Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR,and DFT Approaches

We report X‐ray crystallographic and ¹⁹F NMR studies of the G‐protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O^3G and thence to P^G. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.     

Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR,and DFT Approaches

We report X‐ray crystallographic and ¹⁹F NMR studies of the G‐protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O^3G and thence to P^G. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.     

Mechanism of the Tyrosine Ammonia Lyase Reaction—Tandem Nucleophilic and Electrophilic Enhancement by a Proton Transfer

Quantum mechanics/molecular mechanics calculations in tyrosine ammonia lyase (TAL) ruled out the hypothetical Friedel–Crafts (FC) route for ammonia elimination from L ‐tyrosine due to the high energy of FC intermediates. The calculated pathway from the zwitterionic L ‐tyrosine‐binding state (0.0 kcal mol^?1) to the product‐binding state ((E)‐coumarate+H₂N? MIO; ?24.0 kcal mol^?1; MIO=3,5‐dihydro‐5‐methylidene‐4H‐imidazol‐4‐one) involves an intermediate (IS, ?19.9 kcal mol^?1), which has a covalent bond between the N atom of the substrate and MIO, as well as two transition states (TS1 and TS2). TS1 (14.4 kcal mol^?1) corresponds to a proton transfer from the substrate to the N1 atom of MIO by Tyr300? OH. Thus, a tandem nucleophilic activation of the substrate and electrophilic activation of MIO happens. TS2 (5.2 kcal mol^?1) indicates a concerted C? N bond breaking of the N‐MIO intermediate and deprotonation of the pro‐S β position by Tyr60. Calculations elucidate the role of enzymic bases (Tyr60 and Tyr300) and other catalytically relevant residues (Asn203, Arg303, and Asn333, Asn435), which are fully conserved in the amino acid sequences and in 3D structures of all known MIO‐containing ammonia lyases and 2,3‐aminomutases.     

Theoretical study on the deglycosylation mechanism of rice BGlu1 β‐glucosidase

It is proposed that the catalysis of GH1 enzymes follows a double‐displacement mechanism involving a glycosylation and a deglycosylation steps. In this article, the deglycosylation step was studied using quantum mechanical/molecular mechanical (QM/MM) approach. The calculation results reveal that the nucleophilic water (Wat1) attacks to the anomeric C₁, and the deglycosylation step experiences a barrier of 21.4 kcal/mol from the glycosyl‐enzyme intermediate to the hydrolysis product, in which an oxocarbenium cation‐like transition state (TS) is formed. At the TS, the covalent glycosyl‐enzyme bond is almost broken (distance of 2.45 Å), and the new covalent bond between the attacking oxygen of the water molecule and C₁ is basically established (length of 2.14 Å). In addition, a short hydrogen bridge is observed between the nucleophilic E386 and the C₂? OH of sugar ring (distance of 1.94 Å) at the TS, which facilitates the ring changing from a chair form to half‐chair form, and stabilizes the oxocarbenium cation‐like TS. © 2013 Wiley Periodicals, Inc.     

Boosting Proton Conductivity in Highly Robust 3D Inorganic Cationic Extended Frameworks through Ion Exchange with Dihydrogen Phosphate Anions

The limited long‐term hydrolytic stability of rapidly emerging 3D‐extended framework materials (MOFs, COFs, MOPs, etc.) is still one of major barriers for their practical applications as new solid‐state electrolytes in fuel cells. To obtain hydrolytically stable materials, two H₂PO₄^?‐exchanged 3D inorganic cationic extended frameworks (CEFs) were successfully prepared by a facile anion‐exchange method. Both anion‐exchanged CEFs (YbO(OH)P and NDTBP) show significantly enhanced proton conductivity when compared with the original materials (YbO(OH)Cl and NDTB) with an increase of up to four orders‐of‐magnitude, reaching 2.36×10^?3 and 1.96×10^?2 S cm^?1 at 98 % RH and 85 °C for YbO(OH)P and NDTBP, respectively. These values are comparable to the most efficient proton‐conducting MOFs. In addition, these two anion‐exchanged materials are stable in boiling water, which originates from the strong electrostatic interaction between the H₂PO₄^? anion and the cationic host framework, showing a clear advance over all the acid‐impregnated materials (H₂SO₄@MIL‐101, H₃PO₄@MIL‐101, and H₃PO₄@Tp‐Azo) as practical solid‐state fuel‐cell electrolytes. This work offers a new general and efficient approach to functionalize 3D‐extended frameworks through an anion‐exchange process and achieves water‐stability with ultra‐high proton conductivity above 10^?2 S cm^?1.     

