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This work was supported by the the University of Tours (to IDP and FDG), the University of Montpellier (to SD and EC), the Deutsche Forschungsgemeinschaft (to RTS), the Wellcome Trust project grant 093228 (to TKS) and the Campus France/DAAD PHC PROCOPE 24931RE (to RTS and EC). The funding source has no involvement in the conduct of the research and preparation of the article. Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs. Publisher PDF

Funding: This study was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant BB/M010996/1) via EASTBIO Doctoral Training Partnership studentships to G.F. and B.J.R., and by the University of St Andrews via a StARIS summer research bursary to S.K.L. The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N1-(5-phospho-β-D-ribosyl)-ATP (PRATP) to N1-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5′-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV–VIS spectroscopy and LC–MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall kcat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on kcat and kcat/KM ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N1-(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel. Publisher PDF

Funding: AG acknowledges the UK-MRBT-CDT funded by EPSRC (EP/J500045/1), BEB acknowledges the Wellcome Trust Institutional Strategic Support fund (204821/Z/16/Z), JET acknowledges the Leverhulme Trust (Early Career Fellowship; ECF-2014-005). Nitroxides are a unique class of persistent radicals finding a wide range of applications, from spin probes to polarizing agents, and recently bis-nitroxides have been used as proof-of-concept molecules for quantum information processing. Here we present the syntheses of pyrroline-based nitroxide (NO) radicals and give a comparision of two possible synthetic routes to form two key intermediates, namely 2,2,5,5-tetramethylpyrroline-1-oxyl-3-acetylene (TPA) and 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylic acid (TPC). TPC and TPA were then used as precursors for the synthesis of three model compounds featuring two distant NO groups with a variable degree of conjugation and thus electronic communication between them. Using relatively facile synthetic routes, we produced a number of mono- and bis-nitroxides with the structures of multiple compounds unambiguously characterized by X-ray crystallography, while Continuous Wave Electron Paramagnetic Resonance (CW-EPR) allowed us quantify the electronic communication in the bis-nitroxides. Our study expands the repertoire of mono- and bis-nitroxides with possibilities of exploiting them for studying quantum coherence effects and as polarizing agents. Publisher PDF

Funding: CMC and CJH are funded by the Wellcome Trust (210486/Z/18/Z), additional funding was provided by the University of St Andrews Impact Innovation Fund. MB (Bergkessel) is funded by the University of Dundee/Wellcome Trust Institutional Strategic Support Fund [204816/Z/16/Z] and UKRI Future Leaders Fellowship (funded by the Medical Research Council) [MR/T041811/1]. Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context. Publisher PDF

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Funding: The authors gratefully acknowledge the China Scholarship Council for funding a PhD scholarship for D. W. This work was also funded by the Leverhulme Trust (grant no. RPG-2017-214). I. G. S., W. M. and M. G. were funded by NIH grant R01-GM132595 and Harrison Family Funds. M. P. C. acknowledges the support of a Robert R. Wagner Fellowship at the University of Virginia. R. F. was supported by the Biological and Biotechnological Sciences Research Council (grant no. BB/V014684/1). Serum albumin–Co2+ interactions are of clinical importance. They play a role in mediating the physiological effects associated with cobalt toxicity and are central to the albumin cobalt binding (ACB) assay for diagnosis of myocardial ischemia. To further understand these processes, a deeper understanding of albumin–Co2+ interactions is required. Here, we present the first crystallographic structures of human serum albumin (HSA; three structures) and equine serum albumin (ESA; one structure) in complex with Co2+. Amongst a total of sixteen sites bearing a cobalt ion across the structures, two locations were prominent, and they relate to metal-binding sites A and B. Site-directed mutagenesis and isothermal titration calorimetry (ITC) were employed to characterise sites on HSA. The results indicate that His9 and His67 contribute to the primary (putatively corresponding to site B) and secondary Co2+-binding sites (site A), respectively. The presence of additional multiple weak-affinity Co2+ binding sites on HSA was also supported by ITC studies. Furthermore, addition of 5 molar equivalents of the non-esterified fatty acid palmitate (C16:0) reduced the Co2+-binding affinity at both sites A and B. The presence of bound myristate (C14:0) in the HSA crystal structures provided insight into the fatty acid-mediated structural changes that diminish the affinity of the protein toward Co2+. Together, these data provide further support for the idea that ischemia-modified albumin corresponds to albumin with excessive fatty-acid loading. Collectively, our findings provide a comprehensive understanding of the molecular underpinnings governing Co2+ binding to serum albumin. Publisher PDF

Funding: For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Accepted Author Manuscript version arising. The authors thank the StAnD (St Andrews and Dundee) EPR group for long-standing support and in particular Dr El Mkami for assistance with PDS experiments. J. L.W. acknowledges support by the BBSRC DTP Eastbio (BB/M010996/1). A. G. acknowledges the EPSRC-funded Centre for Doctoral Training in ‘integrated magnetic resonance’, iMR-CDT (EP/J500045/1) B. E. B. and K. A. acknowledge support by the Leverhulme Trust (RPG-2018-397). B. E. B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). Pulse dipolar spectroscopy (PDS) measurements are an important complementary tool in structural biology and are increasingly applied to macromolecular assemblies implicated in human health and disease at physiological concentrations. This requires ever higher sensitivity, and recent advances have driven PDS measurements into the mid-nanomolar concentration regime, though optimization and acquisition of such measurements remains experimentally demanding and time expensive. One important consideration is that constant-time acquisition represents a hard limit for measurement sensitivity, depending on the maximum measured distance. Determining this distance a priori has been facilitated by machine-learning structure prediction (AlphaFold2 and RoseTTAFold) but is often confounded by non-representative behaviour in frozen solution that may mandate multiple rounds of optimization and acquisition. Herein, we endeavour to simultaneously enhance sensitivity and streamline PDS measurement optimization to one-step by benchmarking a variable-time acquisition RIDME experiment applied to CuII-nitroxide and CuII-CuII model systems. Results demonstrate marked sensitivity improvements of both 5- and 6-pulse variable-time RIDME of between 2- and 5-fold over the constant-time analogues. Publisher PDF

Authors thank the Chinese Scholarship Council for funding a Studentship (No. 202008060063) at the University of St. Andrews, U.K. C-2 fluorinated and methylated stereoisomers of the fragrance citronellol 1 and its oxalate esters were prepared from (R)-pulegone 11 and explored as agonists of the human olfactory receptor OR1A1 and assayed also against site-specific mutants. There were clear isomer preferences and C-2 difluorination as in 18 led to the most active compound suggesting an important hydrogen bond donor role for citronellol 1. C-2 methylation and the corresponding oxalate ester analogues were less active. Publisher PDF

