Thermal evaporation as sample preparation for silver-assisted laser desorption/ionization mass spectrometry imaging of cholesterol in amyloid tissues.

Cholesterol plays an important biological role in the body, and its disruption in homeostasis and synthesis has been implicated in several diseases. Mapping the locations of cholesterol is crucial for gaining a better understanding of these conditions. Silver deposition has proven to be an effective method for analyzing cholesterol using mass spectrometry imaging (MSI). We optimized and evaluated thermal evaporation as an alternative deposition technique to sputtering for silver deposition in MSI of cholesterol. A silver layer with a thickness of 6 nm provided an optimal combination of cholesterol signal intensity and mass resolution. The deposition of an ultrathin nanofilm of silver enabled high-resolution MSI with a pixel size of 10 µm. We used this optimized method to visualize the distribution of cholesterol in the senile plaques in the brains of APP/PS1 mice, a model that resembles Alzheimer's disease pathology. We found that cholesterol was evenly distributed across the frontal cortex tissue, with no evidence of plaque-like accumulation. Additionally, we investigated the presence and distribution of cholesterol in myocardial sections of a human heart affected by wild-type ATTR amyloidosis. We identified the presence of cholesterol in areas with amyloid deposition, but complete colocalization was not observed.

Quaternary ammonium fluorides and difluorosilicates as nucleophilic fluorination reagents.

TBAT (tetrabutylammonium difluorotriphenylsilicate) is an excellent homogeneous nucleophilic fluorination reagent, but a high excess of the reagent was reported to be essential. We hence optimized the reaction conditions and compared its nucleophilic fluorination reactivity with that of other common commercial nucleophilic fluorination reagents, such as anhydrous TBAF and TASF (tris(dimethylamino)sulfonium difluorotrimethylsilicate). As the substrates, we employed a standard set of primary and secondary octyl substrates under identical conditions. To eliminate the possibility of hydrogen fluoride elimination in the above reagents, we prepared four quaternary ammonium fluorides lacking ß-elimination possibility in the hydrocarbon chain, transformed them to the corresponding difluorotriphenylsilicates, and compared their reactivity with that of the commercial reagents. Furthermore, attempts to isolate analogous tetrabutylammonium difluoromethyldiphenylsilicate or difluorodimethylphenylsilicate failed, as was confirmed by comparison of the published experimental data with computed 19F NMR spectra. Finally, we studied the transition states of decomposition of various tetramethylammonium methylphenyldifluorosilicates by DFT methods and found that their relative energies increase with an increasing number of phenyl groups. The formation of difluorosilicates is a nearly barrierless process.

Solvent-controlled formation of alkali and alkali-earth-secured cucurbituril/guest trimers.

Cucurbit[7]uril (CB[7]) encapsulates adamantyl and trimethylsilyl substituents of positively charged guests in dimethyl sulfoxide (DMSO). Unlike in water or deuterium oxide, addition of a selection of alkali and alkali-earth cations with van der Waals radii between 1.0 and 1.4 Å (Na+, K+, Ca2+, Sr2+, Ba2+ and Eu3+) to the CB[7]/guest complexes triggers their cation-mediated trimerization, a process that is very slow on the nuclear magnetic resonance (NMR) time scale. Smaller (Li+, Mg2+) or larger cations (Rb+, Cs+ or NH4+) are inert. The trimers display extensive CH-O interactions between the equatorial and pseudo-equatorial hydrogens of CB[7] and the carbonyl rim of the neighboring CB[7] unit in the trimer, and a deeply nested cation between the three interacting carbonylated CB[7] rims; a counteranion is likely perched in the shallow cavity formed by the three outer walls of CB[7] in the trimer. Remarkably, a guest must occupy the cavity of CB[7] for trimerization to take place. Using a combination of semi-empirical and density functional theory techniques in conjunction with continuum solvation models, we showed that trimerization is favored in DMSO, and not in water, because the penalty for the partial desolvation of three of the six CB[7] portals upon aggregation into a trimer is less unfavorable in DMSO compared to water.

Galectin-targeting glycocalix[4]arenes can enter the cells.

