Chemoselective Synthesis and Anti-Kinetoplastidal Properties of 2,6-Diaryl-4H-tetrahydro-thiopyran-4-one S-Oxides: Their Interplay in a Cascade of Redox Reactions from Diarylideneacetones.

2,6-Diaryl-4H-tetrahydro-thiopyran-4-ones and corresponding sulfoxide and sulfone derivatives were designed to lower the major toxicity of their parent anti-kinetoplatidal diarylideneacetones through a prodrug effect. Novel diastereoselective methodologies were developed and generalized from diarylideneacetones and 2,6-diaryl-4H-tetrahydro-thiopyran-4-ones to allow the introduction of a wide substitution profile and to prepare the related S-oxides. The in vitro biological activity and selectivity of diarylideneacetones, 2,6-diaryl-4H-tetrahydro-thiopyran-4-ones, and their S-sulfoxide and sulfone metabolites were evaluated against Trypanosoma brucei brucei, Trypanosoma cruzi, and various Leishmania species in comparison with their cytotoxicity against human fibroblasts hMRC-5. The data revealed that the sulfides, sulfoxides, and sulfones, in which the Michael acceptor sites are temporarily masked, are less toxic against mammal cells while the anti-trypanosomal potency was maintained against T. b. brucei, T. cruzi, L. infantum, and L. donovani, thus confirming the validity of the prodrug strategy. The mechanism of action is proposed to be due to the involvement of diarylideneacetones in cascades of redox reactions involving the trypanothione system. After Michael addition of the dithiol to the double bonds, resulting in an elongated polymer, the latter-upon S-oxidation, followed by syn-eliminations-fragments, under continuous release of reactive oxygen species and sulfenic/sulfonic species, causing the death of the trypanosomal parasites in the micromolar or submicromolar range with high selectivity indexes.

Identification of Serine-Containing Microcystins by UHPLC-MS/MS Using Thiol and Sulfoxide Derivatizations and Detection of Novel Neutral Losses.

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria, and their structural diversity has led to the discovery of more than 300 congeners to date. However, with known amino acid combinations, many more MC congeners are theoretically possible, suggesting many remain unidentified. Herein, two novel serine (Ser)-containing MCs were putatively identified in a Lake Erie cyanobacterial harmful algal bloom (cyanoHAB), using high-resolution UHPLC-MS as well as thiol and sulfoxide derivatization procedures. These MCs contain an α,ß-unsaturated carbonyl on methyl dehydroalanine (Mdha) residue that undergoes Michael addition to produce a thiol-derivatized MC. Derivatization reactions using various thiolation reagents were followed by MS/MS, and two Python codes were used for data analysis and structural elucidation of MCs. Two novel MCs containing Ser at position 1 (i.e., next to Mdha) were putatively identified as [Ser1]MC-RR and [Ser1]MC-YR. Using thiol- and sulfoxide-modified [Ser1]MCs, identifications were confirmed by the observation of specific neutral losses of the oxidized thiols or sulfoxides in CID-MS/MS spectra in both positive and negative electrospray ionization (ESI) modes. These novel neutral losses are unique for MCs with Mdha and an adjacent Ser residue. Data suggest that a gas-phase reaction occurs between oxygen from adjacent Ser residue and sulfur of the Mdha-bonded thiol or sulfoxide, which leads to the formation and detection of stable cyclic MC ions in MS/MS spectra at m/z values corresponding to the loss of oxidized thiols or oxidized sulfoxides from Ser1-containing MCs.

Safrole oxide induced 5-HT neuron-like cell differentiation of bone marrow mesenchymal stem cells by elevating G9a.

In our previous study, we found that safrole oxide (SFO) could induce bone marrow mesenchymal stem cell differentiation into neuron-like cells. However, which kind of neuron cells was induced by SFO was unknown. Here, we found that SFO could induce BMSC differentiation into 5-hydroxytryptamine (5-HT) neuron-like cells. Microarray analysis of BMSCs treated with SFO for 6 h revealed a total of 35 genes changed more than twice. We selected G9a, a histone methyltransferase for further study. The upregulation of G9a was confirmed by RT-PCR and Western blot analysis. Small interfering RNA knockdown of G9a blocked SFO-induced BMSC differentiation. These results demonstrated that G9a was the pivotal factor in SFO-medicated 5-HT neuronal differentiation of BMSCs. Our findings provide a new clue for further investigating the gene control of BMSC differentiation into 5-HT neuron-like cells and provide a putative strategy for depression diseases therapies.

