Detoxification of methylglyoxal by the glyoxalase system is required for glutathione availability and virulence activation in Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive, food-borne pathogen that lives a biphasic lifestyle, cycling between the environment and as a facultative intracellular pathogen of mammals. Upon entry into host cells, L. monocytogenes upregulates expression of glutathione synthase (GshF) and its product, glutathione (GSH), which is an allosteric activator of the master virulence regulator PrfA. Although gshF mutants are highly attenuated for virulence in mice and form very small plaques in host cell monolayers, these virulence defects can be fully rescued by mutations that lock PrfA in its active conformation, referred to as PrfA*. While PrfA activation can be recapitulated in vitro by the addition of reducing agents, the precise biological cue(s) experienced by L. monocytogenes that lead to PrfA activation are not known. Here we performed a genetic screen to identify additional small-plaque mutants that were rescued by PrfA* and identified gloA, which encodes glyoxalase A, a component of a GSH-dependent methylglyoxal (MG) detoxification system. MG is a toxic byproduct of metabolism produced by both the host and pathogen, which if accumulated, causes DNA damage and protein glycation. As a facultative intracellular pathogen, L. monocytogenes must protect itself from MG produced by its own metabolic processes and that of its host. We report that gloA mutants grow normally in broth, are sensitive to exogenous MG and severely attenuated upon IV infection in mice, but are fully rescued for virulence in a PrfA* background. We demonstrate that transcriptional activation of gshF increased upon MG challenge in vitro, and while this resulted in higher levels of GSH for wild-type L. monocytogenes, the glyoxalase mutants had decreased levels of GSH, presumably due to the accumulation of the GSH-MG hemithioacetal adduct. These data suggest that MG acts as a host cue that leads to GSH production and activation of PrfA.

Abiotic Reduction of Organic and Inorganic Compounds by Fe(II)-Associated Reductants: Comprehensive Data Sets and Machine Learning Modeling.

Iron-associated reductants play a crucial role in providing electrons for various reductive transformations. However, developing reliable predictive tools for estimating abiotic reduction rate constants (logk) in such systems has been impeded by the intricate nature of these systems. Our recent study developed a machine learning (ML) model based on 60 organic compounds toward one soluble Fe(II)-reductant. In this study, we built a comprehensive kinetic data set covering the reactivity of 117 organic and 10 inorganic compounds toward four major types of Fe(II)-associated reductants. Separate ML models were developed for organic and inorganic compounds, and the feature importance analysis demonstrated the significance of resonance structures, reducible functional groups, reductant descriptors, and pH in logk prediction. Mechanistic interpretation validated that the models accurately learned the impact of various factors such as aromatic substituents, complexation, bond dissociation energy, reduction potential, LUMO energy, and dominant reductant species. Finally, we found that 38% of the 850,000 compounds in the Distributed Structure-Searchable Toxicity (DSSTox) database contain at least one reducible functional group, and the logk of 285,184 compounds could be reasonably predicted using our model. Overall, the study is a significant step toward reliable predictive tools for anticipating abiotic reduction rate constants in iron-associated reductant systems.

Tunable and Practical Homogeneous Organic Reductants for Cross-Electrophile Coupling.

The syntheses of four new tunable homogeneous organic reductants based on a tetraaminoethylene scaffold are reported. The new reductants have enhanced air stability compared to current homogeneous reductants for metal-mediated reductive transformations, such as cross-electrophile coupling (XEC), and are solids at room temperature. In particular, the weakest reductant is indefinitely stable in air and has a reduction potential of -0.85 V versus ferrocene, which is significantly milder than conventional reductants used in XEC. All of the new reductants can facilitate C(sp2)-C(sp3) Ni-catalyzed XEC reactions and are compatible with complex substrates that are relevant to medicinal chemistry. The reductants span a range of nearly 0.5 V in reduction potential, which allows for control over the rate of electron transfer events in XEC. Specifically, we report a new strategy for controlled alkyl radical generation in Ni-catalyzed C(sp2)-C(sp3) XEC. The key to our approach is to tune the rate of alkyl radical generation from Katritzky salts, which liberate alkyl radicals upon single electron reduction, by varying the redox potentials of the reductant and Katritzky salt utilized in catalysis. Using our method, we perform XEC reactions between benzylic Katritzky salts and aryl halides. The method tolerates a variety of functional groups, some of which are particularly challenging for most XEC transformations. Overall, we expect that our new reductants will both replace conventional homogeneous reductants in current reductive transformations due to their stability and relatively facile synthesis and lead to the development of novel synthetic methods due to their tunability.

