oxSTEF Reagents Are Tunable and Versatile Electrophiles for Selective Disulfide-Rebridging of Native Proteins.

Site-selective disulfide rebridging has emerged as a powerful strategy to modulate the structural and functional properties of proteins. Here, we introduce a novel class of electrophilic reagents, designated oxSTEF, that demonstrate excellent efficiency in disulfide rebridging via double thiol exchange. The oxSTEF reagents are prepared using an efficient synthetic sequence which may be diverted to obtain a range of derivatives allowing for tuning of reactivity or steric bulk. We demonstrate highly selective rebridging of cyclic peptides and native proteins, such as human growth hormone, and the absence of cross-reactivity with other nucleophilic amino acid residues. The oxSTEF conjugates undergo glutathione-mediated disintegration under tumor-relevant glutathione concentrations, which highlights their potential for use in targeted drug delivery. Finally, the α-dicarbonyl motif of the oxSTEF reagents enables "second phase" oxime ligation, which furthermore increases the thiol stability of the conjugates significantly.

α-Lactam Electrophiles for Covalent Chemical Biology.

Electrophilic groups are one of the key pillars of contemporary chemical biology and medicinal chemistry. For instance, 3-membered N-heterocyclic compounds-such as aziridines, azirines, and oxaziridines-possess unique electronic and structural properties which underlie their potential and applicability as covalent tools. The α-lactams are also members of this group of compounds, however, their utility within the field remains unexplored. Here, we demonstrate an α-lactam reagent (AM2) that is tolerant to aqueous buffers while being reactive towards biologically relevant nucleophiles. Interestingly, carboxylesterases 1 and 2 (CES1/2), both serine hydrolases with key roles in endo- and xenobiotic metabolism, were found as primary covalent targets for AM2 in HepG2 liver cancer cells. All in all, this study constitutes the starting point for the further development and exploration of α-lactam-based electrophilic probes in covalent chemical biology.

The covalent reactivity of functionalized 5-hydroxy-butyrolactams is the basis for targeting of fatty acid binding protein 5 (FABP5) by the neurotrophic agent MT-21.

Covalently acting compounds experience a strong interest within chemical biology both as molecular probes in studies of fundamental biological mechanisms and/or as novel drug candidates. In this context, the identification of new classes of reactive groups is particularly important as these can expose novel reactivity modes and, consequently, expand the ligandable proteome. Here, we investigated the electrophilic reactivity of the 3-acyl-5-hydroxy-1,5-dihydro-2H-pyrrole-2-one (AHPO) scaffold, a heterocyclic motif that is e.g. present in various bioactive natural products. Our investigations were focused on the compound MT-21 - a simplified structural analogue of the natural product epolactaene - which is known to have both neurotrophic activity and ability to trigger apoptotic cell death. We found that the central N-acyl hemiaminal group of MT-21 can function as an electrophilic centre enabling divergent reactivity with both amine- and thiol-based nucleophiles, which furthermore translated to reactivity with proteins in both cell lysates and live cells. We found that in live cells MT-21 strongly engaged the lipid transport protein fatty acid-binding protein 5 (FABP5) by direct binding to a cysteine residue in the bottom of the ligand binding pocket. Through preparation of a series of MT-21 derivatives, we probed the specificity of this interaction which was found to be strongly dependent on subtle structural changes. Our study suggests that MT-21 may be employed as a tool compound in future studies of the biology of FABP5, which remains incompletely understood. Furthermore, our study has also made clear that other natural products containing the AHPO-motif may likewise possess covalent reactivity and that this property may underlie their biological activity.

A Reagent for Amine-Directed Conjugation to IgG1 Antibodies.

Functionalized antibodies are an indispensable resource for diagnosis, therapy and as a research tool for chemical biology. However, simpler and better methodologies are often required to improve the labeling of antibodies in terms of selectivity and scalability. Herein, we report the development of an easily available chemical reagent that allows site-directed labeling of native human IgG1 antibodies in good yield and mono-labeling selectivity. The salicylaldehyde moiety of the reagent reacts with surface exposed lysine residues to transiently form an iminium ion, and this positions a semi-reactive ester in proximity of a second lysine residue that reacts with the ester to form an amide. Interestingly, it appears that the formation of the iminium ion also has a significant activating effect of the ester. We use flow cytometry and bio-layer interferometry to confirm that the labeled antibodies retain antigen binding.

Geopolymer/CeO2 as Solid Electrolyte For IT-SOFC.