Elevated levels of galectin-3 are associated with tumorigenesis. Its inhibition with high-affinity carbohydrate ligands opens new therapeutic routes. Targeting of intracellular galectin-3 is challenging for polar inhibitors like carbohydrates. We demonstrate the potential of novel biomedical research tools, glycocalix[4]arenes, to enter epithelial cells, which may allow their interaction with galectin-3.

Tracking Isomerizations of High-Energy Adenine Cation Radicals by UV-Vis Action Spectroscopy and Cyclic Ion Mobility Mass Spectrometry.

We report experimental and computational studies of protonated adenine C-8 σ-radicals that are presumed yet elusive reactive intermediates of oxidative damage to nucleic acids. The radicals were generated in the gas phase by the collision-induced dissociation of C-8-Br and C-8-I bonds in protonated 8-bromo- and 8-iodoadenine as well as by 8-bromo- and 8-iodo-9-methyladenine. Protonation by electrospray of 8-bromo- and 8-iodoadenine was shown by cyclic-ion mobility mass spectrometry (c-IMS) to form the N-1-H, N-9-H and N-3-H, N-7-H protomers in 85:15 and 81:19 ratios, respectively, in accordance with the equilibrium populations of these protomers in water-solvated ions that were calculated by density functional theory (DFT). Protonation of 8-halogenated 9-methyladenines yielded single N-1-H protomers, which was consistent with their thermodynamic stability. The radicals produced from the 8-bromo and 8-iodo adenine cations were characterized by UV-vis photodissociation action spectroscopy (UVPD) and c-IMS. UVPD revealed the formation of C-8 σ-radicals along with N-3-H, N-7-H-adenine π-radicals that arose as secondary products by hydrogen atom migrations. The isomers were identified by matching their action spectra against the calculated vibronic absorption spectra. Deuterium isotope effects were found to slow the isomerization and increase the population of C-8 σ-radicals. The adenine cation radicals were separated by c-IMS and identified by their collision cross sections, which were measured relative to the canonical N-9-H adenine cation radical that was cogenerated in situ as an internal standard. Ab initio CCSD(T)/CBS calculations of isomer energies showed that the adenine C-8 σ-radicals were local energy minima with relative energies at 76-79 kJ mol-1 above that of the canonical adenine cation radical. Rice-Ramsperger-Kassel-Marcus calculations of unimolecular rate constants for hydrogen and deuterium migrations resulting in exergonic isomerizations showed kinetic shifts of 10-17 kJ mol-1, stabilizing the C-8 σ-radicals. C-8 σ-radicals derived from N-1-protonated 9-methyladenine were also thermodynamically unstable and readily isomerized upon formation.

Ozone-enabled fatty acid discovery reveals unexpected diversity in the human lipidome.

Fatty acid isomers are responsible for an under-reported lipidome diversity across all kingdoms of life. Isomers of unsaturated fatty acids are often masked in contemporary analysis by incomplete separation and the absence of sufficiently diagnostic methods for structure elucidation. Here, we introduce a comprehensive workflow, to discover unsaturated fatty acids through coupling liquid chromatography and mass spectrometry with gas-phase ozonolysis of double bonds. The workflow encompasses semi-automated data analysis and enables de novo identification in complex media including human plasma, cancer cell lines and vernix caseosa. The targeted analysis including ozonolysis enables structural assignment over a dynamic range of five orders of magnitude, even in instances of incomplete chromatographic separation. Thereby we expand the number of identified plasma fatty acids two-fold, including non-methylene-interrupted fatty acids. Detection, without prior knowledge, allows discovery of non-canonical double bond positions. Changes in relative isomer abundances reflect underlying perturbations in lipid metabolism.

Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry.

A typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantification, the procedure has limitations. Some domains or parts of the proteins may remain inadequately described due to inefficient detection of certain peptides. This study presents an alternative approach based on sample acetylation and mass spectrometry with atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). These ionizations allowed for improved detection of acetylated peptides obtained via chymotrypsin or glutamyl peptidase I (Glu-C) digestion. APCI and APPI spectra of acetylated peptides often provided sequence information already at the full scan level, while fragmentation spectra of protonated molecules and sodium adducts were easy to interpret. As demonstrated for bovine serum albumin, acetylation improved proteomic analysis. Compared to ESI, gas-phase ionizations APCI and APPI made it possible to detect more peptides and provide better sequence coverages in most cases. Importantly, APCI and APPI detected many peptides which passed unnoticed in the ESI source. Therefore, analytical methods based on chymotrypsin or Glu-C digestion, acetylation, and APPI or APCI provide data complementary to classical bottom-up proteomics.

Silver Ion High-Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry: A Tool for Analyzing Cuticular Hydrocarbons.

Aliphatic hydrocarbons (HCs) are usually analyzed by gas chromatography (GC) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, analyzing long-chain HCs by GC is difficult because of their low volatility and the risk of decomposition at high temperatures. MALDI cannot distinguish between isomeric HCs. An alternative approach based on silver ion high-performance liquid chromatography (Ag-HPLC) is shown here. The separation of HC standards and cuticular HCs was accomplished using two ChromSpher Lipids columns connected in series. A gradient elution of the analytes was optimized using mobile phases prepared from hexane (or isooctane) and acetonitrile, 2-propanol, or toluene. HCs were detected by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Good separation of the analytes according to the number of double bonds, cis/trans geometry, and position of double bonds was achieved. The retention times increased with the number of double bonds, and trans isomers eluted ahead of cis isomers. The mobile phase significantly affected the mass spectra of HCs. Depending on the mobile phase composition, deprotonated molecules, molecular ions, protonated molecules, and various solvent-related adducts of HCs were observed. The optimized Ag-HPLC/APCI-MS was applied for characterizing cuticular HCs from a flesh fly, Neobellieria bullata, and cockroach, Periplaneta americana. The method made it possible to detect a significantly higher number of HCs than previously reported for GC or MALDI-MS. Unsaturated HCs were frequently detected as isomers differing by double-bond position(s). Minor HCs with trans double bonds were found beside the prevailing cis isomers. Ag-HPLC/APCI-MS has great potential to become a new tool in chemical ecology for studying cuticular HCs.

Non-Symmetrical Tetrafluoroalkadienes Synthesized by ROCM of 3,3,4,4-Tetrafluorocyclobutene.

As the first known example of ring-opening cross metathesis (ROCM) of polyfluorinated strained cyclobutenes, ROCM of 3,3,4,4-tetrafluorocyclobutene with electronically rich alkenes, catalyzed by Grubbs or Hoveyda-Grubbs 2nd generation precatalysts, gave a small library of non-symmetrical isolated dienes bearing a tetrafluoroethylene spacer between the double bonds. 1-Butoxy-3,3,4,4-tetrafluorohexa-1,5-diene thus formed underwent subsequent regioselective cross metathesis (CM) with a series of styrenes, catalyzed by Hoveyda-Grubbs 2nd generation precatalyst, leading to non-symmetrically substituted dienes. 6,6-Dibutoxy-3,3,4,4-tetrafluorohex-1-ene, formed by regioselective butoxylation of 1-butoxy-3,3,4,4-tetrafluorohexa-1,5-diene, was dihydroxylated and cyclized to the corresponding 3,3,4,4-tetrafluorohexopyranose.

Characterization of Triacylglycerol Estolide Isomers Using High-Resolution Tandem Mass Spectrometry with Nanoelectrospray Ionization.

Triacylglycerol estolides (TG-EST) are biologically active lipids extensively studied for their anti-inflammatory and anti-diabetic properties. In this work, eight standards of TG-EST were synthesized and systematically investigated by nanoelectrospray tandem mass spectrometry. Mass spectra of synthetic TG-EST were studied with the purpose of enabling the unambiguous identification of these lipids in biological samples. TG-EST glycerol sn-regioisomers and isomers with the fatty acid ester of hydroxy fatty acid (FAHFA) subunit branched in the ω-, α-, or 10-position were used. Ammonium, lithium, and sodium adducts of TG-EST formed by nanoelectrospray ionization were subjected to collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD). Product ion spectra allowed for identification of fatty acid (FA) and FAHFA subunits originally linked to the glycerol backbone and distinguished the α-branching site of the FAHFA from other estolide-branching isomers. The ω- and 10-branching sites were determined by combining CID with ozone-induced dissociation (OzID). Lithium adducts provided the most informative product ions, enabling characterization of FA, hydroxy fatty acid (HFA), and FAHFA subunits. Glycerol sn-regioisomers were distinguished based on the relative abundance of product ions and unambiguously identified using CID/OzID of lithium and sodium adducts.

