UV Lamp as a Facile Ozone Source for Structural Analysis of Unsaturated Lipids Via Electrospray Ionization-Mass Spectrometry

Ozonolysis of alkene functional groups is a type of highly specific and effective chemical reaction, which has found increasing applications in structural analysis of unsaturated lipids via coupling with mass spectrometry (MS). In this work, we utilized a low-pressure mercury lamp (6 W) to initiate ozonolysis inside electrospray ionization (ESI) sources. By placing the lamp near a nanoESI emitter that partially transmits 185 nm ultraviolet (UV) emission from the lamp, dissolved dioxygen in the spray solution was converted into ozone, which subsequently cleaved the double bonds within fatty acyls of lipids. Solvent conditions, such as presence of water and acid solution pH, were found to be critical in optimizing ozonolysis yields. Fast (on seconds time scale) and efficient (50%–100% yield) ozonolysis was achieved for model unsaturated phospholipids and fatty acids with UV lamp-induced ozonolysis incorporated on a static and an infusion nanoESI source. The method was able to differentiate double bond location isomers and identify the geometry of the double bond based on yield. The analytical utility of UV lamp-induced ozonolysis was further demonstrated by implementation on a liquid chromatography (LC)-MS platform. Ozonolysis was effected in a flow microreactor that was made from ozone permeable tubing, so that ambient ozone produced by the lamp irradiation could diffuse into the reactor and induce online ozonolysis post-LC separation and before ESI-MS.

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Using ambient ozone for assignment of double bond position in unsaturated lipids

Unsaturated lipids deposited onto a range of materials are observed to react with the low concentrations of ozone present in normal laboratory air. Parent lipids and ozonolysis cleavage products are both detected directly from surfaces by desorption electrospray ionisation mass spectrometry (DESI-MS) with the resulting mass spectra providing clear evidence of the double bond position within these molecules. This serendipitous process has been coupled with thin-layer chromatography (TLC) to provide a simple but powerful approach for the detailed structural elucidation of lipids present in complex biological extracts. Lipid extracts from human lens were deposited onto normal phase TLC plates and then developed to separate components according to lipid class. Exposure of the developed plates to laboratory air for ca. 1 h prior to DESI-MS analysis gave rise to ozonolysis products allowing for the unambiguous identification of double bond positions in even low abundant, unsaturated lipids. In particular, the co-localization of intact unsaturated lactosylceramides (LacCer) with products from their oxidative cleavage provide the first evidence for the presence of three isomeric LacCer (d18:0/24:1) species in the ocular lens lipidome, i.e., variants with double bonds at the n-9, n-7 and n-5 positions.     

Separation of cis/trans geometrical fatty acid isomers by silver-exchanged zeolite Y

The separation of cis and trans isomers is relevant for biological and nutritional applications, silver-exchanged zeolite Y was prepared and applied for the treatment of mixtures of cis and trans geometrical isomers of mono- and polyunsaturated fatty acid methyl esters (FAMEs). cis FAMEs were adsorbed into zeolite with a high degree of selectivity (cis/trans ratio in the range of 1.9-3.2). The effectiveness was due to the synergism of the π-complexation between the silver ion and the double bonds and the different FAME structures trapped into the zeolite cages. Some indication of the complex stabilities came from theoretical studies using unsaturated lipids. A prototype cartridge was also designed for application in the fractionation of cis/trans FAME mixtures.     

