator (ABCN) at 70°C. The composition of the copolymers was calculated from nitrogen analysis, and the structures were analyzed by IR, ¹H and ¹³C-NMR. The order of relative reactivity (1/r ₁) for the monomers is 4-ethyl (42.6) > 4-butyl (29.4) > 4-t-butyl (26.7) > 4-i-butyl (1.6) > 4-i-propyl (1.29) > 3-methyl (1.26) > 2-methyl (0.8) > 4-methyl (0.4). High T _g of the copolymers, in comparison with that of poly(4-fluorostyrene) indicates a substantial decrease in chain mobility of the copolymer due to the high dipolar character of the trisubstituted ethylene monomer unit. Decomposition of the copolymers in nitrogen occurred in two steps, first in 183–500°C range with residue (5–30% wt.), which then decomposed in the 500–800°C range.     

Mass spectra of ethenol and its deutero analogues

Ethenol, 1-d-ethenol, O-d-ethenol and Z-2-d-ethenol were prepared by pyrolysis of corresponding 5-norbornenols at 800^°C/2 × 10^?6 Torr. The most important fragments in the electron impact mass spectrum of ethenol are [C₂H₃O]⁺ and CHO⁺ and CH₃˙. The hydrogen atom eliminated from the molecular ion comes mainly from the hydroxyl group (68%) and to a lesser extent from C(1) (25%) and C(2) (7%). The loss of the hydroxyl hydrogen is preceded by rate-determining migration of the hydrogen atom from C(1) onto C(2) to yield CH₃C?OH⁺˙ions that decompose to CH₃CO⁺ and H˙. The loss of deuterium from O-d-ethenol shows a very small primary isotope effect (k_H/k_D=1.07), whereas a significant effect is observed for the loss of hydrogen from 1-d-ethenol (k_H/k_D=1.28). The appearance energy of [C₂H₂DO]⁺ from 1-d-ethenol, AE=11.32 eV, gives a critical energy for the hydrogen loss, E=203 kJ mol^?1, which is 90 kJ mol^?1 above the thermochemical threshold for CH₃CO⁺+H˙. The appearance energy of CDO⁺ from 1-d-ethenol was measured as 12.96±0.07 eV, which sets the barrier to isomerization to CH₃CDO⁺˙ at 1121 kJ mol^?1. The ionization energy of ethenol was found to be 9.22±0.03 eV.     

Formation of the bisulfite anion (HSO3–, m/z 81) upon collision‐induced dissociation of anions derived from organic sulfonic acids

In the negative‐ion collision‐induced dissociation mass spectra of most organic sulfonates, the base peak is observed at m/z 80 for the sulfur trioxide radical anion (SO₃^–·). In contrast, the product‐ion spectra of a few sulfonates, such as cysteic acid, aminomethanesulfonate, and 2‐phenylethanesulfonate, show the base peak at m/z 81 for the bisulfite anion (HSO₃^–). An investigation with an extensive variety of sulfonates revealed that the presence of a hydrogen atom at the β‐position relative to the sulfur atom is a prerequisite for the formation of the bisulfite anion. The formation of HSO₃^– is highly favored when the atom at the β‐position is nitrogen, or the leaving neutral species is a highly conjugated molecule such as styrene or acrylic acid. Deuterium‐exchange experiments with aminomethanesulfonate demonstrated that the hydrogen for HSO₃^– formation is transferred from the β‐position. The presence of a peak at m/z 80 in the spectrum of 2‐sulfoacetic acid, in contrast to a peak at m/z 81 in that of 3‐sulfopropanoic acid, corroborated the proposed hydrogen transfer mechanism. For diacidic compounds, such as 4‐sulfobutanoic acid and cysteic acid, the m/z 81 ion can be formed by an alternative mechanism, in which the negative charge of the carboxylate moiety attacks the α‐carbon relative to the sulfur atom. Experiments conducted with deuterium‐exchanged and deuterium‐labeled analogs of sulfocarboxylic acids demonstrated that the formation of the bisulfite anion resulted either from a hydrogen transfer from the β‐carbon, or from a direct attack by the carboxylate moiety on the α‐carbon. Copyright © 2012 John Wiley & Sons, Ltd.     

Rearrangements and isomerism in the molecular ion of o-methoxybenzoic acid

The molecular ion of o-methoxy-d₃-benzoic acid can undergo three different rearrangements and hydrogen/deuterium exchange between the functional groups. This can result in 14 isomers of the molecular ion before it decomposes giving fragments at m/z 105, 106, 107 and 108. Three types of isomer have been identified.     