Funding: The authors thank the EPSRC Centre for Doctoral Training in Critical Resource Catalysis (CRITICAT) for financial support [Ph.D. studentship to S.M.; Grant code: EP/L016419/1]. Aqueous transformations confer many advantages, including decreased environmental impact and increased opportunity for biomolecule modulation. Although several studies have been conducted to enable the cross-coupling of aryl halides in aqueous conditions, until now a process for the cross-coupling of primary alkyl halides in aqueous conditions was missing from the catalytic toolbox and considered impossible. Alkyl halide coupling in water suffers from severe problems. The reasons for this include the strong propensity for β-hydride elimination, the need for highly air- and water-sensitive catalysts and reagents, and the intolerance of many hydrophilic groups to cross-coupling conditions. Here, we report a broadly applicable and readily accessible process for the cross-coupling of water-soluble alkyl halides in water and air by using simple and commercially available bench-stable reagents. The trisulfonated aryl phosphine TXPTS in combination with a water-soluble palladium salt Na2PdCl4 allowed for the Suzuki−Miyaura coupling of water-soluble alkyl halides with aryl boronic acids, boronic esters, and borofluorate salts in mild, fully aqueous conditions. Multiple challenging functionalities, including unprotected amino acids, an unnatural halogenated amino acid within a peptide, and herbicides can be diversified in water. Structurally complex natural products were used as testbeds to showcase the late-stage tagging methodology of marine natural products to enable liquid chromatography−mass spectrometry (LC−MS) detection. This enabling methodology therefore provides a general method for the environmentally friendly and biocompatible derivatization of sp3 alkyl halide bonds. Publisher PDF

Funding: Financial support for this work was provided by the University of St Andrews and EaStCHEM, and the Leverhulme Trust [Grant RPG-2019-155]. Transient states maintained by energy dissipation are an essential feature of dynamic systems where structures and functions are regulated by fluxes of energy and matter through chemical reaction networks. Perfected in biology, chemically fueled dissipative networks incorporating nanoscale components allow the unique properties of nanomaterials to be bestowed with spatiotemporal adaptability and chemical responsiveness. We report the transient dispersion of gold nanoparticles in water, powered by dissipation of a chemical fuel. A dispersed state that is generated under nonequilibrium conditions permits fully reversible solid–liquid or liquid–liquid phase transfer. The molecular basis of the out-of-equilibrium process is reversible covalent modification of nanoparticle-bound ligands by a simple inorganic activator. Activator consumption by a coupled dissipative reaction network leads to autonomous cycling between phases. The out-of-equilibrium lifetime is tunable by adjusting pH, and reversible phase cycling is reproducible over several cycles. Publisher PDF

Funding: The authors acknowledge the Universiti Teknologi MARA for funding under the UMP-IIUM-UiTM Sustainable Research Collaboration Grant [600–RMC/SRC/5/3(043/2020)]. A new isoxazole-based iodo-noium salt, C13 H13 INO5+ ·C2 F3 O2- , has been synthesized and structurally characterized. In the crystal, ions are linked by short I⋯O contacts to form a neutral tetra-ion aggregate. These combine with C-H⋯F and C-H⋯O inter-actions to form double-layered two-dimensional sheets in the (001) plane. Publisher PDF

Funding: For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) license to any Accepted Author Manuscript version arising. They acknowledge support by the Wellcome Trust (204821/Z/16/Z), the British Heart Foundation (PG/15/9/31270 and FS/15/42/31556), and the Leverhulme Trust (RPG-2018–397). J.L.W. acknowledges support by the BBSRC DTP Eastbio. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). Mammalian histidine-rich glycoprotein (HRG) is a highly versatile and abundant blood plasma glycoprotein with a diverse range of ligands that is involved in regulating many essential biological processes, including coagulation, cell adhesion, and angiogenesis. Despite its biomedical importance, structural information on the multi-domain protein is sparse, not least due to intrinsically disordered regions that elude high-resolution structural characterization. Binding of divalent metal ions, particularly ZnII, to multiple sites within the HRG protein is of critical functional importance and exerts a regulatory role. However, characterization of the ZnII binding sites of HRG is a challenge; their number and composition as well as their affinities and stoichiometries of binding are currently not fully understood. In this study, we explored modern electron paramagnetic resonance (EPR) spectroscopy methods supported by protein secondary and tertiary structure prediction to assemble a holistic picture of native HRG and its interaction with metal ions. To the best of our knowledge, this is the first time that this suite of EPR techniques has been applied to count and characterize endogenous metal ion binding sites in a native mammalian protein of unknown structure. Publisher PDF

Funding: This work was supported by the European Research Council (Consolidator Grant 101002326 to J.K.). Ribosomally synthesized and post-translationally modified peptide natural products have provided many highly unusual scaffolds. This includes the intriguing alkaloids crocagins, which possess a tetracyclic core structure and whose biosynthesis has remained enigmatic. Here we use in vitro experiments to demonstrate that three proteins, CgnB, CgnC and CgnE, are sufficient for the production of the hallmark tetracyclic crocagin core from the precursor peptide CgnA. The crystal structures of the homologues CgnB and CgnE reveal them to be the founding members of a peptide-binding protein family and allow us to rationalize their distinct functions. We further show that the hydrolase CgnD liberates the crocagin core scaffold, which is subsequently N-methylated by CgnL. These insights allow us to propose a biosynthetic scheme for crocagins. Bioinformatic analyses based on these data led to the discovery of related biosynthetic pathways that may provide access to a structurally diverse family of peptide-derived pyrroloindoline alkaloids. Publisher PDF

We thank the EPSRC for a studentship (TJP) through the CRITICAT Doctoral Training Centre. FAPESP is also gratefully acknowledged for a studentship (BAP, #2021/09716-5) and a Young Researcher Award (RAC, #2018/03910-1). Concise and general synthesis protocols are reported to generate all-syn mono-, di- and tri-alkylated cyclohexanes where a single fluorine is located on the remaining carbons of the ring. The alkyl groups are positioned to lie equatorially and to have triaxial C–F bonds imparting polarity to these ring systems. Intermolecular electrostatic interactions in the solid-state structure of the trialkylated systems are explored and the resultant supramolecular order opens up prospects for design in soft materials. Publisher PDF

