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Nearly a half-century after Gouterman classified the UV–vis–NIR spectra of porphyrin derivatives as normal, hyper, or hypso, we propose a heretofore unsuspected “mechanism” underlying hypso spectra. Hypsoporphyrins, which exhibit blueshifted optical spectra relative to normal porphyrins (such as Zn porphyrins), typically involve dn transition metal ions, where n > 6. The spectral blueshifts have been traditionally ascribed to elevated porphyrin eg LUMO (lowest unoccupied molecular orbital) energy levels as a result of antibonding interactions with metal dπ orbitals. Herein, we have found instead that the blueshifts reflect a lowering of the a2u HOMO (highest occupied molecular orbital) energy levels. Electronegative metals such as Pd and Pt transfer smaller quantities of electron density to the porphyrin nitrogens, compared to a more electropositive metal such as Zn. With large amplitudes at the porphyrin nitrogens, the a2u HOMOs of Pd(II) and Pt(II) porphyrins accordingly exhibit lower orbital energies than those of Zn(II) porphyrins, thus explaining the hypso effect. Hypso spectra are also observed for corroles: compared with six-coordinate Al(III) corroles, which may be thought of exhibiting normal spectra, Au(III) corroles, for instance, exhibit blueshifted or hypso spectra.

DNA ligases, the enzymes responsible for joining breaks in the phosphodiester backbone of DNA during replication and repair, vary considerably in size and structure. The smallest members of this enzyme class carry out their functions with pared-down protein scaffolds comprising only the core catalytic domains. Here we use sequence similarity network analysis of minimal DNA ligases from all biological super kingdoms, to investigate their evolutionary origins, with a particular focus on bacterial variants. This revealed that bacterial Lig C sequences cluster more closely with Eukaryote and Archaeal ligases, while bacterial Lig E sequences cluster most closely with viral sequences. Further refinement of the latter group delineates a cohesive cluster of canonical Lig E sequences that possess a leader peptide, an exclusively bacteriophage group of T7 DNA ligase homologs and a group with high similarity to the Chlorella virus DNA ligase which includes both bacterial and viral enzymes. The structure and function of the bacterially-encoded Chlorella virus homologs were further investigated by recombinantly producing and characterizing, the ATP-dependent DNA ligase from Burkholderia pseudomallei as well as determining its crystal structure in complex with DNA. This revealed that the enzyme has similar activity characteristics to other ATP-dependent DNA ligases, and significant structural similarity to the eukaryotic virus Chlorella virus including the positioning and DNA contacts of the binding latch region. Analysis of the genomic context of the B. pseudomallei ATP-dependent DNA ligase indicates it is part of a lysogenic bacteriophage present in the B. pseudomallei chromosome representing one likely entry point for the horizontal acquisition of ATP-dependent DNA ligases by bacteria.

We present a fully analytic approach to calculate infrared (IR) and Raman spectra of molecules embedded in complex molecular environments modeled using the fragment-based polarizable embedding (PE) model. We provide the theory for the calculation of analytic second-order geometric derivatives of molecular energies and first-order geometric derivatives of electric dipole moments and dipole-dipole polarizabilities within the PE model. The derivatives are implemented using a general open-ended response theory framework, thus allowing for an extension to higher-order derivatives. The embedding-potential parameters used to describe the environment in the PE model are derived through first-principles calculations, thus allowing a wide variety of systems to be modeled, including solvents, proteins, and other large and complex molecular environments. Here, we present proof-of-principle calculations of IR and Raman spectra of acetone in different solvents. This work is an important step toward calculating accurate vibrational spectra of molecules embedded in realistic environments.

Halogen bonding is a flourishing field of research, but has for long been little recognized. The same goes for its scientific hero, Odd Hassel, who laid the foundations for all current developments. The crystallographic observation of halogen−oxygen interatomic distances shorter than the sum of the van der Waals radii of the involved atoms, and the interpretation of this phenomenon as a charge-transfer interaction, have been ground-breaking. Today, charge-transfer to a polarized halogen is not any longer seen as “odd”, but is commonly referred to as halogen bonding, and is widely exploited in chemistry. Despite the recognition of Hassel's work with a Nobel prize in 1969, surprisingly little appreciation is given to date to the devoted scientist, who established a world-leading laboratory during one of the darkest eras of history. Herein, we wish to revive the legacy and highlight the impact of Odd Hassel's ground-breaking discoveries.

The asymmetric Rh‐catalyzed hydrocarboxylation of α,β‐unsaturated carbonyl compounds was originally developed by Mikami and co‐workers but gives only moderate enantiomeric excesses. In order to understand the factors controlling the enantioselectivity and to propose novel ligands for this reaction, we have used computational and experimental methods to study the Rh‐catalyzed hydrocarboxylation with different bidentate ligands. The analysis of the C−CO2 bond formation transition states with DFT methods shows a preference for outer‐sphere CO2 insertion, where CO2 can undergo a backside or frontside reaction with the nucleophile. The two ligands that prefer a frontside reaction, StackPhos and tBu‐BOX, display an intriguing stacking interaction between CO2 and an N‐heterocyclic ring of the ligand (imidazole or oxazoline). Our experimental results support the computationally predicted low enantiomeric excesses and highlight the difficulty in developing a highly selective version of this reaction.

As part of our efforts to develop rhenium-oxo corroles as photosensitizers for oxygen sensing and photodynamic therapy, we investigated the potential β-perhalogenation of five ReO meso-tris(para-X-phenyl)corroles, Re[TpXPC](O) (X = CF3, H, F, CH3, and OCH3), with elemental chlorine and bromine. With Cl2, β-octachlorinated products Re[Cl8TpXPC](O) were rapidly obtained for X = CF3, H, and CH3, but X = OCH3 resulted in overchlorination on the meso-aryl groups. Full β-octabromination proved slower relative to Cu and Ir corroles, but the desired Re[Br8TpXPC](O) products were finally obtained for X = H and F after a week at room temperature. For X = CH3 and OCH3, these conditions led to undecabrominated products Re[Br11TpXPC](O). Compared to the β-unsubstituted starting materials, the β-octahalogenated products were found to exhibit sharp 1H NMR signals at room temperature, indicating that the aryl groups are locked in place by the β-halogens, and substantially redshifted Soret and Q bands. Single-crystal X-ray structures of Re[Cl8TpCF3PC](O), Re[Cl8TpCH3PC](O), and Re[Br8TpFPC](O) revealed mild saddling for one Cl8 structure and the Br8 structure. These structural variations, however, appear too insignificant to explain the slowness of the β-octabromination protocols, which seems best attributed to the deactivating influence of the high-valent Re center.