Light‐Driven Water Splitting Mediated by Photogenerated Bromine

Light‐driven water splitting was achieved using a dye‐sensitized mesoporous oxide film and the oxidation of bromide (Br^?) to bromine (Br₂) or tribromide (Br₃^?). The chemical oxidant (Br₂ or Br₃^?) is formed during illumination at the photoanode and used as a sacrificial oxidant to drive a water oxidation catalyst (WOC), here demonstrated using [Ru(bda)(pic)₂], ( 1 ; pic=picoline, bda=2,2′‐bipyridine‐6,6′‐dicarboxylate). The photochemical oxidation of bromide produces a chemical oxidant with a potential of 1.09 V vs. NHE for the Br₂/Br^? couple or 1.05 V vs. NHE for the Br₃^?/Br^? couple, which is sufficient to drive water oxidation at 1 (Ru^V/IV≈1.0 V vs. NHE at pH 5.6). At pH 5.6, using a 0.2 m acetate buffer containing 40 mm LiBr and the [Ru(4,4′‐PO₃H₂‐bpy)(bpy)₂]²⁺ ( RuP ²⁺, bpy=2,2′‐bipyridine) chromophore dye on a SnO₂/TiO₂ core–shell electrode resulted in a photocurrent density of around 1.2 mA cm^?2 under approximately 1 Sun illumination and a Faradaic efficiency upon addition of 1 of 77 % for oxygen evolution.     

Mechanism of the Water–Gas Shift Reaction Catalyzed by Efficient Ruthenium‐Based Catalysts: A Computational and Experimental Study

Supported ionic liquid phase (SILP) catalysis enables a highly efficient, Ru‐based, homogeneously catalyzed water‐gas shift reaction (WGSR) between 100 °C and 150 °C. The active Ru‐complexes have been found to exist in imidazolium chloride melts under operating conditions in a dynamic equilibrium, which is dominated by the [Ru(CO)₃Cl₃]^? complex. Herein we present state‐of‐the‐art theoretical calculations to elucidate the reaction mechanism in more detail. We show that the mechanism includes the intermediate formation and degradation of hydrogen chloride, which effectively reduces the high barrier for the formation of the requisite dihydrogen complex. The hypothesis that the rate‐limiting step involves water is supported by using D₂O in continuous catalytic WGSR experiments. The resulting mechanism constitutes a highly competitive alternative to earlier reported generic routes involving nucleophilic addition of hydroxide in the gas phase and in solution.     

Host–Guest Chemistry: Oxoanion Recognition Based on Combined Charge‐Assisted C−H or Halogen‐Bonding Interactions and Anion⋅⋅⋅Anion Interactions Mediated by Hydrogen Bonds

Several bis‐triazolium‐based receptors have been synthesized and their anion‐recognition capabilities have been studied. The central chiral 1,1′‐bi‐2‐naphthol (BINOL) core features either two aryl or ferrocenyl end‐capped side arms with central halogen‐ or hydrogen‐bonding triazolium receptors. NMR spectroscopic data indicate the simultaneous occurrence of several charge‐assisted aliphatic and heteroaromatic C?H noncovalent interactions and combinations of C?H hydrogen and halogen bonding. The receptors are able to selectively interact with HP₂O₇^3?, H₂PO₄^?, and SO₄^2? anions, and the value of the association constant follows the sequence: HP₂O₇^3?>SO₄^2?>H₂PO₄^?. The ferrocenyl end‐capped 7²⁺?2 BF₄ ^? receptor allows recognition and differentiation of H₂PO₄^? and HP₂O₇^3? anions by using different channels: H₂PO₄^? is selectively detected through absorption and emission methods and HP₂O₇^3? by using electrochemical techniques. Significant structural results are the observation of an anion???anion interaction in the solid state (2:2 complex, 6²⁺? [ H₂P₂O₇ ] ^2? ), and a short C?I???O contact is observed in the structure of the complex [ 8 ²⁺][SO₄]_0.5[BF₄].     

