In Situ Generation of 1-Acetylpyrene as a Visible-Light Photocatalyst for the Thia-Paternò-Büchi Reaction.

The thia-Paternò-Büchi reaction represents one of the straightforward approaches to build thietane cores. Unfortunately, the significant instability of thiocarbonyls, particularly thioketones and thioaldehydes, has hitherto rendered this photochemical [2+2]-cycloaddition underexploited. To address this limitation, we report here a visible-light photochemical domino reaction including: the in situ generation of thiocarbonyls, though a Norrish type II fragmentation of pyrenacyl sulfides, and the aforementioned thia-Paternò-Büchi reaction with various non-volatile electron-rich alkenes. The highly efficient synthesis of a wide range of unprecedented thietanes from intrinsically highly unstable thiocabonyls, such as thioaldehydes and aliphatic thioketones, was made possible by the multitasking capability of pyrenacyl sulfides, as a source of thiocarbonyl substrates and as precursors of 1-acetylpyrene, which acts as the photocatalyst for the thia-Paternò-Büchi reaction. The photosensitizer properties of the latter have been experimentally established and a triplet-triplet Dexter energy transfer-based mechanism is proposed.

Deep profiling of plasmalogens by coupling the Paternò-Büchi derivatization with tandem mass spectrometry.

Plasmalogens are a special class of glycerophospholipids characterized by a vinyl ether bond (-C = C-O-) at the sn-1 position of the glycerol backbone. Altered plasmalogen profiles have been observed in neurodegenerative diseases and cancers. Profiling of plasmalogens requires specifying the vinyl ether bond and differentiating them from various types of isobars and isomers. Herein, by coupling C = C derivatization via offline Paternò-Büchi reaction with liquid chromatography-tandem mass spectrometry, we have developed a sensitive workflow for analysis of plasmalogens from biological samples. Using bovine heart lipid extract as a model system, we profiled more than 100 distinct structures of plasmenylethanolamines (PE-Ps) and plasmenylcholines (PC-Ps) at the C = C location level, far exceeding previous reports. Analysis of human glioma and normal brain tissue samples revealed elevated n-10 C = C isomers of PE-Ps in the glioma tissue samples. These findings suggest that the developed workflow holds potential in aiding the study of altered metabolism of plasmalogens in clinical samples.

A Paternò-Büchi Reaction of Aromatics with Quinones under Visible Light Irradiation.

Reported herein is a Paternò-Büchi reaction of aromatic double bonds with quinones under visible light irradiation. The reactions of aromatics with quinones exposed to blue LED irradiation yielded oxetanes at -78 °C, which was attributed to both the activation of double bonds in aromatics and the stabilization of oxetanes by thiadiazole, oxadiazole, or selenadiazole groups. The addition of Cu(OTf)2 to the reaction system at room temperature resulted in the formation of diaryl ethers via the copper-catalyzed ring opening of oxetanes in situ. Notably, the substrate scope was extended to general aromatics.

Alternative fatty acid desaturation pathways revealed by deep profiling of total fatty acids in RAW 264.7 cell line.

In-depth structural characterization of lipids provides a new means to investigate lipid metabolism. In this study, we have conducted deep profiling of total fatty acids (FAs) from RAW 264.7 macrophages by utilizing charge-tagging Paternò-Büchi derivatization of carbon-carbon double bond (C=C) and reversed-phase liquid chromatography-tandem mass spectrometry. A series of FAs exhibiting unusual site(s) of unsaturation was unearthed, with their identities being confirmed by observing anticipated compositional alterations upon desaturase inhibition. The data reveal that FADS2 Δ 6-desaturation can generate n-11 C=C in the odd-chain monounsaturated fatty acids (MUFAs) as well as n-10 and n-12 families of even-chain MUFAs. SCD1 Δ 9-desaturation yields n-6, n-8, and n-10 of odd-chain MUFAs, as well as n-5, n-7, and n-9 families of even-chain MUFAs. Besides n-3 and n-6 families of polyunsaturated fatty acids (PUFAs), the presence of n-7 and n-9 families of PUFAs indicates that the n-7 and n-9 isomers of FA 18:1 can be utilized as substrates for further desaturation and elongation. The n-7 and n-9 families of PUFAs identified in RAW 264.7 macrophages are noteworthy because their C=C modifications are achieved exclusively via de novo lipogenesis. Our discovery outlines the metabolic plasticity in fatty acid desaturation which constitutes an unexplored rewiring in RAW264.7 macrophages.

