Catalytic Processes for the Neutralization of Sulfur Mustard.

Research on the decontamination of the chemical warfare agent sulfur mustard pursues several objectives that include the neutralization of spared ammunition, the cleaning of affected areas, and also the development of protective equipment or tools. Neutralization of vesicant sulfur mustard involves different chemical routes such as hydrolysis, dehydrochlorination, oxidation, or complete mineralization. This review weighs the pros and cons associated with the different systems reported in the literature, with an emphasis on catalytic procedures, to selectively convert sulfur mustard or its simulants into harmless products.

Synthesis of Protected 3,4- and 2,3-Dimercaptophenylalanines as Building Blocks for Fmoc-Peptide Synthesis and Incorporation of the 3,4-Analogue in a Decapeptide Using Solid-Phase Synthesis.

3,4-Dimercaptophenylalanines and 2,3-dimercaptophenylalanines have been synthesized for the first time by nucleophilic substitution of a protected aminomalonate on 3,4- and 2,3-dimercaptobenzyl bromide derivatives. The dithiol functions were protected as thioketals, and the key precursors, diphenylthioketal-protected dimercaptobenzyl bromides, were synthesized via two distinct routes from either dihydroxy benzoates or toluene-3,4-dithiol. Racemic mixtures of the fully protected amino acids were separated by chiral HPLC into the corresponding enantiomers. The absolute configuration of both 3,4- and 2,3-analogues could be assigned based on X-ray crystallography and VCD/DFT measurements. Thioketal groups were deprotected upon reaction with mercury oxide and aqueous tetrafluoroboric acid followed by treatment with H2S gas under an argon atmosphere to obtain the corresponding dimercapto amino acids. The optically pure l-Fmoc-protected 3,4-analogue (S- enantiomer) was successfully incorporated into a decapeptide using standard solid-phase peptide synthesis. Therefore, dithiolene-functionalized peptides are now accessible from a simple synthetic procedure, and this should afford new molecular tools for research into the catalysis, diagnostic, and nanotechnology fields.

Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons.

The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

FeIII and FeII Phosphasalen Complexes: Synthesis, Characterization, and Catalytic Application for 2-Naphthol Oxidative Coupling.

We report on the synthesis and characterization of three iron(III) phosphasalen complexes, [FeIII (Psalen)(X)] differing in the nature of the counter-anion/exogenous ligand (X- =Cl- , NO3 - , OTf- ), as well as the neutral iron(II) analogue, [FeII (Psalen)]. Phosphasalen (Psalen) differs from salen by the presence of iminophosphorane (P=N) functions in place of the imines. All the complexes were characterized by single-crystal X-ray diffraction, UV/Vis, EPR, and cyclic voltammetry. The [FeII (Psalen)] complex was shown to remain tetracoordinated even in coordinating solvent but surprisingly exhibits a magnetic moment in line with a FeII high-spin ground state. For the FeIII complexes, the higher lability of triflate anion compared to nitrate was demonstrated. As they exhibit lower reduction potentials compared to their salen analogues, these complexes were tested for the coupling of 2-naphthol using O2 from air as oxidant. In order to shed light on this reaction, the interaction between 2-naphthol and the FeIII (Psalen) complexes was studied by cyclic voltammetry as well as UV/Vis spectroscopy.

Metal-ion-regulated miniature DNA-binding proteins based on GCN4 and non-native regulation sites.

The design of artificial peptide dimers containing polypyridine switching domains, for which metal-ion coordination is shown to regulate DNA binding, is reported. Short peptides, based on the basic domain of the GCN4 transcription factor (GCN4bd), dimerised with either 2,2'-bipyridine (bipy(GCN4bd)2 ) or 2,2':6',2''-terpyridine (terpy(GCN4bd)2 ) linker units, undergo a conformational rearrangement on Cu(II) and Zn(II) coordination. Depending on the linker substitution pattern, this is proposed to alter the relative alignment of the two peptide moieties, and in turn regulate DNA binding. Circular dichroism and UV-visible spectroscopy reveal that Cu(II) and Zn(II) coordination promotes binding to DNA containing the CRE target site, but to a differing and opposite degree for the two linkers, and that the metal-ion affinity for terpy(GCN4bd)2 is enhanced in the presence of CRE DNA. Binding to DNA containing the shorter AP1 target site, which lacks a single nucleobase pair compared to CRE, as well as half-CRE, which contains only half of the CRE target site, was also investigated. Cu(II) and Zn(II) coordination to terpy(GCN4bd)2 promotes binding to AP1 DNA, and to a lesser extent half-CRE DNA. Whereas, bipy(GCN4bd)2 , for which interpeptide distances are largely independent of metal-ion coordination and less suitable for binding to these shorter sites, displays allosteric ineffective behaviour in these cases. These findings for the first time demonstrate that biomolecular recognition, and specifically sequence-selective DNA binding, can be controlled by metal-ion coordination to designed switching units, non-native regulation sites, in artificial biomolecules. We believe that in the future these could find a wide range of applications in biotechnology.

Rational Design and Characterisation of Novel Mono- and Bimetallic Antibacterial Linde Type A Zeolite Materials.

