Clickable report tags for identification of modified peptides by mass spectrometry

The identification and quantification of modified peptides are critical for the functional characterization of post-translational protein modifications (PTMs) to elucidate their biological function. Nowadays, quantitative mass spectrometry coupled with various bioinformatic pipelines has been successfully used for the determination of a wide range of PTMs. However, direct characterization of low abundant protein PTMs in bottom-up proteomic workflow remains challenging. Here, we present the synthesis and evaluation of tandem mass spectrometry tags (TMT) which are introduced via click-chemistry into peptides bearing alkyne handles. The fragmentation properties of the two mass tags were validated and used for screening in a model system and analysis of AMPylated proteins. The presented tags provide a valuable tool for diagnostic peak generation to increase confidence in the identification of modified peptides and potentially for direct peptide-PTM quantification from various experimental conditions.     

Metal Assisted Synthesis of Cationic Sulfidobismuth Cubanes in Ionic Liquids

Bi₂S₃ was dissolved in the presence of either AuCl/PtCl₂ or AgCl in the ionic liquids [BMIm]Cl ⋅ xAlCl₃ (BMIm=1-n-butyl-3-methylimidazolium; x=4–4.3) through annealing the mixtures at 180 or 200 °C. Upon cooling to room temperature, orange, air-sensitive crystals of [BMIm](Bi₄S₄)[AlCl₄]₅ ( 1 ) or Ag(Bi₇S₈)[S(AlCl₃)₃]₂[AlCl₄]₂ ( 2 ) precipitated, respectively. 1 did not form in the absence of AuCl/PtCl₂, suggesting an essential role of the metal cations. X-ray diffraction on single-crystals of 1 revealed a monoclinic crystal structure that contains (Bi₄S₄)⁴⁺ heterocubanes and [AlCl₄]⁻ tetrahedra as well as [BMIm]⁺ cations. The intercalation of the ionic liquid was confirmed via solid state NMR spectroscopy, revealing unusual coupling behavior. The crystal structure of 2 consists of (Bi₇S₈)⁵⁺ spiro-dicubanes, [S(AlCl₃)₃]²⁻ tetrahedra triples, isolated [AlCl₄]⁻ tetrahedra, and heavily disordered silver(I) cations. No cation ordering took place in 2 upon slow cooling to 100 K.     

Arene Ruthenium(II) Complexes Bearing the κ-P or κ-P,κ-S Ph2P(CH2)3SPh Ligand

Neutral [Ru(η⁶-arene)Cl₂{Ph₂P(CH₂)₃SPh-κP}] (arene = benzene, indane, 1,2,3,4-tetrahydronaphthalene: 2a, 2c and 2d) and cationic [Ru(η⁶-arene)Cl(Ph₂P(CH₂)₃SPh-κP,κS)]X complexes (arene = mesitylene, 1,4-dihydronaphthalene; X = Cl: 3b, 3e; arene = benzene, mesitylene, indane, 1,2,3,4-tetrahydronaphthalene, and 1,4-dihydronaphthalene; X = PF₆: 4a–4e) complexes were prepared and characterized by elemental analysis, IR, ¹H, ¹³C and ³¹P NMR spectroscopy and also by single-crystal X-ray diffraction analyses. The stability of the complexes has been investigated in DMSO. Complexes have been assessed for their cytotoxic activity against 518A2, 8505C, A253, MCF-7 and SW480 cell lines. Generally, complexes exhibited activity in the lower micromolar range; moreover, they are found to be more active than cisplatin. For the most active ruthenium(II) complex, 4b, bearing mesitylene as ligand, the mechanism of action against 8505C cisplatin resistant cell line was determined. Complex 4b induced apoptosis accompanied by caspase activation.     

Quantification of Volatile Aldehydes Deriving from In Vitro Lipid Peroxidation in the Breath of Ventilated Patients

Exhaled aliphatic aldehydes were proposed as non-invasive biomarkers to detect increased lipid peroxidation in various diseases. As a prelude to clinical application of the multicapillary column–ion mobility spectrometry for the evaluation of aldehyde exhalation, we, therefore: (1) identified the most abundant volatile aliphatic aldehydes originating from in vitro oxidation of various polyunsaturated fatty acids; (2) evaluated emittance of aldehydes from plastic parts of the breathing circuit; (3) conducted a pilot study for in vivo quantification of exhaled aldehydes in mechanically ventilated patients. Pentanal, hexanal, heptanal, and nonanal were quantifiable in the headspace of oxidizing polyunsaturated fatty acids, with pentanal and hexanal predominating. Plastic parts of the breathing circuit emitted hexanal, octanal, nonanal, and decanal, whereby nonanal and decanal were ubiquitous and pentanal or heptanal not being detected. Only pentanal was quantifiable in breath of mechanically ventilated surgical patients with a mean exhaled concentration of 13 ± 5 ppb. An explorative analysis suggested that pentanal exhalation is associated with mechanical power—a measure for the invasiveness of mechanical ventilation. In conclusion, exhaled pentanal is a promising non-invasive biomarker for lipid peroxidation inducing pathologies, and should be evaluated in future clinical studies, particularly for detection of lung injury.     