An efficient and sensitive method on the identification of unsaturated fatty acids in biosamples: Total lipid extract from bovine liver as a case study.

Unsaturated fatty acids (UFAs) are fatty acids with one or more carbon-carbon double bonds (C=C). They are building blocks of structurally complex lipids. According to the position of C=C bond in the UFAs, there are multiple isomers with different physiological activities. Therefore, an accurate understanding of UFAs is essential. Combining Paternò-Büchi (PB) reaction with tandem mass spectrometry (MS/MS) analysis has been successfully used to identify unsaturated lipids in bio-samples. However, there are still some challenges, such as low reactive rate and low MS response of PB products. In this study, 3-pyridinecarboxaldehyde (3-PYA) was selected as an efficient PB reagent for the first time. According to the nitrogen rule, introduction of nitrogen did not only make the target MS and MS/MS ions to be easily identified but also increase the ionization efficiency significantly. The developed method in this study demonstrated greater efficiency and sensitivity for the identification of UFAs in total lipid extracts from bovine liver compared with other commonly used PB reagents.

Regio-distribution and double bond locations of unsaturated fatty acids in phospholipids of bovine milk.

Hundreds of phospholipid (PL) species with defined fatty acid (FA) composition have been identified previously in bovine milk using liquid chromatography tandem mass spectrometry (LC-MS/MS). Paterno-Buchi photochemical reaction coupled with LC-MS/MS was applied in this study to further unravel the regio-distribution and double bond (DB) locations of FAs. Using SPE-purified PLs and 2-acetylpyridine as the photochemical derivatization reagent, we were able to reveal the non-specific regio-distribution of unsaturated FAs and the widespread occurrence of regioisomers in milk PLs. Although Δ9 and Δ9,12 were found to be the predominant DB location(s) for C18:1 and C18:2 respectively, other DB positional isomers such as C18:1Δ11, C18:1Δ12 and C18:1Δ13 and C18:2Δ9,11 were widely detected in PL structures, implying that the minor isomers of C18:1 and C18:2 equally participate in the synthesis of PLs. Our study provides novel information on the fine structure of milk PLs and further underlines the complexity of milk lipid composition.

Unsaturated lipid isomeric imaging based on the Paternò-Büchi reaction in the solid phase in ambient conditions.

In recent years, the development of unsaturated lipid isomeric imaging based on the Paternò-Büchi (PB) reaction has improved significantly. However, research on this imaging method in ambient conditions needs to expand. In this paper, a method of PB reaction in the solid phase in ambient conditions is developed, which is combined with air-flow-assisted desorption electrospray ionisation mass spectrometry (AFADESI-MS) to achieve in situ imaging of lipids at an isomeric level. Experiments showed that the efficiency of the PB reaction was much higher when spraying the reagent solution than when sprinkling the reactant powder directly, and it was not lower than that in the liquid phase. This method can simplify the reaction conditions in the imaging process and can be applied to tissue section samples with only 10 min of pre-processing. The study successfully demonstrated the spatial distribution of unsaturated lipid isomers, and the isomeric ratio corresponded to the lesion areas in mouse brain cancer tissues. Due to its simple operation and performance in ambient conditions, this method may be useful for future studies on lipid isomers in tissues.

Azetidines of pharmacological interest.

Azetidines are almost unexplored among nitrogen-containing saturated heterocycles due to difficulties associated with their synthesis. However, over the past few years, attempts have been made by scientists to advance their synthetic feasibility. Compounds with the azetidine moiety display an important and diverse range of pharmacological activities, such as anticancer, antibacterial, antimicrobial, antischizophrenic, antimalarial, antiobesity, anti-inflammatory, antidiabetic, antiviral, antioxidant, analgesic, and dopamine antagonist activities, and are also useful for the treatment of central nervous system disorders and so forth. Owing to its satisfactory stability, molecular rigidity, and chemical and biological properties, azetidine has emerged as a valuable scaffold and it has drawn the attention of medicinal researchers. The present review sheds light on the traditional method of synthesis of azetidine and advancements in synthetic methodology over the past few years, along with its application with various examples, and its biological significance.

Lipidome-wide characterization of phosphatidylinositols and phosphatidylglycerols on CC location level.

