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We herein showcase the ability of NHC-coordinated dinuclear NiI -NiI complexes to override fundamental reactivity limits of mononuclear (NHC)Ni0 catalysts in cross-couplings. This is demonstrated with the development of a chemoselective trifluoromethylselenolation of aryl iodides catalyzed by a NiI dimer. A novel SeCF3 -bridged NiI dimer was isolated and shown to selectively react with Ar-I bonds. Our computational and experimental reactivity data suggest dinuclear NiI catalysis to be operative. The corresponding Ni0 species, on the other hand, suffers from preferred reaction with the product, ArSeCF3 , over productive cross-coupling and is hence inactive.
RESUMEN
Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein's size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstract á .
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Espectrometría de Movilidad Iónica/métodos , Proteínas/química , ARN/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Gases/químicaRESUMEN
Phosphinylidene compounds R(1)R(2)P(O)H are important functionalities in organophosphorus chemistry and display prototropic tautomerism. Quantifying the tautomerization rate is paramount to understanding these compounds' tautomerization behavior, which may impact their reactivities in various reactions. We report a combined theoretical and experimental study of the initial tautomerization rate of a range of phosphinylidene compounds. Initial tautomerization rates are found to decrease in the order H3PO2 > Ph2P(O)H > (PhO)2P(O)H > PhP(O) (OAlk)H > AlkP(O)(OAlk)H ≈ (AlkO)2P(O)H, where "Alk" denotes an alkyl substituent. The combination of computational investigations with experimental validation establishes a quantitative measure for the reactivity of various phosphorus compounds, as well as an accurate predictive tool.
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UNLABELLED: Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies--broad-spectrum antivirals--are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly α-helical structures with small amounts of ß-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCE: The viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.
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Productos del Gen pol/biosíntesis , Productos del Gen pol/química , Virus de la Hepatitis B/enzimología , Algoritmos , Dicroismo Circular , Productos del Gen pol/aislamiento & purificación , Virus de la Hepatitis B/genética , Humanos , Espectrometría de Masas , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismoRESUMEN
RATIONALE: The use of RNAi for new therapeutics is becoming more widespread. To improve the development and quality control of such materials there is a need for rapid, accurate and meaningful analyses. Here, the use of negative ion nano-electrospray ionisation tandem mass spectrometry with ion mobility spectrometry (nESI-MS/MS-IMS-MS) is shown to simplify data interpretation and lead to higher sequence coverage. METHODS: A set of 20-nucleotide RNA molecules was analysed using nESI-MS/MS and their sequences determined manually with the aid of the Simple Oligonucleotide Sequencer (SOS) program. The RNAs were also analysed using nESI-MS/MS-IMS-MS. This incorporates an extra step involving travelling-wave IMS separation of the product ions into groups according to the number of charges that the ions carry. Following this, the RNA sequences were determined from the separated groups of ions. RESULTS: nESI-MS/MS collision-induced dissociation of the RNA sequences produced w, y, a-(Base) and c product ions. Sequence determination resulted in incomplete coverage with bases in the centre of the sequences being unidentifiable because of the plethora of overlapping ions. Sequencing carried out from the nESI-MS/MS-IMS-MS data, whereby individual product ion spectra arising only from ions carrying the same charge were generated, gave full sequence coverage for each nucleotide (except y1 ) with assignment confirmation from a minimum of four different product ions. CONCLUSIONS: Using nESI-MS/MS-IMS-MS to analyse a number of 20-nucleotide RNA molecules produced full sequence coverage with 100% accuracy, in addition to molecular mass confirmation. This method has the potential for automation for higher sample throughput and thus constitutes a robust approach for the quality control of RNAs in therapeutics.
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ARN Interferente Pequeño/química , Análisis de Secuencia de ARN/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Bases , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The room-temperature hydrophosphinylation of unactivated monosubstituted alkenes using phosphinates (ROP(O)H2) and catalytic NiCl2 in the presence of dppe is described. The method is competitive with prior palladium-catalyzed reactions and uses a much cheaper catalyst and simple conditions. The scope of the reaction is quite broad in terms of unactivated terminal olefins, proceeds at room temperature, often avoids chromatographic purification, and allows one-pot conversion to various organophosphorus compounds.