The Nonphysiological Reductant Sodium Dithionite and [FeFe] Hydrogenase: Influence on the Enzyme Mechanism.

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H2). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) covalently attached to a unique [2Fe] subcluster ([2Fe]H), where both sites are redox active. Heterolytic splitting and formation of H2 takes place at [2Fe]H, while [4Fe-4S]H stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state Hox, named HoxH, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. HoxH was previously suggested to have a protonated [4Fe-4S]H. Here, we show that HoxH forms by simple addition of sodium sulfite (Na2SO3, the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO2) is the species involved. Spectroscopy supports binding at or near [4Fe-4S]H, causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that HoxH and its one-electron reduced counterpart Hred'H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the "dithionite (DT) inhibited" states Hox-DTi and Hred-DTi. The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase.

The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it participates in several metabolic pathways in humans. All BADHs known have cysteine in the active site involved in the aldehyde binding, whereas the porcine kidney enzyme (pkBADH) also has a neighborhood cysteine, both sensitive to oxidation. The antineoplastic and immuno-suppressant pre-drug cyclophosphamide (CTX), and its bioactivation products, have two highly oxidating chlorine atoms. This work aimed to analyze the effect of CTX in the activity of porcine kidney betaine aldehyde dehydrogenase. PkBADH was incubated with varying CTX concentration (0 to 2.0 mM) at 25 °C and lost 50 % of its activity with 2.0 mM CTX. The presence of the coenzyme NAD+ (0.5 mM) decreased 95% the activity in 2.0 mM CTX. The substrate betaine aldehyde (0.05 and 0.4 mM, and the products NADH (0.1-0.5 mM) and GB (1 and 10 mM) did not have an effect on the enzyme inactivation by CTX. The reducing agents, dithiothreitol and ß-mercaptoethanol, reverted the pkBADH inactivation, but reduced glutathione (GSH) was unable to restore the enzyme activity. Molecular docking showed that CTX could enter at the enzyme active site, where its chlorine atoms may interact with the catalytic and the neighboring cysteines. The results obtained show that CTX inactivates the pkBADH due to oxidation of the catalytic cysteine or because it oxidizes catalytic and neighborhood cysteine, forming a disulfide bridge with a concomitant decrease in the activity of the enzyme.

The Effect of the Reducing Sugars in the Synthesis of Visible-Light-Active Copper(I) Oxide Photocatalyst.

In the present work, shape tailored Cu2O microparticles were synthesized by changing the nature of the reducing agent and studied subsequently. d-(+)-glucose, d-(+)-fructose, d-(+)xylose, d-(+)-galactose, and d-(+)-arabinose were chosen as reducing agents due to their different reducing abilities. The morpho-structural characteristics were studied by X-ray diffraction (XRD), scanning electron microscopy (SEM), and diffuse reflectance spectroscopy (DRS), while their photocatalytic activity was evaluated by methyl orange degradation under visible light (120 min). The results show that the number of carbon atoms in the sugars affect the morphology and particle size (from 250 nm to 1.2 µm), and differences in their degree of crystallinity and photocatalytic activity were also found. The highest activity was observed when glucose was used as the reducing agent.

Application of a New Dehydroascorbic Acid Reducing Agent in the Analysis of Vitamin C Content in Food.

The analysis of total vitamin C content in food is most frequently performed by reducing dehydroascorbic acid to ascorbic acid, which is then assayed with the technique of high-performance liquid chromatography combined with spectrophotometric detection. Tris(2-carboxyethyl)phosphine is currently the only agent in use that efficiently reduces dehydroascorbic acid at pH < 2. Therefore, there is a continued need to search for new reducing agents that will display a high reactivity and stability in acidic solutions. The objective of the study was to verify the applicability of unithiol and tris(hydroxypropyl)phosphine for a reducing dehydroascorbic acid in an extraction medium with pH < 2. The conducted validation of the newly developed method of determining the total content of vitamin C using tris(hydroxypropyl)phosphine indicates its applicability for food analysis. The method allows obtaining equivalent results compared to the method based on the use of tris(2-carboxyethyl)phosphine. The low efficiency of dehydroascorbic acid reduction with the use of unithiol does not allow its application as a new reducing agent in vitamin C analysis.