As a material for application in the life sciences, a new composite material, geopolymer/CeO2 (GP_CeO2), was synthesized as a potential low-cost solid electrolyte for application in solid oxide fuel cells operating in intermediate temperature (IT-SOFC). The new materials were obtained from alkali-activated metakaolin (calcined clay) in the presence of CeO2 powders (x = 10%). Besides the commercial CeO2 powder, as a source of ceria, two differently synthesized CeO2 powders also were used: CeO2 synthesized by modified glycine nitrate procedure (MGNP) and self-propagating reaction at room temperature (SPRT). The structural, morphological, and electrical properties of pure and GP_CeO2-type samples were investigated by X-ray powder diffraction (XRPD), Fourier transform infrared (FTIR), BET, differential thermal and thermogravimetric analysis (DTA/TGA), scanning electron microscopy (FE-SEM), energy dispersive spectrometer (EDS), and method complex impedance (EIS). XRPD and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) analysis confirmed the formation of solid phase CeO2. The BET, DTA/TGA, FE-SEM, and EDS results indicated that particles of CeO2 were stabile interconnected and form a continuous conductive path, which was confirmed by the EIS method. The highest conductivity of 1.86 × 10-2 Ω-1 cm-1 was obtained for the sample GP_CeO2_MGNP at 700 °C. The corresponding value of activation energy for conductivity was 0.26 eV in the temperature range 500-700 °C.

Positive and negative nano-electrospray mass spectrometry of ruthenated serum albumin supported by docking studies: an integrated approach towards defining metallodrug binding sites on proteins.

Binding of three ruthenium(ii) compounds of general formula mer-[Ru(L3)(N-N)X][Y] (where L3 = 4'-chloro-2,2':6',2''-terpyridine (Cl-tpy); N-N = 1,2-diaminoethane (en), 1,2-diaminocyclohexane (dach) or 2,2'-bipyridine (bipy); X = Cl; Y = Cl) to human serum albumin (HSA) has been investigated by nano-LC/nano-ESI MS and docking studies. A bottom-up proteomics approach has been applied for the structural characterization of metallated proteins and the data were analyzed in both the positive and negative ion mode. The negative ion mode was achieved after the post-column addition of an isopropanol solution of formaldehyde that enabled sample ionization at micro-flow rates. The negative ion mode MS has been proved to be beneficial for the analysis of binding sites on ruthenated protein in terms of ion charge reduction and consequent simplification of target sequence identification based on isotopic differences between ruthenated and non-ruthenated peptides. Moreover, the negative ion mode ESI MS shows the advantage of singly charged ion formation and, unlike MALDI MS, it does not cause complete ligand fragmentation, merging the benefits of each method into a single experiment. Six target sequences were identified for the binding of en and dach compounds, and four sequences for the binding of bipy. All compounds have been found to bind histidine and one aspartate residue. Docking studies showed that the identified sequences are the constituents of five distinct binding sites for en and dach, or two sites for the bipy complex. The selection of binding sites seems to be dependent on the chelate ligand and the form of the complex prior or after hydrolysis of the leaving chloride ligand.

Corrigendum: The Profile and Antimicrobial Activity of Bacillus Lipopeptide Extracts of Five Potential Biocontrol Strains.

[This corrects the article on p. 925 in vol. 8, PMID: 28588570.].

New dinuclear palladium(II) complexes: Studies of the nucleophilic substitution reactions, DNA/BSA interactions and cytotoxic activity.

Six new dinuclear Pd(II) complexes, [{Pd(2,2'-bipy)Cl}2(µ-pz)](ClO4)2 (Pd1), [{Pd(dach)Cl}2(µ-pz)](ClO4)2 (Pd2), [{Pd(en)Cl}2(µ-pz)](ClO4)2 (Pd3), [{Pd(2,2'-bipy)Cl}2(µ-4,4'-bipy)](ClO4)2 (Pd4), [{Pd(dach)Cl}2(µ-4,4'-bipy)](ClO4)2 (Pd5) and [{Pd(en)Cl}2(µ-4,4'-bipy)](ClO4)2 (Pd6) (where 2,2'-bipy=2,2'-bipyridyl, pz=pyrazine, dach=trans-(±)-1,2-diaminocyclohexane, en=ethylenediamine, 4,4'-bipy=4,4'-bipyridyl) have been synthesized and characterized by elemental microanalysis, IR, 1H NMR and MALDI-TOF mass spectrometry. The pKa values of corresponding diaqua complexes were determined by spectrophotometric pH titration. Substitution reactions with thiourea (Tu), l-methionine (l-Met), l-cysteine (l-Cys), l-histidine (l-His) and guanosine-5'-monophosphate (5'-GMP) were studied under the pseudo-first order conditions at pH7.2. Reactions of Pd1 with Tu, l-Met and l-Cys were followed by decomposition of complexes, while structures of dinuclear complexes were preserved during the substitution with nitrogen donors. Interactions with calf-thymus DNA (CT-DNA) were followed by absorption spectroscopy and fluorescence quenching measurements. All complexes can bind to CT-DNA exhibiting high intrinsic binding constants (Kb=104-105M-1). Competitive studies with ethidium bromide (EB) have shown that complexes can displace DNA-bound EB. High values of binding constants towards bovine serum albumin protein (BSA) indicate good binding affinity. Finally, all complexes showed moderate to high cytotoxic activity against HeLa (human cervical epithelial carcinoma cell lines) and MDA-MB-231 (human breast epithelial carcinoma cell lines) tumor cell lines inducing apoptotic type cell death, whereas normal fibroblasts were significantly less sensitive. The impact on cell cycle of these cells was distinctive, where Pd4, Pd5 and Pd6 showed the most prominent effect arresting MDA-MB-231 (human lung fibroblast cell lines) cell in G1/S phase of cell cycle.