This work was financially supported by the Leverhulme Trust (RPG-2017-214) and BBSRC (BB/J006467/1 and BB/V014684/1). We thank Prof. Andrew Riches (University of St. Andrews) for provision of materials, and Dr. Elizabeth Bolitho (University of Warwick) for assistance with cell culture experiments. The role of the extracellular medium in influencing metal uptake into cells has not been described quantitatively. In a chemically defined model system containing albumin, zinc influx into endothelial cells correlates with the extracellular free zinc concentration. Allosteric inhibition of zinc-binding to albumin by free fatty acids increased zinc flux. Publisher PDF

Funding: Authors are grateful to the Chinese Scholarship Council for studentship (Z. F. and C. Y.) support and also the Scientific Research Project of Hubei Education Department (Q20211402) and the open project of Hubei Provincial Key Laboratory of Green Materials for Light Industry (202107B06) for financial support. Stimulated by a suggestion of the late Professor Jack D. Dunitz, that perdeuterated Janus face cycloalkanes may be more polar than their unlabelled forms, the deuterated isotopologue of all cis-1,2,3,4,5,6-hexafluorocyclohexane ([2H6]- 1a ) and all cis-1,2,3,4-tetrafluorocyclopentane ([2H6]- 3a ) were prepared. Computation at the B3LYP−D3 level indicated that [2H6]- 1a is not more polar than its protio form 1, however perdeuterated cyclopentane [2H6]- 3a may indeed be more polar than 3 , although the magnitude is predicted to be small. None-the-less retention time analysis on a reverse phase GC/MS column of an add-mix of 3 and [2H6]- 3a gave some indication that the per-deuterated isotopologue 3a was detected marginally before the unlabelled compound consistent with increased polarity associated with perdeuteration. Publisher PDF

Funding: Authors thank the EPSRC, Leverhulme Trust and the EU Horizon 2020 Sinfonia consortia for financial support. The Chinese Scholarship Council is acknowledged for studentship support (Y.C.). The gene cluster in Streptomyces calvus associated with the biosynthesis of the fluoro- and sulfamyl-metabolite nucleocidin was interrogated by systematic gene knockouts. Out of the 26 gene deletions most did not affect fluorometabolite production, nine abolished sulfamylation but not fluorination, and three precluded fluorination, having no effect on sulfamylation. In addition to nucI, nucG, nucJ, nucK, nucL, nucN, nucO, nucQ and nucP we identified two genes (nucW, nucA), belonging to a phosphoadenosine phosphosulfate (PAPS) gene cluster, as required for sulfamyl assembly. Three genes (orf(-3), orf2 and orf3) were found to be essential for fluorination, although the activities of their protein products are unknown. These genes as well as nucK, nucN, nucO and nucPNP, whose knockouts produced results differing from those described in a recent report, were also deleted in Streptomyces virens - with confirmatory outcomes. This genetic profile should inform biochemistry aimed at uncovering the enzymology behind nucleocidin biosynthesis. Publisher PDF

S.P. thanks the Novo Nordisk Foundation for research grant NNF20OC0065610. EPR Equipment funding by BBSRC to B. E. B. (17 Alert grant BB/R013780/1) is also gratefully acknowledged. We demonstrate by use of continuous wave- and pulse-electron paramagnetic resonance spectroscopy on oriented single crystals of magnetically dilute YbIII ions in Yb0.01Lu0.99(trensal) that molecular entangled two-qubit systems can be constructed by exploiting dipolar interactions between neighboring YbIII centers. Furthermore, we show that the phase memory time and Rabi frequencies of these dipolar-interaction-coupled entangled two-qubit systems are comparable to the ones of the corresponding single qubits. Publisher PDF

Macroscopic electric motors continue to have a large impact on almost every aspect of modern society. Consequently, the effort towards developing molecular motors1–3 that can be driven by electricity could not be more timely. Here we describe an electric molecular motor based on a [3]catenane4,5, in which two cyclobis(paraquat-p-phenylene)6 (CBPQT4+) rings are powered by electricity in solution to circumrotate unidirectionally around a 50-membered loop. The constitution of the loop ensures that both rings undergo highly (85%) unidirectional movement under the guidance of a flashing energy ratchet7,8, whereas the interactions between the two rings give rise to a two-dimensional potential energy surface (PES) similar to that shown by FOF1 ATP synthase9. The unidirectionality is powered by an oscillating10 voltage11,12 or external modulation of the redox potential13. Initially, we focused our attention on the homologous [2]catenane, only to find that the kinetic asymmetry was insufficient to support unidirectional movement of the sole ring. Accordingly, we incorporated a second CBPQT4+ ring to provide further symmetry breaking by interactions between the two mobile rings. This demonstration of electrically driven continual circumrotatory motion of two rings around a loop in a [3]catenane is free from the production of waste products and represents an important step towards surface-bound14 electric molecular motors.

Funding: The China Scholarship Scheme supported Xupeng Yu [201904910787]; some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs [P40 OD010440]. Funding for open access charge: Scottish Higher Education Digital Library Consortium (SHEDL) Read and Publish agreement. Eukaryotic chromosomes typically end in 3′ telomeric overhangs. The safeguarding of telomeric single-stranded DNA overhangs is carried out by factors related to the protection of telomeres 1 (POT1) protein in humans. Of the three POT1-like proteins in Caenorhabditis elegans, POT-3 was the only member thought to not play a role at telomeres. Here, we provide evidence that POT-3 is a bona fide telomere-binding protein. Using a new loss-of-function mutant, we show that the absence of POT-3 causes telomere lengthening and increased levels of telomeric C-circles. We find that POT-3 directly binds the telomeric G-strand in vitro and map its minimal DNA binding site to the six-nucleotide motif, GCTTAG. We further show that the closely related POT-2 protein binds the same motif, but that POT-3 shows higher sequence selectivity. Crucially, in contrast to POT-2, POT-3 prefers binding sites immediately adjacent to the 3′ end of DNA. These differences are significant as genetic analyses reveal that pot-2 and pot-3 do not function redundantly with each other in vivo. Our work highlights the rapid evolution and specialisation of telomere binding proteins and places POT-3 in a unique position to influence activities that control telomere length. Publisher PDF

Funding: Authors thank the Chinese Scholarship Council and FAPESP for studentships (BAP, #2021/09716-5) a Young Researcher Award (RAC, #2018/03910-1) and FAEPEX for a fellowship (RAC, #2479/21). Theory and solution NMR indicate that all-syn 1,3,5-trifluorocyclohexane 5 adopts the expected tri-equatorial conformation, however in the solid state the more polar triaxial conformation is observed. This and the favoured conformations of substituted (Me, OMe, NH(CO)Me, NHBoc) derivatives of 5 are investigated to explore triaxial C–F preferences. Publisher PDF