Antimicrobial resistance, primarily caused by the overuse of antimicrobials such as antibiotics, is becoming an increasing concern to public health. To that end, the global spread of the -lactamase OXA-48 is worrisome, as it readily catalyzes the hydrolysis of -lactam drugs, such as penicillins as well as our “last resort” carbapenems. On the contrary, OXA-48 exhibits only limited catalytic activity against 3rd generations cephalosporins like ceftazidime. However, naturally evolving variants and results from laboratory studies have shown that OXA-48 can expand its substrate profile, conferring increased ceftazidime resistance. Expansion of the substrate profile towards ceftazidime is seen to be accompanied by a trade-off towards carbapenems and penicillins, greatly reducing OXA-48 ability to catalyze the hydrolysis of penicillins and carbapenems. Here, X-ray crystallography, steady-state enzyme kinetics and differential scanning fluorimetry were used to characterize and analyze wild type (wt) OXA-48:wt and two variants, OXA-48:F72L and OXA-48:A33V/K51E/F72L/S212A/T213A (OXA-48:Q5), where the latter two were evolved towards increased ceftazidime resistance. Steady-state enzyme kinetics revealed that the two mutants had increased catalytic ability to hydrolyze ceftazidime. Such increases in kcat/Km hypothesized to arise from increased flexibility of the -loop, which was observed in the OXA-48:Q5 X-ray crystal structure in complex with piperacillin, is in line with previous studies. Further supporting the hypothesis, urea dependent kinetics and thermostability measurements show that these mutants likely exhibit increased dynamical behavior that would aid ceftazidime binding. OXA-48:F72L showed a bigger urea dependence on the enzyme activity with no activity at 4 M urea, whereas OXA-48:wt and OXA-48:Q5 needed 6 M urea to become inactive. This suggested that OXA-48:F72L is more flexible, and that OXA-48:Q5 regains some resistance to chemical denaturing by urea. The pH dependency showed higher piperacillin activity at pH 7.2 compared to 5.2 and 9.2 for all three variants. The increase in ceftazidime activity came along with a functional trade-off against the penicillin piperacillin as well as reduced thermostability of (OXA-48:F72L: -6.5C/OXA-48:Q5: -6.4C) compared to wt OXA-48, which may be caused by sub-optimal substrate positioning within the active site of OXA-48:Q5. This work provides experimental evidence, that during evolution of OXA-48 towards increased ceftazidime activity, structural changes can arise, likely affecting the chemical environment within the active site, causing increased enzyme flexibility, and ultimately shaping functional trade-offs.

The cysteine of HCD (C286) in DYRK1A is involved in disulfide bridge formation with a cysteine (C312) in the DFGSSC sequence. The purpose of this project was to investigate how the state of the disulfide bridge would affect enzyme catalytic and ligand binding properties of the protein kinase. A mutant, DYRK1A C312A, was thus designed to eliminate the disulfide bridge. The mutant was expressed and purified following the same protocol as for DYRK1A wt, including HisTrap purification, TEV cleavage and size exclusion chromatography. Crystallization trials were performed for both the wt and the mutant with the kinase inhibitor Staurosporine. DYRK1A wt with STU crystallized and diffracted with at a resolution of 2.33 Å. The DYRK1A C312A mutant with STU crystallized and diffracted with a resolution of 2.59 Å. The structure was solved by molecular replacement in Molrep (CCP4) and refined by Refmac5 and Phenix. Molecular dynamics (MD) simulations (SCHRODINGER) were performed with the intent to compare diverse disulfide bridge states. Ligand binding and enzyme catalytic properties were analyzed using a combination of techniques, including activity assays, microscale thermophoresis, and isothermal calorimetry. The Thermofluor assay confirmed that both the wt and the mutant bind tightly to STU and AZ-191. It also showed that the mutant consistently has a slightly lower melting temperature than the wt, which would indicate that it is less stable. Solvent accessible surface area (SASA) analysis support the theory of accessibility to conserved cysteine residues.

Benchmarking molecular properties with Gaussian-type orbital (GTO) basis sets can be challenging, because one has to assume that the computed property is at the complete basis set (CBS) limit, without a robust measure of the error. Multiwavelet (MW) bases can be systematically improved with a controllable error, which eliminates the need for such assumptions. In this work, we have used MWs within Kohn–Sham density functional theory to compute static polarizabilities for a set of 92 closed-shell and 32 open-shell species. The results are compared to recent benchmark calculations employing the GTO-type aug-pc4 basis set. We observe discrepancies between GTO and MW results for several species, with open-shell systems showing the largest deviations. Based on linear response calculations, we show that these discrepancies originate from artefacts caused by the field strength, and that several polarizabilies from a previous study were contaminated by higher order responses (hyperpolarizabilities). Based on our MW benchmark results, we can affirm that aug-pc4 is able to provide results close to the CBS limit, as long as finite-difference effects can be controlled. However, we suggest that a better approach is to use MWs, which are able to yield precise finite-difference polarizabilities even with small field strengths.

DNA ligases are diverse enzymes with essential functions in replication and repair of DNA; here we review recent advances in their structure and distribution and discuss how this contributes to understanding their biological roles and technological potential. Recent high-resolution crystal structures of DNA ligases from different organisms, including DNA-bound states and reaction intermediates, have provided considerable insight into their enzymatic mechanism and substrate interactions. All cellular organisms possess at least one DNA ligase, but many species encode multiple forms some of which are modular multifunctional enzymes. New experimental evidence for participation of DNA ligases in pathways with additional DNA modifying enzymes is defining their participation in non-redundant repair processes enabling elucidation of their biological functions. Coupled with identification of a wealth of DNA ligase sequences through genomic data, our increased appreciation of the structural diversity and phylogenetic distribution of DNA ligases has the potential to uncover new biotechnological tools and provide new treatment options for bacterial pathogens.

A porphyrin cis tautomer, where the two central NH protons are on adjacent pyrrole rings, has long been invoked as an intermediate in porphyrin tautomerism. Only recently, however, has such a species been isolated and structurally characterized. Thus, single-crystal X-ray structure determinations of two highly saddled free-base porphyrins, beta-heptakis(trifluoromethyl)-mesotetrakis(p-fluorophenyl)porphyrin, H-2[(CF3)(7)TFPP], and beta-octaiodo- 5,10,15,20- tetrakis(4'-trifluoromethylphenyl) porphyrin, H-2[I8TCF3PP], unambiguously revealed cis tautomeric structures, each stabilized as a termolecular complex with a pair of ROH (R = CH3 or H) molecules that form hydrogen-bonded N-H center dot center dot center dot O-H center dot center dot center dot N straps connecting the central NH groups with the antipodal unprotonated nitrogens. The unusual substitution patterns of these two porphyrins, however, have left open the question how readily such supramolecular assemblies might be engineered, which prompted us to examine the much more synthetically accessible beta-octabromo-meso-tetraphenylporphyrins. Herein, single-crystal Xray structures were obtained for two such compounds, 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(4'-trifluoromethylphenyl)porphyrin, H-2[Br8TCF3PP], and 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(4'-fluorophenyl)porphyrin, H-2[Br8TFPP], and although the central hydrogens could not all be located unambiguously, the electron density could be convincingly modeled as porphyrin cis tautomers, existing in each case as a bis-methanol adduct. In addition, a perusal of the Cambridge Structural Database suggests that there may well be additional examples of porphyrin cis tautomers that have not been recognized as such. We are therefore increasingly confident that porphyrin cis tautomers are readily accessible via supramolecular engineering, involving the simple stratagem of crystallizing a strongly saddled porphyrin from a solvent system containing an amphiprotic species such as water or an alcohol. Research Council of NorwayResearch Council of Norway [262229]; U.S. National Science FoundationNational Science Foundation (NSF) [CHE-1726077] This work was supported by the Research Council of Norway (grant no. 262229) and the U.S. National Science Foundation (grant no. CHE-1726077). We thank Dr. Laura J. McCormickMcPherson for helpful discussions.

