Small molecule MarR modulators potentiate metronidazole antibiotic activity in aerobic E. coli by inducing activation by the nitroreductase NfsA.

Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.

Integrated Experimental and Theoretical Investigation of Copper Active Site Properties of a Lytic Polysaccharide Monooxygenase from Serratia marcescens.

In this paper, we employed a multidisciplinary approach, combining experimental techniques and density functional theory (DFT) calculations to elucidate key features of the copper coordination environment of the bacterial lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens (SmAA10). The structure of the holo-enzyme was successfully obtained by X-ray crystallography. We then determined the copper(II) binding affinity using competing ligands and observed that the affinity of the histidine brace ligands for copper is significantly higher than previously described. UV-vis, advanced electron paramagnetic resonance (EPR), and X-ray absorption spectroscopy (XAS) techniques, including high-energy resolution fluorescence detected (HERFD) XAS, were further used to gain insight into the copper environment in both the Cu(II) and Cu(I) redox states. The experimental data were successfully rationalized by DFT models, offering valuable information on the electronic structure and coordination geometry of the copper center. Finally, the Cu(II)/Cu(I) redox potential was determined using two different methods at ca. 350 mV vs NHE and rationalized by DFT calculations. This integrated approach not only advances our knowledge of the active site properties of SmAA10 but also establishes a robust framework for future studies of similar enzymatic systems.

Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions.

A copper-dependent self-cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46-nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub-structures that flank a highly conserved catalytic core. The DNA self-cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site-specific cleavage of the substrate are two key-aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the "metal-soup" scenario, also known as "super-stoichiometric" ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids-based enzymes. The focus on the endogenous paramagnetic center (Cu2+) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

Syntheses and Electrochemical and EPR Studies of Porphyrins Functionalized with Bulky Aromatic Amine Donors.

A series of nickel(II) porphyrins bearing one or two bulky nitrogen donors at the meso positions were prepared by using Ullmann methodology or more classical Buchwald-Hartwig amination reactions to create the new C-N bonds. For several new compounds, single crystals were obtained, and the X-ray structures were solved. The electrochemical data of these compounds are reported. For a few representative examples, spectroelectrochemical measurements were used to clarify the electron exchange process. In addition, a detailed electron paramagnetic resonance (EPR) study was performed to estimate the extent of delocalization of the generated radical cations. In particular, electron nuclear double resonance spectroscopy (ENDOR) was used to determine the coupling constants. DFT calculations were conducted to corroborate the EPR spectroscopic data.

Multi-Electron Visible Light Photoaccumulation on a Dipyridylamine Copper(II)-Polyoxometalate Conjugate Applied to Photocatalytic Generation of CF3 Radicals.

This article describes the synthesis and characterization of an organic-inorganic hybrid polyoxometalate functionalized by a short link with a tripodal N-based ligand and its copper complex. Upon visible light irradiation, the latter is able to store up to three reducing equivalents. The locus of the reduction is discussed based on physicochemical measurements and DFT calculations. In the presence of Togni's reagent, this complex allows for the photocatalytic generation of CF3 radicals, opening the road to valuable synthetic applications.

Structural Features Governing the Metabolic Stability of Tetraethyl-Substituted Nitroxides in Rat Liver Microsomes.

Nitroxides are potent tools for studying biological systems by electron paramagnetic resonance (EPR). Whatever the application, a certain stability is necessary for successful detection. Since conventional tetramethyl-substituted cyclic nitroxides have insufficient in vivo stability, efforts have recently been made to synthesize more stable, tetraethyl-substituted nitroxides. In our previous study on piperidine nitroxides, the introduction of steric hindrance around the nitroxide moiety successfully increased the resistance to reduction into hydroxylamine. However, it also rendered the carbon backbone susceptible to modifications by xenobiotic metabolism due to increased lipophilicity. Here, we focus on a new series of three nitroxide candidates with tetraethyl substitution, namely with pyrrolidine, pyrroline, and isoindoline cores, to identify which structural features afford increased stability for future probe design and application in in vivo EPR imaging. In the presence of rat liver microsomes, pyrrolidine and pyrroline tetraethyl nitroxides exhibited a higher stability than isoindoline nitroxide, which was studied in detail by HPLC-HRMS. Multiple metabolites suggest that the aerobic transformation of tetraethyl isoindoline nitroxide is initiated by hydrogen abstraction by P450-FeV = O from one of the ethyl groups, followed by rearrangement and further modifications by cytochrome P450, as supported by DFT calculations. Under anaerobic conditions, only reduction by rat liver microsomes was observed with involvement of P450-FeII.