PtCl2-mediated cycloisomerization of unsaturated propargylic carboxylates

The PtCl₂-mediated cycloisomerization of unsaturated propargylic carboxylates yields differently functionalized bicyclo[4.1.0]heptane enol esters from moderate to good yield, in a very diastereoselective manner. We have prepared and submitted to PtCl₂-catalyzed cycloisomerization a series of differently substituted hept-1-en-6-ynes with different O-acyl (acetyl, trichloroacetyl, 3,4,5-trimethoxybenzoyl, etc.) protecting groups at propargylic positions, investigating also the effect of the geometry at the double bond, as well as the effect of the number of substituents at the alkene moiety. As a result, we have found that the O-acetyl migrating group is the best one in terms of simplicity and chemical yields. In this reaction we have isolated mixtures of compounds formed by minor 1-acetoxy-allenes and major bicyclo[4.1.0]heptane derivatives. Major products are the result of a sequential process involving steps of cycloisomerization plus cyclopropanation, followed by acyl migration. The basic methanolysis (K₂CO₃, MeOH) of these intermediates gave mixtures of cis and trans-caran-2-ones. This two-step protocol (cycloisomerization plus basic methanolysis) for the syntheses of α,β-unsaturated cyclopropyl ketones constitutes a synthetic alternative to the usual unfriendly, intramolecular cyclopropanation of unsaturated α-diazocarbonyl derivatives. The formation of these bicyclo[4.1.0]heptane derivatives is a simple, but efficient entry into the skeleton of the ‘carane’ family of natural products.     

Synthesis-freindly chiral α-hydroxymethyl ketones from (-)-carvone

Reactions were studied of ozonolysis and periodate cleavage catalyzed by RuCl₃ of a double bond in (1R,5R)-5-(1-methyl-1-chloroethyl)-2-cyclohexen-1-ol and its acetate. The ozonolysis of the unsaturated alcohol led to the formation of cyclic anomeric methoxyspiranes, and its acetate depending on the conditions and oxidant reagent provided the corresponding α, ω-aldehydes, α, ω-ketoacetals, and α, ω-ketoacids.     

Highly selective Markovnikov hydration of Δ24-sterols and triterpenes by oxymercuration–hydrodemercuration

The reaction of mercuric acetate with unsaturated sterols and tetracyclic triterpenes (oxymercuration), followed by reduction with sodium borohydride/sodium hydroxide in water (hydrodemercuration), is highly selective for the side-chain double bonds 24-methylene or 24 (25). The nuclear double bonds, or those on other positions in the side-chain, do not react. The specificity of mercuration is therefore not directly correlated to the degree of substitution of the double bond, but rather to its steric accessibility. This sequence, which has been tested on some 30 sterols and related tetracyclic triterpenes, can be used to separate conveniently some of the constituents of the often-complex mixtures of sterols and of their tetracyclic triterpene precursors present in the unsaponifiable fraction of plant lipids. To cite this article: L. Elkihel et al., C. R. Chimie 6 (2003) 000–000.     

Localization of cyclopropyl groups and alkenes within glycerophospholipids using gas-phase ion/ion chemistry

Shotgun lipid analysis using electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a common approach for the identification and characterization of glycerophohspholipids GPs. ESI-MS/MS, with the aid of collision-induced dissociation (CID), enables the characterization of GP species at the headgroup and fatty acyl sum compositional levels. However, important structural features that are often present, such as carbon–carbon double bond(s) and cyclopropane ring(s), can be difficult to determine. Here, we report the use of gas-phase charge inversion reactions that, in combination with CID, allow for more detailed structural elucidation of GPs. CID of a singly deprotonated GP, [GP − H]⁻, generates FA anions, [FA − H]⁻. The fatty acid anions can then react with doubly charged cationic magnesium tris-phenanthroline complex, [Mg(Phen)₃]²⁺, to form charge inverted complex cations of the form [FA − H + MgPhen₂]⁺. CID of the complex generates product ion spectral patterns that allow for the identification of carbon–carbon double bond position(s) as well as the sites of cyclopropyl position(s) in unsaturated lipids. This approach to determining both double bond and cyclopropane positions is demonstrated with GPs for the first time using standards and is applied to lipids extracted from Escherichia coli.     

A new fluorescence method for determination of ozone in ambient air

A new simple method for determination of ozone in ambient air is presented. The reaction employed is based on the known ozonolysis of indigo dye. The indigotrisulfonate molecule contains one carbon–carbon double bond (C═C), which reacts with ozone and generates isatinsulfonates and sulfoanthranilate. The quantitatively formed sulfoanthranilate presents fluorescence (λ_ex 245 nm, λ_em 400 nm). Ozone was collected using two cellulose filters coated with 40 μL of 1.0 × 10^{− 3} mol L^{− 1} of indigotrisulfonate. The analytical response was linear in the range 0–150 ppbv ozone, and a detection limit of 7 ppbv was achieved using a sampling time of 15 min and an optimum sampling air flow rate of 0.4 L min^{− 1}. There was no interference from sulfur dioxide, formaldehyde or nitrogen dioxide. The ozonolysis mechanism and the reaction products are discussed.     