Gas Dynamic Virtual Nozzle Sprayer for an Introduction of Liquid Samples in Atmospheric Pressure Ionization Mass Spectrometry.

Electrospray may exhibit inadequate ionization efficiency in some applications. In such cases, atmospheric-pressure chemical ionization (APCI) and photoionization (APPI) can be used. Despite a wide application potential, no APCI and APPI sources dedicated to very low sample flow rates exist on the market. Since the ion source performance depends on the transfer of analytes from the liquid to the gas phase, a nebulizer is a critical component of an ion source. Here, we report on the nebulizer with a gas dynamic virtual nozzle (GDVN) and its applicability in APCI at microliter-per-minute flow rates. Nebulizers differing by geometrical parameters were fabricated and characterized regarding the jet breakup regime, droplet size, droplet velocity, and spray angle for liquid flow rates of 0.75-15.0 µL/min. A micro-APCI source with the GDVN nebulizer behaved as a mass-flow-sensitive detector and provided stable and intense analyte signals. Compared to a classical APCI source, an order of magnitude lower detection limit for verapamil was achieved. Mass spectra recorded with the nebulizer in dripping and jetting modes were almost identical and did not differ from normal APCI spectra. Clogging never occurred during the experiments, indicating the high robustness of the nebulizer. Low-flow-rate APCI and APPI sources with a GDVN sprayer promise new applications for low- and medium-polar analytes.

Glycocalix[4]arenes and their affinity to a library of galectins: the linker matters.

Galectins are lectins that bind ß-galactosides. They are involved in important extra- and intracellular biological processes such as apoptosis, and regulation of the immune system or the cell cycle. High-affinity ligands of galectins may introduce new therapeutic approaches or become new tools for biomedical research. One way of increasing the low affinity of ß-galactoside ligands to galectins is their multivalent presentation, e.g., using calixarenes. We report on the synthesis of glycocalix[4]arenes in cone, partial cone, 1,2-alternate, and 1,3-alternate conformations carrying a lactosyl ligand on three different linkers. The affinity of the prepared compounds to a library of human galectins was determined using competitive ELISA assay and biolayer interferometry. Structure-affinity relationships regarding the influence of the linker and the core structure were formulated. Substantial differences were found between various linker lengths and the position of the triazole unit. The formation of supramolecular clusters was detected by atomic force microscopy. The present work gives a systematic insight into prospective galectin ligands based on the calix[4]arene core.

Alarm communication predates eusociality in termites.

Termites (Blattodea: Isoptera) have evolved specialized defensive strategies for colony protection. Alarm communication enables workers to escape threats while soldiers are recruited to the source of disturbance. Here, we study the vibroacoustic and chemical alarm communication in the wood roach Cryptocercus and in 20 termite species including seven of the nine termite families, all life-types, and all feeding and nesting habits. Our multidisciplinary approach shows that vibratory alarm signals represent an ethological synapomorphy of termites and Cryptocercus. In contrast, chemical alarms have evolved independently in several cockroach groups and at least twice in termites. Vibroacoustic alarm signaling patterns are the most complex in Neoisoptera, in which they are often combined with chemical signals. The alarm characters correlate to phylogenetic position, food type and hardness, foraging area size, and nesting habits. Overall, species of Neoisoptera have developed the most sophisticated communication system amongst termites, potentially contributing to their ecological success.

Advanced high-affinity glycoconjugate ligands of galectins.