J.R. and G.A.B. thank GlaxoSmithKline (GSK) and the Engineering and Physical Sciences Research Council (EPSRC) for an industrial CASE studentship. MMA is supported by a studentship from the government of Libya, and MAU by a studentship from the Petroleum Technology Development Fund (PTDF), Abuja, Nigeria. A selective mono-N-arylation strategy of amidines under Chan-Lam conditions is described. During the reaction optimization phase, the isolation of a mononuclear Cu(II) complex provided unique mechanistic insight into the operation of Chan-Lam mono-N-arylation. The scope of the process is demonstrated, and then applied to access the first mono-N-arylated analogues of pentamidine. Sub-micromolar activity against kinetoplastid parasites was observed for several analogues with no cross-resistance in pentamidine and diminazene-resistant trypanosome strains and against Leishmania mexicana. A fluorescent mono-N-arylated pentamidine analogue revealed rapid cellular uptake, accumulating in parasite nuclei and the kinetoplasts. The DNA binding capability of the mono-N-arylated pentamidine series was confirmed by UV-melt measurements using AT-rich DNA. This work highlights the potential to use Chan-Lam mono-N-arylation to develop therapeutic leads against diamidine-resistant trypanosomiasis and leishmaniasis. Publisher PDF

The flexibility of the zeolite Rho framework offers great potential for tunable molecular sieving. The fully copper‐exchanged form of Rho and mixed Cu,H‐ and Cu,Na‐forms have been prepared. EPR spectroscopy reveals that Cu2+ ions are present in the dehydrated forms and Rietveld refinement shows these prefer S6R sites, away from the d8r windows that control diffusion. Fully exchanged Cu‐Rho remains in an open form upon dehydration, the d8r windows remain nearly circular and the occupancy of window sites is low, so that it adsorbs CO2 rapidly at room temperature. Breakthrough tests with 10 % CO2/40 % CH4 mixtures show that Cu4.9‐Rho is able to produce pure methane, albeit with a relatively low capacity at this pCO2 due to the weak interaction of CO2 with Cu cations. This is in strong contrast to Na‐Rho, where cations in narrow elliptical window sites enable CO2 to be adsorbed with high selectivity and uptake but too slowly to enable the production of pure methane in similar breakthrough experiments. A series of Cu,Na‐Rho materials was prepared to improve uptake and selectivity compared to Cu‐Rho, and kinetics compared to Na‐Rho. Remarkably, Cu,Na‐Rho with >2 Cu cations per unit cell exhibited exsolution, due to the preference of Na cations for narrow S8R sites in distorted Rho and of Cu cations for S6R sites in the centric, open form of Rho. The exsolved Cu,Na‐Rho showed improved performance in CO2/CH4 breakthrough tests, producing pure CH4 with improved uptake and CO2/CH4 selectivity compared to that of Cu4.9‐Rho., Copper cations favour 6‐membered ring (6R) sites in dehydrated zeolite Rho, away from 8R windows that allow adsorption into lta cages. Exchange of Cu cations into Na‐Rho, in which Na cations occupy 8R gating sites between the lta cages, improves CO2 uptake kinetics, while retaining some Na cations enhances CO2/CH4 selectivity. Upon dehydration, Cu,Na‐Rho shows exsolution behaviour, because its framework can adopt distorted and undistorted forms that provide favourable coordination sites for Na and Cu cations, respectively.

Funding: We would like to thank the Engineering and Physical Sciences Research Council, University of St. Andrews, and the EPSRC Centre for Doctoral Training in Critical Resource Catalysis (CRITICAT) for financial support [Ph.D. studentship to MC; Grant code: EP/L016419/1]. Crithidia fasiculata belongs to the trypanosomatidae order of protozoan parasites, bearing close relation to other kinetoplastid parasites such as Trypanosoma brucei and Leishmania spp. As an early diverging lineage of eukaryotes, the study of kinetoplastid parasites has provided unique insights into alternative mechanisms to traditional eukaryotic metabolic pathways. Crithidia are a monogenetic parasite for mosquito species and have two distinct lifecycle stages both taking place in the mosquito gut. These consist of a motile choanomastigote form and an immotile amastigote form morphologically similar to amastigotes in Leishmania. Owing to their close relation to Leishmania, Crithidia are a growing research tool, with continuing interest in its use as a model organism for kinetoplastid research with the added benefit that they are non-pathogenic to humans and can be grown with no special equipment or requirements for biological containment. Although comparatively little research has taken place on Crithidia, similarities to other kinetoplast species has been shown in terms of energy metabolism and genetics. Crithidia also show similarities to kinetoplastids in their production of the monosaccharide D-arabinopyranose similar to Leishmania, which is incorporated into a lipoarabinogalactan a major cell surface GPI-anchored molecule. Additionally, Crithidia have been used as a eukaryotic expression system to express proteins from other kinetoplastids and potentially other eukaryotes including human proteins allowing various co- and post-translational protein modifications to the recombinant proteins. Despite the obvious usefulness and potential of this organism very little is known about its lipid metabolism. Here we describe a detailed lipidomic analyses and demonstrate the possible placidity of Crithidia’s lipid metabolis. This could have important implications for biotechnology approaches and how other kinetoplastids interact with, and scavenge nutrients from their hosts. Publisher PDF

Funding: M.M.H. and A.-L.W. thank the Tromsø Research Foundation and UiT Centre for New Antibacterial Strategies (CANS) for a start-up grant (TFS project ID: 18_CANS). K.H.H. and S.G. thank the Research Council of Norway (No. 300769) and Sigma2 (No. nn9330k and nn4654k), and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 859910. J.E.L. thanks Drs Hassane El Mkami and Robert I. Hunter for technical assistance, the BBSRC (BB/T017740/1) and the Wellcome Trust (099149/Z/12/Z) for the Q-band EPR spectrometer, the Royal Society for a University Research Fellowship and Research Grant RG120645 for the benchtop spectrometer. B.A.L. thanks The Research Council of Norway for the Centre of Excellence and project grants (Grant Nos. 262695 and 274858). Nitroxides are widely used as probes and polarization transfer agents in spectroscopy and imaging. These applications require high stability towards reducing biological environments, as well as beneficial relaxation properties. While the latter is provided by spirocyclic groups on the nitroxide scaffold, such systems are not in themselves robust under reducing conditions. In this work, we introduce a strategy for stability enhancement through conformational tuning, where incorporating additional substituents on the nitroxide ring effects a shift towards highly stable closed spirocyclic conformations, as indicated by X-ray crystallography and density functional theory (DFT) calculations. Closed spirocyclohexyl nitroxides exhibit dramatically improved stability towards reduction by ascorbate, while maintaining long relaxation times in electron paramagnetic resonance (EPR) spectroscopy. These findings have important implications for the future design of new nitroxide-based spin labels and imaging agents. Publisher PDF