Spirocyclic Nitroxides as Versatile Tools in Modern Natural Sciences: From Synthesis to Applications. Part I. Old and New Synthetic Approaches to Spirocyclic Nitroxyl Radicals.

Spirocyclic nitroxyl radicals (SNRs) are stable paramagnetics bearing spiro-junction at a-, b-, or g-carbon atom of the nitroxide fragment, which is part of the heterocyclic system. Despite the fact that the first representatives of SNRs were obtained about 50 years ago, the methodology of their synthesis and their usage in chemistry and biochemical applications have begun to develop rapidly only in the last two decades. Due to the presence of spiro-function in the SNRs molecules, the latter have increased stability to various reducing agents (including biogenic ones), while the structures of the biradicals (SNBRs) comprises a rigid spiro-fused core that fixes mutual position and orientation of nitroxide moieties that favors their use in dynamic nuclear polarization (DNP) experiments. This first review on SNRs will give a glance at various strategies for the synthesis of spiro-substituted, mono-, and bis-nitroxides on the base of six-membered (piperidine, 1,2,3,4-tetrahydroquinoline, 9,9'(10H,10H')-spirobiacridine, piperazine, and morpholine) or five-membered (2,5-dihydro-1H-pyrrole, pyrrolidine, 2,5-dihydro-1H-imidazole, 4,5-dihydro-1H-imidazole, imidazolidine, and oxazolidine) heterocyclic cores.

Biochemical characterization of a partially purified protease from Aspergillus terreus 7461 and its application as an environmentally friendly dehairing agent for leather industry.

Proteases can be used in several biotechnological processes including detergent, food and leather industries. In the leather industry, dehairing is carried out by chemicals, which pollute the environment. Therefore, to make the hair removal process environmentally friendly, a protease produced by Aspergillus terreus has been purified, biochemically characterized and had an efficient ability to remove hair from bovine leather. The protease was produced using 1% wheat bran and was purified 2.3-fold using two chromatographic steps showing a molecular weight of 90 kDa. Optimal temperature and pH were 50 °C and 6.5, respectively. Thermal stability was up to 1 h at 50 °C. Protease was stable to detergents like Tween 80 and to organic solvents. The activity was activated by Ca2+ and inhibited by Hg2+ and Cu2+. The enzyme was classified as serine protease, by the inhibition by PMSF and was stable to reducing agents. It hydrolyzed casein, azocasein, BSA, egg albumin and BTpNA. The Km and Vmax values were 0.65 ± 0.03 mg/mL and 3.66 ± 0.18 µmol/min, respectively. Remarkable properties about temperature, pH, stability to detergents and reducing agents ensure that the protease from A. terreus can be an excellent candidate for industrial applications, particularly in the leather industry.

Green synthesis of metallic nanoparticles using pectin as a reducing agent: a systematic review of the biological activities.

CONTEXT: Pectin is a plant heteropolysaccharide that is biocompatible and biodegradable, enabling it to be an excellent reducing agent (green synthesis) for metallic nanoparticles (MNPs). Nevertheless, in the biological industry, pectin has been left behind in synthesising MNPs, for no known reason. OBJECTIVE: To systematically review the biological activities of pectin synthesised MNPs (Pe-MNPs). METHODS: The databases Springer Link, Scopus, ScienceDirect, Google Scholar, PubMed, Mendeley, and ResearchGate were systematically searched from the date of their inception until 10th February 2020. Pectin, green synthesis, metallic nanoparticles, reducing agent and biological activities were among the key terms searched. The data extraction was focussed on the biological activities of Pe-MNPs and reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations for systematic reviews. RESULTS: A total of 15 studies outlined 7 biological activities of Pe-MNPs in the only three metals that have been explored, namely silver (Ag), gold (Au) and cerium oxide (CeO2). The activities reported from the in vitro and in vivo studies were antimicrobial (9 studies), anticancer (2 studies), drug carrier (3 studies), non-toxic (4 studies), antioxidant (2 studies), wound healing (1 study) and anti-inflammation (1 study). CONCLUSIONS: This systematic review demonstrates the current state of the art of Pe-MNPs biological activities, suggesting that Ag and Au have potent antibacterial and anticancer/chemotherapeutic drug carrier activity, respectively. Further in vitro, in vivo, and clinical research is crucial for a better understanding of the pharmacological potential of pectin synthesised MNPs.