Synthesis,Physical Properties,and Anion Recognition of Two Novel Larger Azaacenes: Benzannelated Hexazaheptacene and Benzannelated N,N′‐Dihydrohexazaheptacene

Two novel larger azaacenes with six or ten N atoms in their backbones, benzannelated 9,11,13,22,24,26‐hexazatetrabenzo[a,c,l,n]heptacene ( HATBH , 1 ) and benzannelated 9,26‐dihydro‐9,11,13,22,24,26‐hexaza‐tetrapyrido[3,2‐a: 2′,3′‐c: 3′′,2′′‐l: 2′′′,3′′′‐n]heptacene ( DHATPH, 2 ), have been successfully synthesized in two steps. The theoretical band gaps estimated through DFT calculations for HATBH ( 1 ) and DHATPH ( 2 ) are 1.949 eV and 2.278 eV, which are close to the experimentally obtained optical band gaps (2.14 eV and 2.39 eV). Interestingly, HATBH ( 1 ) can act as efficient anion sensor for F^? and H₂PO₄^?, while DHATPH ( 2 ) selectively responds to F^? among the ten different anions used (F^?, Cl^?, Br^?, I^?, PF₆^?, HSO₄^?, NO₃^?, BF₄^?, AcO^?, and H₂PO₄^?). Our synthetic strategy could offer a promising and easy way to obtain even larger azaacenes.     

Efficient Noble‐Metal‐Free γ‐Fe2O3@NiO Core–Shell Nanostructured Photocatalysts for Water Oxidation

Flowerlike noble‐metal‐free γ‐Fe₂O₃@NiO core–shell hierarchical nanostructures have been fabricated and examined as a catalyst in the photocatalytic oxidation of water with [Ru(bpy)₃](ClO₄)₂ as a photosensitizer and Na₂S₂O₈ as a sacrificial electron acceptor. An apparent TOF of 0.29 μmols^?1 m^?2 and oxygen yield of 51 % were obtained with γ‐Fe₂O₃@NiO. The γ‐Fe₂O₃@NiO core–shell hierarchical nanostructures could be easily separated from the reaction solution whilst maintaining excellent water‐oxidation activity in the fourth and fifth runs. The surface conditions of γ‐Fe₂O₃@NiO also remained unchanged after the photocatalytic reaction, as confirmed by X‐ray photoelectron spectroscopy (XPS).     

Time‐Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile

The reaction mechanism of nitrile hydratase (NHase) was investigated using time‐resolved crystallography of the mutant NHase, in which βArg56, strictly conserved and hydrogen bonded to the two post‐translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2–1.3 Å). In combination with FTIR analyses of NHase following hydration in H₂¹⁸O, we propose that the metal‐coordinated substrate is nucleophilically attacked by the O(SO^?) atom of αCys114‐SO^?, followed by nucleophilic attack of the S(SO^?) atom by a βArg56‐activated water molecule to release the product amide and regenerate αCys114‐SO^?.     

A Fluorescent Chemosensor for Dihydrogen Phosphate Ion Based on 2‐[2‐Hydroxy‐4‐(diethylamino) phenyl]‐1H‐imidazo[4,5‐b]phenazine‐Fe3+ Ensemble

A long wavelength emission fluorescent (612 nm) chemosensor with high selectivity for H₂PO₄^? ions was designed and synthesized according to the excited state intramolecular proton transfer (ESIPT). The sensor can exist in two tautomeric forms ('keto' and 'enol') in the presence of Fe³⁺ ion, Fe³⁺ may bind with the 'keto' form of the sensor. Furthermore, the in situ generated GY‐Fe³⁺ ensemble could recover the quenched fluorescence upon the addition of H₂PO₄^? anion resulting in an off‐on‐type sensing with a detection limit of micromolar range in the same medium, and other anions, including F^?, Cl^?, Br^?, I^?, AcO^?, HSO₄^?, ClO₄^? and CN^? had nearly no influence on the probing behavior. The test strips based on 2‐[2‐hydroxy‐4‐(diethylamino) phenyl]‐1H‐imidazo[4,5‐b]phenazine and Fe³⁺ metal complex ( GY‐Fe³⁺ ) were fabricated, which could act as convenient and efficient H₂PO₄^? test kits.     