Funding: CJH and CMC are funded by the Wellcome Trust (210486/Z/18/Z) and ES is funded by the Cunningham Trust (PhD-CT-18-41). Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing. Publisher PDF

Funding: Carnegie Trust for the Universities of Scotland (Grant Number(s): 70668). Chinese Scholarship Council. DNA polymerase δ (Pol δ) is a key enzyme for the maintenance of genome integrity in eukaryotic cells, acting in concert with the sliding clamp processivity factor PCNA (proliferating cell nuclear antigen). Three of the four subunits of human Pol δ interact directly with the PCNA homotrimer via a short, conserved protein sequence known as a PCNA interacting protein (PIP) motif. Here, we describe the identification of a PIP motif located towards the N-terminus of the PolD4 subunit of Pol δ (equivalent to human p12) from the thermophilic filamentous fungus Chaetomium thermophilum and present the X-ray crystal structure of the corresponding peptide bound to PCNA at 2.45 Å. Like human p12, the fungal PolD4 PIP motif displays non-canonical binding to PCNA. However, the structures of the human p12 and fungal PolD4 PIP motif peptides are quite distinct, with the fungal PolD4 PIP motif lacking the 310 helical segment that characterises most previously identified PIP motifs. Instead, the fungal PolD4 PIP motif binds PCNA via a conserved glutamine that inserts into the Q-pocket on the surface of PCNA and with conserved leucine and phenylalanine sidechains forming a compact 2-fork plug that inserts into the hydrophobic pocket on PCNA. Despite the unusual binding mode of the fungal PolD4, isothermal calorimetry (ITC) measurements show that its affinity for PCNA is similar to that of its human orthologue. These observations add to a growing body of information on how diverse proteins interact with PCNA and highlight how binding modes can vary significantly between orthologous PCNA partner proteins. Publisher PDF

A.M.S. received Early Stage Research Funding from the European Union’s Seventh Framework Programme FP-7-PEOPLE-2013-ITN through the “MAGnetic Innovation in Catalysis” (MAGIC) Initial Training Network (grant agreement no. 606831). Part of this work was also supported by BBSRC grant: BB/M007065/1. J.L. thanks the Royal Society for a University Research Fellowship, the Carnegie Trust (RIG007510), and the Wellcome Trust for a Multi-User Equipment grant (099149/Z/12/Z). Calmodulin (CaM) is a highly dynamic Ca2+-binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+-replete form has been highly debated. In this study, we use PELDOR (pulsed electron–electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+-bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+-replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+-replete CaM similar to the one observed in early X-ray crystal structures. Publisher PDF

This research was funded, in whole or in part, by the Wellcome Trust (099149/Z/12/Z and 204821/Z/16/Z). A CC BY or equivalent license is applied to the author-accepted manuscript arising from this submission, in accordance with the grant’s open-access conditions. B.E.B. and K.A. acknowledge support by the Leverhulme Trust (RPG-2018-397). J.L.W. was supported by the BBSRC DTP Eastbio. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1). The study of ever more complex biomolecular assemblies implicated in human health and disease is facilitated by a suite of complementary biophysical methods. Pulse Dipolar electron paramagnetic resonance Spectroscopy (PDS) is a powerful tool that provides highly precise geometric constraints in frozen solution, however the drive towards PDS at physiologically relevant sub-μM concentrations is limited by the currently achievable concentration sensitivity. Recently, PDS using a combination of nitroxide and CuII based spin labels allowed measuring 500 nM concentration of a model protein. Using commercial instrumentation and spin labels we demonstrate CuII-CuII and nitroxide-nitroxide PDS measurements at protein concentrations below previous examples reaching 500 and 100 nM, respectively. These results demonstrate the general feasibility of sub-μM PDS measurements at short to intermediate distances (~1.5 - 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting. Postprint

D.S.P. and D.J.F. thank the EPSRC for funding via Prosperity Partnership EP/S035990/1. D.S.P. and D.J.F. thank the ARCHIE-WeSt High-Performance Computing Centre (www.archie-west.ac.uk) for computational resources. The UCL authors thank Prof. Keith Refson for guidance with the phonon calculations, which used the ARCHER U.K. National Supercomputing Service (http://www.archer.ac.uk) as part of the U.K. HEC Materials Chemistry Consortium, which is funded by the EPSRC (EP/L000202, EP/R029431). R.G. was funded by MagnaPharm, a project funded by the European Union’s Horizon 2020 Research and Innovation programme under grant agreement number 736899. We demonstrate that physics-based calculations of intrinsic aqueous solubility can rival cheminformatics-based machine learning predictions. A proof-of-concept was developed for a physics-based approach via a sublimation thermodynamic cycle, building upon previous work that relied upon several thermodynamic approximations, notably the 2RT approximation, and limited conformational sampling. Here, we apply improvements to our sublimation free-energy model with the use of crystal phonon mode calculations to capture the contributions of the vibrational modes of the crystal. Including these improvements with lattice energies computed using the model-potential-based Ψmol method leads to accurate estimates of sublimation free energy. Combining these with hydration free energies obtained from either molecular dynamics free-energy perturbation simulations or density functional theory calculations, solubilities comparable to both experiment and informatics predictions are obtained. The application to coronene, succinic acid, and the pharmaceutical desloratadine shows how the methods must be adapted for the adoption of different conformations in different phases. The approach has the flexibility to extend to applications that cannot be covered by informatics methods. Publisher PDF

Funding: JLW is supported by the BBSRC DTP Eastbio. We thank the Leverhulme Trust for support (RPG-2018-397). This work was supported by equipment funding through the Wellcome Trust (099149/Z/12/Z) and BBSRC (BB/R013780/1). We gratefully acknowledge ISSF support to the University of St Andrews from the Wellcome Trust. Pulse-dipolar EPR is an appealing strategy for structural characterization of complex systems in solution that complements other biophysical techniques. Significantly, the emergence of genetically encoded self-assembling spin labels exploiting exogenously introduced double-histidine motifs in conjunction with CuII-chelates offers high precision distance determination in systems nonpermissive to thiol-directed spin labeling. However, the noncovalency of this interaction exposes potential vulnerabilities to competition from adventitious divalent metal ions, and pH sensitivity. Herein, a combination of room-temperature isothermal titration calorimetry (ITC) and cryogenic relaxation-induced dipolar modulation enhancement (RIDME) measurements are applied to the model protein Streptococcus sp. group G. protein G, B1 domain (GB1). Results demonstrate double-histidine motif spin labeling using CuII-nitrilotriacetic acid (CuII–NTA) is robust against the competitor ligand ZnII–NTA at >1000-fold molar excess, and high nM binding affinity is surprisingly retained under acidic and basic conditions even though room temperature affinity shows a stronger pH dependence. This indicates the strategy is well-suited for diverse biological applications, with the requirement of other metal ion cofactors or slightly acidic pH not necessarily being prohibitive. Publisher PDF