A set of rhenium(V)-oxo meso-triarylcorroles bearing ester and carboxylic acid functionalities were synthesized with a view to determining their potential for photodynamic therapy. Toward this end, we measured their near-IR phosphorescence and their ability to sensitize singlet oxygen formation. The two esters studied, ReVO 5,10,15-tris(meta-carbomethoxyphenyl)corrole and ReVO 5,10,15-tris(para-carbomethoxyphenyl)corrole, were found to exhibit phosphorescence quantum yields of around 1% and fairly long phosphorescence lifetimes of about 60 μs in toluene. The corresponding carboxylic acids, which were examined in ethanolic/aqueous media, in contrast, showed much lower phosphorescence quantum yields on the order of 0.01% and somewhat shorter phosphorescent lifetimes. The quantum yields for singlet oxygen formation, on the other hand, turned out to be equally high (0.72 ± 0.02) for the esters and corresponding carboxylic acids. For the two carboxylic acids, we also carried out photocytotoxicity measurements on rat bladder cancer cells (AY27) and human colon carcinoma cells (WiDr). Cell viability measurements (MTT assays) indicated 50% cell death (LD50) for AY27 cells upon 5 min of blue light exposure with the meta carboxylic acid and upon 7 min of exposure with the para carboxylic acid; complete cell death resulted after 20 min for both compounds. The WiDr cells proved less sensitive, and LD50 values were reached after 8 and 12 min illumination with the meta and para carboxylic acids, respectively.

A range of hitherto unexplored biomass‐derived chemicals have been evaluated as new sustainable solvents for a large variety of CO2‐based carboxylation reactions. Known biomass‐derived solvents (biosolvents) are also included in the study and the results are compared with commonly used solvents for the reactions. Biosolvents can be efficiently applied in a variety of carboxylation reactions, such as Cu‐catalyzed carboxylation of organoboranes and organoboronates, metal‐catalyzed hydrocarboxylation, borocarboxylation, and other related reactions. For many of these reactions, the use of biosolvents provides comparable or better yields than the commonly used solvents. The best biosolvents identified are the so far unexplored candidates isosorbide dimethyl ether, acetaldehyde diethyl acetal, rose oxide, and eucalyptol, alongside the known biosolvent 2‐methyltetrahydrofuran. This strategy was used for the synthesis of the commercial drugs Fenoprofen and Flurbiprofen., Sustainable solvents: The number of biomass‐derived solvents can be expanded. Hitherto unexplored as well as known biomass‐derived solvents can be used for numerous carboxylation reactions with carbon dioxide. Biomass‐derived solvents, such as isosorbide dimethyl ether and acetaldehyde diethyl acetal, are found to be not only as good as but sometimes even better than common organic solvents.

Herein is reported a substantially improved synthesis of 2-substituted benzimidazoles by condensation of 1,2-diaminoarenes and aldehydes using methanol as the reaction medium. The developed method afforded moderate to excellent yields (33–96%) at ambient temperature, displays high functional group tolerance, is conducted open to air, and requires only one minute reaction time under catalyst- and additive-free conditions. Moreover, the efficient protocol permits scale-up to multi-gram scale synthesis of benzimidazoles and will become a method of choice when constructing such heterocyclic systems.

Grouping strategies are needed for per- and polyfluoroalkyl substances (PFAS), in part, because it would be time and resource intensive to test and evaluate the more than 4700 PFAS on the global market on a chemical-by-chemical basis. In this paper we review various grouping strategies that could be used to inform actions on these chemicals and outline the motivations, advantages and disadvantages for each. Grouping strategies are subdivided into (1) those based on the intrinsic properties of the PFAS (e.g.persistence, bioaccumulation potential, toxicity, mobility, molecular size) and (2) those that inform risk assessment through estimation of cumulative exposure and/or effects. The most precautionary grouping approach of those reviewed within this article suggests phasing out PFAS based on their high persistence alone (the so-called "P-sufficient" approach). The least precautionary grouping approach reviewed advocates only grouping PFAS for risk assessment that have the same toxicological effects, modes and mechanisms of action, and elimination kinetics, which would need to be well documented across different PFAS. It is recognised that, given jurisdictional differences in chemical assessment philosophies and methodologies, no one strategy will be generally acceptable. The guiding question we apply to the reviewed grouping strategies is: grouping for what purpose? The motivation behind the grouping (e.g.determining use in productsvs.setting guideline levels for contaminated environments) may lead to different grouping decisions. This assessment provides the necessary context for grouping strategies such that they can be adopted as they are, or built on further, to protect human and environmental health from potential PFAS-related effects., Environmental Science: Processes & Impacts, 22 (7), ISSN:2050-7887, ISSN:2050-7895

The rapid oxidation of carbon black (CB) is a major drawback for its use as a catalyst support in polymer electrolyte fuel cells. Here, we synthesize poly[2,20 -(4,40 -bipyridine)-5,50 -bibenzimidazole] (BiPyPBI) as a conducting polymer and use it to functionalize the surface of CB and homogenously anchor platinum metal nanoparticles (Pt-NPs) on a CB surface. The as-prepared materials were confirmed by different spectroscopic techniques, including nuclear magnetic resonance spectroscopy, energy-dispersive X-ray, thermal gravimetric analysis, and scanning-transmittance microscopy. The as-fabricated polymer-based CB catalyst showed an electrochemical surface area (ECSA) of 63.1 cm2 mgPt1 , giving a catalyst utilization efficiency of 74.3%. Notably, the BiPyPBI-based CB catalyst exhibited remarkable catalytic activity towards oxygen reduction reactions. The onset potential and the diffusion-limiting current density reached 0.66 V and 5.35 mA cm2 , respectively. Furthermore, oxidation stability testing showed a loss of only 16% of Pt-ECSA for BiPyPBI-based CB compared to a 36% loss of Pt-ECSA for commercial Pt/CB after 5000 potential cycles. These improvements were related to the synergetic effect between the nitrogen-rich BiPyPBI polymer, which promoted the catalytic activity through the structural nitrogen atoms, and demolished the degradation of CB via the wrapping process.

Accepted manuscript version, licensed CC BY-NC-ND 4.0. This study was carried out with a view of understanding the temporal dynamics of microcystin concentrations in both algal seston and water samples and the associated public health risk. All the major MC variants, namely MC-LR, MC-YR, and MC-RR, were detected in both the algal seston and water samples. In the majority of the samples, the most potent variant, MC-LR, constituted the greatest proportion of the total MC concentration suggesting extremely high potential public health risk. The exceptionally high concentrations (μg L−1) of all the variants, MC-LR (815), MC-YR (466.6) and MC-RR (265.68), were observed in May. Although the extracellular MCs were relatively less concentrated and less frequently detected, concentrations (μg L−1) of up to 20 of MC-LR, 6.13 of MC-YR, and 1.27 MC-RR were encountered. The strong and significant association between Microcystis abundance and concentration of nitrate (Spearman Rank Order Correlation r = 0.793, p Microcystis, and may have consequently influenced the MC levels in the reservoir.

A combination of quantum chemical calculations and synthetic studies was used to address the possibility of very high (>6) valence states of transition metals in porphyrin‐type complexes. With corrole as a supporting ligand, DFT calculations ruled out Re(VII) and Ir(VII) dioxo complexes as stable species. Attempted rhenium insertion into benzocarbaporphyrin (BCP) ligands on the other hand led to two products with different stoichiometries – Re[BCP]O and Re[BCP]O2. To our surprise, single‐crystal structure determination of one of the complexes of the latter type indicated an ReVO center with a second oxygen bridging the Re−C bond. In other words, although the monooxo complexes Re[BCP]O are oxophilic, the BCP ligand cannot sustain a trans‐ReVII(O)2 center. The search for metal valence states >6 in porphyrin‐type ligand environments must therefore continue.

