Electron paramagnetic resonance as a tool to determine the sodium charge storage mechanism of hard carbon.

Hard carbon is a promising negative electrode material for rechargeable sodium-ion batteries due to the ready availability of their precursors and high reversible charge storage. The reaction mechanisms that drive the sodiation properties in hard carbons and subsequent electrochemical performance are strictly linked to the characteristic slope and plateau regions observed in the voltage profile of these materials. This work shows that electron paramagnetic resonance (EPR) spectroscopy is a powerful and fast diagnostic tool to predict the extent of the charge stored in the slope and plateau regions during galvanostatic tests in hard carbon materials. EPR lineshape simulation and temperature-dependent measurements help to separate the nature of the spins in mechanochemically modified hard carbon materials synthesised at different temperatures. This proves relationships between structure modification and electrochemical signatures in the galvanostatic curves to obtain information on their sodium storage mechanism. Furthermore, through ex situ EPR studies we study the evolution of these EPR signals at different states of charge to further elucidate the storage mechanisms in these carbons. Finally, we discuss the interrelationship between EPR spectroscopy data of the hard carbon samples studied and their corresponding charging storage mechanism.

Ceibinin, a new positional isomer of mangiferin from the inflorescence of Ceiba pentandra (Bombacaceae), elicits similar antioxidant effect but no anti-inflammatory potential compared to mangiferin.

Ceiba pentandra (L.) Gaertn. (Bombacaceae) is popular for the quality of its wood. However, its leaf, stem bark and root bark have been popular in ethnomedicine and, apart from the inflorescence, have been subject of extensive phytochemical investigations. In this study, two compounds were isolated from the crude methanol extract of the inflorescence. Through data from UV, NMR, MS, electrochemical studies, differential scanning calorimetry, and thermogravimetric analysis, the structures were elucidated as 3-C-ß-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (1) and 2-C-ß-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (mangiferin, 2). They were assessed for antioxidant efficacy (DCFDA assay) and for anti-inflammatory efficacy using the lipopolysaccharide (LPS)-induced inflammation model in the RAW 264.7 macrophages (nitrite levels quantified, using Griess Assay, as surrogate for nitric oxide (NO)). Compound 1 (named ceibinin) was established as a novel positional isomer of mangiferin (2). While both 1 and 2 were antioxidant against basal and hydrogen peroxide (100 µM)-induced oxidative stress (6.25 µg/ml abrogated peroxide-induced oxidative stress), ceibinin (1) demonstrated no anti-inflammatory potential, unlike mangiferin (2) which, as previously reported, showed anti-inflammatory effect. Our work reports a positional isomer of mangiferin for the first time in C. pentandra and demonstrates how such isomerism could underlie differences in biological activities and thus the potential for development into therapeutics.

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate.

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label.

Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron-electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to 'knock out' superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00723-021-01350-1.

EPR evidence for α-triphenylstannylvinyl radicals in the O-directed hydrostannation of dialkylacetylenes with Ph3SnH/cat. Et3B/O2.

Here we provide definitive EPR evidence for the existence of α-triphenylstannylvinyl radicals in the low temperature O-directed free radical hydrostannation of dialkyl propargylic alcohols with Ph3SnH/cat. Et3B and O2 in PhMe. Isotropic hyperfine splitting patterns and spectral simulations confirm the assignments made. In the case of the α-triphenylstannylvinyl radical (Z)-2, an isotopic 119/117Sn hyperfine coupling constant of 9.5 mT (95 G) was measured along with a 1Hß hyperfine coupling constant of 1.1 mT.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV =O or FeIV -OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV =O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

Surface Engineering Strategy Using Urea To Improve the Rate Performance of Na2 Ti3 O7 in Na-Ion Batteries.

Na2 Ti3 O7 (NTO) is considered a promising anode material for Na-ion batteries due to its layered structure with an open framework and low and safe average operating voltage of 0.3 V vs. Na+ /Na. However, its poor electronic conductivity needs to be addressed to make this material attractive for practical applications among other anode choices. Here, we report a safe, controllable and affordable method using urea that significantly improves the rate performance of NTO by producing surface defects such as oxygen vacancies and hydroxyl groups, and the secondary phase Na2 Ti6 O13 . The enhanced electrochemical performance agrees with the higher Na+ ion diffusion coefficient, higher charge carrier density and reduced bandgap observed in these samples, without the need of nanosizing and/or complex synthetic strategies. A comprehensive study using a combination of diffraction, microscopic, spectroscopic and electrochemical techniques supported by computational studies based on DFT calculations, was carried out to understand the effects of this treatment on the surface, chemistry and electronic and charge storage properties of NTO. This study underscores the benefits of using urea as a strategy for enhancing the charge storage properties of NTO and thus, unfolding the potential of this material in practical energy storage applications.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV=O or FeIV-OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

Conformational Flexibility of Hybrid [3]- and [4]-Rotaxanes.

