2-Alkylphosphino-1-boraadamantanes.

Boraadamantanes are a privileged class of caged group 13 compounds having a trigonal pyramidal (non-VSEPR) ground-state. The Lewis acid-base chemistry of this type of compound is underdeveloped when compared to acyclic derivatives. This report provides the first examples of bifunctional boraadamantanes with an appended phosphine moiety (also the first boraadamantanes having a phosphorus-boron bond). Using this scaffold, boraadamantane coordination compounds are accessed for the first time.

Fluorinated Dialkyl Chloronium Salts: Synthesis and Reactivity for Fluoroalkylation and Hydride Abstraction.

A new concept for the synthesis of dialkyl chloronium cations [R‒Cl‒R]+ is described (R = CH3, CH2CF3), that allows the formation of fluorinated derivatives. By utilizing the xenonium salt [XeOTeF5][M(OTeF5)n] (M = Sb, n = 6; M = Al, n = 4) chlorine atoms of chloroalkanes or the deactivated chlorofluoroalkane CH2ClCF3 are oxidized and removed as ClOTeF5 leading to the isolation of the corresponding chloronium salt. Since the resulting highly electrophilic cation [Cl(CH2CF3)2]+ is able to alkylate weak nucleophiles, this compound can be utilized for the introduction of a fluorinated alkyl group to those. In addition, the fluorinated alkyl chloronium cation displays a high hydride ion affinity, enabling the activation of linear hydrocarbons by hydride abstraction even at low temperatures ultimately leading to the formation of branched carbocations.

Questing for homoleptic mononuclear manganese complexes with monodentate O-donor ligands.

Compounds containing Mn-O bonds are of utmost importance in biological systems and catalytic processes. Nevertheless, mononuclear manganese complexes containing all O-donor ligands are still rare. Taking advantage of the low tendency of the pentafluoroorthotellurate ligand (teflate, OTeF5) to bridge metal centers, we have synthesized two homoleptic manganese complexes with monomeric structures and an all O-donor coordination sphere. The tetrahedrally distorted MnII anion, [Mn(OTeF5)4]2-, can be described as a high spin d5 complex (S = 5/2), as found experimentally (magnetic susceptibility measurements and EPR spectroscopy) and using theoretical calculations (DFT and CASSCF/NEVPT2). The high spin d4 electronic configuration (S = 2) of the MnIII anion, [Mn(OTeF5)5]2-, was also determined experimentally and theoretically, and a square pyramidal geometry was found to be the most stable one for this complex. Finally, the bonding situation in both complexes was investigated by means of the Interacting Quantum Atoms (IQA) methodology and compared to that of hypothetical mononuclear fluoromanganates. Within each pair of [MnXn]2- (n = 4, 5) species (X = OTeF5, F), the Mn-X interaction is found to be comparable, therefore proving that the similar electronic properties of the teflate and the fluoride are also responsible for the stabilization of these unique species.

A Highly Sterically Encumbered Boron Lewis Acid Enabled by an Organotellurium-Based Ligand.

Lewis acidic boron compounds are ubiquitous in chemistry due to their numerous applications, yet tuning and optimizing their properties towards different purposes is still a challenging field of research. In this work, the boron-based Lewis acid B[OTeF3(C6F5)2]3 was synthesized by reaction of the teflate derivative HOTeF3(C6F5)2 with BCl3 or BCl3 ⋅ SMe2. This new compound presents a remarkably high thermal stability up to 300 °C, as well as one of the most sterically encumbered boron centres known in the literature. Theoretical and experimental methods revealed that B[OTeF3(C6F5)2]3 exhibits a comparable Lewis acidity to that of the well-known B(C6F5)3. The affinity of B[OTeF3(C6F5)2]3 towards pyridine was accessed by Isothermal Titration Calorimetry (ITC) and compared to that of B(OTeF5)3 and B(C6F5)3. The ligand-transfer reactivity of this new boron compound towards different fluorides was demonstrated by the formation of an anionic Au(III) complex and a hypervalent iodine(III) species.

[Xe(OTeF5)(pyF)]+: a strong oxidizing xenonium(II) teflate cation with N-donor bases.

Herein we report on the formation of the adduct salts [Xe(OTeF5)(pyF)][Al(OTeF5)4] (pyF = C5F5N, C5H3F2N) by abstraction of an -OTeF5 group from Xe(OTeF5)2 with the Lewis superacid Al(OTeF5)3 and subsequent adduct formation of the generated [XeOTeF5]+ cation with fluorinated pyridines. These salts represent the first xenonium cations with the weakly coordinating [Al(OTeF5)4]- anion. The strong oxidizing property of these compounds is further assessed.