The development of antimicrobial devices and surfaces requires the setup of suitable materials, able to store and release active principles. In this context, zeolites, which are microporous aluminosilicate minerals, hold great promise, since they are able to serve as a reservoir for metal-ions with antimicrobial properties. Here, we report on the preparation of Linde Type A zeolites, partially exchanged with combinations of metal-ions (Ag+, Cu2+, Zn2+) at different loadings (0.1-11.9 wt.%). We combine X-ray fluorescence, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction to monitor the metal-ion contents, distribution, and conservation of the zeolite structure after exchange. Then, we evaluate their antimicrobial activity, using agar dilution and optical-density monitoring of Escherichia coli cultures. The results indicate that silver-loaded materials are at least 70-fold more active than the copper-, zinc-, and non-exchanged ones. Moreover, zeolites loaded with lower Ag+ concentrations remain active down to 0.1 wt.%, and their activities are directly proportional to the total Ag content. Sequential exchanges with two metal ions (Ag+ and either Cu2+, Zn2+) display synergetic or antagonist effects, depending on the quantity of the second metal. Altogether, this work shows that, by combining analytical and quantitative methods, it is possible to fine-tune the composition of bi-metal-exchanged zeolites, in order to maximise their antimicrobial potential, opening new ways for the development of next-generation composite zeolite-containing antimicrobial materials, with potential applications for the design of dental or bone implants, as well as biomedical devices and pharmaceutical products.

Monitoring of Methionine Sulfoxide Content and Methionine Sulfoxide Reductase Activity.

The sulfur-containing amino acid methionine (Met) plays critical roles in protein synthesis, methylation, and sulfur metabolism. Both in its free form and in the form of an amino acid residue, it can be oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Organisms evolved methionine sulfoxide reductases (MSRs) to reduce MetO to Met, with the MSRs type A (MSRA) and type B (MSRB) being specific for the S and R forms of MetO, respectively. In mammals, the selenoprotein MSRB1 plays an important protein repair function, and its expression is tightly regulated by dietary selenium. In this chapter, we describe a protocol for determining the concentration of protein-based Met-R-O and its analysis in HEK293 cells using a genetically encoded ratiometric fluorescent biosensor MetROx. We also describe the procedure for quantifying MSR activities in cell extracts using specific substrates and a reverse phase HPLC-based method.

Computational and experimental investigations into the conformations of cyclic tetra-α/ß-peptides.

We present a combined computational and experimental study of the energy landscapes of cyclic tetra-α/ß-peptides. We have performed discrete path sampling calculations on a series of cyclic tetra-α/ß-peptides to obtain the relative free energies and barriers to interconversion of their conformers. The most stable conformers of cyclo-[(ß-Ala-Gly)2] contain all-trans peptide groups. The relative energies of the cis isomers and the cis-trans barriers are lower than in acyclic peptides but not as low as in the highly strained cyclic α-peptides. For cyclic tetra-α/ß-peptides containing a single proline residue, of the type cyclo-[ß-Ala-Xaa-ß-Ala-Pro], the energy landscapes show that the most stable isomers containing cis and trans ß-Ala-Pro have similar free energies and are separated by barriers of approximately 15 kcal mol(-1). We show that the underlying energy landscapes of cyclo-[ß-Ala-Lys-ß-Ala-Pro] and cyclo-[ß-Ala-Ala-ß-Ala-Pro] are similar, allowing the substitution of the flexible side chain of Lys with Ala to reduce the computational demand of our calculations. However, the steric bulk of the Val side chain in cyclo-[ß-Ala-Val-ß-Ala-Pro] affects the conformations of the ring, leading to significant differences between its energy landscape and that of cyclo-[ß-Ala-Ala-ß-Ala-Pro]. We have synthesized the cyclic peptide cyclo-[ß-Ala-Lys-ß-Ala-Pro], and NMR spectroscopy shows the presence of conformers that interconvert slowly on the NMR time scale at temperatures up to 80 °C. Calculated circular dichroism (CD) spectra for the proposed major isomer of cyclo-[ß-Ala-Ala-ß-Ala-Pro] are in good agreement with the experimental spectra of cyclo-[ß-Ala-Lys-ß-Ala-Pro], suggesting that the Ala cyclic tetrapeptide is a viable model for the Lys analogue.

Conformational Study of an Artificial Metal-Dependent Regulation Site for Use in Designer Proteins.

This report describes the dimerisation of glutathione, and by extension, other cysteine-containing peptides or protein fragments, with a 5, 5'-disubstituted-2, 2'-bipyridine or 6, 6"-disubstituted-2, 2':6',2"-terpyridine unit. The resulting bipy-GS2 and terpy-GS2 were investigated as potential metal ion dependent switches in aqueous solution, and were found to predominantly adopt the transoïd conformation at physiological pH. Metal complexation with CuII and ZnII at this pH has been studied by UV/Vis, CD, NMR and ion-mobility mass spectrometry. ZnII titrations are consistent with the formation of a 1:1 ZnII:terpy-GS2 complex at pH 7.4, but bipy-GS2 was shown to form both 1:1 and 1:2 complexes with the former being predominant under dilute micromolar conditions. Formation constants for the resulting 1:1 complexes were determined to be log KM 6.86 (bipy-GS2 ) and 6.22 (terpy-GS2 ), consistent with a higher affinity for the unconstrained bipyridine, compared to the strained terpyridine. CuII coordination involves the initial formation of 1:1 complexes, followed by 1.5Cu:1bipy-GS2 and 2Cu:1terpy-GS2 complexes at micromolar concentrations. Binding constants for formation of the 1:1 complexes (log KM 12.5 (bipy-GS2 ); 8.04 and 7.14 (terpy-GS2 )) indicate a higher affinity for CuII than ZnII. Finally, ion-mobility MS studies detected the free ligands in their protonated form, and were consistent with the formation of two different Cu adducts with different conformations in the gas-phase. We illustrate that the bipyridine and terpyridine dimerisation units can behave like conformational switches in response to Cu/Zn complexation, and propose that in future these can be employed in synthetic biology with larger peptide or protein fragments, to control large scale folding and related biological function.