Funding: Leverhulme Trust (Grant Number(s): RPG-2015-308), Leverhulme Trust (Grant Number(s): RPG-2018-362), Engineering and Physical Sciences Research Council (Grant Number(s): EP/R025754/1). Metal‐catalyzed C–N cross‐coupling generally forms C–N bonds by reductive elimination from metal complexes bearing covalent C‐ and N‐ligands. We have identified a Cu‐mediated C–N cross‐coupling that uses a dative N‐ligand in the bond forming event, which, in contrast to conventional methods, generates reactive cationic products. Mechanistic studies suggest the process operates via transmetalation of an aryl organoboron to a Cu(II) complex bearing neutral N‐ligands, such as nitriles or N‐heterocycles. Subsequent generation of a putative Cu(III) complex enables the oxidative C–N coupling to take place, delivering nitrilium intermediates and pyridinium products. The reaction is general for a range of N(sp) and N(sp2) precursors and can be applied to drug synthesis and late‐stage N‐arylation, and the limitations in the methodology are mechanistically evidenced. Publisher PDF

This research was funded, in whole or in part, by the Wellcome Trust (204821/Z/16/Z). BEB and KA acknowledge support by the Leverhulme Trust (RPG-2018–397). JLW acknowledges support by the BBSRC DTP Eastbio. BEB acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). Protein interaction studies often require very low concentrations and highly sensitive biophysical methods. Here, we demonstrate that pulse dipolar electron paramagnetic resonance spectroscopy allows measuring dissociation constants in the nanomolar range. This approach is appealing for concentration-limited biomolecular systems and medium-to-high-affinity binding studies, demonstrated here at 50 nanomolar protein concentration. Publisher PDF

This research was funded by the University of the Basque Country (grant number GIU18/172). The APC was funded by the Basque Government (grant number IT1454‐22). Apurinic apyrimidinic endonuclease 1 (APE1) is a key enzyme of the Base Excision Repair (BER) pathway, which primarily manages oxidative lesions of DNA. Once the damaged base is removed, APE1 recognises the resulting abasic site and cleaves the phosphodiester backbone to allow for the correction by subsequent enzymes of the BER machinery. In spite of a wealth of information on APE1 structure and activity, its regulation mechanism still remains to be understood. Human APE1 consists of a globular catalytic domain preceded by a flexible N-terminal extension, which might be involved in the interaction with DNA. Moreover, the binding of the nuclear chaperone nucleophosmin (NPM1) to this region has been reported to impact APE1 catalysis. To evaluate intra- and inter-molecular conformational rearrangements upon DNA binding, incision, and interaction with NPM1, we used Förster resonance energy transfer (FRET), a fluorescence spectroscopy technique sensitive to molecular distances. Our results suggest that the N-terminus approaches the DNA at the downstream side of the abasic site and enables the building of a predictive model of the full-length APE1/DNA complex. Furthermore, the spatial configuration of the N-terminal tail is sensitive to NPM1, which could be related to the regulation of APE1. Publisher PDF

Funding: This work was supported through a studentship from BBSRC in the EastBio doctoral training programme for E. S.-P. Is-PETase has become an enzyme of significant interest due to its ability to catalyse the degradation of polyethylene terephthalate (PET) at mesophilic temperatures. We performed hybrid quantum mechanics and molecular mechanics (QM/MM) at the DSD-PBEP86-D3/ma-def2-TZVP/CHARMM27//rev-PBE-D3/dev2-SVP/CHARMM level to calculate the energy profile for the degradation of a suitable PET model by this enzyme. Very low overall barriers are computed for serine protease-type hydrolysis steps (as low as 34.1 kJ mol-1). Spontaneous deprotonation of the final product, terephthalic acid, with a high computed driving force indicates that product release could be rate limiting. Publisher PDF

Funding: A. L. S. acknowledges financial support through a Cusanuswerk scholarship. The authors acknowledge support by the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant no INST 40/575-1 FUGG (JUSTUS and JUSTUS 2 cluster). M. C. G. acknowledges support from the Leverhulme Trust (RPG-2016047) and the Alexander von Humboldt Stiftung through the Humboldt-Professorship. A. M. acknowledges support from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 101023743 (PolDev). C. B. acknowledges funding by the BMBF and the Ministry of Culture and Science of the German State of North Rhine-Westphalia (MKW) under the Excellence Strategy of the Federal Government and the Länder. Exciplexes of individual electron donor and acceptor molecules are a promising approach to utilizing otherwise non-emissive triplet states in optoelectronic applications. In this work, we synthesize a series of bis(terpyridine) pyrimidine (BTP) isomers and employ them as electron acceptors in complexes with tris(4-carbazoyl-9-ylphenyl)amine (TCTA). We show that these TCTA : BTP complexes produce thermally activated delayed fluorescence (TADF) by exciplex emission, and we investigate the influence of the nitrogen position in the pyridine on the optical and electronic properties of the exciplex. The molecular arrangement of the complex is studied using scanning tunneling microscopy (STM) as well as classical force field and density functional theory (DFT) simulations. Finally, we fabricate organic light-emitting diodes (OLEDs) with maximum external quantum efficiencies ranging between 0.5% and 2% – depending on the BTP isomer. Postprint Postprint

This is a methodological guide to the use of deep neural networks in the processing of pulsed dipolar spectroscopy (PDS) data encountered in structural biology, organic photovoltaics, photosynthesis research, and other domains featuring long-lived radical pairs and paramagnetic metal ions. PDS uses distance dependence of magnetic dipolar interactions; measuring a single well-defined distance is straightforward, but extracting distance distributions is a hard and mathematically ill-posed problem requiring careful regularisation and background fitting. Neural networks do this exceptionally well, but their “robust black box” reputation hides the complexity of their design and training – particularly when the training dataset is effectively infinite. The objective of this paper is to give insight into training against simulated databases, to discuss network architecture choices, to describe options for handling DEER (double electron-electron resonance) and RIDME (relaxation-induced dipolar modulation enhancement) experiments, and to provide a practical data processing flowchart., Journal of Magnetic Resonance, 338, ISSN:1090-7807