Trifluoromethylthiolation/Selenolation and Lactonization of 2-Alkynylbenzoate: The Application of Benzyl Trifluoromethyl Sulfoxide/Selenium Sulfoxides as SCF3/SeCF3 Reagents.

The combined use of BnS(O)CF3/BnSe(O)CF3 with Tf2O as SCF3/SeCF3 reagents was implemented to realize an efficient synthesis of biologically interesting 4-(trifluoromethylthio/trifluoromethylseleno)isocoumarins from 2-alkynylbenzoates. The mechanistic pathway was postulated to involve formation of the electrophilic SCF3/SeCF3 species via interrupted Pummerer reactions followed by a concerted trifluoromethylthiolation/selenolation and lactonization process.

Formation of volatile sulfur compounds and S-methyl-l-cysteine sulfoxide in Brassica oleracea vegetables.

Besides glucosinolates, Brassica vegetables accumulate sulfur-containing (+)-S-methyl-l-cysteine sulfoxide (SMCSO, methiin), mainly known from Allium vegetables. Such (+)-S-alk(en)yl-l-cysteine sulfoxides can degrade to volatile organosulfur compounds (VOSCs), which have been linked to health beneficial effects. In the present study, the accumulation of SMCSO and the formation of VOSCs was investigated in Brassica oleracea vegetables. SMCSO content of commercially available white and red cabbages was monitored over a three-month period and linked with the formation of VOSCs. S-Methyl methanethiosulfinate was the main VOSC released from SMCSO. Upon heating, it degraded to dimethyltrisulfide and dimethyldisulfide, which were less abundant in fresh homogenates. SMCSO made up approximately 1% of the dry matter of cabbages and the overall contents were similar in white and red cabbages (3.2-10.2 and 3.9-10.3 µmol/g fresh weight, respectively). Using proteome profiling it was shown that recovery of VOSCs correlated with abundance of two isoforms of cystine lyase.

Developing a Targeted Quantitative Strategy for Sulfoxide-Containing MS-Cleavable Cross-Linked Peptides to Probe Conformational Dynamics of Protein Complexes.

In recent years, cross-linking mass spectrometry (XL-MS) has made enormous strides as a technology for probing protein-protein interactions (PPIs) and elucidating architectures of multisubunit assemblies. To define conformational and interaction dynamics of protein complexes under different physiological conditions, various quantitative cross-linking mass spectrometry (QXL-MS) strategies based on stable isotope labeling have been developed. These QXL-MS approaches have effectively allowed comparative analysis of cross-links to determine their relative abundance changes at global scales. Although successful, it remains challenging to consistently obtain quantitative measurements on low-abundant cross-links. Therefore, targeted QXL-MS is needed to enable MS "Western" analysis of cross-links to enhance sensitivity and reliability in quantitation. To this end, we have established a robust parallel reaction monitoring (PRM)-based targeted QXL-MS platform using sulfoxide-containing MS-cleavable cross-linker disuccinimidyl sulfoxide (DSSO), permitting label-free comparative analysis of selected cross-links across multiple samples. In addition, we have applied this methodology to study phosphorylation-dependent conformational dynamics of the human 26S proteasome. The PRM-based targeted QXL-MS analytical platform described here is applicable for all sulfoxide-containing MS-cleavable cross-linkers and can be directly adopted for comparative studies of protein-protein interactions in various cellular contexts.

In vitro and in silico study on consequences of combined exposure to the food-borne alkenylbenzenes estragole and safrole.