The synthesis, structures, and properties of [4]- and [3]-rotaxane complexes are reported where [2]-rotaxanes, formed from heterometallic {Cr7Ni} rings, are bound to a fluoride-centered {CrNi2} triangle. The compounds have been characterized by single-crystal X-ray diffraction and have the formulas [CrNi2(F)(O2CtBu)6]{(BH)[Cr7NiF8(O2CtBu)16]}3 (3) and [CrNi2(F)(O2CtBu)6(THF)]{(BH)[Cr7NiF8(O2CtBu)16]}2 (4), where B = py-CH2CH2NHCH2C6H4SCH3. The [4]-rotaxane 3 is an isosceles triangle of three [2]-rotaxanes bound to the central triangle while the [3]-rotaxane 4 contains only two [2]-rotaxanes bound to the central triangle. Studies of the behavior of 3 and 4 in solution by small-angle X-ray scattering and atomistic molecular dynamic simulations show that the structure of 3 is similar to that found in the crystal but that 4 has a different conformation to the crystal. Continuous wave and pulsed electron paramagnetic resonance spectroscopy was used to study the structures present and demonstrate that in frozen solutions (at 5 K) 4 forms more extended molecules than 3 and with a wider range of conformations.

Heme binding to human CLOCK affects interactions with the E-box.

The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.

Close Encounters of the Weak Kind: Investigations of Electron-Electron Interactions between Dissimilar Spins in Hybrid Rotaxanes.

We report a family of hybrid [2]rotaxanes based on inorganic [Cr7NiF8(O2CtBu)16]- ("{Cr7Ni}") rings templated about organic threads that are terminated at one end with pyridyl groups. These rotaxanes can be coordinated to [Cu(hfac)2] (where Hhfac = 1,1,1,5,5,5-hexafluoroacetylacetone), to give 1:1 or 1:2 Cu:{Cr7Ni} adducts: {[Cu(hfac)2](py-CH2NH2CH2CH2Ph)[Cr7NiF8(O2CtBu)16]}, {[Cu(hfac)2][py-CH2NH2CH2CH3][Cr7NiF8(O2CtBu)16]}, {[Cu(hfac)2]([py-CH2CH2NH2CH2C6H4SCH3][Cr7NiF8(O2CtBu)16])2}, {[Cu(hfac)2]([py-C6H4-CH2NH2(CH2)4Ph][Cr7NiF8(O2CtBu)16])2}, and {[Cu(hfac)2]([3-py-CH2CH2NH2(CH2)3SCH3][Cr7NiF8(O2CtBu)16])2}, the structures of which have been determined by X-ray diffraction. The {Cr7Ni} rings and CuII ions both have electronic spin S = 1/2, but with very different g-values. Continuous-wave EPR spectroscopy reveals the exchange interactions between these dissimilar spins, and hence the communication between the different molecular components that comprise these supramolecular systems. The interactions are weak such that we observe AX or AX2 type spectra. The connectivity between the {Cr7Ni} ring and thread terminus is varied such that the magnitude of the exchange interaction J can be tuned. The coupling is shown to be dominated by through-bond rather than through-space mechanisms.

Mechanism of catalysis and inhibition of Mycobacterium tuberculosis SapM, implications for the development of novel antivirulence drugs.