Identification of cis/trans isomers of methyl ester and oxazoline derivatives of unsaturated fatty acids using GC–FTIR–MS

Identification of cis/trans isomers of unsaturated fatty acids cannot usually be achieved by GC-MS (gas chromatography-mass spectrometry) without reference substances. In this study a GC-FTIR-MS system (gas chromatography-Fourier transform-mass spectrometry) was used to identify fatty acid methyl esters (FAMEs) and differentiate between the cis/trans isomers. Besides methyl esters, 2-alkenyl-4,4-dimethyloxazoline derivatives (DMOX), which have been used to locate double bond positions of unsaturated fatty acids, were examined with respect to their suitability for cis/trans differentiation. A combined GC-FTIR-MS system with a wide band (4000–550 cm^?1) mercury cadmium telluride (MCT) detector was used in series and parallel to identify 31 reference unsaturated fatty acids, including 7 pairs of cis/trans isomers. Serum samples of healthy persons and commercially available fish oil were analyzed as examples of complex mixtures. Using splitless injection the detection limit for the less sensitive IR detector was 25 ng/μl in case of the weak cis and trans bands. In the FTIR spectra cis/trans isomers were identified by analysis of bands arising from C? H out-of-plane (oop) bending: for both the FAME and DMOX derivatives cis-1,2-disubstituted double bonds give a strong band near 720 cm^?1 and the corresponding trans isomers near 967 cm^?1. cis Isomers could be identified further by a band at 3012 cm^?1. With the combined data of the GC-FTIR-MS system it is now possible to identify polyunsaturated fatty acids with regard to the discrimination of cis/trans isomers.     

Studying Interfacial Reactions of Cholesterol Sulfate in an Unsaturated Phosphatidylglycerol Layer with Ozone Using Field Induced Droplet Ionization Mass Spectrometry

Field-induced droplet ionization (FIDI) is a recently developed ionization technique that can transfer ions from the surface of microliter droplets to the gas phase intact. The air-liquid interfacial reactions of cholesterol sulfate (CholSO₄) in a 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol (POPG) surfactant layer with ozone (O₃) are investigated using field-induced droplet ionization mass spectrometry (FIDI-MS). Time-resolved studies of interfacial ozonolysis of CholSO₄ reveal that water plays an important role in forming oxygenated products. An epoxide derivative is observed as a major product of CholSO₄ oxidation in the FIDI-MS spectrum after exposure of the droplet to O₃ for 5 s. The abundance of the epoxide product then decreases with continued O₃ exposure as the finite number of water molecules at the air-liquid interface becomes exhausted. Competitive oxidation of CholSO₄ and POPG is observed when they are present together in a lipid surfactant layer at the air-liquid interface. Competitive reactions of CholSO₄ and POPG with O₃ suggest that CholSO₄ is present with POPG as a well-mixed interfacial layer. Compared with CholSO₄ and POPG alone, the overall ozonolysis rates of both CholSO₄ and POPG are reduced in a mixed layer, suggesting the double bonds of both molecules are shielded by additional hydrocarbons from one another. Molecular dynamics simulations of a monolayer comprising POPG and CholSO₄ correlate well with experimental observations and provide a detailed picture of the interactions between CholSO₄, lipids, and water molecules in the interfacial region.     

Production of radicals in the ozonolysis of propene in air

Radical production in the ozonolysis of propene in air was monitored directly by a peroxy radical chemical amplification (PERCA) instrument at room temperature (298±2 K) and atmospheric pressure (1×105 Pa). The ozonolysis reactions were conducted in a flow tube under pseudo-first-order conditions for ozone. The decay in ozone was calculated based on reaction time tr and effective rate constant keff (keff = k1[C3H6]0)) for the ozone-propene reaction. The total radical yields relative to consumed ozone were d...     