Selectivity of gas‐phase ion/molecule reaction of carbon dioxide with phenide ions

On contrary to the widely accepted conviction that the m/z 93 ion derived from phenol does not react with CO₂, we demonstrate that it makes an adduct with CO₂ to a small but demonstrable extent. For example, the product‐ion mass spectrum recorded for the m/z 98 ion derived from [²H₆]phenol showed a small peak at m/z 142 when CO₂ was used as the collision gas. The formation of an m/z 137 adduct ion from the m/z 93 ion (generated either directly from phenol, or indirectly from salicylic acid by in‐source decarboxylation) was demonstrated also by multiple‐reaction‐monitoring tandem mass spectrometric experiments. According to literature, the m/z 93 ion derived from salicylic acid does not undergo CO₂ addition because it is deemed to exist only in the phenoxide form. This reaction has been previously proposed as a method for differentiating phenoxide ion from its isomeric hydroxyphenide ions. We propose that the m/z 93 ion, albeit small, exists also as the phenide form together with the predominant phenoxide ion. Copyright © 2014 John Wiley & Sons, Ltd.     

Mechanistic study of the decomposition reactions of azobenzene

The electron impact-induced fragmentation of azobenzenes and its d₁, d₂, d₅, d₁₀, and ¹⁵N analogues was studied by mass Spectrometry and ion kinetic energy spectroscopy. The main fragment ions found in the mass spectrum of azobenzene are due to two parallel stepwise processes from the molecular ion: the expulsion of N₂ and two hydrogen radicals producing an ion at m/z 152 having possibly a biphenylene radical cation structure and loss of C₆H₅? and N₂. Except in the elimination of two hydrogen atoms from [M ? N₂]^+· ions, hydrogen scrambling between the phenyl rings does not feature in azobenzene upon electron impact.     

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 H₂and 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 D₂O 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.     

An energy-resolved study of the fragmentation of ionized 1-Penten-3-ol

A detailed energy-resolved study of the fragmentation of CH₂?CHCH(OH)CD₂CD₃ (1-d₅) has been carried out using metastable ion studies and charge exchange techniques, combined with collision-induced dissociation studies to establish the structures of fragment ions. At low internal energies (metastable ions) the molecular ion of 1-d₅ rearranges to the 3-pentanone structure and fragments by loss of C₂H₅ or C₂D₅ leading to the acyl structure, [CH₃CH₂C?O]⁺ or [CD₃CD₂C?O]⁺, for the fragment ion. However, with increasing internal energy of the molecular ion this rearrangement process decreases rapidly in importance and loss of C₂D₅ by direct cleavage, leading to [CH₂?CHCH?OH]⁺, becomes the dominant fragmentation reaction. As a result the [C₃H₅O]⁺ ion seen in the electron impact mass spectrum of 1-penten-3-ol has predominantly the protonated acrolein structure.     

A Distonic Radical-Ion for Detection of Traces of Adventitious Molecular Oxygen (O2) in Collision Gases Used in Tandem Mass Spectrometers

We describe a diagnostic ion that enables rapid semiquantitative evaluation of the degree of oxygen contamination in the collision gases used in tandem mass spectrometers. Upon collision-induced dissociation (CID), the m/z 359 positive ion generated from the analgesic etoricoxib undergoes a facile loss of a methyl sulfone radical [^?SO₂(CH₃); 79-Da] to produce a distonic radical cation of m/z 280. The product-ion spectrum of this m/z 280 ion, recorded under low-energy activation on tandem-in-space QqQ or QqTof mass spectrometers using nitrogen from a generator as the collision gas, or tandem-in-time ion-trap (LCQ, LTQ) mass spectrometers using purified helium as the buffer gas, showed two unexpected peaks at m/z 312 and 295. This enigmatic m/z 312 ion, which bears a mass-to-charge ratio higher than that of the precursor ion, represented an addition of molecular oxygen (O₂) to the precursor ion. The exceptional affinity of the m/z 280 radical cation towards oxygen was deployed to develop a method to determine the oxygen content in collision gases. Figure

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Synthesis,in silico and in vitro Evaluation of Novel Oxazolopyrimidines as Promising Anticancer Agents

