Insignoic acids A - E, unusual α, ß-unsaturated keto fatty acids isolated from the exocarp of Australian rainforest tree Endiandra insignis (Lauraceae).

Anti-inflammatory bioassay-guided compound isolation from the exocarp of the Australian rainforest tree Endiandra insignis (family Lauraceae) has led to the discovery and structural elucidation of unusual α, ß-unsaturated twenty-four carbon fatty acids and their positional isomers, insignoic acids A - E (1a - 5c). The stereochemistry and position of the double bond within the aliphatic chain were independently determined via NMR spectroscopy and Ozone-Induced Dissociation (OzID) Mass Spectrometry, respectively. Compounds (1a - 5c) displayed good to moderate anti-inflammatory activity in the range of 8-84 µM. The low therapeutic index observed when assessing the cell viability in the RAW macrophage cell lines, prompted us to investigate the anticancer potential of these unusual fatty acids. The anti-cancer activity was assessed in A-431 carinoma cell lines and MM649 melanoma cell lines. Insignoic acid C (3a-f) exhibited the highest level of potency with an IC50 value of 5-7 µM against both the cell lines. The insignoic acids are the first of their kind known for incorporating an alpha-beta unsaturated system flanked next to a keto group with an additional level of oxygenation at C-6 in a 24­carbon fatty acid backbone.

Mass Spectrometry Imaging of Lipids Using MALDI Coupled with Plasma-Based Post-Ionization on a Trapped Ion Mobility Mass Spectrometer.

Here we report the development and optimization of a mass spectrometry imaging (MSI) platform that combines an atmospheric-pressure matrix-assisted laser desorption/ionization platform with plasma postionization (AP-MALDI-PPI) and trapped ion mobility spectrometry (TIMS). We discuss optimal parameters for operating the source, characterize the behavior of a variety of lipid classes in positive- and negative-ion modes, and explore the capabilities for lipid imaging using murine brain tissue. The instrument generates high signal-to-noise for numerous lipid species, with mass spectra sharing many similarities to those obtained using laser postionization (MALDI-2). The system is especially well suited for detecting lipids such as phosphatidylethanolamine (PE), as well as numerous sphingolipid classes and glycerolipids. For the first time, the coupling of plasma-based postionization with ion mobility is presented, and we show the value of ion mobility for the resolution and identification of species within rich spectra that contain numerous isobaric/isomeric signals that are not resolved in the m/z dimension alone, including isomeric PE and demethylated phosphatidylcholine lipids produced by in-source fragmentation. The reported instrument provides a powerful and user-friendly approach for MSI of lipids.

Solvent-Mediated Proton-Transfer Catalysis of the Gas-Phase Isomerization of Ciprofloxacin Protomers.

Understanding how neutral molecules become protonated during positive-ion electrospray ionization (ESI) mass spectrometry is critically important to ensure analytes can be efficiently ionized, detected, and unambiguously identified. The ESI solvent is one of several parameters that can alter the dominant site of protonation in polyfunctional molecules and thus, in turn, can significantly change the collision-induced dissociation (CID) mass spectra relied upon for compound identification. Ciprofloxacin─a common fluoroquinolone antibiotic─is one such example whereby positive-ion ESI can result in gas-phase [M + H]+ ions protonated at either the keto-oxygen or the piperazine-nitrogen. Here, we demonstrate that these protonation isomers (or protomers) of ciprofloxacin can be resolved by differential ion mobility spectrometry and give rise to distinctive CID mass spectra following both charge-directed and charge-remote mechanisms. Interaction of mobility-selected protomers with methanol vapor (added via the throttle gas supply) was found to irreversibly convert the piperazine N-protomer to the keto-O-protomer. This methanol-mediated proton-transport catalysis is driven by the overall exothermicity of the reaction, which is computed to favor the O-protomer by 93 kJ mol-1 (in the gas phase). Conversely, gas phase interactions of mobility-selected ions with acetonitrile vapor selectively depletes the N-protomer ion signal as formation of stable [M + H + CH3CN]+ cluster ions skews the apparent protomer population ratio, as the O-protomer is unaffected. These findings provide a mechanistic basis for tuning protomer populations to ensure faithful characterization of multifunctional molecules by tandem mass spectrometry.

Gas phase reactions of iodide and bromide anions with ozone: evidence for stepwise and reversible reactions.