O-silylated enolate phenylthioalkylation: a new synthesis of unsaturated 1,5-dicarbonyl compounds.

The O-silylated enolates of ketones and esters can be phenylthioalkylated by the chlorides (2) and (3) under ZnBr₂-catalysis; ozonolysis and subsequent sulphoxide thermolysis then gives the corresponding unsaturated 1,5-dicarbonyl compounds.     

Chemical modification of poly(hydroxyalkanoates). Copolymers bearing pendant sugars

Copolymers of poly(3‐hydroxyoctanoates) (PHAs) containing repeating units with unsaturated or brominated pendant side chains have been obtained from cultures of Pseudomonas oleovorans grown on mixtures of octanoic acid and undecenoic acid or 11‐bromoundecanoic acid as carbon sources. These polymers, bearing reactive functionalities, have been used to graft acetylated maltosyl units either by anti‐Markovnikov addition to the double bond or S_N2 substitution of the halogen. De‐O‐acetylation of the sugar moieties yielded PHAs with new properties.     

Low-Temperature Ozonolysis of 2-Alkenyl-1,1-dichlorocyclopropanes

Low-temperature ozonolysis of 2-alkenyl-1,1-dichlorocyclopropanes under different conditions (O₃/MeOH–CH₂Cl₂–NaOH, O₃/MeOH or O₃/CH₂Cl₂–AcOH with subsequent treatment with semicarbazide hydrochloride) afforded the corresponding carbonyl and carboxy derivatives at different ratios, depending on the substrate structure and workup procedure. Peroxide products of ozonolysis of 1,1-dichloro-2-ethenylcyclopropane [3-(2,2-dichlorocyclopropyl)-1,2,4-trioxolane and (2,2-dichlorocyclopropyl)(methoxy)methyl hydroperoxide] turned out to be more stable than those derived from 1,1-dichloro-2-ethenyl-2-methylcyclopropane and were isolated in the pure state.     

Secondary organic aerosol formation from limonene ozonolysis: homogeneous and heterogeneous influences as a function of NO(x)

Limonene has a high emission rate both from biogenic sources and from household solvents. Here we examine the limonene + ozone reaction as a source for secondary organic aerosol (SOA). Our data show that limonene has very high potential to form SOA and that NO(x) levels, O(3) levels, and UV radiation all influence SOA formation. High SOA formation is observed under conditions where both double bonds in limonene are oxidized, but those conditions depend strongly on NO(x). At low NO(x), heterogeneous oxidation of the terminal double bond follows the initial limonene ozonolysis (at the endocyclic double bond) almost immediately, making the initial reaction rate limiting. This requires a high uptake coefficient between ozone and the first-generation, unsaturated organic particles. However, at high NO(x), this heterogeneous processing is inhibited and gas-phase oxidation of the terminal double bond dominates. Although this chemistry is slower, it also yields products with low volatility. UV light suppresses production of the lowest volatility products, as we have shown in earlier studies of the alpha-pinene + ozone reaction.     

Studies of structure and mechanism in acetonitrile chemical ionization tandem mass spectrometry of polyunsaturated fatty acid methyl esters

Recently it has been shown that acetonitrile chemical ionization tandem mass spectrometry (CI-MS/MS) is a rapid, on-line means to determine double bond position in fatty acid methyl esters (FAME). The mechanism of this gas phase condensation reaction has been studied. Evidence of the (1-methyleneimino)-1-ethenylium ion (m/z 54), formed upon the reaction of acetonitrile with itself, adding across the double bond in a [2 + 2] cycloaddition reaction is observed. When this nascent complex undergoes collision-induced dissociation, two diagnostic ions emerge. One of these ions results from loss of the hydrocarbon end of the FAME, whereas the other ion results from loss of the methyl ester end, and when considered together, the diagnostic ions localize the positions of the double bonds in the FAME. Several labeling and MS/MS/MS experiments on the two diagnostic ions were performed to determine a plausible fragmentation mechanism of the stable (1-methyleneimino)-1-ethenylium–FAME complex. The first generation product ions, or diagnostic ions, appear to be formed though a charge-driven mechanism, whereas the second generation product ions are formed via charge-remote fragmentations. Plausible mechanisms for the formation and subsequent dissociation of the diagnostic ions are presented for the monounsaturated, diunsaturated, and polyunsaturated (3 or more double bonds) FAME.     