Electrochemical Activation of Diverse Conventional Photoredox Catalysts Induces Potent Photoreductant Activity*.

Herein, we disclose that electrochemical stimulation induces new photocatalytic activity from a range of structurally diverse conventional photocatalysts. These studies uncover a new electron-primed photoredox catalyst capable of promoting the reductive cleavage of strong C(sp2 )-N and C(sp2 )-O bonds. We illustrate several examples of the synthetic utility of these deeply reducing but otherwise safe and mild catalytic conditions. Finally, we employ electrochemical current measurements to perform a reaction progress kinetic analysis. This technique reveals that the improved activity of this new system is a consequence of an enhanced catalyst stability profile.

A Nonheme Mononuclear {FeNO}7 Complex that Produces N2 O in the Absence of an Exogenous Reductant.

A new nonheme iron(II) complex, FeII (Me3 TACN)((OSiPh2 )2 O) (1), is reported. Reaction of 1 with NO(g) gives a stable mononitrosyl complex Fe(NO)(Me3 TACN)((OSiPh2 )2 O) (2), which was characterized by Mössbauer (δ=0.52 mm s-1 , |ΔEQ |=0.80 mm s-1 ), EPR (S=3/2), resonance Raman (RR) and Fe K-edge X-ray absorption spectroscopies. The data show that 2 is an {FeNO}7 complex with an S=3/2 spin ground state. The RR spectrum (λexc =458 nm) of 2 combined with isotopic labeling (15 N, 18 O) reveals ν(N-O)=1680 cm-1 , which is highly activated, and is a nearly identical match to that seen for the reactive mononitrosyl intermediate in the nonheme iron enzyme FDPnor (ν(NO)=1681 cm-1 ). Complex 2 reacts rapidly with H2 O in THF to produce the N-N coupled product N2 O, providing the first example of a mononuclear nonheme iron complex that is capable of converting NO to N2 O in the absence of an exogenous reductant.

A Cysteine-Mediated Synthesis of Red Phosphorus Nanosheets.

Among phosphorus-based nanomaterials, layered black phosphorus and violet phosphorus have been actively explored in the past decade. However, methods for the synthesis of red phosphorus nanosheets (RPNSs) is lacking, even though red phosphorus (RP) is commercially available at low cost and has excellent chemical stability at room temperature. We report an efficient strategy for fabrication of RPNSs and doped RPNSs using cysteine as a reducing reagent. Data from in vitro and in vivo studies suggested that RPNSs can trigger production of reactive oxygen species, DNA damage, and subsequent autophagy-mediated cell death in a shape-dependent manner. Our findings provide a method for construction of layered RP nanomaterials and they present a unique mechanism for the application of phosphorus-based materials in nanomedicines.

Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD+-Dependent Enzymes.

Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, ß-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ1-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD+ and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231-491 M-1 s-1. The reaction is reversible, and nuclear magnetic resonance characterization (1H, 13C, and 31P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD+. We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP+-TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P)+. Because NAD(P)+-dependent oxidoreductases are widespread in nature, our results may be broadly relevant.

Discovery of Oxygen α-Nucleophilic Addition to α,ß-Unsaturated Amides Catalyzed by Redox-Neutral Organic Photoreductant.

The conjugate additions of oxygen-centered nucleophiles to conjugate acceptors are among the most powerful C-O bond formation reactions. The conjugate addition normally takes place at the ß-position carbon to the electron-withdrawing group, resulting in the formation of a stabilized carbanion intermediate that can be quenched by proton or electrophiles to form the ß-addition (i.e., hetero-Michael addition) products. On the contrary, the formation of α-hydroxyl or alkoxyl amides through conjugate addition needs an α,ß-inverse addition. Nevertheless, a regio-inversed nucleophilic α-addition of oxygen-centered nucleophiles to α,ß-unsaturated carbonyl compounds still remains less explored because of the electronic mismatch. In this research, we discovered the first α-specific nucleophilic addition of α,ß-unsaturated amides with oxygen and fluoride nucleophiles. This region-inversed nucleophilic addition is enabled by the catalysis of a novel redox-neutral nondonor-acceptor organic photoreductant (CBZ6). As low as 0.5 mol % of visible light photoreductant was employed. The mechanistic insights were also explored. The oxidative potential of the excited state of CBZ6 is obtained in -1.92 V (vs SCE), presenting a stronger reductive potential than representative metal-cored or organic photoredox catalysts. This feature enabled the umpolung of α,ß-unsaturated amides to take place α-nucleophilic addition other than the normal ß-addition.