Computational Insights of Selective Intramolecular O-atom Transfer Mediated by Bioinspired Copper Complexes.

The stereoselective copper-mediated hydroxylation of intramolecular C-H bonds from tridentate ligands is reinvestigated using DFT calculations. The computational study aims at deciphering the mechanism of C-H hydroxylation obtained after reaction of Cu(I) precursors with dioxygen, using ligands bearing either activated (L1 ) or non-activated (L2 ) C-H bonds. Configurational analysis allows rationalization of the experimentally observed regio- and stereoselectivity. The computed mechanism involves the formation of a side-on peroxide species (P) in equilibrium with the key intermediate bis-(µ-oxo) isomer (O) responsible for the C-H activation step. The P/O equilibrium yields the same activation barrier for the two complexes. However, the main difference between the two model complexes is observed during the C-H activation step, where the complex bearing the non-activated C-H bonds yields a higher energy barrier, accounting for the experimental lack of reactivity of this complex under those conditions.

Quantum Bio-Inorganic Chemistry (QBIC) Society Special Collection.

This Editorial by Guest Editors L. Rulísek, M. Gruden, M. Orio and QBIC Society President R. J. Deeth summarizes the Chemistry Europe Special Collection on quantum bioinorganic chemistry, published in collaboration with the Quantum Bioinorganic Chemistry (QBIC) Society. The Collection, which recognizes of the strides made in this area of research, spans several of our portfolio journals Chemistry-A European Journal, the European Journal of Inorganic Chemistry, ChemPlusChem, ChemBioChem, ChemPhysChem, and ChemistryOpen, includes outstanding work in the field by authors across the world and we hope you enjoy reading them!

Decoding the Ambiguous Electron Paramagnetic Resonance Signals in the Lytic Polysaccharide Monooxygenase from Photorhabdus luminescens.

Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable atomistic interpretation of electron paramagnetic resonance (EPR) data on the Cu(II) active site. Among various LPMO families, the chitin-active PlAA10 shows an intriguing phenomenology with distinct EPR signals, a major rhombic and a minor axial signal. Here, we combine experimental and computational investigations to uncover the structural identity of these signals. X-band EPR spectra recorded at different pH values demonstrate pH-dependent population inversion: the major rhombic signal at pH 6.5 becomes minor at pH 8.5, where the axial signal dominates. This suggests that a protonation change is involved in the interconversion. Precise structural interpretations are pursued with quantum chemical calculations. Given that accurate calculations of Cu g-tensors remain challenging for quantum chemistry, we first address this problem via a thorough calibration study. This enables us to define a density functional that achieves accurate and reliable prediction of g-tensors, giving confidence in our evaluation of PlAA10 LPMO models. Large models were considered that include all parts of the protein matrix surrounding the Cu site, along with the characteristic second-sphere features of PlAA10. The results uniquely identify the rhombic signal with a five-coordinate Cu ion bearing two water molecules in addition to three N-donor ligands. The axial signal is attributed to a four-coordinate Cu ion where only one of the waters remains bound, as hydroxy. Alternatives that involve decoordination of the histidine brace amino group are unlikely based on energetics and spectroscopy. These results provide a reliable spectroscopy-consistent view on the plasticity of the resting state in PlAA10 LPMO as a foundation for further elucidating structure-property relationships and the formation of catalytically competent species. Our strategy is generally applicable to the study of EPR parameters of mononuclear copper-containing metalloenzymes.

From Ligand- to Metal-centered Reactivity: Metal Substitution Effect in Thiosemicarbazone-based Complexes for H2 Production.