Potential consequences of combined exposure to the selected food-borne alkenylbenzenes safrole and estragole or their proximate carcinogenic 1'-hydroxy metabolites were evaluated in vitro and in silico. HepG2 cells were exposed to 1'-hydroxyestragole and 1'-hydroxysafrole individually or in equipotent combination subsequently detecting cytotoxicity and DNA adduct formation. Results indicate that concentration addition adequately describes the cytotoxic effects and no statistically significant differences were shown in the level of formation of the major DNA adducts. Furthermore, physiologically based kinetic modeling revealed that at normal dietary intake the concentration of the parent compounds and their 1'-hydroxymetabolites remain substantially below the Km values for the respective bioactivation and detoxification reactions providing further support for the fact that the simultaneous presence of the two carcinogens or of their proximate carcinogenic 1'-hydroxy metabolites may not affect their DNA adduct formation. Overall, these results point at the absence of interactions upon combined exposure to selected food-borne alkenylbenzenes at realistic dietary levels of intake.

Multicentre, randomised, open-label, phase IV-III study to evaluate the efficacy of cloxacillin plus fosfomycin versus cloxacillin alone in adult patients with methicillin-susceptible Staphylococcus aureus bacteraemia: study protocol for the SAFO trial.

INTRODUCTION: Methicillin-susceptible Staphylococcus aureus (MSSA) bacteraemia is a frequent condition, with high mortality rates. There is a growing interest in identifying new therapeutic regimens able to reduce therapeutic failure and mortality observed with the standard of care of beta-lactam monotherapy. In vitro and small-scale studies have found synergy between cloxacillin and fosfomycin against S. aureus. Our aim is to test the hypothesis that cloxacillin plus fosfomycin achieves higher treatment success than cloxacillin alone in patients with MSSA bacteraemia. METHODS: We will perform a superiority, randomised, open-label, phase IV-III, two-armed parallel group (1:1) clinical trial at 20 Spanish tertiary hospitals. Adults (≥18 years) with isolation of MSSA from at least one blood culture ≤72 hours before inclusion with evidence of infection, will be randomly allocated to receive either cloxacillin 2 g/4-hour intravenous plus fosfomycin 3 g/6-hour intravenous or cloxacillin 2 g/4-hour intravenous alone for 7 days. After the first week, sequential treatment and total duration of antibiotic therapy will be determined according to clinical criteria by the attending physician.Primary endpoints: (1) Treatment success at day 7, a composite endpoint comprising all the following criteria: patient alive, stable or with improved quick-Sequential Organ Failure Assessment score, afebrile and with negative blood cultures for MSSA at day 7. (2) Treatment success at test of cure (TOC) visit: patient alive and no isolation of MSSA in blood culture or at another sterile site from day 8 until TOC (12 weeks after randomisation).We assume a rate of treatment success of 74% in the cloxacillin group. Accepting alpha risk of 0.05 and beta risk of 0.2 in a two-sided test, 183 subjects will be required in each of the control and experimental groups to obtain statistically significant difference of 12% (considered clinically significant). ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Ethics Committee of Bellvitge University Hospital (AC069/18) and from the Spanish Medicines and Healthcare Product Regulatory Agency (AEMPS, AC069/18), and is valid for all participating centres under existing Spanish legislation. The results will be presented at international meetings and will be made available to patients and funders. TRIAL REGISTRATION NUMBER: The protocol has been approved by AEMPS with the Trial Registration Number EudraCT 2018-001207-37. ClinicalTrials.gov Identifier: NCT03959345; Pre-results.

Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing a sulfoxide moiety as anaplastic lymphoma kinase (ALK) inhibition agents.

Anaplastic lymphoma kinase (ALK) targeted therapies have demonstrated remarkable efficacy in ALK-positive lung adenocarcinomas. Here we synthesized and evaluated sixteen new 2,4-diaminopyrimidines bearing a sulfoxide moiety as anaplastic lymphoma kinase (ALK) inhibitors. The optimal compound 9e exhibited excellent antiproliferative activity against non-small cell lung cancer NCI-H2228 cells, which is better than that of Brigatinib and similar to Ceritinib. Mechanism study revealed that the optimal compound 9e decreased the mitochondrial membrane potential and arrested NCI-H2228 cells in the G0/G1 phase, finally resulting in cellular apoptosis. It is interesting that 9e could effectively inhibit the migration of NCI-H2228 cells and may be a promising leading compound for chemotherapy of metastatic cancer.

Metabolomic changes in severe acute malnutrition suggest hepatic oxidative stress: a secondary analysis.