A pH‐responsive cyclopolymer having phospho‐ and sulfopropyl pendents in the same repeating unit: Synthesis,characterization, and its application as an antiscalant

A new zwitterionic monomer 3‐[diallyl{3‐(diethoxyphosphoryl)propyl}ammonio]propane‐1‐sulfonate has been synthesized and cyclopolymerized to give the corresponding polyzwitterion (±) (PZ) bearing both phosphonate and sulfonate functionalities on each repeating unit. phosphonate ester hydrolysis in PZ gave a pH‐responsive dibasic polyzwitterionic acid (±) (PZA) bearing ? PO₃H₂ units. The PZA under pH‐induced transformation was converted into polyzwitterion/anion (± ?) (PZAN) and polyzwitterion/dianion (± =) (PZDAN) having respective ? PO₃H^? and ? PO₃^2? units. The polymers′ interesting solubility and viscosity behaviors have been investigated in detail. The apparent protonation constants in salt‐free water and 0.1 M NaCl of the ? PO₃^2? in (± =) (PZDAN) and ? PO₃H^– in (± ?) (PZAN) as well as in their corresponding monomeric units have been determined. Evaluation of antiscaling properties of the PZA using supersaturated solution of CaSO₄ revealed ≈100% scale inhibition efficiency at a meager concentration of 20 ppm for a duration of 45 h at 40 °C. The PZA has the potential to be used effectively as an antiscalant in reverse osmosis plant. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 5130–5142     

2‐Amino­anilinium phosphite

The solid‐state structure of the title compound, alternatively called 2‐amino­anilinium hydrogen phosphonate, C₆H₉N₂⁺·H₂PO₃^?, shows the monoprotonated di­amine mol­ecule to be multiply hydrogen bonded to HPO₃H^? anions. There is no inter‐phosphite hydrogen bonding, contrary to previous solid‐state observations of the species.     

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

The 1994 structure of a transition‐state analogue with AlF₄⁻ and GDP complexed to G1α, a small G protein, heralded a new field of research into the structure and mechanism of enzymes that manipulate the transfer of phosphoryl (PO₃⁻) groups. The number of enzyme structures in the PDB containing metal fluorides (MF_x) as ligands that imitate either a phosphoryl or a phosphate group was 357 at the end of 2016. They fall into three distinct geometrical classes: 1) Tetrahedral complexes based on BeF₃⁻ that mimic ground‐state phosphates; 2) octahedral complexes, primarily based on AlF₄⁻, which mimic “in‐line” anionic transition states for phosphoryl transfer; and 3) trigonal bipyramidal complexes, represented by MgF₃⁻ and putative AlF₃⁰ moieties, which mimic the geometry of the transition state. The interpretation of these structures provides a deeper mechanistic understanding into the behavior and manipulation of phosphate monoesters in molecular biology. This Review provides a comprehensive overview of these structures, their uses, and their computational development.     

Exchange reactions of alkyl residues in the presence of orthophosphoric and benzenephosphonic monoesters

Experimental data obtained from the study of the behaviour of monoalkyl phosphates ROP(O)(OH)O^? or monoalkyl phosphonates C₆H₅P(O)(OR)O^? in aquo-alcoholic (H₂O + R′OH) solutions suggest: (1) in the case of monoalkyl phosphonates the exchange of R and R′ must proceed by a direct alcoholysis; (2) in the case of monoalkyl phosphates, where the scission of the ester at about pH 4,5 proceeds via the intermediate formation of metaphosphate ion PO₃^?, R′OPO₃H^? can also be formed by the addition of R′OH to PO₃^? (competitive to the addition of water) besides direct alcoholysis of ROPO₃H^?.     

Synthesis and Molecular Recognition of Water‐Soluble S6‐Corona[3]arene[3]pyridazines

We report the efficient and scalable synthesis and molecular‐recognition properties of novel and water‐soluble S₆‐corona[3]arene[3]pyridazines. The synthesis comprises a one‐pot nucleophilic aromatic substitution reaction between diesters of 2,5‐dimercaptoterephthalate and 3,6‐dichlorotetrazine followed by the inverse electron‐demand Diels–Alder reaction of the tetrazine moieties with an enamine and exhaustive saponification of esters. The resulting S₆‐corona[3]arene[3]pyridazines, which adopt a 1,3,5‐alternate conformation in the crystalline state, are able to selectively form stable 1:1 complexes with dicationic guest species in water with association constants ranging from (1.10±0.06)×10³ M ^?1 to (1.18±0.06)×10⁵ M ^?1. The easy availability, large cavity size, strong and selective binding power render the water‐soluble S₆‐corona[3]arene[3]pyridazines useful macrocyclic hosts in various disciplines of supramolecular chemistry.     