The quest to develop and optimize catalysts for H2 production requires a thorough understanding in the possible catalytic mechanisms involved. Transition metals are very often the centers of reactivity in the catalysis, although this can change in the presence of a redox-active ligand. Investigating the differences in catalysis when considering ligand- and metal-centered reactivity is important to find the most optimal mechanisms for hydrogen evolution reaction. Here, we investigated this change of reactivity in two versions of a thiosemicarbazone-based complex, using Co and Ni metal centers. While the Ni version has a ligand-centered reactivity, Co switches it toward a metal-centered one. Comparison between the mechanisms show differences in rate-limiting steps, and shows the importance of identifying those steps in order to optimize the system for hydrogen production.

Self-assembled nickel cubanes as oxygen evolution catalysts.

Ni4O4 cubanes [(µ3-L1O)NiCl(MeOH)]4 (1) and [(µ3-L2O)NiCl(H2O)]4 (2) (L1OH = 1-H-2-benzimidazolylmethanol, L2OH = 1-methyl-2-benzimidazolylmethanol) self-assemble from commercially available 1-H- and 1-methyl-2-benzimidazolylmethanol and NiCl2·6H2O in high yields under mild conditions. Both complexes were characterised spectroscopically and by X-ray crystallography. The cubanes oxidise water electrocatalytically to dioxygen at neutral pH in aqueous potassium phosphate buffer solutions.

Successes, challenges, and opportunities for quantum chemistry in understanding metalloenzymes for solar fuels research.

Quantum chemical approaches today are a powerful tool to study the properties and reactivity of metalloenzymes. In the field of solar fuels research these involve predominantly photosystem II and hydrogenases, which catalyze water oxidation and hydrogen evolution, as well as related biomimetic and bio-inspired models. Theoretical methods are extensively used to better comprehend the nature of catalytic intermediates, establish important structure-function and structure-property correlations, elucidate functional principles, and uncover the catalytic activity of these complex systems by unravelling the key steps of their reaction mechanism. Computations in the field of water oxidation and hydrogen evolution are used as predictive tools to elucidate structures, explain and synthesize complex experimental observations from advanced spectroscopic techniques, rationalize reactivity on the basis of atomistic models and electronic structure, and guide the design of new synthetic targets. This feature article covers recent advances in the application of quantum chemical methods for understanding the nature of catalytic intermediates and the mechanism by which photosystem II and hydrogenases achieve their function, and points at essential questions that remain only partly answered and at challenges that will have to be met by future advances and applications of quantum and computational chemistry.

Unraveling the catalytic mechanisms of H2 production with thiosemicarbazone nickel complexes.

Thiosemicarbazone-based complexes have been explored as a new class of redox-active catalysts H2 production due to their flexibility for extensive optimization. To rationalize the process, we need to understand how these complexes function. In this work, we used DFT calculations to investigate the various mechanisms that could take place for three previously characterized Ni complexes. We found that two possible mechanisms are compatible with previously published experimental data, involving protonation of two adjacent N atoms close to the metal center. The first step likely involves a proton-coupled electron transfer process from a proton source to one of the distal N atoms in the ligand. From here, a second proton can be transferred either to the coordinating N atom situated in between the first protonated atom and the Ni atom, or to the second distal N atom. The former case then has the protons in close distance for H2 production. However, the latter will require a third protonation event to occur, which would fall in one of the N atoms adjacent to the Ni center, resulting in a similar mechanism. Finally, we show that the H-H bond formation is the rate-limiting step, and suggest additional strategies that can be taken into account to further optimize these complexes.

Comparison of Density Functional and Correlated Wave Function Methods for the Prediction of Cu(II) Hyperfine Coupling Constants.