Water-soluble polyketones and esters as the main stable products of ozonolysis of fullerene C60 solutions

Stable ozonolysis products of C₆₀ solutions in CCl₄, toluene, and hexane were studied by elemental analysis, HPLC, and UV and IR spectroscopy. Polyketones and esters were established for the first time to be the main stable products, whose content increased during the whole ozonolysis time (1 h). Epoxides C₆₀O _n (n = 1—6) are accumulated within 1—3 min, and after 5 min of ozonolysis their concentration decreases to zero. Fullerene C₆₀ disappears from the reaction solution due to its conversion to oxides and mechanical capturing of C₆₀ by these oxides to form a precipitate. The oxidation of C₆₀ is completed in the solid phase by the formation of the C₆₀O₁₆ oxide in which 9.68 O atoms fall on fullerene polyketones, 6 O atoms are attributed to esters, and 0.32 O atoms fall per epoxides. The optimum medium for preparation of the C₆₀ oxides is CCl₄ rather than traditional toluene, which reacts with ozone in the side reaction to form products containing active oxygen. The C₆₀ cage is raptured during ozonolysis because of the C=C bond cleavage to form two C=O groups at the ends of the open hexagon. Ozonolysis of C₆₀ solutions in CCl₄ is efficient for synthesis of water-soluble fullerene oxides due to the high yield and solubility of polyketones and esters in water.     

Effect of Bio-based Catalyst in Biodiesel Synthesis

A cost-effective and environmentally friendly biodiesel synthesis has drawn attention in recent research activities. Used cooking oil which is known as waste is used in this study. The objectives of this research were to study an effect of biobased-catalyst which is used as supporting catalyst in simultaneous ozonolysis and transesterification for biodiesel synthesis and to study the effect of two steps process in biodiesel synthesis. The bio-based catalyst used in this process was empty palm bunch ash which was used as supporting catalyst for KOH. Two steps reaction were designed, the first step was run in a reactor at 30 °C with a continuous supply of ozone gas for 3 hours to cleave the unsaturated fatty acids at the double bonds. The second step was a follow up process after the first step without a supply of ozone gas, the temperature was increased up to 60 °C and the reaction continue for two hours. The second step aimed to convert saturated fatty acid which was not yet fully converted at the first step. Results of this study showed that 1.5% of KOH gave better performance in producing short chain methyl esters compared to 1% of KOH in the first step process at various percent weight of ash. The highest short chain methyl esters and long chain methyl esters produced in the first step process were 85.722 mg/liter and 655.286 mg/liter respectively, which was used 17.3 weight % ash and 1.5 weight % KOH. Short chain methyl esters were produced as a result of unsaturated fatty acid cracked by ozonolysis. It is confirmed that a simultaneous ozonolysis and transesterification occurred in the first step process. In conclusion, the presence of bio-based catalyst as supporting catalyst for KOH to produce higher total methyl esters has been effective. The second step process in this experiment was not effective since the effect of reaction time can enhance the hydrolysis of esters as a reverse reaction of transesterification, resulted in loss of esters.     

Ozone production and losses in N<Subscript>2</Subscript>/O<Subscript>2</Subscript> mixtures in an ozone generator

Nonunique ozone concentrations at the output of an ozone generator under identical external conditions of barrier discharge activation of N₂/O₂ mixtures but with different prehistories of operating practice and employed gas mixtures are investigated theoretically. An analytical approach is developed to determine the ozone yield with regard for its heterogeneous loss. Plasma-chemical and electron kinetics in the N₂/O₂-mixtures are calculated numerically. The results of numerical calculations are compared to experimental data obtained by the authors. It is noted that the heterogeneous loss of ozone is the probable reason for the observed variety of behavior of О₃ concentrations, depending on prehistory of ozone generator operation, along with the N₂ and O₂ gas flow rates and the specific active power.