New potential bioactive oxazolopyrimidines have been synthesized using two main approaches: the pyrimidine ring annulation on a functionalized oxazole and the benzoyl bromide trimerization followed by rearrangement and formation of the oxazolo[5,4-d]pyrimidine scaffold. The docking analyzes have shown that 7-piperazine substituted oxazolo[4,5-d]pyrimidines 8a – 8c could be potential VEGFR2 inhibitors with high free energy of ligand–protein complex formation (ΔG: −10.1, −9.6, −9.8 kcal/mol, respectively). In vitro antitumor assays confirmed theoretical predictions that oxazolo[4,5-d]pyrimidines 8a – 8c containing positively charged piperazine moiety should demonstrate significantly higher cytotoxic effects. 4-[5-(4-Chlorophenyl)-2-phenyl[1,3]oxazolo[4,5-d]pyrimidin-7-yl]piperazin-1-ium trifluoroacetate ( 8c ) exhibited a slightly higher antiproliferative effect (IC₅₀=0.21 μm ) than doxorubicin (IC₅₀=0.36 μm ) on MDA-MB-231 cell line and has relatively good results on OVCAR-3 (IC₅₀=1.7 μm ) and HCT-116 (IC₅₀=0.24 μm ) cells.     

Fragmentation of the metastable molecular ion of methyl lactate: The formation of oxygen-protonated methanol [CH3OH2]+ involving double hydrogen atom transfers

The spontaneous unimolecular dissociation reaction of methyl lactate (1) ionized by electron impact was investigated by a combination of mass-analyzed ion kinetic energy spectrometry and deuterium labeling. The metastable ions 1^+· decompose in a variety of ways: four fragment peaks are observed at m/z 89, 76, 61, and 45, which correspond to the losses of ?H₃, CO, CH₃?O, and ?OOCH₃, respectively. Double hydrogen atom transfer occurs in the third reaction. The source-generated m/z 61 ions decompose into oxygen-protonated methanols at m/z 33 ([CH₃OH ₂ ⁺ ]) by the loss of CO with double hydrogen atom migration. Both hydroxyl and methyne hydrogen atoms in 1 ^+· are present in the resultant protonated methanols.     

Ion cyclotron resonance mass spectrometric study of ion—molecule reactions in toluene—pyridine mixtures

Ion—molecule reactions in toluene, toluene-d₈, pyridine, 4-methyl-, 4-ethyl- and 4-terr-butylpyridine and quinoline and their mixtures were studied by ICR mass spectrometry at 10^-7 Torr (1 Torr = 133.3 Pa). The reactions of benzyl (m/z 91), benzyl-d₇ (m/z 98), methylbenzyl (m/z 105), and azabenzyl (m/z 92) ions with pyridines and quinoline were observed. The reactions of protonated pyridines with unprotonated molecules leading to homoconjugated ions (BHB)⁺ were detected.     

Dissociation of the diphenylnitrile imine radical cation into benzonitrile and [phenylnitrene]+˙

The collision induced dissociation/mass analysed ion kinetic energy mass spectra of 2,5-diphenyltetrazole demonstrate the decay sequence [diphenyltetrazole]^+˙→ [diphenylnitrile imine]^+˙→ m/z 91. The m/z 91 ion was shown to be identical to the ion formed by loss of N₂ from the phenyl azide radical cation, thus suggesting the phenylnitrene structure for the m/z 91 ion.     

A feature of the fragmentation mechanism of exo-2-norbornyl chloride. Stereoselective hydrogen atom abstraction revealed by metastable ion studies

The position of deuterium in the products of the addition of HCL and DCI to exo 5, 6-d₂ norbornene has been determined mass spectrometrically by measuring metastable ion abundances. The results show a stereo-selective hydrogen atom abstraction when the molecular ion of exo-2-norbornyl chloride fragements by loss of a chloroethyl radical.     

Chemical discrimination of multilayered paint cross sections for potential forensic applications using time‐of‐flight secondary ion mass spectrometry

Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) equipped with a bismuth imaging source and an argon gas cluster ion beam (GCIB) was used to image polished cross‐sections of four automotive multilayer paint samples. Secondary ion mass spectrometry chemical imaging of the individual layers was possible after a GCIB sputter ion dose of (7 × 10¹⁵) ions/cm² was applied for the removal of polishing residue, at which point the chemical composition of the individual clear coats could be distinguished using principal components analysis. For the differentiation of the four clear coat chemistries, only four secondary ion peaks were necessary; C₂H₅O⁺ (m/z 45.04), C₉H₉NO₂⁺ (m/z 163.09), and C₁₀H₁₁NO₂⁺ (m/z 177.10) that appeared to be fragments of the carbamate‐based clear coat, and C₇H₁₁⁺ (m/z 95.09) that was strongly associated with the polyurethane‐based clear coat. Clear identification of the four paint samples based on this short peak list highlights the strength of the SIMS technique as a potential forensic approach to discriminate automotive paints and suggests that many more variables could be included in the multivariate and statistical analysis to differentiate a wider range of clear coat chemistries.     