Galectins are proteins of the family of human lectins. By binding terminal galactose units of cell surface glycans, they moderate biological and pathological processes such as cell signaling, cell adhesion, apoptosis, fibrosis, carcinogenesis, and metabolic disorders. The binding of monovalent glycans to galectins is usually relatively weak. Therefore, the presentation of carbohydrate ligands on multivalent scaffolds can efficiently increase and/or discriminate the affinity of the glycoconjugate to different galectins. A library of glycoclusters and glycodendrimers with various structural presentations of the common functionalized N-acetyllactosamine ligand was prepared to evaluate how the mode of presentation affects the affinity and selectivity to the two most abundant galectins, galectin-1 (Gal-1) and galectin-3 (Gal-3). In addition, the effect of a one- to two-unit carbohydrate spacer on the affinity of the glycoconjugates was determined. A new design of the biolayer interferometry (BLI) method with specific AVI-tagged constructs was used to determine the affinity to galectins, and compared with the gold-standard method of isothermal titration calorimetry (ITC). This study reveals new routes to low nanomolar glycoconjugate inhibitors of galectins of interest for biomedical research.

Selectively Halogenated Flavonolignans-Preparation and Antibacterial Activity.

A library of previously unknown halogenated derivatives of flavonolignans (silybins A and B, 2,3-dehydrosilybin, silychristin A, and 2,3-dehydrosilychristin A) was prepared. The effect of halogenation on the biological activity of flavonolignans was investigated. Halogenated derivatives had a significant effect on bacteria. All prepared derivatives inhibited the AI-2 type of bacterial communication (quorum sensing) at concentrations below 10 µM. All prepared compounds also inhibited the adhesion of bacteria (Staphyloccocus aureus and Pseudomonas aeruginosa) to the surface, preventing biofilm formation. These two effects indicate that the halogenated derivatives are promising antibacterial agents. Moreover, these derivatives acted synergistically with antibiotics and reduced the viability of antibiotic-resistant S. aureus. Some flavonolignans were able to reverse the resistant phenotype to a sensitive one, implying that they modulate antibiotic resistance.

Sulfation of Phenolic Acids: Chemoenzymatic vs. Chemical Synthesis.

Phenolic acids are known flavonoid metabolites, which typically undergo bioconjugation during phase II of biotransformation, forming sulfates, along with other conjugates. Sulfated derivatives of phenolic acids can be synthesized by two approaches: chemoenzymatically by 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferases or PAPS-independent aryl sulfotransferases such as those from Desulfitobacterium hafniense, or chemically using SO3 complexes. Both approaches were tested with six selected phenolic acids (2-hydroxyphenylacetic acid (2-HPA), 3-hydroxyphenylacetic acid (3-HPA), 4-hydroxyphenylacetic acid (4-HPA), 3,4-dihydroxyphenylacetic acid (DHPA), 3-(4-hydroxyphenyl)propionic acid (4-HPP), and 3,4-dihydroxyphenylpropionic acid (DHPP)) to create a library of sulfated metabolites of phenolic acids. The sulfates of 3-HPA, 4-HPA, 4-HPP, DHPA, and DHPP were all obtained by the methods of chemical synthesis. In contrast, the enzymatic sulfation of monohydroxyphenolic acids failed probably due to enzyme inhibition, whereas the same reaction was successful for dihydroxyphenolic acids (DHPA and DHPP). Special attention was also paid to the counterions of the sulfates, a topic often poorly reported in synthetic works. The products obtained will serve as authentic analytical standards in metabolic studies and to determine their biological activity.

Preparation of Synthetic and Natural Derivatives of Flavonoids Using Suzuki-Miyaura Cross-Coupling Reaction.

Herein, we report the use of the Suzuki-Miyaura cross-coupling reaction for the preparation of a library of synthetic derivatives of flavonoids for biological activity assays. We have investigated the reactivity of halogenated flavonoids with aryl boronates and with boronyl flavonoids. This reaction was used to prepare new synthetic derivatives of flavonoids substituted at C-8 with aryl, heteroaryl, alkyl, and boronate substituents. The formation of flavonoid boronate enabled a cross-coupling reaction with halogenated flavones yielding biflavonoids connected at C-8. This method was used for the preparation of natural compounds including C-8 prenylated compounds, such as sinoflavonoid NB. Flavonoid boronates were used for the preparation of rare C-8 hydroxyflavonoids (natural flavonoids gossypetin and hypolaetin). A series of previously unknown derivatives of quercetin and luteolin were prepared and fully characterized.