N, N-dimethyltryptamine forms oxygenated metabolites via CYP2D6 - an in vitro investigation.

N, N-dimethyltryptamine (DMT) is a psychedelic compound that has shown potential in the treatment of depression. Aside from the primary role of monoamine oxidase A (MAO-A) in DMT metabolism, the metabolic pathways are poorly understood. Increasing this understanding is an essential aspect of ensuring safe and efficacious use of DMT.This work aimed to investigate the cytochrome 450 (CYP) mediated metabolism of DMT by incubating DMT with recombinant human CYP enzymes and human liver microsomes (HLM) followed by analysis using high-resolution mass spectrometry for metabolite identification.DMT was rapidly metabolised by CYP2D6, while stable with all other investigated CYP enzymes. The metabolism of DMT in HLM was reduced after inclusion of harmine and SKF-525A whereas quinidine did not affect the metabolic rate, likely due to MAO-A residues present in HLM. Analysis of the CYP2D6 incubates showed formation of mono-, di- and tri-oxygenated metabolites, likely as a result of hydroxylation on the indole core.More research is needed to investigate the role of this metabolic pathway in vivo and any pharmacological activity of the proposed metabolites. Our findings may impact on safety issues following intake of ayahuasca in slow CYP2D6 metabolizers or with concomitant use of CYP2D6 inhibitors.

Air-stable aryl derivatives of pentafluoroorthotellurate.

We report on two different sets of air-stable derivatives of pentafluoroorthotellurate containing fluorinated and non-fluorinated aryl groups. The acid cis-PhTeF4OH was obtained in gram scale and further transformed to Ag[cis-PhTeF4O], which was used as a cis-PhTeF4O transfer reagent to obtain [PPh4][cis-PhTeF4O]. Furthermore, the synthesis of trans-(C6F5)2TeF3OH was achieved by a selective hydrolysis of trans-(C6F5)2TeF4 in the presence of KF and subsequent protonation by aHF. Quantum-chemical calculations show a higher acidity and robustness against fluoride abstraction for trans-(C6F5)2TeF3OH compared to cis-PhTeF4OH.

Unravelling the Role of the Pentafluoroorthotellurate Group as a Ligand in Nickel Chemistry.

The pentafluoroorthotellurate group (teflate, OTeF5 ) is able to form species, for which only the fluoride analogues are known. Despite nickel fluorides being widely investigated, nickel teflates have remained elusive for decades. By reaction of [NiCl4 ]2- and neat ClOTeF5 , we have synthesized the homoleptic [Ni(OTeF5 )4 ]2- anion, which presents a distorted tetrahedral structure, unlike the polymeric [NiF4 ]2- . This high-spin complex has allowed the study of the electronic properties of the teflate group, which can be classified as a weak/medium-field ligand, and therefore behaves as the fluoride analogue also in ligand-field terms. The teflate ligands in [NEt4 ]2 [Ni(OTeF5 )4 ] are easily substituted, as shown by the formation of [Ni(NCMe)6 ][OTeF5 ]2 by dissolving it in acetonitrile. Nevertheless, careful reactions with other conventional ligands have enabled the crystallization of nickel teflate complexes with different coordination geometries, i.e. [NEt4 ]2 [trans-Ni(OEt2 )2 (OTeF5 )4 ] or [NEt4 ][Ni(bpyMe2 )(OTeF5 )3 ].

Insights on the Lewis Superacid Al(OTeF5 )3 : Solvent Adducts, Characterization and Properties.

Preparation and characterization of the dimeric Lewis superacid [Al(OTeF5 )3 ]2 and various solvent adducts is presented. The latter range from thermally stable adducts to highly reactive, weakly bound species. DFT calculations on the ligand affinity of these Lewis acids were performed in order to rank their remaining Lewis acidity. An experimental proof of the Lewis acidity is provided by the reaction of solvent-adducts of Al(OTeF5 )3 with [PPh4 ][SbF6 ] and OPEt3 , respectively. Furthermore, their reactivity towards chloride and pentafluoroorthotellurate salts as well as (CH3 )3 SiCl and (CH3 )3 SiF is shown. This includes the formation of the dianion [Al(OTeF5 )5 ]2- .

The Tris(pentafluorophenyl)methylium Cation: Isolation and Reactivity.