Despite the impacts - both positive and negative - of atmospheric ozone for life on Earth, there remain significant gaps in our knowledge of the products, mechanisms and rates of some of its most fundamental gas phase reactions. This incomplete understanding is largely due to the experimental challenges involved in the study of gas-phase reactions of ozone and, in particular, the identification of short-lived reaction intermediates. Here we report direct observation of the stepwise reaction of the halide anions iodide (I-) and bromide (Br-) with ozone to produce XO3- (where X = I and Br, respectively). These results substantially revise the rate constant for the I- + O3 reaction to 1.1 (± 0.5) × 10-12 cm3 molecule-1 s-1 (0.13% efficiency) and the Br- + O3 reaction to 6.2 (± 0.4) × 10-15 cm3 molecule-1 s-1 (0.001% efficiency). Exploiting five-orders of temporal dynamic range on a linear ion trap mass spectrometer enabled explicit measurement of the rate constants for the highly efficient intermediate, XO- + O3 and XO2- + O3, reactions thus confirming a stepwise addition of three oxygen atoms (i.e., X- + 3O3 → XO3- + 3O2) with the first addition representing the rate determining step. Evidence is also presented for (i) slow reverse reactions of XO- and XO2-, but not XO3-, with molecular oxygen and (ii) the photodissociation of IO-, IO2- and IO3- to release I-. Collectively, these results suggest relatively short lifetimes for Br- and I- in the tropospere with direct gas-phase oxidation by ozone playing a role in both the formation of atmospheric halogen oxides and, conversely, in the ozone depletion associated with springtime polar bromine explosion events.

Selecting and identifying gas-phase protonation isomers of nicotineH+ using combined laser, ion mobility and mass spectrometry techniques.

The detection and assignment of protonation isomers, termed protomers, of gas-phase ions remains a challenge in mass spectrometry. With the emergence of ion-mobility techniques combined with tuneable-laser photodissociation spectroscopy, new experimental combinations are possible to now meet this challenge. In this paper, the differences in fragmentation and electronic spectroscopy of singly protonated (S)-nicotine (nicH+) ions are analysed using action spectroscopy in the ultraviolet region and field asymmetric ion mobility spectrometry (FAIMS). Experiments are supported by quantum chemical calculations (DFT, TD-DFT and CC2) of both spectroscopic and thermochemical properties. Electrospray ionisation (ESI) of (S)-nicotine from different solvents leads to different populations of two nicH+ protomers corresponding to protonation on the pyridine nitrogen and pyrrolidine nitrogen, respectively. FAIMS gives partial resolution of these protomers and enables characteristic product ions to be identified for each isomer as verified directly by analysis of product-ion specific action spectroscopy. It is shown that while characteristic, these product ions are not exclusive to each protomer. Calculations of vertical electronic transitions assist in rationalising the photodissociation action spectra. The integration of photodissociation action spectroscopy with FAIMS-mass spectrometry is anticipated to be a useful approach for separating and assigning protonation isomers of many other small molecular ions.

Reaction of ionised steryl esters with ozone in the gas phase.

Cholesterol is an ubiquitous membrane lipid, that also serves as a precursor to many steroid hormones. The 5,6 carbon-carbon double bond on the tetracyclic carbon backbone of cholesterol is an attractive target for ozone with the reaction giving rise to a wide range of possibly bioactive molecules. Despite this, little is known about the ozonolysis of cholesterol esters, which often possess an additional double bond(s) on the fatty acyl chain. Understanding the intrinsic gas phase reaction of ozone with the two disparate double bond positions on cholesteryl esters can inform our understanding of these processes in vivo, particularly reactions occurring at the air-water interface (e.g., tear film lipid layer) and on the surfaces of the body where these cholesterol and cholesteryl esters may be present (e.g., sebum). In the present work we describe the gas phase ozonolysis of lithium and sodium cations formed from three steryl esters: two isomeric for double bond position (cholestanyl oleate and cholesteryl stearate), and a third with carbon-carbon double bonds present in both the sterol ring system and fatty acyl chain (cholesteryl oleate). We confirm the enhanced reactivity of the endocyclic carbon-carbon double bond with ozone over double bonds present in the acyl chain, and elucidate competitive interactions between the two double bond positions during ozonolysis. Elucidation of the mechanisms underlying this interaction is important for both understanding these processes in vivo and for deploying ozonolysis chemistry in analytical strategies for lipidomics.

Differential-Mobility Spectrometry of 1-Deoxysphingosine Isomers: New Insights into the Gas Phase Structures of Ionized Lipids.