We investigate the effect of relativity on harmonic vibrational frequencies. Density functional theory (DFT) calculations using the four-component Dirac–Coulomb Hamiltonian have been performed for 15 hydrides (H2X, X = O, S, Se, Te, Po; XH3, X = N, P, As, Sb, Bi; and XH4, X = C, Si, Ge, Sn, Pb) as well as for HC≡CPbH3. The vibrational frequencies have been calculated using finite differences of the molecular energy with respect to geometrical distortions of the nuclei. The influences of the choice of basis set, exchange–correlation functional, and step length for the numerical differentiation on the calculated harmonic vibrational frequencies have been tested, and the method has been found to be numerically robust. Relativistic effects are noticeable for the heavier congeners H2Te and H<2Po, SbH3 and BiH3, and SnH4 and PbH4 and are much more pronounced for the vibrational modes with higher frequencies. Spin–orbit effects constitute a very small fraction of the total relativistic effects, except for H2Te and H2Po. For HC≡CPbH3 we find that only the frequencies of the modes with large contributions from Pb displacements are significantly affected by relativity.

The synthesis of novel 5,7-diaryl and diheteroaryl indoles has been explored via efficient double Suzuki–Miyaura coupling. The method notably employs a low catalyst loading of Pd(PPh3)4 (1.5 mol%/coupling) and water as the reaction solvent to obtain 5,7-diarylated indoles without using N-protecting groups in up to 91% yield. The approach is also suitable for N-protected and 3-substituted indoles and constitutes an important green and convenient arylation strategy for the benzenoid ring of indoles. The synthesized diarylindoles are fluorescent.

The determination of the temperature dependence of enzyme catalysis has traditionally been a labourious undertaking. We have developed a new approach to the classical Arrhenius parameter estimation by fitting the change in velocity under a gradual change in temperature. The evaluation with a simulated dataset shows that the approach is valid. The approach is demonstrated as a useful tool by characterizing the Bacillus pumilus LipA enzyme. Our results for the lipase show that the enzyme is psychrotolerant, with an activation energy of 15.3 kcal/mol for the chromogenic substrate para-nitrophenyl butyrate. Our results demonstrate that this can produce equivalent curves to the traditional approach while requiring significantly less sample, labour and time. Our method is further validated by characterizing three α-amylases from different species and habitats. The experiments with the α-amylases show that the approach works over a wide range of temperatures and clearly differentiates between psychrophilic, mesophilic and thermophilic enzymes. The methodology is released as an open-source implementation in Python, available online or used locally. This method of determining the activation parameters can make studies of the temperature dependence of enzyme catalysis more widely adapted to understand how enzymes have evolved to function in extreme environments. Moreover, the thermodynamic parameters that are estimated serve as functional validations of the empirical valence bond calculations of enzyme catalysis.

The development of green Buchwald–Hartwig aminations has long been considered challenging, due to the high sensitivity of the reaction to the environment. Here we show that food-grade and waste vegetable oils, triglycerides originating from animals, and natural waxes can serve as excellent green solvents for Buchwald–Hartwig amination. We further demonstrate that amphiphiles and trace ingredients present in triglycerides as additives have a decisive effect on the yields of Buchwald–Hartwig aminations.

A combination of Pt L3-edge X-ray absorption spectroscopy (EXAFS and XANES) and DFT (TPSS) calculations have been performed on powder samples of the archetypal platinum porphyrinoid complexes PtII[TpCF3PP], PtIV[TpCF3PP]Cl2, and PtIV[TpCF3PC](Ar)(py), where TpCF3PP2- = meso-tetrakis(p-trifluoromethylphenyl)porphyrinato and TpCF3PC3- = meso-tris(p-trifluoromethylphenyl)corrolato. The three complexes yielded Pt L3-edge energies of 11 566.0 eV, 11 567.2 eV, and 11 567.6 eV, respectively. The 1.2 eV blueshift from the Pt(II) to the Pt(IV) porphyrin derivative is smaller than expected for a formal two-electron oxidation of the metal center. A rationale was provided by DFT-based Hirshfeld which showed that the porphyrin ligand in the Pt(IV) complex is actually substantially oxidized relative to that in the Pt(II) complex. The much smaller blueshift of 0.4 eV, going from PtIV[TpCF3PP]Cl2, and PtIV[TpCF3PC](Ar)(py), is ascribable to the significantly stronger ligand field in the latter compound.

Extensive DFT calculations with several exchange–correlation functionals indicate that molybdenum-dichlorido Viking helmet corroles are noninnocent with significant MoIV-corrole˙2− character. The effect is mediated by a Mo(4d)-corrole(π) orbital interaction similar to that postulated for MnCl, FeCl and FeNO corroles. The effect also appears to operate in tungsten-dichlorido corroles but is weaker relative to that for Mo. In contrast, MoO triarylcorroles do not exhibit a significant degree of corrole radical character. Furthermore, the Soret absorption maxima of a series of MoCl2 tris(para-X-phenyl)corrole derivatives were found to redshift dramatically with increasing electron-donating character of the para substituent X, essentially clinching the case for a noninnocent macrocycle in MoCl2 corroles.

The Lacinutrix genus was discovered in 2005 and includes 12 Gram-negative bacterial species. To the best of our knowledge, the secondary metabolite production potential of this genus has not been explored before, and examination of Lacinutrix species may reveal novel chemistry. As part of a screening project of Arctic marine bacteria, the Lacinutrix sp. strain M09B143 was cultivated, extracted, fractionated and tested for antibacterial and cytotoxic activities. One fraction had antibacterial activity and was subjected to mass spectrometry analysis, which revealed two compounds with elemental composition that did not match any known compounds in databases. This resulted in the identification and isolation of two novel isobranched lyso-ornithine lipids, whose structures were elucidated by mass spectrometry and NMR spectroscopy. Lyso-ornithine lipids consist of a 3-hydroxy fatty acid linked to the alpha amino group of an ornithine amino acid through an amide bond. The fatty acid chains were determined to be iso-C15:0 (1) and iso-C16:0 (2). Compound 1 was active against the Gram-positive S. agalactiae, while 2 showed cytotoxic activity against A2058 human melanoma cells.

Possible mechanisms are demonstrated to explain why the antibiotic-resistance enzyme OXA-436 has higher catalytic rates towards last-resort antibiotics under physiological conditions compared with its more widespread homolog OXA-48., The crystal structure of the class D β-lactamase OXA-436 was solved to a resolution of 1.80 Å. Higher catalytic rates were found at higher temperatures for the clinically important antibiotic imipenem, indicating better adaptation of OXA-436 to its mesophilic host than OXA-48, which is believed to originate from an environmental source. Furthermore, based on the most populated conformations during 100 ns molecular-dynamics simulations, it is postulated that the modulation of activity involves conformational shifts of the α3–α4 and β5–β6 loops. While these changes overall do not cause clinically significant shifts in the resistance profile, they show that antibiotic-resistance enzymes exist in a continuum. It is believed that these seemingly neutral differences in the sequence exist on a path leading to significant changes in substrate selectivity.

Development of safe, renewable, cheap and versatile solvents is a longstanding challenge in chemistry. We show here that vegetable oils and related systems can become prominent solvents for organic synthesis. Suzuki–Miyaura, Hiyama, Stille, Sonogashira and Heck cross-couplings proceed with quantitative yields in a range of vegetable oils, fish oil, butter and waxes used as solvents. Appropriate methodologies for high-throughput screening and sustainable isolation techniques applicable for vegetable oils and related lipids are presented.