Mycobacterium tuberculosis (Mtb) SapM is a secreted virulence factor critical for intracellular survival of the pathogen. The role of SapM in phagosome maturation arrest in host macrophages suggests its potential as a drug target to assist in the clearance of tuberculosis infection. However, the mechanism of action of SapM at the molecular level remains unknown. In this study, we provide new insights into the mechanism of catalysis, substrate specificity and inhibition of SapM, and we identify the critical residues for catalysis and substrate binding. Our findings demonstrate that SapM is an atypical monoester alkaline phosphatase, with a serine-based mechanism of catalysis probably metal-dependent. Particularly relevant to SapM function and pathogenesis, is its activity towards PI(4,5)P2 and PI3P, two phosphoinositides that function at the early stages of microbial phagocytosis and phagosome formation. This suggests that SapM may have a pleiotropic role with a wider importance on Mtb infection than initially thought. Finally, we have identified two inhibitors of SapM, L-ascorbic acid and 2-phospho-L-ascorbic, which define two different mechanisms by which the catalytic activity of this phosphatase could be regulated. Critically, we demonstrate that 2-phospho-L-ascorbic reduces mycobacterial survival in macrophage infections, hence confirming the potential of SapM as a therapeutic drug target.

Photochemical Spin Dynamics of the Vitamin B12 Derivative, Methylcobalamin.

Derivatives of vitamin B12 are six-coordinate cobalt corrinoids found in humans, other animals, and microorganisms. By acting as enzymatic cofactors and photoreceptor chromophores, they serve vital metabolic and photoprotective functions. Depending on the context, the chemical mechanisms of the biologically active derivatives of B12-methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl)-can be very different from one another. The extent to which this chemistry is tuned by the upper axial ligand, however, is not yet clear. Here, we have used a combination of time-resolved Fourier transform-electron paramagnetic resonance (FT-EPR), magnetic field effect experiments, and spin dynamic simulations to reveal that the upper axial ligand alone only results in relatively minor changes to the photochemical spin dynamics of B12. By studying the photolysis of MeCbl, we find that, similar to AdoCbl, the initial (or "geminate") radical pairs (RPs) are born predominantly in the singlet spin state and thus originate from singlet excited-state precursors. This is in contrast to the triplet RPs and precursors proposed previously. Unlike AdoCbl, the extent of geminate recombination is limited following MeCbl photolysis, resulting in significant distortions to the FT-EPR signal caused by polarization from spin-correlated methyl-methyl radical "f-pairs" formed following rapid diffusion. Despite the photophysical mechanism that precedes photolysis of MeCbl showing wavelength dependence, the subsequent spin dynamics appear to be largely independent of excitation wavelength, again similar to AdoCbl. Our data finally provide clarity to what in the literature to date has been a confused and contradictory picture. We conclude that, although the upper axial position of MeCbl and AdoCbl does impact their reactivity to some extent, the remarkable biochemical diversity of these fascinating molecules is most likely a result of tuning by their protein environment.

In situ electrochemical electron paramagnetic resonance spectroscopy as a tool to probe electrical double layer capacitance.

Electron paramagnetic resonance (EPR) spectroscopy is applied in situ to monitor the electrochemical capacitance of activated carbon in aqueous solutions, thereby revealing aspects of the charge storage mechanism. The EPR signal of activated carbon increases during the charging process and returns reversibly when discharged. Simulation of the spectral lineshape and its temperature dependence indicate that two kinds of spins exist: spin at defects giving rise to a narrow signal, and spins associated with surface-bound aromatic moieties causing a broad signal. A potential-dependent response of the narrow feature is seen in each of the electrolyte solutions used, while changes of the broad feature occur only at higher electrolyte concentrations. The results suggest that the observed increase of unpaired electron density on activated carbon is due to the formation of radical species due to reduction of functional groups. The potential dependence of the broad feature at higher electrolyte concentrations may be related to the further adsorption of ions into the deep porous structure of activated carbon.

A Bifunctional Spin Label for Ligand Recognition on Surfaces.

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Electron paramagnetic resonance (EPR) of biomolecules spin-labeled with nitroxides can offer uniquely sensitive and selective insights into these processes, but new spin-labeling strategies are needed. The synthesis and study of a bromoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported. The first examples of mannose and biotin ligands coupled to aqueous carboxy-functionalized gold nanoparticles through a spin label are presented. EPR spectra were obtained for the spin-labeled ligands both free in solution and attached to nanoparticles. The labels were recognized by the mannose-binding lectin, Con A, and the biotin-binding protein avidin-peroxidase. Binding gave quantifiable changes in the EPR spectra from which binding profiles could be obtained that reflect the strength of binding in each case.

Measuring Spin⋠⋠⋠Spin Interactions between Heterospins in a Hybrid [2]Rotaxane.

Use of molecular electron spins as qubits for quantum computing will depend on the ability to produce molecules with weak but measurable interactions between the qubits. Here we demonstrate use of pulsed EPR spectroscopy to measure the interaction between two inequivalent spins in a hybrid rotaxane molecule.