Herein, we present two different routes for the synthesis of the perfluorinated trityl cation, which allowed the handling of the free, uncoordinated species in organic solvents for the first time. The usage of the weakly coordinating anion [Al(OTeF5 )4 ]- and its derivatives allows the characterization of this species by NMR spectroscopy and most importantly by single-crystal X-ray diffraction. The high hydride ion affinity of the cation is shown by hydrogen abstraction from isobutane. Furthermore, cyclic voltammetry reveals its oxidative potential which is supported by the reaction with tris(4-bromophenyl)amine, giving rise to the formation of the ammoniumyl radical cation, also known as "magic blue".

Development and application of a highly sensitive LC-MS/MS method for simultaneous quantification of N,N-dimethyltryptamine and two of its metabolites in human plasma.

A highly sensitive LC-MS/MS method for the quantification of N,N-dimethyltryptamine (DMT) and its metabolites indole-3-acetic acid and DMT N-oxide in human plasma has been developed and validated. Chromatography was performed using a diphenyl column with gradient elution (0.1% formic acid in methanol/water). The mass spectrometer was operated in multiple reaction monitoring mode. A methanolic solution containing internal standards 2-methylindole 3-acetic acid and deuterated DMT, was added to plasma samples, followed by protein precipitation with acetonitrile. The samples were centrifuged and supernatants transferred to new tubes and evaporated to dryness before reconstitution in aqueous mobile phase. The method was validated with regards to accuracy, precision, sensitivity, selectivity, recovery, matrix effects, stability, carry-over and dilution integrity. The validated linear range was 0.25-200 nM for DMT and 15-250 nM for DMT N-oxide. For the endogenous compound indole-3-acetic acid a different approach was taken due to its significant presence in blank samples. The change in signal response from a blank sample was used when constructing the calibration curve with linearity demonstrated between elevations of 500-5000 nM above the blank. Applicability of the described method was demonstrated through analysis of plasma samples from healthy volunteers having received intravenous injections of DMT. The presented method for rapid and sensitive quantification of DMT and its metabolites in human plasma can be applied to future studies aiming to characterize DMT disposition and its relationship to immediate psychedelic or long-term antidepressive effects.

Structural proof of a [C-F-C]+ fluoronium cation.

Organic fluoronium ions can be described as positively charged molecules in which the most electronegative and least polarizable element fluorine engages in two partially covalent bonding interactions to two carbon centers. While recent solvolysis experiments and NMR spectroscopic studies on a metastable [C-F-C]+ fluoronium ion strongly support the divalent fluoronium structure over the alternative rapidly equilibrating classical carbocation, the model system has, to date, eluded crystallographic analysis to confirm this phenomenon in the solid state. Herein, we report the single crystal structure of a symmetrical [C-F-C]+ fluoronium cation. Besides its synthesis and crystallographic characterization as the [Sb2F11]- salt, vibrational spectra are discussed and a detailed analysis concerning the nature of the bonding situation in this fluoronium ion and its heavier halonium homologues is performed, which provides detailed insights on this molecular structure.

Factors Affecting the Pharmacokinetics of Pyrazinamide and Its Metabolites in Patients Coinfected with HIV and Implications for Individualized Dosing.

Pyrazinamide is a first-line drug used in the treatment of tuberculosis. High exposure to pyrazinamide and its metabolites may result in hepatotoxicity, whereas low exposure to pyrazinamide has been correlated with treatment failure of first-line antitubercular therapy. The aim of this study was to describe the pharmacokinetics and metabolism of pyrazinamide in patients coinfected with tuberculosis and HIV. We further aimed to identify demographic and clinical factors which affect the pharmacokinetics of pyrazinamide and its metabolites in order to suggest individualized dosing regimens. Plasma concentrations of pyrazinamide, pyrazinoic acid, and 5-hydroxypyrazinamide from 63 Rwandan patients coinfected with tuberculosis and HIV were determined by liquid chromatography-tandem mass spectrometry followed by nonlinear mixed-effects modeling. Females had a close to 50% higher relative pyrazinamide bioavailability compared to males. The distribution volumes of pyrazinamide and both metabolites were lower in patients on concomitant efavirenz-based HIV therapy. Furthermore, there was a linear relationship between serum creatinine and oral clearance of pyrazinoic acid. Simulations indicated that increasing doses from 25 mg/kg of body weight to 35 mg/kg and 50 mg/kg in females and males, respectively, would result in adequate exposure with regard to suggested thresholds and increase probability of target attainment to >0.9 for a MIC of 25 mg/liter. Further, lowering the dose by 40% in patients with high serum creatinine would prevent accumulation of toxic metabolites. Individualized dosing is proposed to decrease variability in exposure to pyrazinamide and its metabolites. Reducing the variability in exposure may lower the risk of treatment failure and resistance development.