Separation and structural identification of lipids remain a major challenge for contemporary lipidomics. Regioisomeric lipids differing only in position(s) of unsaturation are often not differentiated by conventional liquid chromatography-mass spectrometry approaches leading to the incomplete, or sometimes incorrect, assignation of molecular structure. Here we describe an investigation of the gas phase separations by differential-mobility spectrometry (DMS) of a series of synthetic analogues of the recently described 1-deoxysphingosine. The dependence of the DMS behavior on the position of the carbon-carbon double bond within the ionized lipid is systematically explored and compared to trends from complementary investigations, including collision cross-sections measured by drift tube ion mobility, reaction efficiency with ozone, and molecular dynamics simulations. Consistent trends across these modes of interrogation point to the importance of direct, through-space interactions between the charge site and the carbon-carbon double bond. Differences in the geometry and energetics of this intramolecular interaction underpin DMS separations and influence reactivity trends between regioisomers. Importantly, the disruption and reformation of these intramolecular solvation interactions during DMS are proposed to be the causative factor in the observed separations of ionized lipids which are shown to have otherwise identical collision cross-sections. These findings provide key insights into the strengths and limitations of current ion-mobility technologies for lipid isomer separations and can thus guide a more systematic approach to improved analytical separations in lipidomics.

Determination of ester position in isomeric (O-acyl)-hydroxy fatty acids by ion trap mass spectrometry.

RATIONALE: (O-acyl)-hydroxy fatty acids (OAHFAs) are a recently discovered class of endogenous lipids, generating significant interest for their correlation with enhanced glucose tolerance. Structural variants that differ in the position of the ester linkage have been described, including the ω-OAHFA sub-class, that plays a key role in stabilizing the human tear film. Developing analytical tools for rapid and unambiguous structural elucidation of OAHFAs is essential to understanding their diverse physiological functions. METHODS: Commercially available and synthesized OAHFA standards were dissolved in chloroform and subsequently diluted into methanol with 1.5 mM ammonium acetate. Negative ion collision-induced dissociation (CID) MSn spectra were acquired using chip-based nano-electrospray ionization (Advion TriVersa NanoMate) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). RESULTS: Major product ions observed during CID of [OAHFA - H]- ions readily identify the constituent fatty acid and hydroxy fatty acid; however, isomers are not easily distinguished. Interrogation of the hydroxy fatty acid and dehydrated hydroxy fatty acid product ions by MSn and ion-molecule reactions yielded diagnostic ions that readily pinpoint hydroxylation position and, thus, the OAHFA ester location. Conversely, these ions are characteristically absent in the MS3 spectra of ω-OAHFAs. Unimolecular dissociation mechanisms are proposed, which are shown to be consistent with prior isotopic labelling experiments. CONCLUSIONS: A mechanistic rationale is provided to explain the unimolecular dissociation of [OAHFA - H]- ions in an ion trap mass spectrometer, thus enabling near-complete de novo structural elucidation of OAHFAs in shotgun lipidomics workflows, even if synthetic standards are unavailable for comparison. Copyright © 2016 John Wiley & Sons, Ltd.

Elucidating the chemical structure of native 1-deoxysphingosine.

The 1-deoxysphingolipids (1-deoxySLs) are formed by an alternate substrate usage of the enzyme, serine-palmitoyltransferase, and are devoid of the C1-OH-group present in canonical sphingolipids. Pathologically elevated 1-deoxySL levels are associated with the rare inherited neuropathy, HSAN1, and diabetes type 2 and might contribute to ß cell failure and the diabetic sensory neuropathy. In analogy to canonical sphingolipids, it was assumed that 1-deoxySLs also bear a (4E) double bond, which is normally introduced by sphingolipid delta(4)-desaturase 1. This, however, was never confirmed. We therefore supplemented HEK293 cells with isotope-labeled D3-1-deoxysphinganine and compared the downstream formed D3-1-deoxysphingosine (1-deoxySO) to a commercial synthetic SPH m18:1(4E)(3OH) standard. Both compounds showed the same m/z, but differed in their RPLC retention time and atmospheric pressure chemical ionization in-source fragmentation, suggesting that the two compounds are structural isomers. Using dimethyl disulfide derivatization followed by MS(2) as well as differential-mobility spectrometry combined with ozone-induced dissociation MS, we identified the carbon-carbon double bond in native 1-deoxySO to be located at the (Δ14) position. Comparing the chromatographic behavior of native 1-deoxySO to chemically synthesized SPH m18:1(14Z) and (14E) stereoisomers assigned the native compound to be SPH m18:1(14Z). This indicates that 1-deoxySLs are metabolized differently than canonical sphingolipids.