Severe acute malnutrition (SAM), due to poor energy and/or protein intake, is associated with poor growth, depressed immune function, and long-term impacts on metabolic function. As the liver is a major metabolic organ and malnutrition poses metabolic stress, we hypothesize that SAM will be associated with alterations in the hepatic metabolome reflective of oxidative stress, gluconeogenesis, and ketogenesis. Thus, the purpose of this secondary analysis was to understand how SAM alters hepatic metabolism using a piglet model. Weanling piglets were feed either a reference (REF) or protein-energy deficient diet (MAL) for 5 weeks. After dietary treatment MAL piglets were severely underweight (weight-for-age Z-score of -3.29, Welch's t test, P = .0007), moderately wasted (weight-for-length Z-score of-2.49, Welch's t test, P = .003), and tended toward higher hepatic triglyceride content (Welch's t test, P = .07). Hematologic and blood biochemical measurements were assessed at baseline and after dietary treatment. The hepatic metabolome was investigated using 1H-NMR spectroscopy. Hepatic concentrations of betaine, cysteine, and glutathione tended to be lower in MAL (Welch's t test with FDR correction, P < .1), while inosine, lactate, and methionine sulfoxide concentrations were higher in MAL (inosine: P = .0448, lactate: P = .0258, methionine sulfoxide: P = .0337). These changes suggest that SAM is associated with elevated hepatic oxidative stress, increased gluconeogenesis, and alterations in 1-carbon metabolism.

Feasibility study on recycled vegetable oil waste and recycled polyethylene for the modification of aged asphalt.

The application of reclaimed asphalt pavement has been widely encouraged due to its significant economic and environmental benefits. However, it is necessary to add rejuvenators to ensure its performance. Currently, bio-oil-based regenerants have attracted attention owing to their advantages of renewability and cost savings. The purpose of this paper is to study the use of recycled vegetable oil waste (R-oil) and recycled polyethylene particles for the regeneration and modification of aged asphalt. Physical, rheological, and chemical tests were used to figure out their influence on the pavement performance of aged asphalt. According to the physical test indices (penetration, softening point, and ductility), the performance of the rejuvenated asphalt was better than that of virgin asphalt. The workability and low-temperature performance of the rejuvenated asphalt were basically the same as those of virgin asphalt, and its fatigue and high-temperature performance were better. Infrared spectroscopy showed that R-oil diluted the high-polarity sulfoxide base of aged asphalt. Gel permeation chromatography showed that its molecular weight dispersion was better than that of aged asphalt. Therefore, R-oil and polyethylene can improve the pavement performance and chemical properties of aged asphalt.

A chemical covalent tactic for bio-thiol sensing and protein labeling agent design.

Discovering novel chemical reactions is important for bioanalysis. Herein, we report a tactic for bio-thiol sensing and protein labeling agent design by the installation of a sulfoxide group onto the skeleton of various fluorophores, and powerfully validate its abilities, which may shed light on the development of specific protein tags to give insight into their biological functions.

Exploring Spacer Arm Structures for Designs of Asymmetric Sulfoxide-Containing MS-Cleavable Cross-Linkers.

Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MS-cleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multistage mass spectrometry (MSn). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of disuccinimidyl sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics, and MS identification of cross-linked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS2-based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MSn analysis. This work has established a solid foundation for further development of sulfoxide-containing MS-cleavable cross-linkers with new functionalities.

Selective Electroenzymatic Oxyfunctionalization by Alkane Monooxygenase in a Biofuel Cell.

Aliphatic synthetic intermediates with high added value are generally produced from alkane sources (e.g., petroleum) by inert carbon-hydrogen (C-H) bond activation using classical chemical methods (i.e. high temperature, rare metals). As an alternative approach for these reactions, alkane monooxygenase from Pseudomonas putida (alkB) is able to catalyze the difficult terminal oxyfunctionalization of alkanes selectively and under mild conditions. Herein, we report an electrosynthetic system using an alkB biocathode which produces alcohols, epoxides, and sulfoxides through bioelectrochemical hydroxylation, epoxidation, sulfoxidation, and demethylation. The capacity of the alkB binding pocket to protect internal functional groups is also demonstrated. By coupling our alkB biocathode with a hydrogenase bioanode and using H2 as a clean fuel source, we have developed and characterized a series of enzymatic fuel cells capable of oxyfunctionalization while simultaneously producing electricity.