The rising prevalence of diabetes is threatening global health. It is known not only for the occurrence of severe complications but also for the SARS-Cov-2 pandemic, which shows that it exacerbates susceptibility to infections. Current therapies focus on artificially maintaining insulin homeostasis, and a durable cure has not yet been achieved. We demonstrate that our set of small molecule inhibitors of DYRK1A kinase potently promotes β-cell proliferation, enhances long-term insulin secretion, and balances glucagon level in the organoid model of the human islets. Comparable activity is seen in INS-1E and MIN6 cells, in isolated mice islets, and human iPSC-derived β-cells. Our compounds exert a significantly more pronounced effect compared to harmine, the best-documented molecule enhancing β-cell proliferation. Using a body-like environment of the organoid, we provide a proof-of-concept that small–molecule–induced human β-cell proliferation via DYRK1A inhibition is achievable, which lends a considerable promise for regenerative medicine in T1DM and T2DM treatment.

Helminth parasitic infections are common in small ruminants in Norway; infection is usually treated with anthelmintic drugs, but anthelmintic resistance is an increasing problem. It is necessary to identify strategies to reduce the use of anthelmintic drugs and mitigate the impact of anthelmintic resistance. Condensed tannin (CT)-rich forages have been shown to reduce the helminth burden in small ruminants, but these forages have limited cultivation potential in Scandinavia. A good source for CT in cold climatic regions may be the bark of several commercially utilized tree species. In the present study, we determined the content and characterized the type of CT in bark extracts of pine (Pinus sylvestris L.), spruce (Picea abies L.), and birch (Betula pubescens). Extracts of selected bark samples were tested for their anthelmintic efficacy against the ovine infectious nematode Teladorsagia circumcincta. Total CT content was higher in the bark from younger (10–40 years old) pine and spruce trees; it decreased with tree age in pine, whereas it remained relatively stable in the bark of spruce and birch. Pine trees consisted of 100% procyanidins, whereas prodelphinins were present in most spruce (4–17%) and all birch samples (5–34%). Our studies clearly showed that there is variation in the anthelmintic activity of water and acetone extracts of bark samples collected from various sites around Norway, as this was measured with two independent in vitro assays, the egg hatch and larvae motility assays. The anthelmintic activity of some extracts was consistent between the two assays; for example, extracts from the three samples with the highest CT content showed very high activity in both assays, whereas the extract from the sample with the lowest CT content showed the lowest activity in both assays. For other extracts, activity was not consistent across the assays, which could be attributed to the susceptibility of the different stages of the parasitic life cycle. We demonstrated that bark extracts from commercially used trees in Scandinavia have the potential to be used as alternatives to anthelmintics. Further work should focus on refining the associations between bark extracts and anthelmintic activity to identify the best strategies to reduce the input of anthelmintic drugs in livestock production systems. Copyright © 2021 Athanasiadou, Almvik, Hellström, Madland, Simic and Steinshamn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that can be manufactured sustainably and represent a promising green alternative to petrochemical‐based plastics. Here, we describe the complete genome of a new marine PHA‐producing bacterium—Photobacterium ganghwense (strain C2.2), which we have isolated from the Black Sea seashore. This new isolate is psychrotolerant and accumulates PHA when glycerol is provided as the main carbon source. Transmission electron microscopy, specific staining with Nile Red visualized via epifluorescence microscopy and gas chromatography analysis confirmed the accumulation of PHA. This is the only PHA‐producing Photobacterium for which we now have a complete genome sequence, allowing us to investigate the pathways for PHA production and other secondary metabolite synthesis pathways. The de novo assembly genome, obtained using open‐source tools, comprises two chromosomes (3.5, 2 Mbp) and a megaplasmid (202 kbp). We identify the entire PHA synthesis gene cluster that encodes a class I PHA synthase, a phasin, a 3‐ketothiolase, and an acetoacetyl‐CoA reductase. No conventional PHA depolymerase was identified in strain C2.2, but a putative lipase with extracellular amorphous PHA depolymerase activity was annotated, suggesting that C2.2 is unable to degrade intracellular PHA. A complete pathway for the conversion of glycerol to acetyl‐CoA was annotated, in accordance with its ability to convert glycerol to PHA. Several secondary metabolite biosynthetic gene clusters and a low number of genes involved in antibiotic resistance and virulence were also identified, indicating the strain's suitability for biotechnological applications., We present the first complete genome of a polyhydroxyalkanoate (PHA) producing member of the Photobacterium genus. Photobacterium ganghwense strain C2.2 was isolated from the Black Sea shoreline and was able to accumulate PHAs using glycerol as the sole carbon source. The biotechnological potential of our strain is emphasized by the complete PHA synthesis operon annotation coupled with the naturally occurring lack of a known phaZ depolymerase gene in the strain genome.

Reprinted with permission from Pomogaev VA, Avramov, Ruud K. Photo-transformation trajectories of Nitro-Spiropyran in Hybrid Compounds with [60]Fullerene. Journal of Physical Chemistry C. 2019;123(30):18215-18221. Copyright 2019 American Chemical Society. Photo- and thermo- isomerization trajectories of various conversion pathways between nitro-spiropyran and its trans–trans–cis merocyanine form were produced and the role of nπ* states was investigated along the corresponding potential energy surface calculated using the ωB97XD functional and the cc-pVDZ basis set. The nondissociative nπ* states on the photoisomerization trajectories can switch from/to the dissociative photoactive ππ* state at two intersections between their energy surfaces. The photochromic properties inherited in hybrid compounds of nitro-spiropyran-containing [60]fullerene are interpreted due to a reversible “dual energy bypass” ππ*↔nπ* mechanism in terms of both adiabatic absorption and highly effective nonadiabatic dissipative transitions between the excited states of the photochromic fragment, that prevents energy loss through the [60]fullerene and in this way keeps the photochromic properties intact.

Acid-catalyzed condensation of pyrrole, 4-trifluoromethylbenzaldehyde, and azulene, followed by DDQ oxidation, has resulted in the isolation of the novel macrocycle azulicorrole, arguably the first example of a carbacorrole aside from N-confused corrole. Despite poor yields (

Publisher's statement (option C): https://pubs.acs.org/page/4authors/authorchoice/options.html A series of metal–metal bonded osmium corrole dimers, {Os[TpXPC]}2, were synthesized in reasonably good yields (35–46%) via the interaction of the corresponding free-base meso-tris(p-X-phenyl)corroles (H3[TpXPC], X = CF3, H, CH3, and OCH3), Os3(CO)12, and potassium carbonate in 1,2,4-trichlorobenzene under an inert atmosphere at 180 °C over several hours. The complexes are only the second class of Os corroles reported to date (the first being OsVIN corroles) and also the second class of metal–metal bonded metallocorrole dimers (the other being Ru corrole dimers). Comparison of the X-ray structures, redox potentials, and optical spectra of analogous Ru and Os corrole dimers, along with scalar-relativistic DFT calculations, has provided an experimentally calibrated account of relativistic effects in these complexes. Three of the Os corrole dimers (X = CF3, H, and OCH3) were analyzed with single-crystal X-ray diffraction analysis, revealing inversion-related corrole rings with eclipsed Os–N bonds and Os–Os distances of ∼2.24 Å that are ∼0.06 Å longer than the Ru–Ru distances in the analogous Ru corrole dimers. Interestingly, a comparison of scalar-relativistic and nonrelativistic DFT calculations indicates that this difference in metal–metal bond distance does not, in fact, reflect a differential relativistic effect. For a given corrole ligand, the Ru and Os corrole dimers exhibit nearly identical oxidation potentials but dramatically different reduction potentials, with the Os values ∼0.5 V lower relative to Ru, suggesting that whereas oxidation occurs in a ligand-centered manner, reduction is substantially metal-centered, which indeed was confirmed by scalar-relativistic calculations. The calculations further indicate that approximately a third of the ∼0.5 V difference in reduction potentials can be ascribed to relativity. The somewhat muted value of this relativistic effect appears to be related to the finding that reduction of an Os corrole dimer is not exclusively metal-based but that a significant amount of spin density is delocalized over to the corrole ligand; in contrast, reduction of an Ru corrole dimer occurs exclusively on the Ru–Ru linkage. For isoelectronic complexes, the Ru and Os corrole dimers exhibit substantially different UV–vis spectra. A key difference is a strong near-UV feature of the Os series, which in energy terms is blue-shifted by ∼0.55 V relative to the analogous feature of the Ru series. TDDFT calculations suggest that this difference may be related to higher-energy Os(5d)-based LUMOs in the Os case relative to analogous MOs for Ru.