Phosphatidylglycerol (PG) and phosphatidylinositol (PI) are two essential classes of glycerophospholipids (GPs), playing versatile roles such as signalling messengers and lipid-protein interaction ligands in cell. Although a majority of PG and PI molecular species contain unsaturated fatty acyl chain(s), conventional tandem mass spectrometry (MS/MS) methods cannot discern isomers different in carbon-carbon double bond (CC) locations. In this work, we paired phosphate methylation with acetone Paternò-Büchi (PB) reaction, aiming to provide a solution for sensitive and structurally informative analysis of these two important classes of GPs down to the location of CC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow was established. Offline methylated PG or PI mixtures were subjected to hydrophilic interaction chromatographic separation, online acetone PB reaction, and MS/MS via collision-induced dissociation (CID) for CC location determination in positive ion mode. This method was sensitive, offering limit of identification at 5 nM for both PG and PI standards down to CC locations. On molecular species level, 49 PI and 31 PG were identified from bovine liver, while 61 PIs were identified from human plasma. This workflow also enabled ratiometric comparisons of CC location isomers (C18:1 Δ9 vs. Δ11) of a series of PIs from type 2 diabetes (T2D) plasma to that of normal plasma samples. PI 16:0_18:1 and PI 18:0_18:1 were found to exhibit significant changes in CC isomeric ratios between T2D and normal plasma samples. The above results demonstrate that the developed LC-PB-MS/MS workflow is applicable to different classes of lipids and compatible with other established lipid derivatization methods to achieve comprehensive lipid analysis.

Double bond localization in unsaturated rhamnolipid precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids by liquid chromatography-mass spectrometry applying online Paternò-Büchi reaction.

Lipids are biomolecules with a broad variety of chemical structures, which renders them essential not only for various biological functions but also interestingly for biotechnological applications. Rhamnolipids are microbial glycolipids with surface-active properties and are widely used biosurfactants. They are composed of one or two L-rhamnoses and up to three hydroxy fatty acids. Their biosynthetic precursors are 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs). The latter are also present in cell supernatants as complex mixtures and are extensively studied for their potential to replace synthetically derived surfactants. The carbon chain lengths of HAAs determine their physical properties, such as their abilities to foam and emulsify, and their critical micelle concentration. Despite growing biotechnological interest, methods for structural elucidation are limited and often rely on hydrolysis and analysis of free hydroxy fatty acids losing the connectivity information. Therefore, a high-performance liquid chromatography-mass spectrometry method was developed for comprehensive structural characterization of intact HAAs. Information is provided on chain length and number of double bonds in each hydroxy fatty acid and their linkage by tandem mass spectrometry (MS/MS). Post-column photochemical derivatization by online Paternὸ-Büchi reaction and MS/MS fragmentation experiments generated diagnostic fragments allowing structural characterization down to the double bond position level. Furthermore, the presented experiments demonstrate a powerful approach for structure elucidation of complex lipids by tailored fragmentation.

Identification of polyunsaturated triacylglycerols and CC location isomers in sacha inchi oil by photochemical reaction mass spectrometry combined with nuclear magnetic resonance spectroscopy.

Sacha inchi oil is derived from the seeds of Plukenetia volubilis L. and has great nutritional value due to its high contents of active polyunsaturated triacylglycerols (PUTAGs). In this study, we developed a methodology combined Paternò-Büchi reaction nanoelectrospray ionization mass spectrometry (PB-nanoESI-MS) and nuclear magnetic resonance (NMR) to identify CC locations and isomers of PUTAGs in sacha inchi oil. Benzophenone was used as the PB reagent, and the optimized solvent composition (methanol:chloroform = 9:1) allowed for PUTAGs and their PB products to be detected with higher intensities. In addition, we made efforts to interpret the MS2 spectra for identification lipid species. A series of C57-PUTAGs and C59-PUTAGs were detected and identified via high-resolution PB-nanoESI-MS, and the predominant PUTAGs were TAG 18:1(Δ9)_18:3(Δ9,12,15)_18:3(Δ9,12,15) and TAG 18:2(Δ9,12)_18:2(Δ9,12)_18:3 (Δ9,12,15), which demonstrated that the PB-nanoESI-MS approach in this study provides help in promoting the development of structural determination of triacylglycerols in food chemistry.

Olefinic reagents tested for peptide derivatization with switchable properties: Stable upon collision induced dissociation and cleavable by in-source Paternò-Büchi reactions.