The reliable prediction of Cu(II) hyperfine coupling constants remains a challenge for quantum chemistry. Until recently only density functional theory (DFT) could target this property for systems of realistic size. However, wave function based methods become increasingly applicable. In the present work, we define a large set of Cu(II) complexes with experimentally known hyperfine coupling constants and use it to investigate the performance of modern quantum chemical methods for the prediction of this challenging spectroscopic parameter. DFT methods are evaluated against orbital-optimized second-order Møller-Plesset (OO-MP2) theory and coupled cluster calculations including singles and doubles excitations, driven by the domain-based local pair natural orbital approach (DLPNO-CCSD). Special attention is paid to the definition of a basis set that converges adequately toward the basis set limit for the given property for all methods considered in this study, and a specifically optimized basis set is proposed for this purpose. The results suggest that wave function based methods can supplant but do not outcompete DFT for the calculation of Cu(II) hyperfine coupling constants. Mainstream hybrid functionals such as B3PW91 remain on average the best choice.

Hydrogen evolution reaction mediated by an all-sulfur trinuclear nickel complex.

We report the synthesis and the characterization of a trinuclear nickel complex. Solid state and solution studies using X-ray diffraction, NMR and UV-vis spectroscopy highlight the square planar geometries around the metal centers and an all-sulfur coordination sphere. It exhibits significant electrocatalytic activity for hydrogen evolution in DMF using Et3NHCl as the proton source. DFT studies suggest that sulfur atoms act as proton relay, as proposed in [NiFe] hydrogenases.

Unexpected rapid aerobic transformation of 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical by cytochrome P450 in the presence of NADPH: Evidence against a simple reduction of the nitroxide moiety to the hydroxylamine.

Aminoxyl radicals (nitroxides) are a class of compounds with important biomedical applications, serving as antioxidants, spin labels for proteins, spin probes of oximetry, pH, or redox status in electron paramagnetic resonance (EPR), or as contrast agents in magnetic resonance imaging (MRI). However, the fast reduction of the radical moiety in common tetramethyl-substituted cyclic nitroxides within cells, yielding diamagnetic hydroxylamines, limits their use in spectroscopic and imaging studies. In vivo half-lives of commonly used tetramethyl-substituted nitroxides span no more than a few minutes. Therefore, synthetic efforts have focused on enhancing the nitroxide stability towards reduction by varying the electronic and steric environment of the radical. Tetraethyl-substitution at alpha position to the aminoxyl function proved efficient in vitro against reduction by ascorbate or cytosolic extracts. Moreover, 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical (TEEPONE) was used successfully for tridimensional EPR and MRI in vivo imaging of mouse head, with a reported half-life of over 80 min. We decided to investigate the stability of tetraethyl-substituted piperidine nitroxides in the presence of hepatic microsomal fractions, since no detailed study of their "metabolic stability" at the molecular level had been reported despite examples of the use of these nitroxides in vivo. In this context, the rapid aerobic transformation of TEEPONE observed in the presence of rat liver microsomal fractions and NADPH was unexpected. Combining EPR, HPLC-HRMS, and DFT studies on a series of piperidine nitroxides - TEEPONE, 4-oxo-2,2,6,6-tetramethyl(piperidin-1-yloxyl) (TEMPONE), and 2,2,6,6-tetraethyl-4-hydroxy(piperidin-1-yloxyl) (TEEPOL), we propose that the rapid loss in paramagnetic character of TEEPONE is not due to reduction to hydroxylamine but is a consequence of carbon backbone modification initiated by hydrogen radical abstraction in alpha position to the carbonyl by the P450-Fe(V)=O species. Besides, hydrogen radical abstraction by P450 on ethyl substituents, leading to dehydrogenation or hydroxylation products, leaves the aminoxyl function intact but could alter the linewidth of the EPR signal and thus interfere with methods relying on measurement of this parameter (EPR oximetry).

Ligand-based electronic effects on the electrocatalytic hydrogen production by thiosemicarbazone nickel complexes.

This work reports on the synthesis and characterization of a series of mononuclear thiosemicarbazone nickel complexes that display significant catalytic activity for hydrogen production in DMF using trifluoroacetic acid as the proton source. The ligand framework was chemically modified by varying the electron-donating abilities of the para substituents on the phenyl rings, which was expected to impact the capability of the resulting complexes to reduce protons into hydrogen. Over the four nickel complexes that were obtained, the one with the thiomethyl substituent, NiSCH3, was found to overtake the catalytic performances of the parent complex NiOCH3 featuring lower overpotential values and similar maximum turnover frequencies. These results confirm the electronic effects of the ligand on HER when using thiosemicarbazone nickel complexes and support that chemical modifications can tune the catalytic performances of such systems.