Simultaneous quantification of four first line antitubercular drugs and metabolites in human plasma by hydrophilic interaction chromatography and tandem mass spectrometry.

Co-infection of tuberculosis in HIV-patients is a major health concern worldwide and especially so in Sub-Saharan Africa. To enhance the study of potential drug-drug interactions when simultaneously treating the two infections, a liquid chromatography tandem mass spectrometry method was developed for the quantitation of the four first line anti-tuberculosis drugs isoniazid, rifampicin, pyrazinamide, ethambutol and four of their major metabolites in human plasma. Analytes were extracted from 200 µL of plasma using a sequential liquid-liquid extraction with ethyl acetate at neutral and acidic pH. The combined extracts were analyzed by liquid chromatography with mass spectrometric detection in a multiple reaction monitoring mode. The chromatographic separation was performed on a hydrophilic interaction column using a stepwise gradient with two mobile phases consisting of water with 0.3% formic acid and methanol with 0.3% formic acid, respectively. The total run time of each analysis was 4 min. The lower limit of quantification applied was 40 ng/mL for ethambutol, acetylisoniazid and 25-desacetylrifampicin, 60 ng/mL for 5-hydroxypyrazinamide, 80 ng/mL for isoniazid and isonicotinic acid, 200 ng/mL for rifampicin and 320 ng/mL for pyrazinamide. The method was validated according to US Food and Drug Administration guidance. The method exhibited adequate accuracy (87.1-114.9%), precision (CV < 12.8%) and specificity. Recovery and matrix effect were consistent (CV < 11.9%). The extracted samples were stable in the autosampler at 8 °C for up to 24 h as well as after three freeze-thaw cycles (recovery > 86.3%). The method has been shown to be robust for the analysis of the stated drugs and metabolites in human plasma obtained from 73 patients receiving these four first line anti-tuberculosis drugs.

Inhibition of CYP3A by Antimalarial Piperaquine and Its Metabolites in Human Liver Microsomes With IVIV Extrapolation.

The potential of the antimalarial piperaquine and its metabolites to inhibit CYP3A was investigated in pooled human liver microsomes. CYP3A activity was measured by liquid chromatography-tandem mass spectrometry as the rate of 1'-hydroxymidazolam formation. Piperaquine was found to be a reversible, potent inhibitor of CYP3A with the following parameter estimates (%CV): IC50 = 0.76 µM (29), Ki = 0.68 µM (29). In addition, piperaquine acted as a time-dependent inhibitor with IC50 declining to 0.32 µM (28) during 30-min pre-incubation. Time-dependent inhibitor estimates were kinact = 0.024 min-1 (30) and KI = 1.63 µM (17). Metabolite M2 was a highly potent reversible inhibitor with estimated IC50 and Ki values of 0.057 µM (17) and 0.043 µM (3), respectively. M1 and M5 metabolites did not show any inhibitory properties within the limits of assay used. Average (95th percentile) simulated in vivo areas under the curve of midazolam increased 2.2-fold (3.7-fold) on the third which is the last day of piperaquine dosing, whereas for its metabolite M2, areas under the curve of midazolam increased 7.7-fold (13-fold).

LC-MS/MS quantitation of antimalarial drug piperaquine and metabolites in human plasma.

PURPOSE: This study aimed to develop a sensitive, quantitative assay for the antimalarial piperaquine (PQ) and its metabolites M1 and M2 in human plasma. RESULTS: Analytes were gradiently separated on a C18 column and detected with a Sciex API 4000 MS/MS with an ESI source operated in the positive ion mode with deuterated PQ as internal standard. The response was linear in the range 3.9-2508nM with a runtime of 7.0min per sample. The method was applied to clinical samples from healthy volunteers. CONCLUSION: This LC-MS/MS method for the simultaneous quantitation of PQ and two of its metabolites in plasma may prove helpful for assessment of metabolite safety issues in vivo.

Shuffling Active Site Substate Populations Affects Catalytic Activity: The Case of Glucose Oxidase.