Ion–molecule reactions of fragment ions derived from protonated allyl methyl ether

Unusual behaviour has been noted for allyl methyl ether (1) chemically ionized in a high-pressure ion source. Tandem mass spectrometry indicates the formation of methylcyclopentadienyl and methoxy-1-propenylcarbenium ions (d, m/z 81 and e, m/z 85). The origin of these unexpected ions has been elucidated using conventional and Fourier transform ion cyclotron resonance experiments: primary fragment ions derived from protonated 1 (allyl ions a and methoxymethyl cations b) generate collision complexes with neutral 1, giving rise to the ions d and e, respectively, after methanol elimination.     

The substituent effect on the single and double hydrogen atom transfer reactions in para-substituted benzoic acid isobutyl esters

The substituent effect on the single and double hydrogen atom transfer reactions in para-substituted benzoic acid isobutyl esters has been investigated by electron impact mass spectrometry. Electron-donating substituents favour formation of the [M? C₄H₈]⁺˙ ion generated by single hydrogen atom transfer reaction (McLafferty rearrangement), whereas electron-withdrawing substituents favour formation of the [M? C₄H₇]⁺ ion generated by double hydrogen atom transfer reaction. In the case of the latter compounds, the m/z56 ([C₄H₈]⁺˙) ion, which is generated by single hydrogen atom transfer reaction with charge migration, is very intense, while in the former compounds, the m/z56 ion is very weak. These observations can be reasonably explained on thermochemical grounds based on the sum of the standard heats of formation of the fragments.     

Backbone and side-chain specific dissociations of <Emphasis Type="Italic">z</Emphasis> ions from non-tryptic peptides

Backbone z-type fragment ions formed by electron-transfer dissociation (ETD) of doubly protonated peptides AAHAL, AHDAL, and AHADL were subjected to collisional activation and their dissociation products were studied by ETD-CID-MS³ and MS⁴. Electron structure theory calculations were performed to elucidate ion structures and reaction mechanisms. All z ions showed competitive eliminations of C₃H₇ and C₄H₈ from the C-terminal Leu side chain. The energetics and kinetics of these dissociations were studied computationally for the z₄ ion from AAHAL, and optimized structures are reported for several intermediates and transition states. RRKM calculations on the combined B3LYP and PMP2/6-311++G(2d,p) potential energy surface provided unimolecular rate constants that closely reproduced the experimental branching ratios for C₃H₇ and C₄H₈ eliminations. Mechanisms were also studied for the loss of CO₂ from z ions generated by ETD of AHDAL and AHADL and for a specific radical-induced Asp-C_α-CO backbone cleavage. CID of the z ions under study did not produce any fragment ions that would indicate cascade backbone dissociations triggered by the radical sites. In contrast, the majority of backbone dissociations occurred at bonds that were remote from the radical sites (spin-remote dissociations) and were triggered by proton migrations that were analogous to those considered for standard peptide ion fragmentations.     

Identification of Antifungal Peptides from Bacillus subtilis BS-918

Bacillus subtilis BS-918 shows strong activity against a broad spectrum of plant and postharvest pathogenic fungi. Antifungal compounds produced by BS-918 were isolated by extraction and gel chromatography, and structural identification was performed by electrospray ionization coupled with collision induced dissociation mass spectrometry. Two kinds of precursor ions belonging to iturin and fengycin were revealed. The first precursor ion at m/z 1071.7 was analyzed by collision induced dissociation mass spectrometry and was identified to be iturin A with a C₁₆ β-amino fatty acid chain. Collision induced dissociation mass spectrometry of the second precursor showed the presence of two fengycins. Ions at m/z 1449.5 and m/z 1463.9 were identified to homologs of fengycin A with C₁₅ and C₁₆ β-hydroxy fatty acid chains, whereas ions at m/z 1477.8 and m/z 1491.9 were homologs of fengycin B with C₁₅ and C₁₆ fatty acid chains.