We propose a modified coupled cluster Monte Carlo algorithm that stochastically samples connected terms within the truncated Baker--Campbell--Hausdorff expansion of the similarity transformed Hamiltonian by construction of coupled cluster diagrams on the fly. Our new approach -- diagCCMC -- allows propagation to be performed using only the connected components of the similarity-transformed Hamiltonian, greatly reducing the memory cost associated with the stochastic solution of the coupled cluster equations. We show that for perfectly local, noninteracting systems, diagCCMC is able to represent the coupled cluster wavefunction with a memory cost that scales linearly with system size. The favorable memory cost is observed with the only assumption of fixed stochastic granularity and is valid for arbitrary levels of coupled cluster theory. Significant reduction in memory cost is also shown to smoothly appear with dissociation of a finite chain of helium atoms. This approach is also shown not to break down in the presence of strong correlation through the example of a stretched nitrogen molecule. Our novel methodology moves the theoretical basis of coupled cluster Monte Carlo closer to deterministic approaches., Comment: 31 pages, 6 figures

As pioneered by Corey and Chaykovsky, sulfoxonium ylides have had widespread application in organic synthesis for more than a half century. In most of the reactions, sulfoxonium ylides were used to react with electrophiles. Under suitable reaction conditions these ylides can generate metal carbenoids and react with nucleophiles. By combining the typical reactivity of sulfoxonium ylides with transition-metal catalysis, a growing number of investigations have expanded their application in organic synthesis. This review provides an update on the preparation of sulfoxonium ylides and their applications in carbenoid transfer reactions.1 Introduction2 Preparation of Sulfoxonium Ylides3 Investigation for Carbenoid Formation from Sulfoxonium Ylide 4 X–H (X = N, O, S, C) Functionalization Reactions5 Polymerizaton of Carbenoids Generated from Sulfoxonium Ylides6 Conclusion and Perspective

A highly efficient and catalyst-free protocol is reported for the synthesis of quinoxalines via the classical cyclocondensation reaction between aryldiamines and dicarbonyl compounds. Remarkably simple and green reaction conditions employing methanol as solvent afforded medium to excellent yield of quinoxalines after only one-minute reaction time at ambient temperature. The conditions allow at least 10 gram scale synthesis of quinoxalines and should be a preferred starting point for optimization and method of choice for applications in the synthetic community.

The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism., eLife digest Bacteria can be infected by viruses known as bacteriophages. These viruses inject their genetic material into bacterial cells and use the bacteria’s own machinery to build the proteins they need to survive and infect other cells. To protect themselves, bacteria produce a molecule called S-adenosyl methionine, or SAM for short, which deposits marks on the bacteria’s DNA. These marks help the bacteria distinguish their own genetic material from the genetic material of foreign invaders: any DNA not bearing the mark from SAM will be immediately broken down by the bacterial cell. This system helps to block many types of bacteriophage infections, but not all. Some bacteriophages carry genes that code for enzymes called SAMases, which can break down SAM, switching off the bacteria’s defenses. The most well-known SAMase was first discovered in the 1960s in a bacteriophage called T3. Chemical studies of this SAMase suggested that it works as a 'hydrolase', meaning that it uses water to break SAM apart. New SAMases have since been discovered in bacteriophages from environmental water samples, which, despite being able to degrade SAM, are genetically dissimilar to one another and the SAMase in T3. This brings into question whether these enzymes all use the same mechanism to break SAM down. To gain a better understanding of how these SAMases work, Guo, Söderholm, Kanchugal, Isaksen et al. solved the crystal structure of one of the newly discovered enzymes called Svi3-3. This revealed three copies of the Svi3-3 enzyme join together to form a unit that SAM binds to at the border between two of the enzymes. Computer simulations of this structure suggested that Svi3-3 holds SAM in a position where it cannot interact with water, and that once in the grip of the SAMase, SAM instead reacts with itself and splits into two. Experiments confirmed these predictions for Svi3-3 and the other tested SAMases. Furthermore, the SAMase from bacteriophage T3 was also found to degrade SAM using the same mechanism. This shows that this group of SAMases are not hydrolases as originally thought, but in fact ‘lyases’: enzymes that break molecules apart without using water. These findings form a starting point for further investigations into how SAM lyases help bacteriophages evade detection. SAM has various different functions in other living organisms, and these lyases could be used to modulate the levels of SAM in future studies investigating its role.

From previous work, it was identified two analytes of interest, produced by Lacinutrix sp. The two analytes are iso-branched lyso-ornithine lipids with only one CH2 group differing between the two compounds, in the hydrocarbon chain (lysoC15:0 and C16:0). The bacterium was isolated from the marine sponge Halicondria sp. that was collected beside Bjørnøya, on a research cruise in 2009. The analytes were identified and nominated for isolation based on bioactivity of fractionated extract from the bacterium. Unfortunately, the isolated quantities of the two were too low to allow bioactivity profiling. In the current project, the aim was therefore to generate more of the pure compounds to further perform bioactivity profiling. This was done by performing a large-scale fermentation of the bacterium. Further, the exudates of the bacterium were extracted, fractionated, and the targeted compounds were purified then tested for bioactivity in a dose-response manner on a variety of assays, with the aim to generate bioactivity data. The isolation is therefore targeted, meaning that we already knew what compounds that are to be isolated. The bioactivity results showed an antimicrobial activity against S. agalactiae and a modest antimicrobial activity against E.faecialis and S. aureus, only for compound 1. No antimicrobial activity was displayed against the tested Gram-negative bacteria for both compounds. Cytotoxic assay was also run for the cell line A2058 (human melanoma), and activity was observed only for compound 2. No cytotoxic activity was observed against the cell line MRC-5 (non-malignant cells). These surprising results of almost identical structures indicate that the length of the hydrocarbon chain contribute to differences in activity, as the results display a selective activity against bacterial cells for compound 1 and a selective activity against human melanoma cells for compound 2. No cytotoxic activity was observed against the cell line MRC-5 (non-malignant cells)