Radical Formation in the Gas-Phase Ozonolysis of Deprotonated Cysteine.

Although the deleterious effects of ozone on the human respiratory system are well-known, many of the precise chemical mechanisms that both cause damage and afford protection in the pulmonary epithelial lining fluid are poorly understood. As a key first step to elucidating the intrinsic reactivity of ozone with proteins, its reactions with deprotonated cysteine [Cys-H](-) are examined in the gas phase. Reaction proceeds at near the collision limit to give a rich set of products including 1) sequential oxygen atom abstraction reactions to yield cysteine sulfenate, sulfinate and sulfonate anions, and significantly 2) sulfenate radical anions formed by ejection of a hydroperoxy radical. The free-radical pathway occurs only when both thiol and carboxylate moieties are available, implicating electron-transfer as a key step in this reaction. This novel and facile reaction is also observed in small cys-containing peptides indicating a possible role for this chemistry in protein ozonolysis.

Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry.

Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag](+) and [PC (18:1/16:0) + Ag](+)} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation.

Characterisation of sphingolipids in the human lens by thin layer chromatography-desorption electrospray ionisation mass spectrometry.

The lipidome of the human lens is unique in that cholesterol and dihydrosphingomyelin are the dominant classes. Moreover, the lens lipidome is not static with dramatic changes in several sphingolipid classes associated with both aging and cataract. Accordingly, there is a clear need to expand knowledge of the molecular species that constitute the human lens sphingolipidome. In this study, human lens lipids have been extracted and separated by thin-layer chromatography (TLC). Direct analysis of the TLC plates by desorption electrospray ionisation-mass spectrometry (DESI-MS) allowed the detection over 30 species from 11 classes of sphingolipids. Significantly, novel classes of lens lipids including sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates were identified.

Antibacterial anthranilic acid derivatives from Geijera parviflora.

Five anthranilic acid derivatives, a mixture I of three new compounds 11'-hexadecenoylanthranilic acid (1), 9'-hexadecenoylanthranilic acid (2), and 7'-hexadecenoylanthranilic acid (3), as well as a new compound 9,12,15-octadecatrienoylanthranilic acid (4) together with a new natural product, hexadecanoylanthranilic acid (5), were isolated from Geijera parviflora Lindl. (Rutaceae). Their structures were elucidated by extensive spectroscopic measurements, and the positions of the double bonds in compounds 1-3 of the mixture I were determined by tandem mass spectrometry employing ozone-induced dissociation. The mixture I and compound 5 showed good antibacterial activity against several Gram-positive strains.

Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: the next step towards "top-down" lipidomics.

The complete structural elucidation of complex lipids, including glycerophospholipids, using only mass spectrometry represents a major challenge to contemporary analytical technologies. Here, we demonstrate that product ions arising from the collision-induced dissociation (CID) of the [M + Na](+) adduct ions of phospholipids can be isolated and subjected to subsequent gas-phase ozonolysis - known as ozone-induced dissociation (OzID) - in a linear ion-trap mass spectrometer. The resulting CID/OzID experiment yields abundant product ions that are characteristic of the acyl substitution on the glycerol backbone (i.e., sn-position). This approach is shown to differentiate sn-positional isomers, such as the regioisomeric phosphatidylcholine pair of PC 16:0/18:1 and PC 18:1/16:0. Importantly, CID/OzID provides a sensitive diagnostic for the existence of an isomeric mixture in a given sample. This is of very high value for the analysis of tissue extracts since CID/OzID analyses can reveal changes in the relative abundance of isomeric constituents even within different tissues from the same animal. Finally, we demonstrate the ability to assign carbon-carbon double bond positions to individual acyl chains at specific backbone positions by adding subsequent CID and/or OzID steps to the workflow and that this can be achieved in a single step using a hybrid triple quadrupole-linear ion trap mass spectrometer. This unique approach represents the most complete and specific structural analysis of lipids by mass spectrometry demonstrated to date and is a significant step towards comprehensive top-down lipidomics.

Direct observation of photodissociation products from phenylperoxyl radicals isolated in the gas phase.