This contribution is part of our ongoing efforts to develop innovative cross-linking (XL) reagents and protocols for facilitated peptide mixture analysis and efficient assignment of cross-linked peptide products. In this report, we combine in-source Paternò-Büchi (PB) photo-chemistry with a tandem mass spectrometry approach to selectively address the fragmentation of a tailor-made cross-linking reagent. The PB photochemistry, so far exclusively used for the identification of unsaturation sites in lipids and in lipidomics, is now introduced to the field of chemical cross-linking. Based on trans-3-hexenedioic acid, an olefinic homo bifunctional amine reactive XL reagent was designed and synthesized for this proof-of-principle study. Condensation products of the olefinic reagent with a set of exemplary peptides are used to test the feasibility of the concept. Benzophenone is photochemically reacted in the nano-electrospray ion source and forms oxetane PB reaction products. Subsequent CID-MS triggered retro-PB reaction of the respective isobaric oxetane molecular ions and delivers reliably and predictably two sets of characteristic fragment ions of the cross-linker. Based on these signature ion sets, a straightforward identification of covalently interconnected peptides in complex digests is proposed. Furthermore, CID-MSn experiments of the retro-PB reaction products deliver peptide backbone characteristic fragment ions. Additionally, the olefinic XL reagents exhibit a pronounced robustness upon CID-activation, without previous UV-excitation. These experiments document that a complete backbone fragmentation is possible, while the linker-moiety remains intact. This feature renders the new olefinic linkers switchable between a stable, noncleavable cross-linking mode and an in-source PB cleavable mode.

In-depth structural characterization of phospholipids by pairing solution photochemical reaction with charge inversion ion/ion chemistry.

Shotgun lipid analysis based on electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in lipidomic studies. One challenge for the shotgun approach is the discrimination of lipid isomers and isobars. Gas-phase charge inversion via ion/ion reactions has been used as an effective method to identify multiple isomeric/isobaric components in a single MS peak by exploiting the distinctive functionality of different lipid classes. In doing so, fatty acyl chain information can be obtained without recourse to condensed-phase separations or derivatization. This method alone, however, cannot provide carbon-carbon double bond (C=C) location information from fatty acyl chains. Herein, we provide an enhanced method pairing photochemical derivatization of C=C via the Paternò-Büchi reaction with charge inversion ion/ion tandem mass spectrometry. This method was able to provide gas-phase separation of phosphatidylcholines and phosphatidylethanolamines, the fatty acyl compositions, and the C=C location within each fatty acyl chain. We have successfully applied this method to bovine liver lipid extracts and identified 40 molecular species of glycerophospholipids with detailed structural information including head group, fatty acyl composition, and C=C location. Graphical Abstract ᅟ.

An On-Tissue Paternò-Büchi Reaction for Localization of Carbon-Carbon Double Bonds in Phospholipids and Glycolipids by Matrix-Assisted Laser-Desorption-Ionization Mass-Spectrometry Imaging.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) visualizes the distribution of phospho- and glycolipids in tissue sections. However, C=C double-bond (db) positional isomers generally cannot be distinguished. Now an on-tissue Paternò-Büchi (PB) derivatization procedure that exploits benzaldehyde as a MALDI-MSI-compatible reagent is introduced. Laser-induced postionization (MALDI-2) was used to boost the yields of protonated PB products. Collision-induced dissociation of these species generated characteristic ion pairs, indicative of C=C position, for numerous singly and polyunsaturated phospholipids and glycosphingolipids in mouse brain tissue. Several db-positional isomers of phosphatidylcholine and phosphatidylserine species were expressed with highly differential levels in the white and gray matter areas of cerebellum. Our PB-MALDI-MS/MS procedure could help to better understand the physiological role of these db-positional isomers.

Benzophenone used as the photochemical reagent for pinpointing C=C locations in unsaturated lipids through shotgun and liquid chromatography-mass spectrometry approaches.

Unsaturated lipids exhibit different physiological significances due to the different locations of the carbon-carbon double bond (C=C). Identifying lipid isomers with mass-based methods remains challenging. Xia's group has been successfully employed Paternò-Büchi (PB) reaction, a photochemical reaction with UV irradiation, coupled with tandem mass spectrometry (MS/MS) to identify and quantify unsaturated lipids in complex mixtures. However, the existing PB reagents possess certain demerits. In this regard, a new PB reagent that is compatible with various lipidomic analysis platforms must be screened. In this study, we comprehensively evaluated the conditions of the PB reaction and screened benzophenone as a new PB reagent. Benzophenone possesses unique advantages, such as a relative high PB yield; the PB products could be readily distinguished from the reacted lipids based on the added high molecular weight (182 Da); and the benzophenone does not affect the lipids appearance interval for reversed-phase column separation. Furthermore, we optimized the reaction conditions by using benzophenone as a PB reagent and summarized the molecular formulas of the diagnostic ions according to the fragment rules. The proposed PB method has been implemented in shotgun and LC-MS lipidomics. To our best knowledge, this work is the first to report the integrated PB reaction with LC-MS lipidomics for identification of lipid isomers.