Although several hypotheses have been proposed, the significance of secondary genetic elements remains uncertain. In an effort to extend our current knowledge on multipartite genomes we studied how different gene categories are distributed and expressed in Pseudoalteromonas. Our data support that secondary replicons are open and flexible in nature, and likely serve a variety of specialized roles in bacteria. This work also adds to the growing body of evidence demonstrating that bacterial cells are highly organized at all levels., Bacterial genomes typically consist of one large chromosome, but can also include secondary replicons. These so-called multipartite genomes are scattered on the bacterial tree of life with the majority of cases belonging to Proteobacteria. Within the class gamma-proteobacteria, multipartite genomes are restricted to the two families Vibrionaceae and Pseudoalteromonadaceae. Whereas the genome of vibrios is well studied, information on the Pseudoalteromonadaceae genome is much scarcer. We have studied Pseudoalteromonadaceae with respect to the origin of the chromid, how pangene categories are distributed, how genes are expressed relative to their genomic location, and identified chromid hallmark genes. We calculated the Pseudoalteromonadaceae pangenome based on 25 complete genomes and found that core/softcore are significantly overrepresented in late replicating sectors of the chromid, regardless of how the chromid is replicated. On the chromosome, core/softcore and shell/cloud genes are only weakly overrepresented at the chromosomal replication origin and termination sequences, respectively. Gene expression is trending downwards with increasing distance from the chromosomal oriC, whereas the chromidal expression pattern is more complex. Moreover, we identified 78 chromid hallmark genes, and BLASTp searches suggest that the majority of them were acquired from the ancestral gene pool of Alteromonadales. Finally, our data strongly suggest that the chromid originates from a plasmid that was acquired in a relatively recent event. In summary, this study extends our knowledge on multipartite genomes, and helps us understand how and why secondary replicons are acquired, why they are maintained, and how they are shaped by evolution.

This is the peer reviewed version of the following article: Silk, Price, Mohanty, Leiros, Lund, Thompson, Chalmers. Side-Chain Interactions in d/l Peptide Nanotubes: Studies by Crystallography, NMR Spectroscopy and Molecular Dynamics. Chemistry - A European Journal. 2021, which has been published in final form at https://doi.org/10.1002/chem.202102106. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited. Our understanding of the factors affecting the stability of cyclic d / l peptide (CP) nanotubes remains underdeveloped. In this work, we investigate the impact of side chain alignment, hydrophobicity and charge on CP nanotube stability through X-ray crystallography, NMR spectroscopy and molecular dynamics (MD) simulations. We characterise the distinct CP-CP alignments that can form and identify stable and unstable dimers by MD simulation. We measure H-bond half-lives of synthesised CPs by 1 H-D exchange experiments and find good correlation with predicted CP-CP stabilities. We find that hydrophobic amino acids improve CP dimer stability but experimentally reduce solubility. Charged amino acids either increase or decrease CP dimer stability depending on the relative orientation and composition of charged groups. X-ray crystal structures are solved for two CPs, revealing non-tubular folded conformations. Ultimately, this work will assist the educated design of stable tubular structures for potential applications in biomedicine.

Down syndrome (DS) is a complex genetic disorder associated with substantial physical, cognitive, and behavioral challenges. Due to better treatment options for the physical co-morbidities of DS, the life expectancy of individuals with DS is beginning to approach that of the general population. However, the cognitive deficits seen in individuals with DS still cannot be addressed pharmacologically. In young individuals with DS, the level of intellectual disability varies from mild to severe, but cognitive ability generally decreases with increasing age, and all individuals with DS have early onset Alzheimer’s disease (AD) pathology by the age of 40. The present study introduces a novel inhibitor for the protein kinase DYRK1A, a key controlling kinase whose encoding gene is located on chromosome 21. The novel inhibitor is well characterized for use in mouse models and thus represents a valuable tool compound for further DYRK1A research.

Medical devices with an effective anti-colonization surface are important tools for combatting healthcare-associated infections. Here, we investigated the anti-colonization efficacy of antimicrobial peptides covalently attached to a gold model surface. The gold surface was modified by a self-assembled polyethylene glycol monolayer with an acetylene terminus. The peptides were covalently connected to the surface through a copper-catalyzed [3 + 2] azide-acetylene coupling (CuAAC). The anti-colonization efficacy of the surfaces varied as a function of the antimicrobial activity of the peptides, and very effective surfaces could be prepared with a 6 log unit reduction in bacterial colonization.

Background - Para-nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs. Methods - Cyclohexylamine inhibited four different APs. Co-crystallization with the cold-active Vibrio AP (VAP) was performed and the structure solved. Results - Inhibition of VAP fitted a non-competitive kinetic model (Km unchanged, Vmax reduced) with IC50 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC50 27.9 mM at pH 8.0, where the addition of 0.5 M NaCl altered the inhibition to the level observed at pH 9.8. APs from E. coli and calf intestines were less sensitive to cyclohexylamine, whereas an Antarctic bacterial AP was similar to VAP in this respect. X-ray crystallography at 2.3 Å showed two binding sites, one in the active site channel and another at the surface close to dimer interface. Antarctic bacterial AP and VAP have Trp274 in common in their active-sites, that takes part in binding cyclohexylamine. VAP variants W274A, W274K, and W274H gave IC50 values of 179 mM, 188 mM and 187 mM, respectively, at pH 9.8. Conclusions - The binding of cyclohexylamine in locations at the dimeric interface and/or in the active site of APs may delay product release or reduce the rate of catalytic step(s) involving conformational changes and intersubunit communications. General significance - Cyclohexylamine is a common chemical in industries and used as a counterion in substrates for alkaline phosphatase, a clinically important and common enzyme in the biosphere.

1H NMR spectroscopy has become an important technique for the characterization of transition-metal hydride complexes, whose metal-bound hydrides are often difficult to locate by X-ray diffraction. In this regard, the accurate prediction of 1H NMR chemical shifts provides a useful, but challenging, strategy to help in the interpretation of the experimental spectra. In this work, we establish a density-functional-theory protocol that includes relativistic, solvent, and dynamic effects at a high level of theory, allowing us to report an accurate and reliable interpretation of 1H NMR hydride chemical shifts of iridium polyhydride complexes. In particular, we have studied in detail the hydride chemical shifts of the [Ir6(IMe)8(CO)2H14]2+ complex in order to validate previous assignments. The computed 1H NMR chemical shifts are strongly dependent on the relativistic treatment, the choice of the DFT exchange–correlation functional, and the conformational dynamics. By combining a fully relativistic four-component electronic-structure treatment with ab initio molecular dynamics, we were able to reliably model both the terminal and bridging hydride chemical shifts and to show that two NMR hydride signals were inversely assigned in the experiment., A new strategy by computational 1H NMR spectroscopy was used to predict and support the characterization of complex metal polyhydrides. A density-functional-theory protocol including relativistic, solvent, and dynamic effects at a high level of theory was established for the [Ir6(IMe)8(CO)2H14]2+ complex. The combination of a relativistic four-component electronic-structure treatment with ab initio molecular dynamics provided a means to accurately model both terminal and bridging hydride shifts, showing that two signals were inversely assigned in the NMR experiment.

This study highlights the importance of a hydrogen bonding network between the subunits in a homodimeric enzyme involving a large loop for maintaining a high catalytic rate. Although increased local flexibility in the opposite subunit increased when the network was broken by producing a loop variant, the catalytic rate was halved, contrary to expectations., The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability. Here, we designed several different variants of VAP with the aim of removing intersubunit interactions at the dimer interface. Breaking the intersubunit contacts from one residue in particular (Arg336) reduced the temperature stability of the catalytically potent conformation and caused a 40% drop in catalytic rate. The high catalytic rates of enzymes from cold‐adapted organisms are often associated with increased dynamic flexibility. Comparison of the relative B‐factors of the R336L crystal structure to that of the wild‐type confirmed surface flexibility was increased in a loop on the opposite monomer, but not in the large loop. The increase in flexibility resulted in a reduced catalytic rate. The large loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half‐of‐sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl group affinity (inactive).