Gas phase peroxyl radicals are central to our chemical understanding of combustion and atmospheric processes and are typically characterized by strong absorption in the UV (λ(max) ≈ 240 nm). The analogous maximum absorption feature for arylperoxyl radicals is predicted to shift to the visible but has not previously been characterized nor have any photoproducts arising from this transition been identified. Here we describe the controlled synthesis and isolation in vacuo of an array of charge-substituted phenylperoxyl radicals at room temperature, including the 4-(N,N,N-trimethylammonium)methyl phenylperoxyl radical cation (4-Me3N([+])CH2-C6H4OO(•)), using linear ion-trap mass spectrometry. Photodissociation mass spectra obtained at wavelengths ranging from 310 to 500 nm reveal two major photoproduct channels corresponding to homolysis of aryl-OO and arylO-O bonds resulting in loss of O2 and O, respectively. Combining the photodissociation yields across this spectral window produces a broad (FWHM ≈ 60 nm) but clearly resolved feature centered at λ(max) = 403 nm (3.08 eV). The influence of the charge-tag identity and its proximity to the radical site are investigated and demonstrate no effect on the identity of the two dominant photoproduct channels. Electronic structure calculations have located the vertical B ← X transition of these substituted phenylperoxyl radicals within the experimental uncertainty and further predict the analogous transition for unsubstituted phenylperoxyl radical (C6H5OO(•)) to be 457 nm (2.71 eV), nearly 45 nm shorter than previous estimates and in good agreement with recent computational values.

Ozone-induced dissociation of conjugated lipids reveals significant reaction rate enhancements and characteristic odd-electron product ions.

Ozone-induced dissociation (OzID) is an alternative ion activation method that relies on the gas phase ion-molecule reaction between a mass-selected target ion and ozone in an ion trap mass spectrometer. Herein, we evaluated the performance of OzID for both the structural elucidation and selective detection of conjugated carbon-carbon double bond motifs within lipids. The relative reactivity trends for [M + X](+) ions (where X = Li, Na, K) formed via electrospray ionization (ESI) of conjugated versus nonconjugated fatty acid methyl esters (FAMEs) were examined using two different OzID-enabled linear ion-trap mass spectrometers. Compared with nonconjugated analogues, FAMEs derived from conjugated linoleic acids were found to react up to 200 times faster and to yield characteristic radical cations. The significantly enhanced reactivity of conjugated isomers means that OzID product ions can be observed without invoking a reaction delay in the experimental sequence (i.e., trapping of ions in the presence of ozone is not required). This possibility has been exploited to undertake neutral-loss scans on a triple quadrupole mass spectrometer targeting characteristic OzID transitions. Such analyses reveal the presence of conjugated double bonds in lipids extracted from selected foodstuffs. Finally, by benchmarking of the absolute ozone concentration inside the ion trap, second order rate constants for the gas phase reactions between unsaturated organic ions and ozone were obtained. These results demonstrate a significant influence of the adducting metal on reaction rate constants in the fashion Li > Na > K.

Photo-tautomerization of acetaldehyde to vinyl alcohol: a potential route to tropospheric acids.

Current atmospheric models underestimate the production of organic acids in the troposphere. We report a detailed kinetic model of the photochemistry of acetaldehyde (ethanal) under tropospheric conditions. The rate constants are benchmarked to collision-free experiments, where extensive photo-isomerization is observed upon irradiation with actinic ultraviolet radiation (310 to 330 nanometers). The model quantitatively reproduces the experiments and shows unequivocally that keto-enol photo-tautomerization, forming vinyl alcohol (ethenol), is the crucial first step. When collisions at atmospheric pressure are included, the model quantitatively reproduces previously reported quantum yields for photodissociation at all pressures and wavelengths. The model also predicts that 21 ± 4% of the initially excited acetaldehyde forms stable vinyl alcohol, a known precursor to organic acid formation, which may help to account for the production of organic acids in the troposphere.

Near-threshold H/D exchange in CD3CHO photodissociation.

Measuring the isotopic abundance of hydrogen versus deuterium atoms is a key method for interrogating reaction pathways in chemistry. H/D 'scrambling' is the intramolecular rearrangement of labile isotopes of hydrogen atoms and when it occurs through unanticipated pathways can complicate the interpretation of such experiments. Here, we investigate H/D scrambling in acetaldehyde at the energetic threshold for breaking the formyl C-H bond and reveal an unexpected unimolecular mechanism. Laser photolysis experiments of CD3CHO show that up to 17% of the products have undergone H/D exchange to give CD2H + DCO. Transition-state theory calculations reveal that the dominant mechanism involves four sequential H- or D-shifts to form CD2HCDO, which then undergoes conventional C-C bond cleavage. At the lowest energy the molecule undergoes an average of 20 H- or D-shifts before products are formed, evincing significant scrambling of H and D atoms. Analogous photochemically induced isomerizations and isotope scrambling are probably important in both atmospheric chemistry and combustion reactions.