Breaking a Couple: Disulfide Reducing Agents.

Cysteine is present in a large number of natural and synthetic (bio)molecules. Although the thiol side chain of Cys can be in a free form, in most cases it forms a disulfide bond either with a second Cys (bridge) or with another thiol, as in the case of protecting groups. Efficient reduction of these disulfide bridges is a requirement for many applications of Cys-containing molecules in the fields of chemistry and biochemistry. Here we review reducing methods for disulfide bonds, taking into consideration the solubility of the substrates when selecting the appropriate reducing reagent.

Reducing Agent-Mediated Nonenzymatic Conversion of 2-Oxoglutarate to Succinate: Implications for Oxygenase Assays.

l-Ascorbate (l-Asc) is often added to assays with isolated FeII - and 2-oxoglutarate (2OG)-dependent oxygenases to enhance activity. l-Asc is proposed to be important in catalysis by some 2OG oxygenases in vivo. We report observations on the nonenzymatic conversion of 2OG to succinate, which is mediated by hydrogen peroxide generated by the reaction of l-Asc and dioxygen. Slow nonenzymatic oxidation of 2OG to succinate occurs with some, but not all, other reducing agents commonly used in 2OG oxygenase assays. We intend these observations will help in the robust assignment of substrates and inhibitors for 2OG oxygenases.

Vitamin C measurement in critical illness: challenges, methodologies and quality improvements.

Background There is renewed interest in high-dose vitamin C interventions in clinical medicine due to its antioxidant properties, safe use and cost-effectiveness. Yet, randomised control trials (RCTs) employing these interventions are failing to include robust analytical methodology and proper sample handling and processing techniques. Consequently, comparisons between studies becomes impossible as there is no metrological traceability and results may be prone to pre-analytical errors. Content Through published vitamin C stability studies, method comparison papers and data from vitamin C external quality assurance programs, an assessment was made on the functionality of current methods for critically ill patient samples. Summary Data was obtained from two external quality assurance programs, two papers assessing sample stability and interlaboratory agreement and a publication on vitamin C method comparisons. A shift from spectrophotometric and enzymatic methodologies to high performance liquid chromatography (HPLC) greatly improved the variability and interlaboratory agreement. Therefore, the current analytical performance of vitamin C HPLC methodologies are acceptable for the requirements of a high-dose vitamin C RCTs. Outlook Recommendations across the total testing process of vitamin C have been provided to improve the quality of the results. The harmonisation of sample handling and processing procedures will further improve the reliability of current analytical methodologies.

Efficient switching of mCherry fluorescence using chemical caging.

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with ß-mercaptoethanol (ßME). The molecules can be recovered to the red fluorescent state by washing out the ßME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the ßME-induced fluorescence quenching of mCherry occurs both via the direct addition of ßME to the chromophore and through ßME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores.

Effect of Reducing Agent on Solution Synthesis of Li3V2(PO4)3 Cathode Material for Lithium Ion Batteries.

In this study, Li3V2(PO4)3 (LVP) powders are prepared by a solution synthesis method. The effects of two reducing agents on crystal structure and morphology and electrochemical properties are investigated. Preliminary studies on reducing agents such as oxalic acid and citric acid, are used to reduce the vanadium (V) precursor. The oxalic acid-assisted synthesis induces smaller particles (30 nm) compared with the citric acid-assisted synthesis (70 nm). The LVP powders obtained by the oxalic acid exhibit a higher specific capacity (124 mAh g-1 at 1C) and better cycling performance (122 mAh g-1 following 50 cycles at 1C rate) than those for the citric acid. This is due to their higher electronic conductivity caused by carbon coating and downsizing the particles. The charge-discharge plateaus obtained from cyclic voltammetry are in good agreement with galvanostatic cycling profiles.