Turbulence transition and the edge of chaos in pipe flow

The linear stability of pipe flow implies that only perturbations of sufficient strength will trigger the transition to turbulence. In order to determine this threshold in perturbation amplitude we study the edge of chaos which separates perturbations that decay towards the laminar profile and perturbations that trigger turbulence. Using the lifetime as an indicator and methods developed in Skufca et al., Phys. Rev. Lett. 96, 174101 (2006), we show that superimposed on an overall 1/Re scaling predicted and studied previously there are small, nonmonotonic variations reflecting folds in the edge of chaos. By tracing the motion in the edge we find that it is formed by the stable manifold of a unique flow field that is dominated by a pair of downstream vortices, asymmetrically placed towards the wall. The flow field that generates the edge of chaos shows intrinsic chaotic dynamics.     

Line tension at fluid membrane domain boundaries measured by micropipette aspiration

Line tension is a determinant of fluid phase domain formation kinetics and morphology in lipid bilayer membranes, which are models for biological membrane heterogeneity. We describe the first direct measurement of this line tension by micropipette aspiration. Our data are analyzed with a model that does not rely on independently measured (and composition dependent) secondary parameters, such as bending stiffness or membrane viscosities. Line tension is strongly composition dependent and decreases towards a critical consolute point in a quasiternary room temperature phase diagram.     

Development and Molecular Understanding of a Pd-Catalyzed Cyanation of Aryl Boronic Acids Enabled by High-Throughput Experimentation and Data Analysis

A synthetic method for the palladium-catalyzed cyanation of aryl boronic acids using bench stable and non-toxic N-cyanosuccinimide has been developed. High-throughput experimentation facilitated the screen of 90 different ligands and the resultant statistical data analysis identified that ligand σ-donation, π-acidity and sterics are key drivers that govern yield. Categorization into three ligand groups – monophosphines, bisphosphines and miscellaneous – was performed before the analysis. For the monophosphines, the yield of the reaction increases for strong σ-donating, weak π-accepting ligands, with flexible pendant substituents. For the bisphosphines, the yield predominantly correlates with ligand lability. The applicability of the designed reaction to a wider substrate scope was investigated, showing good functional group tolerance in particular with boronic acids bearing electron-withdrawing substituents. This work outlines the development of a novel reaction, coupled with a fast and efficient workflow to gain understanding of the optimal ligand properties for the design of improved palladium cross-coupling catalysts.     

Stable and Storable N(CF3)2 Transfer Reagents

Fluorinated groups are essential for drug design, agrochemicals, and materials science. The bis(trifluoromethyl)amino group is an example of a stable group that has a high potential. While the number of molecules containing perfluoroalkyl, perfluoroalkoxy, and other fluorinated groups is steadily increasing, examples with the N(CF₃)₂ group are rare. One reason is that transfer reagents are scarce and metal-based storable reagents are unknown. Herein, a set of Cu^I and Ag^I bis(trifluoromethyl)amido complexes stabilized by N- and P-donor ligands with unprecedented stability are presented. The complexes are stable solids that can even be manipulated in air for a short time. They are bis(trifluoromethyl)amination reagents as shown by nucleophilic substitution and Sandmeyer reactions. In addition to a series of benzylbis(trifluoromethyl)amines, 2-bis(trifluoromethyl)amino acetate was obtained, which, upon hydrolysis, gives the fluorinated amino acid N,N-bis(trifluoromethyl)glycine.     

Guided Antitumoural Drugs: (Imidazol-2-ylidene)(L)gold(I) Complexes Seeking Cellular Targets Controlled by the Nature of Ligand L

Three [1,3-diethyl-4-(p-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)imidazol-2-ylidene](L)gold(I) complexes, 4 a (L=Cl), 5 a (L=PPh₃), and 6 a (L=same N-heterocyclic carbene (NHC)), and their fluorescent [4-(anthracen-9-yl)-1,3-diethyl-5-phenylimidazol-2-ylidene](L)gold(I) analogues, 4 b , 5 b , and 6 b , respectively, were studied for their localisation and effects in cancer cells. Despite their identical NHC ligands, the last three accumulated in different compartments of melanoma cells, namely, the nucleus ( 4 b ), mitochondria ( 5 b ), or lysosomes ( 6 b ). Ligand L was also more decisive for the site of accumulation than the NHC ligand because the couples 4 a / 4 b , 5 a / 5 b , and 6 a / 6 b , carrying different NHC ligands, afforded similar results in cytotoxicity tests, and tests on targets typically found at their sites of accumulation, such as DNA in nuclei, reactive oxygen species and thioredoxin reductase in mitochondria, and lysosomal membranes. Regardless of the site of accumulation, cancer cell apoptosis was eventually induced. The concept of guiding a bioactive complex fragment to a particular subcellular target by secondary ligand L could reduce unwanted side effects.     

Fluorinated Azobenzenes Switchable with Red Light

Molecular photoswitches triggered with red or NIR light are optimal for photomodulation of complex biological systems, including efficient penetration of the human body for therapeutic purposes (“therapeutic window”). Yet, they are rarely reported, and even more rarely functional under aqueous conditions. In this work, fluorinated azobenzenes are shown to exhibit efficient E→Z photoisomerization with red light (PSS_660nm >75 % Z) upon conjugation with unsaturated substituents. Initially demonstrated for aldehyde groups, this effect was also observed in a more complex structure by incorporating the chromophore into a cyclic dipeptide with propensity for self-assembly. Under physiological conditions, the latter molecule formed a supramolecular material that reversibly changed its viscosity upon irradiation with red light. Our observation can lead to design of new photopharmacology agents or phototriggered materials for in vivo use.