Funding: We thank EPSRC (EP/R013799/1) for a grant (DB) and the University of St Andrews for a Studentship (XF). The isolation of three adenosine based metabolites 6–8 from Streptomyces calvus is reported. The metabolites are structurally related to the fluorine containing antibiotic nucleocidin 1 and two recently identified glycosylated fluoroadenosines 2 and 3, however in this case the three metabolites do not contain a fluorine, suggesting that the biosynthetic enzymes to the fluorometabolites also process their non-fluorinated counterparts. Publisher PDF

We thank EPSRC for a grant (EP/R013799/1) and for a Studentship (JC) through the CRITICAT Centre for Doctoral training (CDT). FAPESP is also gratefully acknowledged for a studentship (BAP, #2019/03855-3), and a Young Research Award (RAC, #2018/03910-1). CENAPAD-SP, CESUP and SDumont are acknowledged for computational clusters used in theory calculations. This study uses X-ray crystallography, theory and Langmuir isotherm analysis to explore the conformations and molecular packing of alkyl all-cis 2,3,4,5,6-pentafluorocyclohexyl motifs, which are prepared by direct aryl hydrogenations from alkyl- or vinyl- pentafluoroaryl benzenes. Favoured conformations retain the more polar triaxial C-F bond arrangement of the all-cis 2,3,4,5,6-pentafluorocyclohexyl ring systems with the alkyl substituent adopting an equatorial orientation, and accommodating strong supramolecular interactions between rings. Langmuir isotherm analysis on a water subphase of a long chain fatty acid and alcohol carrying terminal all-cis 2,3,4,5,6-pentafluorocyclohexyl rings do not show any indication of monolayer assembly relative to their cyclohexane analogues, instead the molecules appear to aggregate and form higher molecular assemblies prior to compression. The study indicates the power and potential of this ring system as a motif for ordering supramolecular assembly. Publisher PDF

The development of mild, aqueous conditions for the cross-coupling of highly functionalized (hetero)aryl chlorides or bromides is attractive, enabling their functionalization and diversification. Herein, we report a general method for Suzuki–Miyaura cross-coupling at 37 °C in aqueous media in the presence of air. We demonstrate application of this general methodology for derivatisation of (poly)chlorinated, medicinally active compounds and halogenated amino acids. The approach holds the potential to be a useful tool for late-stage functionalization or analogue generation., Simple, aqueous and direct cross-coupling of diverse and complex (hetero)aromatic halides and active pharmaceutical agents.

We report the synthesis of all‐cis 1,2,4,5‐tetrakis (trifluoromethyl)‐ and all‐cis 1,2,3,4,5,6‐hexakis (trifluoromethyl)‐ cyclohexanes by direct hydrogenation of precursor tetrakis‐ or hexakis‐ (trifluoromethyl)benzenes. The resultant cyclohexanes have a stereochemistry such that all the CF3 groups are on the same face of the cyclohexyl ring. All‐cis 1,2,3,4,5,6‐hexakis(trifluoromethyl)cyclohexane is the most sterically demanding of the all‐cis hexakis substituted cyclohexanes prepared to date, with a barrier (ΔG) to ring inversion calculated at 27 kcal mol−1. The X‐ray structure of all‐cis 1,2,3,4,5,6‐hexakis(trifluoromethyl)cyclohexane displays a flattened chair conformation and the electrostatic profile of this compound reveals a large diffuse negative density on the fluorine face and a focused positive density on the hydrogen face. The electropositive hydrogen face can co‐ordinate chloride (K≈103) and to a lesser extent fluoride and iodide ions. Dehydrofluorination promoted decomposition occurs with fluoride ion acting as a base., All‐cis 1,2,4,5‐tetrakis (trifluoromethyl)‐ and all‐cis 1,2,3,4,5,6‐hexakis (trifluoromethyl)‐ cyclohexanes were prepared by direct aryl hydrogenation. All‐cis 1,2,3,4,5,6‐hexakis(trifluoromethyl)cyclohexane has a high barrier to ring inversion (ΔG≠=27 kcal mol−1) and a Janus face profile with a large diffuse negative density on the fluorine face, and a focused positive density on the hydrogen face which coordinates carbonyls and halides.

We report counter‐intuitive axial preferences in non‐stereochemically biased, selectively fluorinated methoxycyclohexanes. These pseudo‐anomeric effects are apparent when electronegative CF2 groups are placed at the C‐2, C‐4 and C‐6 positions of the cyclohexane ring to render the C‐3/5 axial hydrogen atoms electropositive. The electrostatic interaction between these axial hydrogen atoms and the ‐OMe oxygen is stabilising. The effect is explored using high‐level ab initio and DFT calculations in the framework of NBO, QTAIM and NCI analysis across a range of derivatives, and experimentally (19F{1H}‐NMR at −80 °C) for some illustrative examples. The effect is significant in energy terms for a weak interaction, and illustrates a new stereoelectronic aspect attributed to selective fluorine substitution in organic chemistry., Fluorine chemistry: Selective fluorination in methoxycyclohexanes induces an electrostatic 1,3‐diaxial interaction favouring a counter‐intuitive axial preference.

Authors thank the Diamond Light Source for beam time allocation and beam line staff for assistance with data collection. Funding for these studies was provided by BBSRC (BB/T006161/1 & BB/T006188/1 to J. H. N. & N. G. J. R., respectively), and the National Institutes of Health (R01 GM129793 to V. d. C.-L.) The enzyme ForT catalyzes C–C bond formation between 5′-phosphoribosyl-1′-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of formycin A 5′-phosphate by Streptomyces kaniharaensis. We report the 2.5 Å resolution structure of the ForT/PRPP complex and locate active site residues critical for PRPP recognition and catalysis. Publisher PDF

This project was supported by a BBSRC grant (BB/S018069/1) to C.P., who was supported by the Royal Society of Edinburgh, Tenovus (T15/41) and Carnegie Trust (OS000256), at the initial stages of this project. Further C.P. acknowledges support from the University of St Andrews for the C.K. studentship and the University of Leeds and the Chinese Scholarship Council for the Y.M. studentship. B.E.B. and C.P. acknowledge support by the Leverhulme Trust (RPG-2018–397). This work was also supported by previous Wellcome Trust [099149/Z/12/Z] and BBSRC equipment grants (BB/R013780/1). The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as structural model for rationalizing functional observations in multiple MscL orthologues. Although these orthologues adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nano-pockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels. Here we modify the nano-pocket regions of MscL from Escherichia coli and Mycobacterium tuberculosis and employ PELDOR/DEER distance and 3pESEEM deuterium accessibility measurements to interrogate channel structure within lipids, in which both channels adopt a closed conformation. Significant in-lipid structural differences between the two constructs suggest a more compact EcMscL at the membrane inner-leaflet, as a consequence of a rotated TM2 helix. Observed differences within lipids could explain EcMscL’s higher tension sensitivity and should be taken into account in extrapolated models used for MscL gating rationalization. Publisher PDF