A fluorescent turn-on probe for detection of hypochlorus acid and its bioimaging in living cells.

Hypochlorous acid has played several functions in the biological system. However, excess HOCl can cause damage to biomolecules and result in some diseases. Accordingly, a new fluorescent probe, BSP, has been developed for fast recognition of HOCl through the HOCl-induced oxidation of methyl phenyl sulfide to sulfoxide. The reaction of BSP with HOCl caused a 22-fold fluorescence enhancement (quantum yield increase from 0.006 to 0.133). The detection limit of HOCl is found to be 30 nM (S/N = 3). The fluorescence enhancement is due to the suppression of the photo-induced electron transfer from the methyl phenyl sulfide moiety to BODIPY. Eventually, the cellular fluorescence imaging experiment showed that BSP could be effectively used for monitoring HOCl in living cells.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Enantioselective Sulfoxidation of Thioanisole by Cascading a Choline Oxidase and a Peroxygenase in the Presence of Natural Deep Eutectic Solvents.

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride-based deep eutectic solvents serving as co-solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15 mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.

A Click-Chemistry-Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry.

Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein-protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter- and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low-abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS2 and potentially for MS3 experiments. Thus, the new crosslinking reagent, in combination with high-end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases.

S­allyl­cysteine sulfoxide (alliin) alleviates myocardial infarction by modulating cardiomyocyte necroptosis and autophagy.

S­allyl­cysteine sulfoxide (alliin) is the main organosulfur component of garlic and its preparations. The present study aimed to examine the protective effect of alliin on cardiac function and the underlying mechanism in a mouse model of myocardial infarction (MI). Notably, alliin treatment preserved heart function, attenuated the area of infarction in the myocardium of mice and reduced lesions in the myocardium, including cardiomyocyte fibrosis and death. Further mechanistic experiments revealed that alliin inhibited necroptosis but promoted autophagy in vitro and in vivo. Cell viability assays showed that alliin dose­dependently reduced the necroptotic index and inhibited the expression of necroptosis­related receptor­interacting protein 1, receptor­interacting protein 3 and tumor necrosis factor receptor­associated factor 2, whereas the levels of Beclin 1 and microtubule­associated protein 1 light chain 3, which are associated with autophagy, exhibited an opposite trend upon treatment with alliin. In addition, the level of peroxisome proliferator­activated receptor γ was increased by alliin. Collectively, these findings demonstrate that alliin has the potential to protect cardiomyocytes from necroptosis following MI and that this protective effect occurs via the enhancement of autophagy.

New insights into the molecular physiology of sulfoxide reduction in bacteria.

Sulfoxides occur in biology as products of the S-oxygenation of small molecules as well as in peptides and proteins and their formation is often associated with oxidative stress and can affect biological function. In bacteria, sulfoxide damage can be reversed by different types of enzymes. Thioredoxin-dependent peptide methionine sulfoxide reductases (MSR proteins) repair oxidized methionine residues and are found in all Domains of life. In bacteria MSR proteins are often found in the cytoplasm but in some bacteria, including pathogenic Neisseria, Streptococci, and Haemophilus they are extracytoplasmic. Mutants lacking MSR proteins are often sensitive to oxidative stress and in pathogens exhibit decreased virulence as indicated by reduced survival in host cell or animal model systems. Molybdenum enzymes are also known to reduce S-oxides and traditionally their physiological role was considered to be in anaerobic respiration using dimethylsulfoxide (DMSO) as an electron acceptor. However, it now appears that some enzymes (MtsZ) of the DMSO reductase family of Mo enzymes use methionine sulfoxide as preferred physiological substrate and thus may be involved in scavenging/recycling of this amino acid. Similarly, an enzyme (MsrP/YedY) of the sulfite oxidase family of Mo enzymes has been shown to be involved in repair of methionine sulfoxides in periplasmic proteins. Again, some mutants deficient in Mo-dependent sulfoxide reductases exhibit reduced virulence, and there is evidence that these Mo enzymes and some MSR systems are induced by hypochlorite produced by the innate immune system. This review describes recent advances in the understanding of the molecular microbiology of MSR systems and the broadening of the role of Mo-dependent sulfoxide reductase to encompass functions beyond anaerobic respiration.