Anion Recognition Using Novel and Colorimetric Tweezer Receptors:1,3-Phenylenedi(carbonylhydrazone) in Aqueous-organic Binary Solvents

本文设计合成了2种新型的间苯二甲酰腙类钳形受体。在DMSO和DMSO-H2O混合溶液中,通过紫外可见光谱分别考察了受体分子3a对F-, Cl-, Br-, I-, AcO-, HSO4-, H2PO4-和ClO4-的相互作用。结果表明,在DMSO溶液中,受体3a对F-,CH3COO-和H2PO4-有显著识别效果,溶液颜色由无色变为黄色,实现裸眼检测。在15%H2O-85%DMSO含水体系中,3a可高选择性识别CH3COO-。1H NMR滴定表明过量F-的加入使受体分子3a发生脱质子作用,探讨了主客体之间的作用机理。并直接用于水相中无机醋酸盐的直接显色检测。     

Influence of Decreasing Solvent Polarity (1,4‐Dioxane/Water Mixtures) on the Acid–Base and Copper(II)‐Binding Properties of Guanosine 5′‐Diphosphate

The acidity constants of twofold protonated guanosine 5′‐diphosphate, H₂(GDP)^?, and the stability constants of the [Cu(H;GDP)] and [Cu(GDP)]^? complexes were determined in H₂O as well as in 30 or 50% (v/v) 1,4‐dioxane/H₂O by potentiometric pH titrations (25°; I=0.1M , NaNO₃). The results showed that in H₂O one of the two protons of H₂(GDP)^? is located mainly at the N(7) site and the other one at the terminal β‐phosphate group. In contrast, for 50% 1,4‐dioxane/H₂O solutions, a micro acidity‐constant evaluation evidenced that ca. 75% of the H₂(GDP)^? species have both protons phosphate‐bound, because the basicity of pyridine‐type N sites decreases with decreasing solvent polarity whereas the one of phosphate groups increases. In the [Cu(H;GDP)] complex, the proton and the metal ion are in all three solvents overwhelmingly phosphate‐bound, and the release of this proton is inhibited by decreasing polarity of the solvent. Based on previously determined straight‐line plots of log K vs. pK (where R represents a non‐interacting residue in simple diphosphate monoesters ROP(O^?)(?O)? O? P(?O)(O^?)₂, R? DP^3?), which were now extended to mixed solvents (based on analogies), it is concluded that, in all three solvents, the [Cu(GDP)]^? complex is more stable than expected based on the basicity of the diphosphate residue. This increased stability is attributed to macrochelate formation of the phosphate‐coordinated Cu²⁺ with N(7) of the guanine residue. The formation degree of this macrochelate amounts in aqueous solution to ca. 75% (being thus higher than that of the Cu²⁺ complex of adenosine 5′‐diphosphate) and in 50% (v/v) 1,4‐dioxane/H₂O to ca. 60%, i.e., the formation degree of the macrochelate is only relatively little affected by the change in solvent, though it needs to be emphasized that the overall stability of the [Cu(GDP)]^? complex increases with decreasing solvent polarity. By including previously studied systems in the considerations, the biological implications are shortly discussed, and it is concluded that Nature has here a tool to alter the structure of complexes by shifting them on a protein surface from a polar to an apolar region and vice versa.     

Single‐Site Copper(II) Water Oxidation Electrocatalysis: Rate Enhancements with HPO42− as a Proton Acceptor at pH 8

The complex Cu^II(Py₃P) ( 1 ) is an electrocatalyst for water oxidation to dioxygen in H₂PO₄^?/HPO₄^2? buffered aqueous solutions. Controlled potential electrolysis experiments with 1 at pH 8.0 at an applied potential of 1.40 V versus the normal hydrogen electrode resulted in the formation of dioxygen (84 % Faradaic yield) through multiple catalyst turnovers with minimal catalyst deactivation. The results of an electrochemical kinetics study point to a single‐site mechanism for water oxidation catalysis with involvement of phosphate buffer anions either through atom–proton transfer in a rate‐limiting O? O bond‐forming step with HPO₄^2? as the acceptor base or by concerted electron–proton transfer with electron transfer to the electrode and proton transfer to the HPO₄^2? base.