Carbapenemases are enzymes able to hydrolyze the last resort b-lactam antibiotics (carbapenems), which are used for the treatment of infections caused by resistant bacteria. Carbapenemases are structurally and mechanistically classified into serine-b-lactamases (SBLs) and metallo-b-lactamases (MBLs). In order to combat the hydrolytic activity of these enzymes, combination therapy of b-lactam with b-lactamase inhibitor have been clinically successful. Nevertheless, clinically approved inhibitors for a number of important carbapenemases are still missing and resistance against some of the clinically successful combinations have been already reported. Therefore, there is an urgent need to find new effective inhibitors that could potentially reach clinical use. The approach targeted in this thesis is to design new inhibitors against carbapenemases that could be used in the combination therapy with a carbapenem antibiotic to restore its effect. The goal of my work was to develop synthetic methods for the synthesis of inhibitors targeting two clinically relevant carbapenemases - the serine-b-lactamase oxacillinase 48 (OXA-48) and the metallo-b-lactamase Verona integron-encoded metallo-b-lactamase (VIM-2). For the design and development of inhibitors, a fragment-based approach based on previously discovered inhibitory fragments and structural data of the fragments in complex with the target enzymes was choosen. In this thesis I discuss the developed synthetic strategy towards unsymmetrical 3,5-disubstituted benzoic acids using selective Suzuki-Miyaura cross-coupling. Applying the developed method, I synthesized a small extended fragment library of both symmetrical and unsymmetrical 3,5-disubstituted benzoic acids targeting OXA-48. The aim of synthesizing these extended fragments was to target two directions in the binding pocket as suggested by overlaying structural data of smaller fragments in complex with OXA-48. I also developed a synthetic strategy towards 2-aroylbenzoic acid analogues via carbonylative Suzuki coupling using CO in a safe fashion. 2-Aroylbenzoic acids were known to inhibit the carbapenemase VIM-2. Through my investigations about a general synthetic strategy towards 2-aroylbenzoic acid, I found some limitations about substrates with ionizable functional groups and sterically hindered substrates. I then extended my investigation to find better reaction conditions for carbonylative coupling reactions. I also introduced sustainability to the project by using renewable solvents aiming for better reactivity in palladium-catalyzed C-C, C-O, C-N bond forming carbonylative couplings. In summary, through the presented work a range of carbapenemase (OXA-48 and VIM-2) inhibitors have been synthesized. Additionally, the developed synthetic strategies are considered to be a starting point to build a general approach to synthesize a wide range of potent inhibitors against carbapenemases. The work resulted in three publications (Paper I, II, III).

The design and synthesis of copper complexes that can reduce nitrite to NO has attracted considerable interest. They have been guided by the structural information on the catalytic Cu centre of the widespread enzymes Cu nitrite reductases but the chemically novel side-on binding of NO observed in all crystallographic studies of these enzymes has been questioned in terms of its functional relevance. We show conversion of NO2− to NO in the crystal maintained at 170 K and present ‘molecular movies’ defining events during enzyme turnover including the formation of side-on Cu-NO intermediate. DFT modelling suggests that both true {CuNO}11 and formal {CuNO}10 states may occur as side-on forms in an enzymatic active site with the stability of the {CuNO}10 side-on form governed by the protonation state of the histidine ligands. Formation of a copper-nitrosyl intermediate thus needs to be accommodated in future design templates for functional synthetic Cu-NiR complexes., Observation of side-on copper-nitrosyl intermediate and its confirmation by DFT during catalysis of copper nitrite reductases.

Background The genome of Vibrionaceae bacteria, which consists of two circular chromosomes, is replicated in a highly ordered fashion. In fast-growing bacteria, multifork replication results in higher gene copy numbers and increased expression of genes located close to the origin of replication of Chr 1 (ori1). This is believed to be a growth optimization strategy to satisfy the high demand of essential growth factors during fast growth. The relationship between ori1-proximate growth-related genes and gene expression during fast growth has been investigated by many researchers. However, it remains unclear which other gene categories that are present close to ori1 and if expression of all ori1-proximate genes is increased during fast growth, or if expression is selectively elevated for certain gene categories. Results We calculated the pangenome of all complete genomes from the Vibrionaceae family and mapped the four pangene categories, core, softcore, shell and cloud, to their chromosomal positions. This revealed that core and softcore genes were found heavily biased towards ori1, while shell genes were overrepresented at the opposite part of Chr 1 (i.e., close to ter1). RNA-seq of Aliivibrio salmonicida and Vibrio natriegens showed global gene expression patterns that consistently correlated with chromosomal distance to ori1. Despite a biased gene distribution pattern, all pangene categories contributed to a skewed expression pattern at fast-growing conditions, whereas at slow-growing conditions, softcore, shell and cloud genes were responsible for elevated expression. Conclusion The pangene categories were non-randomly organized on Chr 1, with an overrepresentation of core and softcore genes around ori1, and overrepresentation of shell and cloud genes around ter1. Furthermore, we mapped our gene distribution data on to the intracellular positioning of chromatin described for V. cholerae, and found that core/softcore and shell/cloud genes appear enriched at two spatially separated intracellular regions. Based on these observations, we hypothesize that there is a link between the genomic location of genes and their cellular placement.

Solvents constitute the largest component for many chemical processes and substitution of nonrenewable solvents is a longstanding goal for green chemistry. Here, we show that Pd-catalyzed carbonylative couplings, such as carbonylative crosscouplings, aminocarbonylations, and alkoxycarbonylations, can be successfully realized using renewable solvents. The present research covers not only well-established renewable solvents, such as 2-methyltetrahydrofuran (2MeTHF), limonene, and dimethyl carbonate, but also recently introduced biomass-derived 1,1-diethoxyethane, isosorbide dimethyl ether, eucalyptol, rose oxide, γ-terpinene, and α-pinene. The carbonylative coupling of boronic acids and aryl bromides works well in limonene. Aminocarbonylation gave excellent results in dimethyl carbonate, α-pinene, and limonene, while alkoxycarbonylation was successful in 2MeTHF, α-pinene, γ-terpinene, and dimethyl carbonate. The developed methods based on renewable solvents can be used for the synthesis of commercial drug Trimetozine and an analogue of Itopride.

Methane oxidizing bacteria (methanotrophs) within the genus Methylobacter constitute the biological filter for methane (CH4) in many Arctic soils. Multiple Methylobacter strains have been identified in these environments but we seldom know the ecological significance of the different strains. High-Arctic peatlands in Svalbard are heavily influenced by herbivory, leading to reduced vascular plant and root biomass. Here, we have measured potential CH4 oxidation rates and identified the active methantrophs in grazed peat and peat protected from grazing by fencing (exclosures) for 18 years. Grazed peat sustained a higher water table, higher CH4 concentrations and lower oxygen (O2) concentrations than exclosed peat. Correspondingly, the highest CH4 oxidation potentials were closer to the O2 rich surface in the grazed than in the protected peat. A comparison of 16S rRNA genes showed that the majority of methanotrophs in both sites belong to the genus Methylobacter. Further analyses of pmoA transcripts revealed that several Methylobacter OTUs were active in the peat but that different OTUs dominated the grazed peat than the exclosed peat. We conclude that grazing influences soil conditions, the active CH4 filter and that different Methylobacter populations are responsible for CH4 oxidation depending on the environmental conditions., Grazing in high-Arctic peatlands leads to soil ecosystem changes selecting for niche-adapted methanotrophs.