Modulation of Antimalarial Activity at a Putative Bisquinoline Receptor In Vivo Using Fluorinated Bisquinolines.

Antimalarials can interact with heme covalently, by π⋅⋅⋅π interactions or by hydrogen bonding. Consequently, the prototropy of 4-aminoquinolines and quinoline methanols was investigated by using quantum mechanics. Calculations showed mefloquine protonated preferentially at the piperidine and was impeded at the endocyclic nitrogen because of electronic rather than steric factors. In gas-phase calculations, 7-substituted mono- and bis-4-aminoquinolines were preferentially protonated at the endocyclic quinoline nitrogen. By contrast, compounds with a trifluoromethyl substituent on both the 2- and 8-positions, reversed the order of protonation, which now favored the exocyclic secondary amine nitrogen at the 4-position. Loss of antimalarial efficacy by CF3 groups simultaneously occupying the 2- and 8-positions was recovered if the CF3 group occupied the 7-position. Hence, trifluoromethyl groups buttressing the quinolinyl nitrogen shifted binding of antimalarials to hematin, enabling switching from endocyclic to the exocyclic N. Both theoretical calculations (DFT calculations: B3LYP/BS1) and crystal structure of (±)-trans-N1 ,N2 -bis-(2,8-ditrifluoromethylquinolin-4-yl)cyclohexane-1,2-diamine were used to reveal the preferred mode(s) of interaction with hematin. The order of antimalarial activity in vivo followed the capacity for a redox change of the iron(III) state, which has important implications for the future rational design of 4-aminoquinoline antimalarials.

Direct Evidence of an Excited-State Triplet Species upon Photoactivation of the Chlorophyll Precursor Protochlorophyllide.

The chlorophyll precursor protochlorophyllide (Pchlide), which is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase, has unique excited-state properties that facilitate photocatalysis. Previous time-resolved spectroscopy measurements have implied that a long-lived triplet state is formed during the excited-state relaxation of Pchlide, although direct evidence of its existence is still lacking. Here we use time-resolved electron paramagnetic resonance (EPR) in combination with time-resolved absorption measurements at a range of temperatures (10-290 K), solvents, and oxygen concentrations to provide a detailed characterization of the triplet state of Pchlide. The triplet decays in a biphasic, oxygen-dependent manner, while the first reported EPR signature of a Pchlide triplet displays both emissive and absorptive features and an antisymmetric spectrum similar to other porphyrin triplet states. This work demonstrates that the Pchlide triplet is accessible to various cryogenic spectroscopic probes over a range of time scales and paves the way for understanding its potential role in catalysis.

The Organic Secondary Building Unit: Strong Intermolecular π Interactions Define Topology in MIT-25, a Mesoporous MOF with Proton-Replete Channels.

The structure-directing role of the inorganic secondary building unit (SBU) is key for determining the topology of metal-organic frameworks (MOFs). Here we show that organic building units relying on strong π interactions that are energetically competitive with the formation of common inorganic SBUs can also play a role in defining the topology. We demonstrate the importance of the organic SBU in the formation of Mg2H6(H3O)(TTFTB)3 (MIT-25), a mesoporous MOF with the new ssp topology. A delocalized electronic hole is critical in the stabilization of the TTF triad organic SBUs and exemplifies a design principle for future MOF synthesis.

Detection of Ligand-induced Conformational Changes in the Activation Loop of Aurora-A Kinase by PELDOR Spectroscopy.

The structure of protein kinases has been extensively studied by protein crystallography. Conformational movement of the kinase activation loop is thought to be crucial for regulation of activity; however, in many cases the position of the activation loop in solution is unknown. Protein kinases are an important class of therapeutic target and kinase inhibitors are classified by their effect on the activation loop. Here, we report the use of pulsed electron double resonance (PELDOR) and site-directed spin labeling to monitor conformational changes through the insertion of MTSL [S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1 H-pyrrol-3-yl)methyl methanesulfonothioate] on the dynamic activation loop and a stable site on the outer surface of the enzyme. The action of different ligands such as microtubule-associated protein (TPX2) and inhibitors could be discriminated as well as their ability to lock the activation loop in a fixed conformation. This study provides evidence for structural adaptations that could be used for drug design and a methodological approach that has potential to characterize inhibitors in development.