Optimizing Group Transfer Catalysis by Copper Complex with Redox-Active Ligand in an Entatic State.

Metalloenzymes use earth-abundant non-noble metals to perform high-fidelity transformations in the biological world. To ensure chemical efficiency, metalloenzymes have acquired evolutionary reactivity-enhancing tools. Among these, the entatic state model states that a strongly distorted geometry induced by ligands around a metal center gives rise to an energized structure called entatic state, strongly improving the reactivity. However, the original definition refers both to the transfer of electrons or chemical groups, whereas the chemical application of this concept in synthetic systems has mostly focused on electron transfer, therefore eluding chemical transformations. Here we report that a highly strained redox-active ligand enables a copper complex to perform catalytic nitrogen- and carbon-group transfer in as fast as 2 min, thus exhibiting a strong increase in reactivity compared with its unstrained analogue. This report combines two reactivity-enhancing features from metalloenzymes, entasis and redox cofactors, applied to group-transfer catalysis.

Tuning Inner-Sphere Electron Transfer in a Series of Copper/Nitrosoarene Adducts.

A series of copper/nitrosoarene complexes was created that mimics several steps in biomimetic O2 activation by copper(I). The reaction of the copper(I) complex of N,N,N',N'-tetramethypropylenediamine with a series of para-substituted nitrosobenzene derivatives leads to adducts in which the nitrosoarene (ArNO) is reduced by zero, one, or two electrons, akin to the isovalent species dioxygen, superoxide, and peroxide, respectively. The geometric and electronic structures of these adducts were characterized by means of X-ray diffraction, vibrational analysis, ultraviolet-visible spectroscopy, NMR, electrochemistry, and density functional theory (DFT) calculations. The bonding mode of the NO moiety depends on the oxidation state of the ArNO moiety: κN for ArNO, mononuclear η2-NO and dinuclear µ-η2:η1 for ArNO•-, and dinuclear µ-η2:η2 for ArNO2-. 15N isotopic labeling confirms the reduction state by measuring the NO stretching frequency (1392 cm-1 for κN-ArNO, 1226 cm-1 for η2-ArNO•-, 1133 cm-1 for dinuclear µ-η2:η1-ArNO•-, and 875 cm-1 for dinuclear µ-η2:η2 for ArNO2-). The 15N NMR signal disappears for the ArNO•- species, establishing a unique diagnostic for the radical state. Electrochemical studies indicate reduction waves that are consistent with one-electron reduction of the adducts and are compared with studies performed on Cu-O2 analogues. DFT calculations were undertaken to confirm our experimental findings, notably to establish the nature of the charge-transfer transitions responsible for the intense green color of the complexes. In fine, this family of complexes is unique in that it walks through three redox states of the ArNO moiety while keeping the metal and its supporting ligand the same. This work provides snapshots of the reactivity of the toxic nitrosoarene molecules with the biologically relevant Cu(I) ion.

When Light and Acid Play Tic-Tac-Toe with a Nine-State Molecular Switch.

Combining different molecular switching functions in a single molecule is a simple strategy to develop commutable molecules featuring more than two commutation states. The present study reports on two molecular systems consisting of two indolino-oxazolidine (Box) moieties connected to an aromatic bridge (phenyl or bithiophene) by ethylenic junctions. Such systems, referenced as BiBox, are expected to show up multiaddressable and multiresponsive behaviors. On one hand, the oxazolidine ring opening/closure of Box moieties can be addressed by chemical stimuli, and on the other hand, the trans-to-cis isomerization of the ethylenic junctions is induced by visible light irradiation (with a thermal back conversion). NMR and UV-visible spectroscopies allowed to characterize up to nine out of the ten theoretically expected commutation states as well as to measure the kinetics of the interconversions. Also, steady state fluorescence spectroscopy measurements highlighted the strong influence of the open/closed states of the Box moieties on their emission properties.