Photochemical formation of HCO and CH3 on the ground S0 (1A') state of CH3CHO.

The dynamics of the photodissociation of CH(3)CHO into CH(3) + HCO products have been investigated at energies between 30,953 and 31,771 cm(-1), spanning the threshold for radical production on the triplet (T(1)) surface. A barrierless pathway to CH(3) + HCO radical products formed on the ground state (S(0)) surface was discovered and established to be an important reaction channel in acetaldehyde photodissociation throughout this wavelength range. HCO laser induced fluorescence (LIF) spectra recorded from CH(3)CHO dissociated above and below the T(1) barrier energy are quite different; HCO produced on S(0) yields a more congested LIF spectrum with sharp rotational transitions, while HCO formed on the T(1) surface displays fewer, more intense, Doppler-broadened lines. These differences have been further explored in the populations of the HCO K(a) = 1 doublets. Despite the upper and lower levels being almost isoenergetic, HCO formed on T(1) preferentially populates the upper K(c) state due to the geometry of the T(1) transition state structure. In contrast, HCO formed on S(0) produces equal population in each of the upper and lower K(a) = 1 components. Product state distributions (PSDs) showed that HCO formed on S(0) is born with an approximately statistical distribution of population in the available product states, modeled well by phase space theory. HCO formed on the T(1) surface, in contrast, has a PSD that can be characterized as arising from "impulsive" dynamics. Previous discrepancies in the height of the T(1) barrier are discussed following the observation that, once the T(1) channel is energetically accessible, there is competition between the S(0) and T(1) pathways, with the dominance of the triplet channel increasing with increasing photolysis energy.

Unimolecular thermal fragmentation of ortho-benzyne.

The ortho-benzyne diradical, o-C(6)H(4) has been produced with a supersonic nozzle and its subsequent thermal decomposition has been studied. As the temperature of the nozzle is increased, the benzyne molecule fragments: o-C(6)H(4)+Delta--> products. The thermal dissociation products were identified by three experimental methods: (i) time-of-flight photoionization mass spectrometry, (ii) matrix-isolation Fourier transform infrared absorption spectroscopy, and (iii) chemical ionization mass spectrometry. At the threshold dissociation temperature, o-benzyne cleanly decomposes into acetylene and diacetylene via an apparent retro-Diels-Alder process: o-C(6)H(4)+Delta-->HC triple bond CH+HC triple bond C-C triple bond CH. The experimental Delta(rxn)H(298)(o-C(6)H(4)-->HC triple bond CH+HC triple bond C-C triple bond CH) is found to be 57+/-3 kcal mol(-1). Further experiments with the substituted benzyne, 3,6-(CH(3))(2)-o-C(6)H(2), are consistent with a retro-Diels-Alder fragmentation. But at higher nozzle temperatures, the cracking pattern becomes more complicated. To interpret these experiments, the retro-Diels-Alder fragmentation of o-benzyne has been investigated by rigorous ab initio electronic structure computations. These calculations used basis sets as large as [C(7s6p5d4f3g2h1i)H(6s5p4d3f2g1h)] (cc-pV6Z) and electron correlation treatments as extensive as full coupled cluster through triple excitations (CCSDT), in cases with a perturbative term for connected quadruples [CCSDT(Q)]. Focal point extrapolations of the computational data yield a 0 K barrier for the concerted, C(2v)-symmetric decomposition of o-benzyne, E(b)(o-C(6)H(4)-->HC triple bond CH+HC triple bond C-C triple bond CH)=88.0+/-0.5 kcal mol(-1). A barrier of this magnitude is consistent with the experimental results. A careful assessment of the thermochemistry for the high temperature fragmentation of benzene is presented: C(6)H(6)-->H+[C(6)H(5)]-->H+[o-C(6)H(4)]-->HC triple bond CH+HC triple bond C-C triple bond CH. Benzyne may be an important intermediate in the thermal decomposition of many alkylbenzenes (arenes). High engine temperatures above 1500 K may crack these alkylbenzenes to a mixture of alkyl radicals and phenyl radicals. The phenyl radicals will then dissociate first to benzyne and then to acetylene and diacetylene.