Authors thank the Chinese Scholarship Council for a studentship (ZF) and the EPSRC Mass Spectroscopy Service at Swansea for analysis. This paper describes the synthesis of a series of organic liquid crystals (LC) containing selectively fluorinated cyclopropanes at their termini. The syntheses used difluorocarbene additions to olefin precursors, an approach which proved straightforward such that these liquid crystal candidates could be efficiently prepared. Their physical and thermodynamic properties were evaluated and depending on individual structures, they either displayed positive or negative dielectric anisotropy. The study gives some guidance into effective structure property relationships for the design of LCs containing selectively fluorinated cyclopropane motifs. Publisher PDF

Funding: National Institutes of Health (NIH) [R35GM118160 to M.P.T., R01GM097330 to S.B. and 1F31GM125365 toK.F.]; Biotechnology and Biological Sciences Research Council [REF: BB/S000313/1 to M.F.W.]. Funding for open access charge: NIH grant. Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity. Publisher PDF

Syngenta (Case Award to DJP) and a Philip Leverhulme Prize for funding (SLC, AE). RKM and PHW are grateful to the American Chemical Society Petroleum Research Fund and National Science Foundation (NSF-MRI: CHE-1429616). The importance of 1,5-O···chalcogen (Ch) interactions in isochalcogenourea catalysis (Ch = O, S, Se) is investigated. Conformational analyses of N-acyl isochalcogenouronium species and comparison with kinetic data demonstrate the significance of 1,5-O···Ch interactions in enantioselective catalysis. Importantly, the selenium analogue demonstrates enhanced rate and selectivity profiles across a range of reaction processes including nitronate conjugate addition and formal [4+2] cycloadditions. A gram-scale synthesis of the most active selenium analogue was developed using a previously unreported seleno-Hugerschoff reaction, allowing the challenging kinetic resolutions of tertiary alcohols to be performed at 500 ppm catalyst loading. Density Functional Theory (DFT) and natural bond orbital (NBO) calculations support the role of orbital delocalization (occurring by intramolecular chalcogen bonding) in determining the conformation, equilibrium population, and reactivity of N-acylated intermediates. Postprint

Funding: AG was supported by the EPSRC funded Centre for Doctoral Training in ‘integrated magnetic resonance’, iMR-CDT (EP/J500045/1) the time the research was conducted. BEB is supported by the Leverhulme Trust (RPG-2018–397) and equipment funding from BBSRC (BB/R013780/1 and BB/T017740/1). Pulse dipolar electron paramagnetic resonance spectroscopy (PDS) is continuously furthering the understanding of chemical and biological assemblies through distance measurements in the nanometer range. New paramagnets and pulse sequences can provide structural insights not accessible through other techniques. In the pursuit of alternative spin centers for PDS, we synthesized a low-spin CoII complex bearing a nitroxide (NO) moiety, where both the CoII and NO have an electron spin S of 1/2. We measured CoII-NO distances with the well-established double electron–electron resonance (DEER aka PELDOR) experiment, as well as with the five- and six-pulse relaxation-induced dipolar modulation enhancement (RIDME) spectroscopies at Q-band frequencies (34 GHz). We first identified challenges related to the stability of the complex in solution via DEER and X-ray crystallography and showed that even in cases where complex disproportionation is unavoidable, CoII-NO PDS measurements are feasible and give good signal-to-noise (SNR) ratios. Specifically, DEER and five-pulse RIDME exhibited an SNR of ~100, and while the six-pulse RIDME exhibited compromised SNR, it helped us minimize unwanted signals from the RIDME traces. Last, we demonstrated RIDME at a 10 μM sample concentration. Our results demonstrate paramagnetic CoII to be a feasible spin center in medium magnetic fields with opportunities for PDS studies involving CoII ions. Publisher PDF

Funding: This work was supported by a China Scholarship Council-University of St Andrews PhD Fellowship (GX) and an IBioIC funded PhD studentship in collaboration with Scottish Forestry and the Scottish Forestry Trust (GS). Members of the karrikin family of bioactive small molecules are known to promote germination of a range of plants following large scale fires. As a result, they are relevant and interesting compounds. This report describes their synthesis from a biomass-derived product stream. During work to fractionate biomass with the goal of obtaining high quality lignins, an interesting co-product stream derived from the hemicellulose in the biomass, was obtained. Whilst many applications of this co-product stream can be proposed, in this case the major monosaccharides have been converted to relevant karrikins in short reaction sequences. Key results include a highly selective catalytic oxidation reaction, conversion of the resulting ketone to a butenolide by two alternative approaches, a selective acetal reductive opening reaction, X-ray crystallographic analysis of two compounds and detailed comparison of the final products with previous literature reports. Only through successful use of all the components generated during biomass refining, can economic sustainability be potentially achieved. Publisher PDF

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant BB/M010996/1) via EASTBIO Doctoral Training Partnership studentships to B.J.R. and G.F., and by the Engineering and Physical Sciences Research Council (EPSRC) [grant number EP/L016419/1] via a CRITICAT Centre for Doctoral Training studentship to T.F.G.M. ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis in bacteria, namely, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate. Catalytic (HisGS) and regulatory (HisZ) subunits assemble in a hetero-octamer where HisZ activates HisGS and mediates allosteric inhibition by histidine. In Acinetobacter baumannnii, HisGS is necessary for the bacterium to persist in the lung during pneumonia. Inhibition of ATPPRT is thus a promising strategy for specific antibiotic development. Here, A. baumannii ATPPRT is shown to follow a rapid equilibrium random kinetic mechanism, unlike any other ATPPRT. Histidine noncompetitively inhibits ATPPRT. Binding kinetics indicates histidine binds to free ATPPRT and to ATPPRT:PRPP and ATPPRT:ATP binary complexes with similar affinity following a two-step binding mechanism, but with distinct kinetic partition of the initial enzyme:inhibitor complex. The dipeptide histidine-proline inhibits ATPPRT competitively and likely uncompetitively, respectively, against PRPP and ATP. Rapid kinetics analysis shows His-Pro binds to the ATPPRT:ATP complex via a two-step binding mechanism. A related HisZ that shares 43% sequence identity with A. baumannii HisZ is a tight-binding allosteric inhibitor of A. baumannii HisGS. These findings lay the foundation for inhibitor design against A. baumannii ATPPRT. Postprint