The Profile and Antimicrobial Activity of Bacillus Lipopeptide Extracts of Five Potential Biocontrol Strains.

In this study the efficacy of two different methods for extracting lipopeptides produced by five Bacillus strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction-was investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of Xanthomonas arboricola and Pseudomonas syringae phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving in situ its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with Bacillus amyloliquefaciens, while SS-10.7 was more closely related to Bacillus pumilus.

High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions.

Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) is routinely used in proteomics research. Mass spectrometry-based peptide analysis is performed de facto in positive-ion mode, except for the analysis of some post-translationally modified peptides (e.g., phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce, because of a lack of negatively charged amino acid side-chain residues that would enable efficient ionization (i.e., on average, every 10th amino acid residue is negatively charged). Also, several phenomena linked to negative ionization, such as corona discharge, arcing, and electrospray destabilization, because of the presence of polar mobile-phase solutions or acidic mobile-phase additives (e.g., formic or trifluoroacetic acid), reduce its use. Named phenomena influence microflow and nanoflow electrospray ionization (ESI) of peptides in a way that prevents the formation of negatively charged peptide ions. In this work, we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2,2-dimethylpropanal, ethyl methanoate, and 2-phenyl-2-oxoethanal as the negative-ion-mode mobile-phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions through oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.

Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin.

Hyphenated mass spectrometry (MS) techniques have attained an important position in analysis of covalent and non-covalent interactions of metal complexes with peptides and proteins. The aim of the present study was to qualitatively and quantitatively determine ruthenium binding sites on a protein using tandem mass spectrometry and allied techniques, i.e. liquid chromatography (LC) and inductively coupled plasma optical emission spectrometry (ICP-OES). For that purpose, two newly synthesized Ru(II) complexes of a meridional geometry, namely mer-[Ru(4' Cl-tpy)(en)Cl](+) (1) and mer-[Ru(4' Cl-tpy)(dach)Cl](+) (2) (where 4' Cl-tpy=4'-chloro-2,2':6',2″-terpyridine, en=1,2-diaminoethane and dach=1,2-diaminocyclohexane), and bovine serum albumin were used. The binding of the complexes to the protein was investigated by means of size exclusion- and reversed phase-LC, ICP OES, matrix-assisted laser desorption ionization MS and MS/MS. Ruthenated peptide sequence and a binding target amino acid were revealed through accurate elucidation of MS/MS spectra. The results obtained in this study suggest a high binding capacity of the protein towards both complexes, with up to 5.77±0.14 and 6.95±0.43mol of 1 and 2 bound per mol of protein, respectively. The proposed binding mechanism for the selected complexes includes the release of Cl ligand, its replacement with water molecule and further coordination to electron donor histidine residue.

Sensitivity and accuracy of organic matrix-assisted laser desorption and ionisation mass spectrometry of FeCl3 is higher than in in matrix-free approach.

We compare the quality and reliability of laser desorption and ionisation mass spectra of FeCl3 acquired without the assistance of the matrix with the spectra acquired with different organic matrix molecules. Generally, inorganic salts tend to form clusters upon laser irradiation, the signals of which can be easily distinguished from ions arising from the matrix. In the presence of a matrix, cluster ions are, however, mostly suppressed. We have compared the number of analyte signals, accuracy of determination of isotope composition of the analyte and the sensitivity of FeCl3 detection between different approaches. The results obtained imply that the sensitivity of mass spectrometric analysis of FeCl3 is somewhat higher when matrices are applied than in the matrix-free approach. Among all matrices tested in this work, F20TPP seems to be the most promising for further applications as a matrix for mass spectrometry of inorganic salts.