MALDI Mass Spectrometry Imaging of Lipids on Free-Floating Brain Sections and Immunohistochemically Colocalized Markers of Neurodegeneration.

In mass spectrometry imaging (MSI), the essential steps in sample preparation include collection and storage. The most widely used preservation procedure for MSI consists in freezing samples and storing them at temperatures below -80 °C. On the other hand, the most common method for preserving biological samples in clinical practice is their fixation in paraformaldehyde. The storage of free-floating sections is a particular type of the preservation of paraformaldehyde-fixed tissues that is used in immunohistochemistry. This chapter describes the approach of the multimodal imaging of free-floating brain sections using the MSI of lipids and the immunohistochemistry of neurodegeneration markers.

Structural Characterization of Unusual Fatty Acid Methyl Esters with Double and Triple Bonds Using HPLC/APCI-MS2 with Acetonitrile In-Source Derivatization.

Double and triple bonds have significant effects on the biological activities of lipids. Determining multiple bond positions in their molecules by mass spectrometry usually requires chemical derivatization. This work presents an HPLC/MS method for pinpointing the double and triple bonds in fatty acids. Fatty acid methyl esters were separated by reversed-phase HPLC with an acetonitrile mobile phase. In the APCI source, acetonitrile formed reactive species, which added to double and triple bonds to form [M + C3H5N]+• ions. Their collisional activation in an ion trap provided fragments helpful in localizing the multiple bond positions. This approach was applied to fatty acids with isolated, cumulated, and conjugated double bonds and triple bonds. The fatty acids were isolated from the fat body of early-nesting bumblebee Bombus pratorum and seeds or seed oils of Punicum granatum, Marrubium vulgare, and Santalum album. Using the method, the presence of the known fatty acids was confirmed, and new ones were discovered.

A simple and high-yield route to iridium, rhodium, osmium and ruthenium nido-6-metalladecaborane compounds.

We report a high-yield heterogeneous solid/liquid phase synthetic method to a series of nido-6-metalladecaboranes. The hydridoirida- and hydridorhoda-decaboranes, [6,6,6-H(PPh3)2-nido-6-MB9H13] [M = Ir (1), Rh (2)] are isolatable in 98% yields from the reaction of the square-planar M(I) complexes, [MCl(PPh3)3] (M = Rh, Ir), with K[B9H14]. The same synthetic procedure, but using [MCl(CO)H(PPh3)3] (M = Ru, Os) as metal starting reagents produces the CO-ligated clusters, [6,6,6-(CO)(PPh3)2-nido-6-MB9H13] [M = Ru (3), Os (4)], in yields of 83% and 95%, respectively. These highly convenient syntheses permit the investigation of the reaction chemistry of the new nido-6-metalladecaboranes. Thus, the CO-ligated compounds, 3 and 4, react with the square-planar platinum(II) complex, [PtCl2(PMe2Ph)2], in the presence of potassium triethylborohydride, to give the bimetallic clusters, [1,1,1-(CO)H(PPh3)-isocloso-1-RuB9H8-µ-(1,2)-{Pt(PMe2Ph)2}] (5) and [7,7-(PMe2Ph)2-9,9,9-(CO)(PPh3)2-nido-7,9-PtOsB9H11] (6), and the monometallic nido-5-osamadecaborane, [5,5,5-(PPh3)2(CO)-nido-5-OsB9H13] (7). This reactivity illustrates the potential of polyhedral boron-based clusters as molecular scaffolds ("B-frames") for the construction of multimetallic species. Single-crystal X-ray diffraction analyses have revealed the molecular structures of 3, 5, 6 and 7; the compounds are also studied by multielement NMR spectroscopy, mass spectrometry, IR spectroscopy, and in some cases computationally. Futhermore, the rotation of the {M(X)(PR3)2} moiety (X = H, CO), as PH3-ligated models, is studied by means of DFT-calculated relaxed potential energy surface scans, giving some insight into the lability of the metal-to-borane fragment interaction and of the exo-polyhedral ligands.