Glucose oxidase has wide applications in the pharmaceutical, chemical, and food industries. Many recent studies have enhanced key properties of this enzyme using directed evolution, yet without being able to reveal why these mutations are actually beneficial. This work presents a synergistic combination of experimental and computational methods, indicating how mutations, even when distant from the active site, positively affect glucose oxidase catalysis. We have determined the crystal structures of glucose oxidase mutants containing molecular oxygen in the active site. The catalytically important His516 residue has been previously shown to be flexible in the wild-type enzyme. The molecular dynamics simulations performed in this work allow us to quantify this floppiness, revealing that His516 exists in two states: catalytic and noncatalytic. The relative populations of these two substates are almost identical in the wild-type enzyme, with His516 readily shuffling between them. In the glucose oxidase mutants, on the other hand, the mutations enrich the catalytic His516 conformation and reduce the flexibility of this residue, leading to an enhancement in their catalytic efficiency. This study stresses the benefit of active site preorganization with respect to enzyme conversion rates by reducing molecular reorientation needs. We further suggest that the computational approach based on Hamiltonian replica exchange molecular dynamics, used in this study, may be a general approach to screening in silico for improved enzyme variants involving flexible catalytic residues.

Epigenetic program and transcription factor circuitry of dendritic cell development.

Dendritic cells (DC) are professional antigen presenting cells that develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Multipotent progenitors (MPP) are committed to DC restricted common DC progenitors (CDP), which differentiate into specific DC subsets, classical DC (cDC) and plasmacytoid DC (pDC). To determine epigenetic states and regulatory circuitries during DC differentiation, we measured consecutive changes of genome-wide gene expression, histone modification and transcription factor occupancy during the sequel MPP-CDP-cDC/pDC. Specific histone marks in CDP reveal a DC-primed epigenetic signature, which is maintained and reinforced during DC differentiation. Epigenetic marks and transcription factor PU.1 occupancy increasingly coincide upon DC differentiation. By integrating PU.1 occupancy and gene expression we devised a transcription factor regulatory circuitry for DC commitment and subset specification. The circuitry provides the transcription factor hierarchy that drives the sequel MPP-CDP-cDC/pDC, including Irf4, Irf8, Tcf4, Spib and Stat factors. The circuitry also includes feedback loops inferred for individual or multiple factors, which stabilize distinct stages of DC development and DC subsets. In summary, here we describe the basic regulatory circuitry of transcription factors that drives DC development.

The three-dimensional structure of VIM-31--a metallo-ß-lactamase from Enterobacter cloacae in its native and oxidized form.

The metallo-ß-lactamase VIM-31 differs from VIM-2 by only two Tyr224His and His252Arg substitutions. Located close to the active site, the Tyr224His substitution is also present in VIM-1, VIM-4, VIM-7 and VIM-12. The VIM-31 variant was reported in 2012 from Enterobacter cloacae and kinetically characterized. It exhibits globally lower catalytic efficiencies than VIM-2. In the present study, we report the three-dimensional structures of VIM-31 in its native (reduced) and oxidized forms. The so-called 'flapping-loop' (loop 1) and loop 3 of VIM-31 were not positioned as in VIM-2 but instead were closer to the active site as in VIM-4, resulting in a narrower active site in VIM-31. Also, the presence of His224 in VIM-31 disrupts hydrogen-bonding networks close to the active site. Moreover, a third zinc-binding site, which also exists in VIM-2 structures, could be identified as a structural explanation for the decreased activity of VIM-MBLs at high zinc concentrations.

Enzyme-substrate complex structures of CYP154C5 shed light on its mode of highly selective steroid hydroxylation.

CYP154C5 from Nocardia farcinica is a bacterial cytochrome P450 monooxygenase active on steroid molecules. The enzyme has recently been shown to exhibit exclusive regioselectivity and stereoselectivity in the conversion of various pregnans and androstans, yielding 16α-hydroxylated steroid products. This makes the enzyme an attractive candidate for industrial application in steroid hormone synthesis. Here, crystal structures of CYP154C5 in complex with four different steroid molecules were solved at resolutions of up to 1.9 Å. These are the first reported P450 structures from the CYP154 family in complex with a substrate. The active site of CYP154C5 forms a flattened hydrophobic channel with two opposing polar regions, perfectly resembling the size and polarity distribution of the steroids and thus resulting in highly specific steroid binding with Kd values in the range 10-100 nM. Key enzyme-substrate interactions were identified that accounted for the exclusive regioselectivity and stereoselectivity of the enzyme. Additionally, comparison of the four CYP154C5-steroid structures revealed distinct structural differences, explaining the observed variations in kinetic data obtained for this P450 with the steroids pregnenolone, dehydroepiandrosterone, progesterone, androstenedione, testosterone and nandrolone. This will facilitate the generation of variants with improved activity or altered selectivity in the future by means of protein engineering.