Entropy-Driven Diastereoselectivity Improvement in the Paternò-Büchi Reaction of 1-Naphthyl Aryl Ethenes with a Chiral Cyanobenzoate through Remote Alkylation.

The precise stereocontrol of photocycloaddition reactions is still a significant challenge owing to their mechanistic complexity and the involvement of highly reactive and short-lived intermediates. Attempts have hitherto been made through structural modifications, mostly by introducing steric conflicts, to increase the difference between the enthalpic barriers. Herein, we show that entropy plays a crucial role in influencing the diastereoselectivity of a Paternò-Büchi reaction. Remote meta alkylation of the donor caused nominal changes in its photophysical properties as well as those of the exciplexes derived thereof. Nevertheless, the diastereomeric excess of the oxetane product was greatly improved by about 40 %. This enhancement, which is not accompanied by any significant changes in the photophysical properties, is difficult to rationalize by conventional enthalpic control concepts based on repulsive steric and/or attractive intermolecular interactions as well as electronic perturbations. Differential activation parameters and compensatory enthalpy-entropy relationships revealed that the diastereoselectivity enhancement is not simply enthalpic but also entropic in origin.

Realizing an Aza Paternò-Büchi Reaction.

Intramolecular atropselective aza Paternò-Büchi reaction involving atropisomeric enamide and imine functionalities under sensitized irradiation leads to azetidine products in good yield and selectivity (ee >96 %). A mechanistic model based on detailed photophysical and isomerization kinetic studies is provided that shed light into the reactivity of enamides leading to aza Paternò-Büchi reaction.

Identification and quantitation of lipid C=C location isomers: A shotgun lipidomics approach enabled by photochemical reaction.

The field of lipidomics has been significantly advanced by mass spectrometric analysis. The distinction and quantitation of the unsaturated lipid isomers, however, remain a long-standing challenge. In this study, we have developed an analytical tool for both identification and quantitation of lipid C=C location isomers from complex mixtures using online Paternò-Büchi reaction coupled with tandem mass spectrometry (MS/MS). The potential of this method has been demonstrated with an implementation into shotgun lipid analysis of animal tissues. Among 96 of the unsaturated fatty acids and glycerophospholipids identified from rat brain tissue, 50% of them were found as mixtures of C=C location isomers; for the first time, to our knowledge, the quantitative information of lipid C=C isomers from a broad range of classes was obtained. This method also enabled facile cross-tissue examinations, which revealed significant changes in C=C location isomer compositions of a series of fatty acids and glycerophospholipid (GP) species between the normal and cancerous tissues.

Pinpointing double bonds in lipids by Paternò-Büchi reactions and mass spectrometry.

The positions of double bonds in lipids play critical roles in their biochemical and biophysical properties. In this study, by coupling Paternò-Büchi (P-B) reaction with tandem mass spectrometry, we developed a novel method that can achieve confident, fast, and sensitive determination of double bond locations within various types of lipids. The P-B reaction is facilitated by UV irradiation of a nanoelectrospray plume entraining lipids and acetone. Tandem mass spectrometry of the on-line reaction products via collision activation leads to the rupture of oxetane rings and the formation of diagnostic ions specific to the double bond location.

Photocycloaddition of aromatic and aliphatic aldehydes to isoxazoles: Cycloaddition reactivity and stability studies.

The first photocycloadditions of aromatic and aliphatic aldehydes to methylated isoxazoles are reported. The reactions lead solely to the exo-adducts with high regio- and diastereoselectivities. Ring methylation of the isoxazole substrates is crucial for high conversions and product stability. The 6-arylated bicyclic oxetanes 9a-9c were characterized by X-ray structure analyses and showed the highest thermal stabilities. All oxetanes formed from isoxazoles were highly acid-sensitive and also thermally unstable. Cleavage to the original substrates is dominant and the isoxazole derived oxetanes show type T photochromism.